http://2013.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=100&target=BGI+K2&year=&month=2013.igem.org - User contributions [en]2024-03-29T10:22:55ZFrom 2013.igem.orgMediaWiki 1.16.5http://2013.igem.org/World_Championship_Jamboree/Practice_SessionsWorld Championship Jamboree/Practice Sessions2013-10-26T06:13:28Z<p>BGI K2: </p>
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<p class="contentheader">Practice Sessions - November 01</p><br />
<p>Use this sign-up sheet to sign up for a practice session slot on Friday night (<b>November 1st</b>) to practice your talk. Note that there will NOT be any A/V (audio/visual) support on staff. All classrooms will be unlocked and you should use them and leave them as you found them. Be sure to bring necessary computer equipment with you, such as chargers and adapters, as these will not be provided.</p><br />
<br />
<p>There are a limited number of time slots available on a first-come first-serve basis so please only choose one slot. We cannot match the room that you will ultimately give your presentation in with the practice room (please see the <a href="https://2013.igem.org/World_Championship_Jamboree/Map">campus map</a> for building locations). This should, however, give you a chance to practice your talk in a new environment. Please keep in mind that there will be teams waiting to use the room after you, so make sure that your practice finishes on time.</p><br />
<br />
<p>Also, pre-registration will be available on Friday November 1st, starting at 3pm at <b>Building 12, Room 156</b>. Conference services will be on-site to pass out team registration boxes (see the <a href="https://2013.igem.org/World_Championship_Jamboree/Handbook">Jamboree Handbook</a>).</p> <br />
<br />
<p><strong>Note</strong>: Use the wiki edit button to add your team to the schedule (the markup is located at the bottom of the page). Additional rooms may be added in the coming weeks.<br />
</p><br />
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<thead><br />
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<th style="width:100px;">Time</th><br />
<th>RM 32-123 </th><br />
<th>RM 32-141 </th><br />
<th>RM 32-155 </th><br />
<th>RM 34-101 </th><br />
<th>RM 56-114 </th><br />
<th>RM 56-154 </th><br />
<th>RM 66-144 </th><br />
<th>RM 66-168 </th><br />
</tr><br />
</thead><br />
<tbody><br />
<tr class="even"><br />
<th>5:30 - 6:00PM</th><br />
<td>Freiburg</td><br />
<td>Valencia Biocampus</td><br />
<td>TU-Eindhoven</td><br />
<td>Tokyo_Tech</td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
</tr><br />
<tr class="even"><br />
<th>6:00 - 6:30PM</th><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td>ZJU-China</td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
</tr><br />
<tr class="odd"><br />
<th>6:30 - 7:00PM</th><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td>NJU China</td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
</tr><br />
<tr class="even"><br />
<th>7:00 - 7:30PM</th><br />
<td></td><br />
<td></td><br />
<td>XMU Software</td><br />
<td>Peking</td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
</tr><br />
<tr class="even"><br />
<th>7:30 - 8:00PM</th><br />
<td>XMU China</td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
</tr><br />
<tr class="odd"><br />
<th>8:00 - 8:30PM</th><br />
<td></td><br />
<td></td><br />
<td>USTC-Software</td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
</tr><br />
<tr class="even"><br />
<th>8:30 - 9:00PM</th><br />
<td>TU-Munich</td><br />
<td>ETH Zurich</td><br />
<td>Shenzhen_BGIC_0101</td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
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<tr class="odd"><br />
<th>9:00 - 9:30PM</th><br />
<td></td> <br />
<td></td><br />
<td>Shenzhen_BGIC_ATCG</td><br />
<td>Paris_Bettencourt</td><br />
<td>Penn</td><br />
<td></td><br />
<td></td><br />
<td>UCSF</td><br />
</tr><br />
</tbody><br />
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<img src="https://static.igem.org/mediawiki/2013/f/f6/2013WCJ_practice_sessions_map.png" /><br />
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</html></div>BGI K2http://2013.igem.org/World_Championship_Jamboree/Practice_SessionsWorld Championship Jamboree/Practice Sessions2013-10-26T06:13:02Z<p>BGI K2: </p>
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<td><br />
<p class="contentheader">Practice Sessions - November 01</p><br />
<p>Use this sign-up sheet to sign up for a practice session slot on Friday night (<b>November 1st</b>) to practice your talk. Note that there will NOT be any A/V (audio/visual) support on staff. All classrooms will be unlocked and you should use them and leave them as you found them. Be sure to bring necessary computer equipment with you, such as chargers and adapters, as these will not be provided.</p><br />
<br />
<p>There are a limited number of time slots available on a first-come first-serve basis so please only choose one slot. We cannot match the room that you will ultimately give your presentation in with the practice room (please see the <a href="https://2013.igem.org/World_Championship_Jamboree/Map">campus map</a> for building locations). This should, however, give you a chance to practice your talk in a new environment. Please keep in mind that there will be teams waiting to use the room after you, so make sure that your practice finishes on time.</p><br />
<br />
<p>Also, pre-registration will be available on Friday November 1st, starting at 3pm at <b>Building 12, Room 156</b>. Conference services will be on-site to pass out team registration boxes (see the <a href="https://2013.igem.org/World_Championship_Jamboree/Handbook">Jamboree Handbook</a>).</p> <br />
<br />
<p><strong>Note</strong>: Use the wiki edit button to add your team to the schedule (the markup is located at the bottom of the page). Additional rooms may be added in the coming weeks.<br />
</p><br />
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<table class="calendar" align="center"><br />
<thead><br />
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<th style="width:100px;">Time</th><br />
<th>RM 32-123 </th><br />
<th>RM 32-141 </th><br />
<th>RM 32-155 </th><br />
<th>RM 34-101 </th><br />
<th>RM 56-114 </th><br />
<th>RM 56-154 </th><br />
<th>RM 66-144 </th><br />
<th>RM 66-168 </th><br />
</tr><br />
</thead><br />
<tbody><br />
<tr class="even"><br />
<th>5:30 - 6:00PM</th><br />
<td>Freiburg</td><br />
<td>Valencia Biocampus</td><br />
<td>TU-Eindhoven</td><br />
<td>Tokyo_Tech</td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
</tr><br />
<tr class="even"><br />
<th>6:00 - 6:30PM</th><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td>ZJU-China</td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
</tr><br />
<tr class="odd"><br />
<th>6:30 - 7:00PM</th><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td>NJU China</td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
</tr><br />
<tr class="even"><br />
<th>7:00 - 7:30PM</th><br />
<td></td><br />
<td></td><br />
<td>XMU Software</td><br />
<td>Peking</td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
</tr><br />
<tr class="even"><br />
<th>7:30 - 8:00PM</th><br />
<td>XMU China</td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
</tr><br />
<tr class="odd"><br />
<th>8:00 - 8:30PM</th><br />
<td></td><br />
<td></td><br />
<td>USTC-Software</td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
</tr><br />
<tr class="even"><br />
<th>8:30 - 9:00PM</th><br />
<td>TU-Munich</td><br />
<td>ETH Zurich</td><br />
<td></td><br />
<td>Shenzhen_BGIC_0101</td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
</tr><br />
<tr class="odd"><br />
<th>9:00 - 9:30PM</th><br />
<td></td> <br />
<td></td><br />
<td>Shenzhen_BGIC_ATCG</td><br />
<td>Paris_Bettencourt</td><br />
<td>Penn</td><br />
<td></td><br />
<td></td><br />
<td>UCSF</td><br />
</tr><br />
</tbody><br />
</table><br />
<br /><br />
<img src="https://static.igem.org/mediawiki/2013/f/f6/2013WCJ_practice_sessions_map.png" /><br />
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</html></div>BGI K2http://2013.igem.org/Team:Shenzhen_BGIC_ATCG/resultsTeam:Shenzhen BGIC ATCG/results2013-09-28T01:24:49Z<p>BGI K2: </p>
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<div><!----------------------<br />
<br />
<br />
Title: Results<br />
Team: Shenzhen_BGIC_ATCG<br />
Author: BGI_K2<br />
Twitter: @BGI_K2<br />
Email: im.ss.kk@gmail.com<br />
<br />
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<div class="navleftholder"><br />
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<li><a id="navleftsub1" class="navleftsub" href="#board1">The Magic</a></li><br />
<li><a id="navleftsub2" class="navleftsub" href="#board2">Promoter Verification</a></li><br />
<li><a id="navleftsub3" class="navleftsub" href="#board3">Reporter Locating</a></li><br />
<li><a id="navleftsub4" class="navleftsub" href="#board4">Degradation Rate</a></li><br />
<li><a id="navleftsub5" class="navleftsub" href="#board5">Cell Synchronization</a></li><br />
<li><a id="navleftsub6" class="navleftsub" href="#board6">Alternative Splicing</a></li><br />
</ul></div><br />
<br />
<div id="board1" class="board"></div><br />
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<br />
<ul id="myGallery1"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/></li><br />
<li><img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" title="Degradation Tags" description="Degradation tags testing device for E. coli."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" title="Microfluidics" description="Microfluidics device for degradation test."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/2/23/Sync.jpg" title="Synchronization" description="All cells are synchronized to G1 phase by using the microfluidics method."/></li><br />
</ul></div><br />
<div id="box1" class="box"><br />
<h3>The Magic</h3><br />
<p>Our project “Cell Magic", a complex work has been completed partly,due to the time limitation. The results we have gotten are listed below:</p><br />
<p>We verified some of the cyclin promoters. The clb6 promoter, which is supposed to be expressed in G1 phase, has been verified perfectly.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." width=50%/><p>Figure. clb6 promoter for G1 phase has been verified</p><br />
<p>Targeting peptides to three locations have been verified. Others are still being constructed and tested.</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. " width=50%/><p>Figure. Test for targeting peptides to mitochondria, nucleus and vacuolar. </P><br />
<p>We got good data for degradation tags for <i>E. coli</i>. Construction of degradation devices for yeast have been finished and being tested. Degradation rate calculated from both methods mates well.</p><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" width=50%/><p>Figure. Pictures for degradation tag testing devices with positive control.</p><br />
<p>Microfluidics device has been successfully developed for measurement and cell synchronization.</p><br />
<img src="https://static.igem.org/mediawiki/2013/f/fc/Microfluidic.gif" /><p>Figure. Cells being captured by microfluidic chip.</p><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" width=50%/><br />
<p>Figure. Degradation tags were tested in Microfluidic</p><br />
<p>Cell synchronization has been successful by changing the method to microfluidics based one.</p><br />
<img src="https://static.igem.org/mediawiki/2013/2/23/Sync.jpg" title="Synchronization" description="All cells are synchronized to G1 phase by using the microfluidics method." width=50%/><br />
<p>Figure. All cells are synchronized to G1 phase by using the microfluidics method.</p><br />
<p>Alternative splicing device are still in construction, related results will be updated later.</p><br />
<br />
</div><br />
<br />
<div id="board2" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery2"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/1/19/Flu.jpg" title="S Phase Promoter" description="cln3 promoter for S phase has been verified using flow cytometry." /></li><br />
</ul></div><br />
<div id="box2" class="box"><br />
<h3>Promoter Verification</h3><br />
<h4>Achievements</h4><br />
<p> Using the GFP as reporter and morphological alteration as cell cycle representation, we verified the Clb6 can be activated in G1 phase in the yeast.</P><br />
<p> As shown in the picture in normal lights, there are some yeast were budding. These budded yeast cells and small size budding yeasts (within the red circles) are assumed in the G1 phase of cell cycle. And the next picture was taken under activation light, thus the green lights can representing the Clb6 promoter expressing. The two pictures indicated the Clb6 were expressed in the G1 phase as we expected when designing the experiments </p><br />
<p> PS: we modified the contrast ratio to lower the lights of neighbor cells, thus our results looking better.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" /><p>Figure. clb6 promoter for G1 phase has been verified.</p><br />
<p>We also captured fluorescence on cln3 promoter (S phase) test device, for the fluorescence is quite weak, we utilize micro plate reader and flow cytometry to verify the promoter.</p><br />
<img src="https://static.igem.org/mediawiki/2013/1/19/Flu.jpg" /><p>Figure. Flow cytometry to verify S phase promoter cln3.</p><br />
<h4>Problems</h4><br />
<h5>Yeast Version</h5><br />
<p>While the promoters are natural ones without optimization, and our shuttle plasmid is pSR416 which is a single copied one, fluorescence is too weak to be captured by microfluidics device. That is a main reason for why we currently cannot show you a perfect cell magic movie. We are now constructing all devices with high copy number vectors, and try to optimize the junction near kozak sequence to make a better magic.</p><br />
<h5><i>E. coli</i> Version</h5><br />
<p>Promoters for <i>E. coli</i> seems not to be successful yet. In our first version, original 5' utr of promoter's downstream gene is included in BioBrick's sequences. The wild-type promoter and RBS may be too weak, or the junction of RFC[23] may have negative effect to the RBS. So in the present version, original 5' utr has been removed and strong RBS B0030 is used. We will update related results later.</p><br />
</div><br />
<br />
<div id="board3" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery3"><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/0/0d/Reporter.jpg" title="Modified Reporters" description="Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose."/></li><br />
</ul></div><br />
<div id="box3" class="box"><br />
<h3>Reporter Locating</h3><br />
<h4>Reporter Modification</h4><br />
<p>Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose.</p><br />
<img src="https://static.igem.org/mediawiki/2013/0/0d/Reporter.jpg" title="Modified Reporters" description="Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose."/><p>Figure. Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose</p><br />
<h4>Targeting Peptides</h4><br />
<p> The four pictures shows the reporters can rightly locate to Mitochondria, Nucleus and Vacuole,respectively. Picture A is the negative control, all yeast cells are lighted with GFP. And figure B is the reporter to the mitochondria, we can saw there are several light spots in one cells. Figure C is the reporter located in nucleus, the green spots are small and there is only one in a yeast cell.The last picture shows the reporter of Vacuolar membrane, the green lights were discrete in cells which was as expected.</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/><p>Figure. Test for targeting peptides to mitochondria, nucleus and vacuolar.</p><br />
</div><br />
<br />
<div id="board4" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery4"><br />
<li><img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" title="E. coli Version" description="Degradation tags testing device for E. coli"/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" title="IPTG Repressed" description="Microfluidics device for half life test with IPTG input (promoter of RFP repressed)."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/e9/Degladder.jpg" title="RFP Ladder" description="RFP ladder with different degradation tags)."/></li><br />
<br />
</ul></div><br />
<div id="box4" class="box"><br />
<h3>Degradation Rate</h3><br />
<p>In every curcuits measurement, we firstly test the growth curve through detecting the absorbance. Taken small amount of bateria, inoculate into 400ul LB medium, ensuring the initial concentration between 0.02-0.05 (OD600). Cultured in the room temperature, detect the OD600 value every 20 minutes. Y axis represents the logarithm of bacteria number, X axis represents the growth time. </p><br />
<img src="https://static.igem.org/mediawiki/2013/6/68/Growth-Curve.jpg" /><p>Figure. Growth curves have been drawn for all constructed devices.</p><br />
<p>In E.coli, the adaptor SspB tethers ssrA tagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. Which means, a variation of the WT SsrA tag sequence (K1051206, K1051207 and K1051208) will accelerate the degradation of proteins when fused to their C-terminal. Thus the degradation rates are dependent on concentration of proteases and binding mediators.</p><br />
<h4>Achievements</h4><br />
<p>We constructed the measurement pathway of each tag (K1051257, K1051258 and K1051259) to test the rates of degradation of tagged proteins respectively. J04450 was used as positive control because of the same promoter and fluorescent protein. </p><br />
<img src="https://static.igem.org/mediawiki/2013/e/e9/Degladder.jpg" /><br />
<p>Figure. Fluorescence ladder for degradation test devices. From left, the negative control,BBa_K1051257, BBa_K1051258, BBa_K1051259, J04450 as Positive Control. </p><br />
<p>As the pictures showed, the lights of RFP within three degradation tags are decreasing. </p><br />
<!----------------------<br />
<img src="https://static.igem.org/mediawiki/2013/a/a6/IMG_0804.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/c/ca/IMG_0817.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/M0050bright.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/0/06/M0050strong.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/8/89/M0050weak.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/6/63/Flo.jpg" /><br />
<br />
--------------------><br />
<p>Enzyme - labelled meter detect the fluorescent protein intensity</p><br />
<p>First of all, take a certain amount of bacteria liquid, recovery to around OD0.6(600), ensuring the bacteria in the logarithmic growth phase. Diluted and then the bacteria transferred to 96 well plates, measured their fluorescence intensity. Red fluorescent protein using an excitation wavelength of 584nm, and its emission wavelength is 607nm.When measuring, we first detect the OD600 of each strain, removing the factor of bacteria number difference. Thus the fluorescence intensity cannot be altered by bacteria quantity. Then the measurement mode switching for measurement of fluorescence, fluorescence intensity.</p><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" /><br />
<p>Figure. The test results for K1051258 and positive control. A. K1051258 in bright field; B. K1051258 in exciting lights; C. Positive control in bright field; D. K1051258 in exciting lights.</p><br />
<br />
<p>Images are also available for K1051257 and K1051259. </p><br />
<img src="https://static.igem.org/mediawiki/parts/6/6a/DT.png" title="Fluorescence Intensity" description="Fluorescence intensity of positive control and three tested degradation tags." /><p>Figure. Fluorescence intensity calculated by ImageJ positive control and three tested degradation tags.</p><br />
<p>The degradation efficiency of three degradation tags are obvious.</p><br />
<p>While in simulation, we obtained the degradation rate by calculating with [P1_P]/[P2_P]:</p><br />
<img src="https://static.igem.org/mediawiki/2013/b/ba/Simulation_result.png"> (Calculated from microscope data, K1051258)<br />
<br />
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<img src="https://static.igem.org/mediawiki/2013/8/83/0917_t00_white_60mm.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/7/72/0917_t15_white_60mm.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/4/4e/0917_t30_white_60mm.jpg" /><br />
--------------------><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg"/><br />
<p>Figure. The test results of BBa_K1051258 in chip. A,LB medium,O minuts; B, IPTG medium,9minutes; C, IPTG medium, 15 minutes</p><br />
<br />
<br />
<p>Using the IPTG medium, the new RFP expression was stopped and we can regard the lights as degradation tags' efficiency. Obviously, the lights are decreasing along with the time. Thus, it indicates that the degradation tags work effectively.</p><br />
<img src="https://static.igem.org/mediawiki/parts/1/1e/Average_flurescence_intensity_of_K1051258_measurement.jpg" title="Half Life" description="RFP with degradation tag's half life tested by microfluidics." /><br />
<p>Figure. RFP with degradation tag's half life tested by microfluidics and fit by modeling.</p><br />
<p>Calculated by ImageJ and from experimental fitting curve we got its half-life of our protein 6.88 min. So the degradation rate should be: </p><br />
<img src="https://static.igem.org/mediawiki/2013/9/94/Hl-rate.png"> (Calculated from microfluidics data)<br />
<p>Simulation results calculated and fit from both microscope and microfluidics mates well, which has verified our experimental results.</p><br />
<br />
<h4>Problems</h4><br />
<h5>Yeast Version</h5><br />
<p>The test for yeast degradation tags has not been fully successful. Fluorescence has been captured in some occasions but not stable. It may caused by the same problem with promoter test devices - plasmid copy number. </p><br />
<h5><i>E. coli</i> Version</h5><br />
<p>Data collected from microscope for different degradation tags did not fully meet performance expectation. Reasons may be multiple to increase systematic errors.</p><br />
<p>- Fluorescence is too weak for calculating</p><br />
<p>- Quenching of fluorescence, especially in RFP</p><br />
<p>- Sample size is small for ImageJ calculating</p><br />
<p>We will have more data detected by microfluidics and flow cytometry. Results will be updated later.</p><br />
</div><br />
<br />
<div id="board5" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery5"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/4a/Capture.jpg" title="E. coli captured" description="E. coli cells captured by microfluidics device."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/3/3f/Sc_bright_20.jpg" title="E. coli captured" description="Yeast cells captured by microfluidics device."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/2/23/Sync.jpg" title="Synchronization" description="All cells are synchronized to G1 phase by using the microfluidics method."/></li><br />
</ul></div><br />
<div id="box5" class="box"><br />
<h3>Cell Synchronization</h3><br />
<h4>Microfluidics Device</h4><br />
<p>We made the chip as a platform for watching and synchronize the cells. First step is capture the cells by the chip. As showed in the two figures, both E.coli and budding yeast can be captured by the chip successfully.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/4a/Capture.jpg" /><br />
<p>Figure. <i>E. coli</i> and yeast cells have been captured by the chip successfully.</p><br />
<p>As Sic1 method has not been successful, we tried another way by changing medium by microfluidics. Protocols can be found in Notes page.</p><br />
<img src="https://static.igem.org/mediawiki/2013/2/23/Sync.jpg" title="Synchronization" description="All cells are synchronized to G1 phase by using the microfluidics method."/><br />
<p>Figure. All cells are synchronized to G1 phase by using the microfluidics method.</p><br />
<p>All yeast cells in microfluidics chip are in G1 phase, after budding.</p><br />
<br />
<h4>Sic1 Synchronization</h4><br />
<p>Seven phosphorylation sites are mutated by PCR to make Sic1 mutant. However, it has been taking us too long time that we have been still working on right clones. Experiments results will be updated later. <br />
<br />
</div><br />
<br />
<div id="board6" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery6"><br />
<li><img src="https://static.igem.org/mediawiki/2013/b/b9/Blank-01.png" /><li><br />
</ul></div><br />
<div id="box6" class="box"><br />
<h3>Alternative Splicing</h3><br />
<h4>Original Design</h4><br />
<p>In our project, we attempt to use intron as a switch. However, according to previous research, there are two splicing forms of SRC1 intron: one is complete splicing (5’S) while the other can leave 4bp at its 5’ end (5’L). The remaining 4-bp causes frame-shift and a stop codon in the region of adaptor, thus the following exon will not be translated. However, this kind of switch is not complete – the ratio of 5’L and 5’S varied between 40-60 and 85-15.</p><br />
<p>In order to make a complete switch, we abandoned the remaining 4bp of SRC1 intron to avoid producing 5’L mRNA. So the result comes out to be: when Hub1p are expressed, they bind to spliceosome to modify it, resulting to easy recognizing 5’S splice site and its splicing; when inhibit the expression of HUB1 by CRISPRi system, spliceosome can hardly recognize 5’S splice site, therefore no intron can be spliced. </p><br />
<h4>Further Plans</h4><br />
<p>However, things didn’t go as we expected. Our experiment showed that this 4bp plays important role in SRC1 intron splicing. So, to achieve our goal, we redesigned another three versions of SRC1 intron:<br />
</p><br />
<p>1. Intron with GG at its 5’ end</p><br />
<p>2. Intron with CGG at its 5’ end</p><br />
<p>3. Intron with its original 6bp at both end</p><br />
<p>Due to the lack of time, we are still constructing these parts to test devices.</p><br />
<br />
</div><br />
<br />
</div></div>BGI K2http://2013.igem.org/Team:Shenzhen_BGIC_ATCG/resultsTeam:Shenzhen BGIC ATCG/results2013-09-28T01:23:35Z<p>BGI K2: </p>
<hr />
<div><!----------------------<br />
<br />
<br />
Title: Results<br />
Team: Shenzhen_BGIC_ATCG<br />
Author: BGI_K2<br />
Twitter: @BGI_K2<br />
Email: im.ss.kk@gmail.com<br />
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<div class="navleftholder"><br />
<ul class="navleft"><br />
<li><a id="navleftsub1" class="navleftsub" href="#board1">The Magic</a></li><br />
<li><a id="navleftsub2" class="navleftsub" href="#board2">Promoter Verification</a></li><br />
<li><a id="navleftsub3" class="navleftsub" href="#board3">Reporter Locating</a></li><br />
<li><a id="navleftsub4" class="navleftsub" href="#board4">Degradation Rate</a></li><br />
<li><a id="navleftsub5" class="navleftsub" href="#board5">Cell Synchronization</a></li><br />
<li><a id="navleftsub6" class="navleftsub" href="#board6">Alternative Splicing</a></li><br />
</ul></div><br />
<br />
<div id="board1" class="board"></div><br />
<div class="media"><br />
<br />
<ul id="myGallery1"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/></li><br />
<li><img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" title="Degradation Tags" description="Degradation tags testing device for E. coli."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" title="Microfluidics" description="Microfluidics device for degradation test."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/2/23/Sync.jpg" title="Synchronization" description="All cells are synchronized to G1 phase by using the microfluidics method."/></li><br />
</ul></div><br />
<div id="box1" class="box"><br />
<h3>The Magic</h3><br />
<p>Our project “Cell Magic", a complex work has been completed partly,due to the time limitation. The results we have gotten are listed below:</p><br />
<p>We verified some of the cyclin promoters. The clb6 promoter, which is supposed to be expressed in G1 phase, has been verified perfectly.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." width=50%/><p>Figure. clb6 promoter for G1 phase has been verified</p><br />
<p>Targeting peptides to three locations have been verified. Others are still being constructed and tested.</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. " width=50%/><p>Figure. Test for targeting peptides to mitochondria, nucleus and vacuolar. </P><br />
<p>We got good data for degradation tags for <i>E. coli</i>. Construction of degradation devices for yeast have been finished and being tested. Degradation rate calculated from both methods mates well.</p><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" width=50%/><p>Figure. Pictures for degradation tag testing devices with positive control.</p><br />
<p>Microfluidics device has been successfully developed for measurement and cell synchronization.</p><br />
<img src="https://static.igem.org/mediawiki/2013/f/fc/Microfluidic.gif" /><p>Figure. Cells being captured by microfluidic chip.</p><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" width=50%/><br />
<p>Figure. Degradation tags were tested in Microfluidic</p><br />
<p>Cell synchronization has been successful by changing the method to microfluidics based one.</p><br />
<img src="https://static.igem.org/mediawiki/2013/2/23/Sync.jpg" title="Synchronization" description="All cells are synchronized to G1 phase by using the microfluidics method." width=50%/><br />
<p>Figure. All cells are synchronized to G1 phase by using the microfluidics method.</p><br />
<p>Alternative splicing device are still in construction, related results will be updated later.</p><br />
<br />
</div><br />
<br />
<div id="board2" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery2"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/1/19/Flu.jpg" title="S Phase Promoter" description="cln3 promoter for S phase has been verified using flow cytometry." /></li><br />
</ul></div><br />
<div id="box2" class="box"><br />
<h3>Promoter Verification</h3><br />
<h4>Achievements</h4><br />
<p> Using the GFP as reporter and morphological alteration as cell cycle representation, we verified the Clb6 can be activated in G1 phase in the yeast.</P><br />
<p> As shown in the picture in normal lights, there are some yeast were budding. These budded yeast cells and small size budding yeasts (within the red circles) are assumed in the G1 phase of cell cycle. And the next picture was taken under activation light, thus the green lights can representing the Clb6 promoter expressing. The two pictures indicated the Clb6 were expressed in the G1 phase as we expected when designing the experiments </p><br />
<p> PS: we modified the contrast ratio to lower the lights of neighbor cells, thus our results looking better.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" /><p>Figure. clb6 promoter for G1 phase has been verified.</p><br />
<p>We also captured fluorescence on cln3 promoter (S phase) test device, for the fluorescence is quite weak, we utilize micro plate reader and flow cytometry to verify the promoter.</p><br />
<img src="https://static.igem.org/mediawiki/2013/1/19/Flu.jpg" /><p>Figure. Flow cytometry to verify S phase promoter cln3.</p><br />
<h4>Problems</h4><br />
<h5>Yeast Version</h5><br />
<p>While the promoters are natural ones without optimization, and our shuttle plasmid is pSR416 which is a single copied one, fluorescence is too weak to be captured by microfluidics device. That is a main reason for why we currently cannot show you a perfect cell magic movie. We are now constructing all devices with high copy number vectors, and try to optimize the junction near kozak sequence to make a better magic.</p><br />
<h5><i>E. coli</i> Version</h5><br />
<p>Promoters for <i>E. coli</i> seems not to be successful yet. In our first version, original 5' utr of promoter's downstream gene is included in BioBrick's sequences. The wild-type promoter and RBS may be too weak, or the junction of RFC[23] may have negative effect to the RBS. So in the present version, original 5' utr has been removed and strong RBS B0030 is used. We will update related results later.</p><br />
</div><br />
<br />
<div id="board3" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery3"><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/0/0d/Reporter.jpg" title="Modified Reporters" description="Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose."/></li><br />
</ul></div><br />
<div id="box3" class="box"><br />
<h3>Reporter Locating</h3><br />
<h4>Reporter Modification</h4><br />
<p>Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose.</p><br />
<img src="https://static.igem.org/mediawiki/2013/0/0d/Reporter.jpg" title="Modified Reporters" description="Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose."/><p>Figure. Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose</p><br />
<h4>Targeting Peptides</h4><br />
<p> The four pictures shows the reporters can rightly locate to Mitochondria, Nucleus and Vacuole,respectively. Picture A is the negative control, all yeast cells are lighted with GFP. And figure B is the reporter to the mitochondria, we can saw there are several light spots in one cells. Figure C is the reporter located in nucleus, the green spots are small and there is only one in a yeast cell.The last picture shows the reporter of Vacuolar membrane, the green lights were discrete in cells which was as expected.</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/><p>Figure. Test for targeting peptides to mitochondria, nucleus and vacuolar.</p><br />
</div><br />
<br />
<div id="board4" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery4"><br />
<li><img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" title="E. coli Version" description="Degradation tags testing device for E. coli"/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" title="IPTG Repressed" description="Microfluidics device for half life test with IPTG input (promoter of RFP repressed)."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/e9/Degladder.jpg" title="RFP Ladder" description="RFP ladder with different degradation tags)."/></li><br />
<br />
</ul></div><br />
<div id="box4" class="box"><br />
<h3>Degradation Rate</h3><br />
<p>In every curcuits measurement, we firstly test the growth curve through detecting the absorbance. Taken small amount of bateria, inoculate into 400ul LB medium, ensuring the initial concentration between 0.02-0.05 (OD600). Cultured in the room temperature, detect the OD600 value every 20 minutes. Y axis represents the logarithm of bacteria number, X axis represents the growth time. </p><br />
<img src="https://static.igem.org/mediawiki/2013/6/68/Growth-Curve.jpg" /><p>Figure. Growth curves have been drawn for all constructed devices.</p><br />
<p>In E.coli, the adaptor SspB tethers ssrA tagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. Which means, a variation of the WT SsrA tag sequence (K1051206, K1051207 and K1051208) will accelerate the degradation of proteins when fused to their C-terminal. Thus the degradation rates are dependent on concentration of proteases and binding mediators.</p><br />
<h4>Achievements</h4><br />
<p>We constructed the measurement pathway of each tag (K1051257, K1051258 and K1051259) to test the rates of degradation of tagged proteins respectively. J04450 was used as positive control because of the same promoter and fluorescent protein. </p><br />
<img src="https://static.igem.org/mediawiki/2013/e/e9/Degladder.jpg" /><br />
<p>Figure. Fluorescence ladder for degradation test devices. From left, the negative control,BBa_K1051257, BBa_K1051258, BBa_K1051259, J04450 as Positive Control. </p><br />
<p>As the pictures showed, the lights of RFP within three degradation tags are decreasing. </p><br />
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<img src="https://static.igem.org/mediawiki/2013/a/a6/IMG_0804.jpg" /><br />
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<img src="https://static.igem.org/mediawiki/2013/a/a8/M0050bright.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/0/06/M0050strong.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/8/89/M0050weak.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/6/63/Flo.jpg" /><br />
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<p>Enzyme - labelled meter detect the fluorescent protein intensity</p><br />
<p>First of all, take a certain amount of bacteria liquid, recovery to around OD0.6(600), ensuring the bacteria in the logarithmic growth phase. Diluted and then the bacteria transferred to 96 well plates, measured their fluorescence intensity. Red fluorescent protein using an excitation wavelength of 584nm, and its emission wavelength is 607nm.When measuring, we first detect the OD600 of each strain, removing the factor of bacteria number difference. Thus the fluorescence intensity cannot be altered by bacteria quantity. Then the measurement mode switching for measurement of fluorescence, fluorescence intensity.</p><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" /><br />
<p>Figure. The test results for K1051258 and positive control. A. K1051258 in bright field; B. K1051258 in exciting lights; C. Positive control in bright field; D. K1051258 in exciting lights.</p><br />
<br />
<p>Images are also available for K1051257 and K1051259. </p><br />
<img src="https://static.igem.org/mediawiki/parts/6/6a/DT.png" title="Fluorescence Intensity" description="Fluorescence intensity of positive control and three tested degradation tags." /><p>Figure. Fluorescence intensity calculated by ImageJ positive control and three tested degradation tags.</p><br />
<p>The degradation efficiency of three degradation tags are obvious.</p><br />
<p>While in simulation, we obtained the degradation rate by calculating with [P1_P]/[P2_P]:</p><br />
<img src="https://static.igem.org/mediawiki/2013/b/ba/Simulation_result.png"> (Calculated from microscope data, K1051258)<br />
<br />
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<img src="https://static.igem.org/mediawiki/2013/8/83/0917_t00_white_60mm.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/7/72/0917_t15_white_60mm.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/4/4e/0917_t30_white_60mm.jpg" /><br />
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<img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg"/><br />
<p>Figure. The test results of BBa_K1051258 in chip. A,LB medium,O minuts; B, IPTG medium,9minutes; C, IPTG medium, 15 minutes</p><br />
<br />
<br />
<p>Using the IPTG medium, the new RFP expression was stopped and we can regard the lights as degradation tags' efficiency. Obviously, the lights are decreasing along with the time. Thus, it indicates that the degradation tags work effectively.</p><br />
<img src="https://static.igem.org/mediawiki/parts/1/1e/Average_flurescence_intensity_of_K1051258_measurement.jpg" title="Half Life" description="RFP with degradation tag's half life tested by microfluidics." /><br />
<p>Figure. RFP with degradation tag's half life tested by microfluidics and fit by modeling.</p><br />
<p>Calculated by ImageJ and from experimental fitting curve we got its half-life of our protein 6.88 min. So the degradation rate should be: </p><br />
<img src="https://static.igem.org/mediawiki/2013/9/94/Hl-rate.png"> (Calculated from microfluidics data)<br />
<p>Simulation results calculated and fit from both microscope and microfluidics mates well, which has verified our experimental results.</p><br />
<br />
<h4>Problems</h4><br />
<h5>Yeast Version</h5><br />
<p>The test for yeast degradation tags has not been fully successful. Fluorescence has been captured in some occasions but not stable. It may caused by the same problem with promoter test devices - plasmid copy number. </p><br />
<h5><i>E. coli</i> Version</h5><br />
<p>Data collected from microscope for different degradation tags did not fully meet performance expectation. Reasons may be multiple to increase systematic errors.</p><br />
<p>- Fluorescence is too weak for calculating</p><br />
<p>- Quenching of fluorescence, especially in RFP</p><br />
<p>- Sample size is small for ImageJ calculating</p><br />
<p>We will have more data detected by microfluidics and flow cytometry. Results will be updated later.</p><br />
</div><br />
<br />
<div id="board5" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery5"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/4a/Capture.jpg" title="E. coli captured" description="E. coli cells captured by microfluidics device."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/3/3f/Sc_bright_20.jpg" title="E. coli captured" description="Yeast cells captured by microfluidics device."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/2/23/Sync.jpg" title="Cell Synchronized" description="All cells are synchronized to G1 phase by using the microfluidics method."/></li><br />
</ul></div><br />
<div id="box5" class="box"><br />
<h3>Cell Synchronization</h3><br />
<h4>Microfluidics Device</h4><br />
<p>We made the chip as a platform for watching and synchronize the cells. First step is capture the cells by the chip. As showed in the two figures, both E.coli and budding yeast can be captured by the chip successfully.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/4a/Capture.jpg" /><br />
<p>Figure. <i>E. coli</i> and yeast cells have been captured by the chip successfully.</p><br />
<p>As Sic1 method has not been successful, we tried another way by changing medium by microfluidics. Protocols can be found in Notes page.</p><br />
<img src="https://static.igem.org/mediawiki/2013/2/23/Sync.jpg" title="Cell Synchronized" description="All cells are synchronized to G1 phase by using the microfluidics method."/><br />
<p>Figure. All cells are synchronized to G1 phase by using the microfluidics method.</p><br />
<p>All yeast cells in microfluidics chip are in G1 phase, after budding.</p><br />
<br />
<h4>Sic1 Synchronization</h4><br />
<p>Seven phosphorylation sites are mutated by PCR to make Sic1 mutant. However, it has been taking us too long time that we have been still working on right clones. Experiments results will be updated later. <br />
<br />
</div><br />
<br />
<div id="board6" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery6"><br />
<li><img src="https://static.igem.org/mediawiki/2013/b/b9/Blank-01.png" /><li><br />
</ul></div><br />
<div id="box6" class="box"><br />
<h3>Alternative Splicing</h3><br />
<h4>Original Design</h4><br />
<p>In our project, we attempt to use intron as a switch. However, according to previous research, there are two splicing forms of SRC1 intron: one is complete splicing (5’S) while the other can leave 4bp at its 5’ end (5’L). The remaining 4-bp causes frame-shift and a stop codon in the region of adaptor, thus the following exon will not be translated. However, this kind of switch is not complete – the ratio of 5’L and 5’S varied between 40-60 and 85-15.</p><br />
<p>In order to make a complete switch, we abandoned the remaining 4bp of SRC1 intron to avoid producing 5’L mRNA. So the result comes out to be: when Hub1p are expressed, they bind to spliceosome to modify it, resulting to easy recognizing 5’S splice site and its splicing; when inhibit the expression of HUB1 by CRISPRi system, spliceosome can hardly recognize 5’S splice site, therefore no intron can be spliced. </p><br />
<h4>Further Plans</h4><br />
<p>However, things didn’t go as we expected. Our experiment showed that this 4bp plays important role in SRC1 intron splicing. So, to achieve our goal, we redesigned another three versions of SRC1 intron:<br />
</p><br />
<p>1. Intron with GG at its 5’ end</p><br />
<p>2. Intron with CGG at its 5’ end</p><br />
<p>3. Intron with its original 6bp at both end</p><br />
<p>Due to the lack of time, we are still constructing these parts to test devices.</p><br />
<br />
</div><br />
<br />
</div></div>BGI K2http://2013.igem.org/Team:Shenzhen_BGIC_ATCG/resultsTeam:Shenzhen BGIC ATCG/results2013-09-28T01:22:27Z<p>BGI K2: </p>
<hr />
<div><!----------------------<br />
<br />
<br />
Title: Results<br />
Team: Shenzhen_BGIC_ATCG<br />
Author: BGI_K2<br />
Twitter: @BGI_K2<br />
Email: im.ss.kk@gmail.com<br />
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<div class="navleftholder"><br />
<ul class="navleft"><br />
<li><a id="navleftsub1" class="navleftsub" href="#board1">The Magic</a></li><br />
<li><a id="navleftsub2" class="navleftsub" href="#board2">Promoter Verification</a></li><br />
<li><a id="navleftsub3" class="navleftsub" href="#board3">Reporter Locating</a></li><br />
<li><a id="navleftsub4" class="navleftsub" href="#board4">Degradation Rate</a></li><br />
<li><a id="navleftsub5" class="navleftsub" href="#board5">Cell Synchronization</a></li><br />
<li><a id="navleftsub6" class="navleftsub" href="#board6">Alternative Splicing</a></li><br />
</ul></div><br />
<br />
<div id="board1" class="board"></div><br />
<div class="media"><br />
<br />
<ul id="myGallery1"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/></li><br />
<li><img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" title="Degradation Tags" description="Degradation tags testing device for E. coli."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" title="Microfluidics" description="Microfluidics device for degradation test."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/2/23/Sync.jpg" title="Synchronization" description="All cells are synchronized to G1 phase by using the microfluidics method."/></li><br />
</ul></div><br />
<div id="box1" class="box"><br />
<h3>The Magic</h3><br />
<p>Our project “Cell Magic", a complex work has been completed partly,due to the time limitation. The results we have gotten are listed below:</p><br />
<p>We verified some of the cyclin promoters. The clb6 promoter, which is supposed to be expressed in G1 phase, has been verified perfectly.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." width=50%/><p>Figure. clb6 promoter for G1 phase has been verified</p><br />
<p>Targeting peptides to three locations have been verified. Others are still being constructed and tested.</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. " width=50%/><p>Figure. Test for targeting peptides to mitochondria, nucleus and vacuolar. </P><br />
<p>We got good data for degradation tags for <i>E. coli</i>. Construction of degradation devices for yeast have been finished and being tested. Degradation rate calculated from both methods mates well.</p><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" width=50%/><p>Figure. Pictures for degradation tag testing devices with positive control.</p><br />
<p>Microfluidics device has been successfully developed for measurement and cell synchronization.</p><br />
<img src="https://static.igem.org/mediawiki/2013/f/fc/Microfluidic.gif" /><p>Figure. Cells being captured by microfluidic chip.</p><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" width=50%/><br />
<p>Figure. Degradation tags were tested in Microfluidic</p><br />
<p>Cell synchronization has been successful by changing the method to microfluidics based one.</p><br />
<img src="https://static.igem.org/mediawiki/2013/2/23/Sync.jpg" title="Synchronization" description="All cells are synchronized to G1 phase by using the microfluidics method." width=50%/><br />
<p>Figure. All cells are synchronized to G1 phase by using the microfluidics method.</p><br />
<p>Alternative splicing device are still in construction, related results will be updated later.</p><br />
<br />
</div><br />
<br />
<div id="board2" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery2"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/1/19/Flu.jpg" title="S Phase Promoter" description="cln3 promoter for S phase has been verified using flow cytometry." /></li><br />
</ul></div><br />
<div id="box2" class="box"><br />
<h3>Promoter Verification</h3><br />
<h4>Achievements</h4><br />
<p> Using the GFP as reporter and morphological alteration as cell cycle representation, we verified the Clb6 can be activated in G1 phase in the yeast.</P><br />
<p> As shown in the picture in normal lights, there are some yeast were budding. These budded yeast cells and small size budding yeasts (within the red circles) are assumed in the G1 phase of cell cycle. And the next picture was taken under activation light, thus the green lights can representing the Clb6 promoter expressing. The two pictures indicated the Clb6 were expressed in the G1 phase as we expected when designing the experiments </p><br />
<p> PS: we modified the contrast ratio to lower the lights of neighbor cells, thus our results looking better.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" /><p>Figure. clb6 promoter for G1 phase has been verified.</p><br />
<p>We also captured fluorescence on cln3 promoter (S phase) test device, for the fluorescence is quite weak, we utilize micro plate reader and flow cytometry to verify the promoter.</p><br />
<img src="https://static.igem.org/mediawiki/2013/1/19/Flu.jpg" /><p>Figure. Flow cytometry to verify S phase promoter cln3.</p><br />
<h4>Problems</h4><br />
<h5>Yeast Version</h5><br />
<p>While the promoters are natural ones without optimization, and our shuttle plasmid is pSR416 which is a single copied one, fluorescence is too weak to be captured by microfluidics device. That is a main reason for why we currently cannot show you a perfect cell magic movie. We are now constructing all devices with high copy number vectors, and try to optimize the junction near kozak sequence to make a better magic.</p><br />
<h5><i>E. coli</i> Version</h5><br />
<p>Promoters for <i>E. coli</i> seems not to be successful yet. In our first version, original 5' utr of promoter's downstream gene is included in BioBrick's sequences. The wild-type promoter and RBS may be too weak, or the junction of RFC[23] may have negative effect to the RBS. So in the present version, original 5' utr has been removed and strong RBS B0030 is used. We will update related results later.</p><br />
</div><br />
<br />
<div id="board3" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery3"><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/0/0d/Reporter.jpg" title="Modified Reporters" description="Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose."/></li><br />
</ul></div><br />
<div id="box3" class="box"><br />
<h3>Reporter Locating</h3><br />
<h4>Reporter Modification</h4><br />
<p>Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose.</p><br />
<img src="https://static.igem.org/mediawiki/2013/0/0d/Reporter.jpg" title="Modified Reporters" description="Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose."/><p>Figure. Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose</p><br />
<h4>Targeting Peptides</h4><br />
<p> The four pictures shows the reporters can rightly locate to Mitochondria, Nucleus and Vacuole,respectively. Picture A is the negative control, all yeast cells are lighted with GFP. And figure B is the reporter to the mitochondria, we can saw there are several light spots in one cells. Figure C is the reporter located in nucleus, the green spots are small and there is only one in a yeast cell.The last picture shows the reporter of Vacuolar membrane, the green lights were discrete in cells which was as expected.</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/><p>Figure. Test for targeting peptides to mitochondria, nucleus and vacuolar.</p><br />
</div><br />
<br />
<div id="board4" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery4"><br />
<li><img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" title="E. coli Version" description="Degradation tags testing device for E. coli"/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" title="IPTG Repressed" description="Microfluidics device for half life test with IPTG input (promoter of RFP repressed)."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/e9/Degladder.jpg" title="RFP Ladder" description="RFP ladder with different degradation tags)."/></li><br />
<br />
</ul></div><br />
<div id="box4" class="box"><br />
<h3>Degradation Rate</h3><br />
<p>In every curcuits measurement, we firstly test the growth curve through detecting the absorbance. Taken small amount of bateria, inoculate into 400ul LB medium, ensuring the initial concentration between 0.02-0.05 (OD600). Cultured in the room temperature, detect the OD600 value every 20 minutes. Y axis represents the logarithm of bacteria number, X axis represents the growth time. </p><br />
<img src="https://static.igem.org/mediawiki/2013/6/68/Growth-Curve.jpg" /><p>Figure. Growth curves have been drawn for all constructed devices.</p><br />
<p>In E.coli, the adaptor SspB tethers ssrA tagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. Which means, a variation of the WT SsrA tag sequence (K1051206, K1051207 and K1051208) will accelerate the degradation of proteins when fused to their C-terminal. Thus the degradation rates are dependent on concentration of proteases and binding mediators.</p><br />
<h4>Achievements</h4><br />
<p>We constructed the measurement pathway of each tag (K1051257, K1051258 and K1051259) to test the rates of degradation of tagged proteins respectively. J04450 was used as positive control because of the same promoter and fluorescent protein. </p><br />
<img src="https://static.igem.org/mediawiki/2013/e/e9/Degladder.jpg" /><br />
<p>Figure. Fluorescence ladder for degradation test devices. From left, the negative control,BBa_K1051257, BBa_K1051258, BBa_K1051259, J04450 as Positive Control. </p><br />
<p>As the pictures showed, the lights of RFP within three degradation tags are decreasing. </p><br />
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<img src="https://static.igem.org/mediawiki/2013/a/a6/IMG_0804.jpg" /><br />
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<img src="https://static.igem.org/mediawiki/2013/a/a8/M0050bright.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/0/06/M0050strong.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/8/89/M0050weak.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/6/63/Flo.jpg" /><br />
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<p>Enzyme - labelled meter detect the fluorescent protein intensity</p><br />
<p>First of all, take a certain amount of bacteria liquid, recovery to around OD0.6(600), ensuring the bacteria in the logarithmic growth phase. Diluted and then the bacteria transferred to 96 well plates, measured their fluorescence intensity. Red fluorescent protein using an excitation wavelength of 584nm, and its emission wavelength is 607nm.When measuring, we first detect the OD600 of each strain, removing the factor of bacteria number difference. Thus the fluorescence intensity cannot be altered by bacteria quantity. Then the measurement mode switching for measurement of fluorescence, fluorescence intensity.</p><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" /><br />
<p>Figure. The test results for K1051258 and positive control. A. K1051258 in bright field; B. K1051258 in exciting lights; C. Positive control in bright field; D. K1051258 in exciting lights.</p><br />
<br />
<p>Images are also available for K1051257 and K1051259. </p><br />
<img src="https://static.igem.org/mediawiki/parts/6/6a/DT.png" title="Fluorescence Intensity" description="Fluorescence intensity of positive control and three tested degradation tags." /><p>Figure. Fluorescence intensity calculated by ImageJ positive control and three tested degradation tags.</p><br />
<p>The degradation efficiency of three degradation tags are obvious.</p><br />
<p>While in simulation, we obtained the degradation rate by calculating with [P1_P]/[P2_P]:</p><br />
<img src="https://static.igem.org/mediawiki/2013/b/ba/Simulation_result.png"> (Calculated from microscope data)<br />
<br />
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<img src="https://static.igem.org/mediawiki/2013/8/83/0917_t00_white_60mm.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/7/72/0917_t15_white_60mm.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/4/4e/0917_t30_white_60mm.jpg" /><br />
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<img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg"/><br />
<p>Figure. The test results of BBa_K1051258 in chip. A,LB medium,O minuts; B, IPTG medium,9minutes; C, IPTG medium, 15 minutes</p><br />
<br />
<br />
<p>Using the IPTG medium, the new RFP expression was stopped and we can regard the lights as degradation tags' efficiency. Obviously, the lights are decreasing along with the time. Thus, it indicates that the degradation tags work effectively.</p><br />
<img src="https://static.igem.org/mediawiki/parts/1/1e/Average_flurescence_intensity_of_K1051258_measurement.jpg" title="Half Life" description="RFP with degradation tag's half life tested by microfluidics." /><br />
<p>Figure. RFP with degradation tag's half life tested by microfluidics and fit by modeling.</p><br />
<p>Calculated by ImageJ and from experimental fitting curve we got its half-life of our protein 6.88 min. So the degradation rate should be: </p><br />
<img src="https://static.igem.org/mediawiki/2013/9/94/Hl-rate.png"> (Calculated from microfluidics data)<br />
<p>Simulation results calculated and fit from both microscope and microfluidics mates well, which has verified our experimental results.</p><br />
<br />
<h4>Problems</h4><br />
<h5>Yeast Version</h5><br />
<p>The test for yeast degradation tags has not been fully successful. Fluorescence has been captured in some occasions but not stable. It may caused by the same problem with promoter test devices - plasmid copy number. </p><br />
<h5><i>E. coli</i> Version</h5><br />
<p>Data collected from microscope for different degradation tags did not fully meet performance expectation. Reasons may be multiple to increase systematic errors.</p><br />
<p>- Fluorescence is too weak for calculating</p><br />
<p>- Quenching of fluorescence, especially in RFP</p><br />
<p>- Sample size is small for ImageJ calculating</p><br />
<p>We will have more data detected by microfluidics and flow cytometry. Results will be updated later.</p><br />
</div><br />
<br />
<div id="board5" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery5"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/4a/Capture.jpg" title="E. coli captured" description="E. coli cells captured by microfluidics device."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/3/3f/Sc_bright_20.jpg" title="E. coli captured" description="Yeast cells captured by microfluidics device."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/2/23/Sync.jpg" title="Cell Synchronized" description="All cells are synchronized to G1 phase by using the microfluidics method."/></li><br />
</ul></div><br />
<div id="box5" class="box"><br />
<h3>Cell Synchronization</h3><br />
<h4>Microfluidics Device</h4><br />
<p>We made the chip as a platform for watching and synchronize the cells. First step is capture the cells by the chip. As showed in the two figures, both E.coli and budding yeast can be captured by the chip successfully.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/4a/Capture.jpg" /><br />
<p>Figure. <i>E. coli</i> and yeast cells have been captured by the chip successfully.</p><br />
<p>As Sic1 method has not been successful, we tried another way by changing medium by microfluidics. Protocols can be found in Notes page.</p><br />
<img src="https://static.igem.org/mediawiki/2013/2/23/Sync.jpg" title="Cell Synchronized" description="All cells are synchronized to G1 phase by using the microfluidics method."/><br />
<p>Figure. All cells are synchronized to G1 phase by using the microfluidics method.</p><br />
<p>All yeast cells in microfluidics chip are in G1 phase, after budding.</p><br />
<br />
<h4>Sic1 Synchronization</h4><br />
<p>Seven phosphorylation sites are mutated by PCR to make Sic1 mutant. However, it has been taking us too long time that we have been still working on right clones. Experiments results will be updated later. <br />
<br />
</div><br />
<br />
<div id="board6" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery6"><br />
<li><img src="https://static.igem.org/mediawiki/2013/b/b9/Blank-01.png" /><li><br />
</ul></div><br />
<div id="box6" class="box"><br />
<h3>Alternative Splicing</h3><br />
<h4>Original Design</h4><br />
<p>In our project, we attempt to use intron as a switch. However, according to previous research, there are two splicing forms of SRC1 intron: one is complete splicing (5’S) while the other can leave 4bp at its 5’ end (5’L). The remaining 4-bp causes frame-shift and a stop codon in the region of adaptor, thus the following exon will not be translated. However, this kind of switch is not complete – the ratio of 5’L and 5’S varied between 40-60 and 85-15.</p><br />
<p>In order to make a complete switch, we abandoned the remaining 4bp of SRC1 intron to avoid producing 5’L mRNA. So the result comes out to be: when Hub1p are expressed, they bind to spliceosome to modify it, resulting to easy recognizing 5’S splice site and its splicing; when inhibit the expression of HUB1 by CRISPRi system, spliceosome can hardly recognize 5’S splice site, therefore no intron can be spliced. </p><br />
<h4>Further Plans</h4><br />
<p>However, things didn’t go as we expected. Our experiment showed that this 4bp plays important role in SRC1 intron splicing. So, to achieve our goal, we redesigned another three versions of SRC1 intron:<br />
</p><br />
<p>1. Intron with GG at its 5’ end</p><br />
<p>2. Intron with CGG at its 5’ end</p><br />
<p>3. Intron with its original 6bp at both end</p><br />
<p>Due to the lack of time, we are still constructing these parts to test devices.</p><br />
<br />
</div><br />
<br />
</div></div>BGI K2http://2013.igem.org/Team:Shenzhen_BGIC_ATCG/resultsTeam:Shenzhen BGIC ATCG/results2013-09-28T01:19:25Z<p>BGI K2: </p>
<hr />
<div><!----------------------<br />
<br />
<br />
Title: Results<br />
Team: Shenzhen_BGIC_ATCG<br />
Author: BGI_K2<br />
Twitter: @BGI_K2<br />
Email: im.ss.kk@gmail.com<br />
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<div class="navleftholder"><br />
<ul class="navleft"><br />
<li><a id="navleftsub1" class="navleftsub" href="#board1">The Magic</a></li><br />
<li><a id="navleftsub2" class="navleftsub" href="#board2">Promoter Verification</a></li><br />
<li><a id="navleftsub3" class="navleftsub" href="#board3">Reporter Locating</a></li><br />
<li><a id="navleftsub4" class="navleftsub" href="#board4">Degradation Rate</a></li><br />
<li><a id="navleftsub5" class="navleftsub" href="#board5">Cell Synchronization</a></li><br />
<li><a id="navleftsub6" class="navleftsub" href="#board6">Alternative Splicing</a></li><br />
</ul></div><br />
<br />
<div id="board1" class="board"></div><br />
<div class="media"><br />
<br />
<ul id="myGallery1"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/></li><br />
<li><img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" title="Degradation Tags" description="Degradation tags testing device for E. coli."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" title="Microfluidics" description="Microfluidics device for degradation test."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/2/23/Sync.jpg" title="Synchronization" description="All cells are synchronized to G1 phase by using the microfluidics method."/></li><br />
</ul></div><br />
<div id="box1" class="box"><br />
<h3>The Magic</h3><br />
<p>Our project “Cell Magic", a complex work has been completed partly,due to the time limitation. The results we have gotten are listed below:</p><br />
<p>We verified some of the cyclin promoters. The clb6 promoter, which is supposed to be expressed in G1 phase, has been verified perfectly.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." width=50%/><p>Figure. clb6 promoter for G1 phase has been verified</p><br />
<p>Targeting peptides to three locations have been verified. Others are still being constructed and tested.</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. " width=50%/><p>Figure. Test for targeting peptides to mitochondria, nucleus and vacuolar. </P><br />
<p>We got good data for degradation tags for <i>E. coli</i>. Construction of degradation devices for yeast have been finished and being tested. Degradation rate calculated from both methods mates well.</p><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" width=50%/><p>Figure. Pictures for degradation tag testing devices with positive control.</p><br />
<p>Microfluidics device has been successfully developed for measurement and cell synchronization.</p><br />
<img src="https://static.igem.org/mediawiki/2013/f/fc/Microfluidic.gif" /><p>Figure. Cells being captured by microfluidic chip.</p><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" width=50%/><br />
<p>Figure. Degradation tags were tested in Microfluidic</p><br />
<p>Cell synchronization has been successful by changing the method to microfluidics based one.</p><br />
<img src="https://static.igem.org/mediawiki/2013/2/23/Sync.jpg" title="Synchronization" description="All cells are synchronized to G1 phase by using the microfluidics method." width=50%/><br />
<p>Figure. All cells are synchronized to G1 phase by using the microfluidics method.</p><br />
<p>Alternative splicing device are still in construction, related results will be updated later.</p><br />
<br />
</div><br />
<br />
<div id="board2" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery2"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/1/19/Flu.jpg" title="S Phase Promoter" description="cln3 promoter for S phase has been verified using flow cytometry." /></li><br />
</ul></div><br />
<div id="box2" class="box"><br />
<h3>Promoter Verification</h3><br />
<h4>Achievements</h4><br />
<p> Using the GFP as reporter and morphological alteration as cell cycle representation, we verified the Clb6 can be activated in G1 phase in the yeast.</P><br />
<p> As shown in the picture in normal lights, there are some yeast were budding. These budded yeast cells and small size budding yeasts (within the red circles) are assumed in the G1 phase of cell cycle. And the next picture was taken under activation light, thus the green lights can representing the Clb6 promoter expressing. The two pictures indicated the Clb6 were expressed in the G1 phase as we expected when designing the experiments </p><br />
<p> PS: we modified the contrast ratio to lower the lights of neighbor cells, thus our results looking better.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" /><p>Figure. clb6 promoter for G1 phase has been verified.</p><br />
<p>We also captured fluorescence on cln3 promoter (S phase) test device, for the fluorescence is quite weak, we utilize micro plate reader and flow cytometry to verify the promoter.</p><br />
<img src="https://static.igem.org/mediawiki/2013/1/19/Flu.jpg" /><p>Figure. Flow cytometry to verify S phase promoter cln3.</p><br />
<h4>Problems</h4><br />
<h5>Yeast Version</h5><br />
<p>While the promoters are natural ones without optimization, and our shuttle plasmid is pSR416 which is a single copied one, fluorescence is too weak to be captured by microfluidics device. That is a main reason for why we currently cannot show you a perfect cell magic movie. We are now constructing all devices with high copy number vectors, and try to optimize the junction near kozak sequence to make a better magic.</p><br />
<h5><i>E. coli</i> Version</h5><br />
<p>Promoters for <i>E. coli</i> seems not to be successful yet. In our first version, original 5' utr of promoter's downstream gene is included in BioBrick's sequences. The wild-type promoter and RBS may be too weak, or the junction of RFC[23] may have negative effect to the RBS. So in the present version, original 5' utr has been removed and strong RBS B0030 is used. We will update related results later.</p><br />
</div><br />
<br />
<div id="board3" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery3"><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/0/0d/Reporter.jpg" title="Modified Reporters" description="Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose."/></li><br />
</ul></div><br />
<div id="box3" class="box"><br />
<h3>Reporter Locating</h3><br />
<h4>Reporter Modification</h4><br />
<p>Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose.</p><br />
<img src="https://static.igem.org/mediawiki/2013/0/0d/Reporter.jpg" title="Modified Reporters" description="Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose."/><p>Figure. Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose</p><br />
<h4>Targeting Peptides</h4><br />
<p> The four pictures shows the reporters can rightly locate to Mitochondria, Nucleus and Vacuole,respectively. Picture A is the negative control, all yeast cells are lighted with GFP. And figure B is the reporter to the mitochondria, we can saw there are several light spots in one cells. Figure C is the reporter located in nucleus, the green spots are small and there is only one in a yeast cell.The last picture shows the reporter of Vacuolar membrane, the green lights were discrete in cells which was as expected.</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/><p>Figure. Test for targeting peptides to mitochondria, nucleus and vacuolar.</p><br />
</div><br />
<br />
<div id="board4" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery4"><br />
<li><img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" title="E. coli Version" description="Degradation tags testing device for E. coli"/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" title="IPTG Repressed" description="Microfluidics device for half life test with IPTG input (promoter of RFP repressed)."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/e9/Degladder.jpg" title="RFP Ladder" description="RFP ladder with different degradation tags)."/></li><br />
<br />
</ul></div><br />
<div id="box4" class="box"><br />
<h3>Degradation Rate</h3><br />
<p>In every curcuits measurement, we firstly test the growth curve through detecting the absorbance. Taken small amount of bateria, inoculate into 400ul LB medium, ensuring the initial concentration between 0.02-0.05 (OD600). Cultured in the room temperature, detect the OD600 value every 20 minutes. Y axis represents the logarithm of bacteria number, X axis represents the growth time. </p><br />
<img src="https://static.igem.org/mediawiki/2013/6/68/Growth-Curve.jpg" /><p>Figure. Growth curves have been drawn for all constructed devices.</p><br />
<p>In E.coli, the adaptor SspB tethers ssrA tagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. Which means, a variation of the WT SsrA tag sequence (K1051206, K1051207 and K1051208) will accelerate the degradation of proteins when fused to their C-terminal. Thus the degradation rates are dependent on concentration of proteases and binding mediators.</p><br />
<p>We constructed the measurement pathway of each tag (K1051257, K1051258 and K1051259) to test the rates of degradation of tagged proteins respectively. J04450 was used as positive control because of the same promoter and fluorescent protein. </p><br />
<h4>Achievements</h4><br />
<p>Enzyme - labelled meter detect the fluorescent protein intensity</p><br />
<p>First of all, take a certain amount of bacteria liquid, recovery to around OD0.6(600), ensuring the bacteria in the logarithmic growth phase. Diluted and then the bacteria transferred to 96 well plates, measured their fluorescence intensity. Red fluorescent protein using an excitation wavelength of 584nm, and its emission wavelength is 607nm.When measuring, we first detect the OD600 of each strain, removing the factor of bacteria number difference. Thus the fluorescence intensity cannot be altered by bacteria quantity. Then the measurement mode switching for measurement of fluorescence, fluorescence intensity.</p><br />
<img src="https://static.igem.org/mediawiki/2013/e/e9/Degladder.jpg" /><br />
<p>Figure. Fluorescence ladder for degradation test devices. From left, the negative control,BBa_K1051257, BBa_K1051258, BBa_K1051259, J04450 as Positive Control. </p><br />
<p>As the pictures showed, the lights of RFP within three degradation tags are decreasing. </p><br />
<!----------------------<br />
<img src="https://static.igem.org/mediawiki/2013/a/a6/IMG_0804.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/c/ca/IMG_0817.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/M0050bright.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/0/06/M0050strong.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/8/89/M0050weak.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/6/63/Flo.jpg" /><br />
<br />
--------------------><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" /><br />
<p>Figure. The test results for K1051258 and positive control. A. K1051258 in bright field; B. K1051258 in exciting lights; C. Positive control in bright field; D. K1051258 in exciting lights.</p><br />
<p>Images are also available for K1051257 and K1051259. While in simulation, we obtained the degradation rate by calculating with [P1_P]/[P2_P]:</p><br />
<img src="https://static.igem.org/mediawiki/2013/b/ba/Simulation_result.png"> (Calculated from microscope data)<br />
<br />
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<img src="https://static.igem.org/mediawiki/2013/8/83/0917_t00_white_60mm.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/7/72/0917_t15_white_60mm.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/4/4e/0917_t30_white_60mm.jpg" /><br />
--------------------><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg"/><br />
<p>Figure. The test results of BBa_K1051258 in chip. A,LB medium,O minuts; B, IPTG medium,9minutes; C, IPTG medium, 15 minutes</p><br />
<img src="https://static.igem.org/mediawiki/parts/6/6a/DT.png" title="Fluorescence Intensity" description="Fluorescence intensity of positive control and three tested degradation tags." /><p>Figure. Fluorescence intensity calculated by ImageJ positive control and three tested degradation tags.</p><br />
<p>The degradation efficiency of three degradation tags are obvious.</p><br />
<br />
<p>Using the IPTG medium, the new RFP expression was stopped and we can regard the lights as degradation tags' efficiency. Obviously, the lights are decreasing along with the time. Thus, it indicates that the degradation tags work effectively.</p><br />
<img src="https://static.igem.org/mediawiki/parts/1/1e/Average_flurescence_intensity_of_K1051258_measurement.jpg" title="Half Life" description="RFP with degradation tag's half life tested by microfluidics." /><br />
<p>Figure. RFP with degradation tag's half life tested by microfluidics and fit by modeling.</p><br />
<p>Calculated by ImageJ and from experimental fitting curve we got its half-life of our protein 6.88 min. So the degradation rate should be: </p><br />
<img src="https://static.igem.org/mediawiki/2013/9/94/Hl-rate.png"> (Calculated from microfluidics data)<br />
<p>Simulation results calculated and fit from both microscope and microfluidics mates well, which has verified our experimental results.</p><br />
<br />
<h4>Problems</h4><br />
<h5>Yeast Version</h5><br />
<p>The test for yeast degradation tags has not been fully successful. Fluorescence has been captured in some occasions but not stable. It may caused by the same problem with promoter test devices - plasmid copy number. </p><br />
<h5><i>E. coli</i> Version</h5><br />
<p>Data collected from microscope for different degradation tags did not fully meet performance expectation. Reasons may be multiple to increase systematic errors.</p><br />
<p>- Fluorescence is too weak for calculating</p><br />
<p>- Quenching of fluorescence, especially in RFP</p><br />
<p>- Sample size is small for ImageJ calculating</p><br />
<p>We will have more data detected by microfluidics and flow cytometry. Results will be updated later.</p><br />
</div><br />
<br />
<div id="board5" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery5"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/4a/Capture.jpg" title="E. coli captured" description="E. coli cells captured by microfluidics device."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/3/3f/Sc_bright_20.jpg" title="E. coli captured" description="Yeast cells captured by microfluidics device."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/2/23/Sync.jpg" title="Cell Synchronized" description="All cells are synchronized to G1 phase by using the microfluidics method."/></li><br />
</ul></div><br />
<div id="box5" class="box"><br />
<h3>Cell Synchronization</h3><br />
<h4>Microfluidics Device</h4><br />
<p>We made the chip as a platform for watching and synchronize the cells. First step is capture the cells by the chip. As showed in the two figures, both E.coli and budding yeast can be captured by the chip successfully.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/4a/Capture.jpg" /><br />
<p>Figure. <i>E. coli</i> and yeast cells have been captured by the chip successfully.</p><br />
<p>As Sic1 method has not been successful, we tried another way by changing medium by microfluidics. Protocols can be found in Notes page.</p><br />
<img src="https://static.igem.org/mediawiki/2013/2/23/Sync.jpg" title="Cell Synchronized" description="All cells are synchronized to G1 phase by using the microfluidics method."/><br />
<p>Figure. All cells are synchronized to G1 phase by using the microfluidics method.</p><br />
<p>All yeast cells in microfluidics chip are in G1 phase, after budding.</p><br />
<br />
<h4>Sic1 Synchronization</h4><br />
<p>Seven phosphorylation sites are mutated by PCR to make Sic1 mutant. However, it has been taking us too long time that we have been still working on right clones. Experiments results will be updated later. <br />
<br />
</div><br />
<br />
<div id="board6" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery6"><br />
<li><img src="https://static.igem.org/mediawiki/2013/b/b9/Blank-01.png" /><li><br />
</ul></div><br />
<div id="box6" class="box"><br />
<h3>Alternative Splicing</h3><br />
<h4>Original Design</h4><br />
<p>In our project, we attempt to use intron as a switch. However, according to previous research, there are two splicing forms of SRC1 intron: one is complete splicing (5’S) while the other can leave 4bp at its 5’ end (5’L). The remaining 4-bp causes frame-shift and a stop codon in the region of adaptor, thus the following exon will not be translated. However, this kind of switch is not complete – the ratio of 5’L and 5’S varied between 40-60 and 85-15.</p><br />
<p>In order to make a complete switch, we abandoned the remaining 4bp of SRC1 intron to avoid producing 5’L mRNA. So the result comes out to be: when Hub1p are expressed, they bind to spliceosome to modify it, resulting to easy recognizing 5’S splice site and its splicing; when inhibit the expression of HUB1 by CRISPRi system, spliceosome can hardly recognize 5’S splice site, therefore no intron can be spliced. </p><br />
<h4>Further Plans</h4><br />
<p>However, things didn’t go as we expected. Our experiment showed that this 4bp plays important role in SRC1 intron splicing. So, to achieve our goal, we redesigned another three versions of SRC1 intron:<br />
</p><br />
<p>1. Intron with GG at its 5’ end</p><br />
<p>2. Intron with CGG at its 5’ end</p><br />
<p>3. Intron with its original 6bp at both end</p><br />
<p>Due to the lack of time, we are still constructing these parts to test devices.</p><br />
<br />
</div><br />
<br />
</div></div>BGI K2http://2013.igem.org/Team:Shenzhen_BGIC_ATCG/resultsTeam:Shenzhen BGIC ATCG/results2013-09-28T01:16:30Z<p>BGI K2: </p>
<hr />
<div><!----------------------<br />
<br />
<br />
Title: Results<br />
Team: Shenzhen_BGIC_ATCG<br />
Author: BGI_K2<br />
Twitter: @BGI_K2<br />
Email: im.ss.kk@gmail.com<br />
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<div class="navleftholder"><br />
<ul class="navleft"><br />
<li><a id="navleftsub1" class="navleftsub" href="#board1">The Magic</a></li><br />
<li><a id="navleftsub2" class="navleftsub" href="#board2">Promoter Verification</a></li><br />
<li><a id="navleftsub3" class="navleftsub" href="#board3">Reporter Locating</a></li><br />
<li><a id="navleftsub4" class="navleftsub" href="#board4">Degradation Rate</a></li><br />
<li><a id="navleftsub5" class="navleftsub" href="#board5">Cell Synchronization</a></li><br />
<li><a id="navleftsub6" class="navleftsub" href="#board6">Alternative Splicing</a></li><br />
</ul></div><br />
<br />
<div id="board1" class="board"></div><br />
<div class="media"><br />
<br />
<ul id="myGallery1"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/></li><br />
<li><img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" title="Degradation Tags" description="Degradation tags testing device for E. coli."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" title="Microfluidics" description="Microfluidics device for degradation test."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/2/23/Sync.jpg" title="Synchronization" description="All cells are synchronized to G1 phase by using the microfluidics method."/></li><br />
</ul></div><br />
<div id="box1" class="box"><br />
<h3>The Magic</h3><br />
<p>Our project “Cell Magic", a complex work has been completed partly,due to the time limitation. The results we have gotten are listed below:</p><br />
<p>We verified some of the cyclin promoters. The clb6 promoter, which is supposed to be expressed in G1 phase, has been verified perfectly.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." width=50%/><p>Figure. clb6 promoter for G1 phase has been verified</p><br />
<p>Targeting peptides to three locations have been verified. Others are still being constructed and tested.</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. " width=50%/><p>Figure. Test for targeting peptides to mitochondria, nucleus and vacuolar. </P><br />
<p>We got good data for degradation tags for <i>E. coli</i>. Construction of degradation devices for yeast have been finished and being tested. Degradation rate calculated from both methods mates well.</p><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" width=50%/><p>Figure. Pictures for degradation tag testing devices with positive control.</p><br />
<p>Microfluidics device has been successfully developed for measurement and cell synchronization.</p><br />
<img src="https://static.igem.org/mediawiki/2013/f/fc/Microfluidic.gif" /><p>Figure. Cells being captured by microfluidic chip.</p><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" width=50%/><br />
<p>Figure. Degradation tags were tested in Microfluidic</p><br />
<p>Cell synchronization has been successful by changing the method to microfluidics based one.</p><br />
<img src="https://static.igem.org/mediawiki/2013/2/23/Sync.jpg" title="Synchronization" description="All cells are synchronized to G1 phase by using the microfluidics method." width=50%/><br />
<p>Figure. All cells are synchronized to G1 phase by using the microfluidics method.</p><br />
<p>Alternative splicing device are still in construction, related results will be updated later.</p><br />
<br />
</div><br />
<br />
<div id="board2" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery2"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/1/19/Flu.jpg" title="S Phase Promoter" description="cln3 promoter for S phase has been verified using flow cytometry." /></li><br />
</ul></div><br />
<div id="box2" class="box"><br />
<h3>Promoter Verification</h3><br />
<h4>Achievements</h4><br />
<p> Using the GFP as reporter and morphological alteration as cell cycle representation, we verified the Clb6 can be activated in G1 phase in the yeast.</P><br />
<p> As shown in the picture in normal lights, there are some yeast were budding. These budded yeast cells and small size budding yeasts (within the red circles) are assumed in the G1 phase of cell cycle. And the next picture was taken under activation light, thus the green lights can representing the Clb6 promoter expressing. The two pictures indicated the Clb6 were expressed in the G1 phase as we expected when designing the experiments </p><br />
<p> PS: we modified the contrast ratio to lower the lights of neighbor cells, thus our results looking better.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" /><p>Figure. clb6 promoter for G1 phase has been verified.</p><br />
<p>We also captured fluorescence on cln3 promoter (S phase) test device, for the fluorescence is quite weak, we utilize micro plate reader and flow cytometry to verify the promoter.</p><br />
<img src="https://static.igem.org/mediawiki/2013/1/19/Flu.jpg" /><p>Figure. Flow cytometry to verify S phase promoter cln3.</p><br />
<h4>Problems</h4><br />
<h5>Yeast Version</h5><br />
<p>While the promoters are natural ones without optimization, and our shuttle plasmid is pSR416 which is a single copied one, fluorescence is too weak to be captured by microfluidics device. That is a main reason for why we currently cannot show you a perfect cell magic movie. We are now constructing all devices with high copy number vectors, and try to optimize the junction near kozak sequence to make a better magic.</p><br />
<h5><i>E. coli</i> Version</h5><br />
<p>Promoters for <i>E. coli</i> seems not to be successful yet. In our first version, original 5' utr of promoter's downstream gene is included in BioBrick's sequences. The wild-type promoter and RBS may be too weak, or the junction of RFC[23] may have negative effect to the RBS. So in the present version, original 5' utr has been removed and strong RBS B0030 is used. We will update related results later.</p><br />
</div><br />
<br />
<div id="board3" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery3"><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/0/0d/Reporter.jpg" title="Modified Reporters" description="Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose."/></li><br />
</ul></div><br />
<div id="box3" class="box"><br />
<h3>Reporter Locating</h3><br />
<h4>Reporter Modification</h4><br />
<p>Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose.</p><br />
<img src="https://static.igem.org/mediawiki/2013/0/0d/Reporter.jpg" title="Modified Reporters" description="Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose."/><p>Figure. Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose</p><br />
<h4>Targeting Peptides</h4><br />
<p> The four pictures shows the reporters can rightly locate to Mitochondria, Nucleus and Vacuole,respectively. Picture A is the negative control, all yeast cells are lighted with GFP. And figure B is the reporter to the mitochondria, we can saw there are several light spots in one cells. Figure C is the reporter located in nucleus, the green spots are small and there is only one in a yeast cell.The last picture shows the reporter of Vacuolar membrane, the green lights were discrete in cells which was as expected.</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/><p>Figure. Test for targeting peptides to mitochondria, nucleus and vacuolar.</p><br />
</div><br />
<br />
<div id="board4" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery4"><br />
<li><img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" title="E. coli Version" description="Degradation tags testing device for E. coli"/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" title="IPTG Repressed" description="Microfluidics device for half life test with IPTG input (promoter of RFP repressed)."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/e9/Degladder.jpg" title="RFP Ladder" description="RFP ladder with different degradation tags)."/></li><br />
<br />
</ul></div><br />
<div id="box4" class="box"><br />
<h3>Degradation Rate</h3><br />
<p>In every curcuits measurement, we firstly test the growth curve through detecting the absorbance. Taken small amount of bateria, inoculate into 400ul LB medium, ensuring the initial concentration between 0.02-0.05 (OD600). Cultured in the room temperature, detect the OD600 value every 20 minutes. Y axis represents the logarithm of bacteria number, X axis represents the growth time. </p><br />
<img src="https://static.igem.org/mediawiki/2013/6/68/Growth-Curve.jpg" /><p>Figure. Growth curves have been drawn for all constructed devices.</p><br />
<p>In E.coli, the adaptor SspB tethers ssrA tagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. Which means, a variation of the WT SsrA tag sequence (K1051206, K1051207 and K1051208) will accelerate the degradation of proteins when fused to their C-terminal. Thus the degradation rates are dependent on concentration of proteases and binding mediators.</p><br />
<p>We constructed the measurement pathway of each tag (K1051257, K1051258 and K1051259) to test the rates of degradation of tagged proteins respectively. J04450 was used as positive control because of the same promoter and fluorescent protein. </p><br />
<h4>Achievements</h4><br />
<img src="https://static.igem.org/mediawiki/2013/e/e9/Degladder.jpg" /><br />
<p>Figure. Fluorescence ladder for degradation test devices. From left, the negative control,BBa_K1051257, BBa_K1051258, BBa_K1051259, J04450 as Positive Control. </p><br />
<p>As the pictures showed, the lights of RFP within three degradation tags are decreasing. </p><br />
<!----------------------<br />
<img src="https://static.igem.org/mediawiki/2013/a/a6/IMG_0804.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/c/ca/IMG_0817.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/M0050bright.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/0/06/M0050strong.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/8/89/M0050weak.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/6/63/Flo.jpg" /><br />
<br />
--------------------><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" /><br />
<p>Figure. The test results for K1051258 and positive control. A. K1051258 in bright field; B. K1051258 in exciting lights; C. Positive control in bright field; D. K1051258 in exciting lights.</p><br />
<p>Images are also available for K1051257 and K1051259. While in simulation, we obtained the degradation rate by calculating with [P1_P]/[P2_P]:</p><br />
<img src="https://static.igem.org/mediawiki/2013/b/ba/Simulation_result.png"> (Calculated from microscope data)<br />
<p>Enzyme - labelled meter detect the fluorescent protein intensity</p><br />
<p>First of all, take a certain amount of bacteria liquid, recovery to around OD0.6(600), ensuring the bacteria in the logarithmic growth phase. Diluted and then the bacteria transferred to 96 well plates, measured their fluorescence intensity. Red fluorescent protein using an excitation wavelength of 584nm, and its emission wavelength is 607nm.When measuring, we first detect the OD600 of each strain, removing the factor of bacteria number difference. Thus the fluorescence intensity cannot be altered by bacteria quantity. Then the measurement mode switching for measurement of fluorescence, fluorescence intensity.</p><br />
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<img src="https://static.igem.org/mediawiki/2013/8/83/0917_t00_white_60mm.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/7/72/0917_t15_white_60mm.jpg" /><br />
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<img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg"/><br />
<p>Figure. The test results of BBa_K1051258 in chip. A,LB medium,O minuts; B, IPTG medium,9minutes; C, IPTG medium, 15 minutes</p><br />
<img src="https://static.igem.org/mediawiki/parts/6/6a/DT.png" title="Fluorescence Intensity" description="Fluorescence intensity of positive control and three tested degradation tags." /><p>Figure. Fluorescence intensity calculated by ImageJ positive control and three tested degradation tags.</p><br />
<p>The degradation efficiency of three degradation tags are obvious.</p><br />
<br />
<p>Using the IPTG medium, the new RFP expression was stopped and we can regard the lights as degradation tags' efficiency. Obviously, the lights are decreasing along with the time. Thus, it indicates that the degradation tags work effectively.</p><br />
<img src="https://static.igem.org/mediawiki/parts/1/1e/Average_flurescence_intensity_of_K1051258_measurement.jpg" title="Half Life" description="RFP with degradation tag's half life tested by microfluidics." /><br />
<p>Figure. RFP with degradation tag's half life tested by microfluidics and fit by modeling.</p><br />
<p>Calculated by ImageJ and from experimental fitting curve we got its half-life of our protein 6.88 min. So the degradation rate should be: </p><br />
<img src="https://static.igem.org/mediawiki/2013/9/94/Hl-rate.png"> (Calculated from microfluidics data)<br />
<p>Simulation results calculated and fit from both microscope and microfluidics mates well, which has verified our experimental results.</p><br />
<br />
<h4>Problems</h4><br />
<h5>Yeast Version</h5><br />
<p>The test for yeast degradation tags has not been fully successful. Fluorescence has been captured in some occasions but not stable. It may caused by the same problem with promoter test devices - plasmid copy number. </p><br />
<h5><i>E. coli</i> Version</h5><br />
<p>Data collected from microscope for different degradation tags did not fully meet performance expectation. Reasons may be multiple to increase systematic errors.</p><br />
<p>- Fluorescence is too weak for calculating</p><br />
<p>- Quenching of fluorescence, especially in RFP</p><br />
<p>- Sample size is small for ImageJ calculating</p><br />
<p>We will have more data detected by microfluidics and flow cytometry. Results will be updated later.</p><br />
</div><br />
<br />
<div id="board5" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery5"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/4a/Capture.jpg" title="E. coli captured" description="E. coli cells captured by microfluidics device."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/3/3f/Sc_bright_20.jpg" title="E. coli captured" description="Yeast cells captured by microfluidics device."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/2/23/Sync.jpg" title="Cell Synchronized" description="All cells are synchronized to G1 phase by using the microfluidics method."/></li><br />
</ul></div><br />
<div id="box5" class="box"><br />
<h3>Cell Synchronization</h3><br />
<h4>Microfluidics Device</h4><br />
<p>We made the chip as a platform for watching and synchronize the cells. First step is capture the cells by the chip. As showed in the two figures, both E.coli and budding yeast can be captured by the chip successfully.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/4a/Capture.jpg" /><br />
<p>Figure. <i>E. coli</i> and yeast cells have been captured by the chip successfully.</p><br />
<p>As Sic1 method has not been successful, we tried another way by changing medium by microfluidics. Protocols can be found in Notes page.</p><br />
<img src="https://static.igem.org/mediawiki/2013/2/23/Sync.jpg" title="Cell Synchronized" description="All cells are synchronized to G1 phase by using the microfluidics method."/><br />
<p>Figure. All cells are synchronized to G1 phase by using the microfluidics method.</p><br />
<p>All yeast cells in microfluidics chip are in G1 phase, after budding.</p><br />
<br />
<h4>Sic1 Synchronization</h4><br />
<p>Seven phosphorylation sites are mutated by PCR to make Sic1 mutant. However, it has been taking us too long time that we have been still working on right clones. Experiments results will be updated later. <br />
<br />
</div><br />
<br />
<div id="board6" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery6"><br />
<li><img src="https://static.igem.org/mediawiki/2013/b/b9/Blank-01.png" /><li><br />
</ul></div><br />
<div id="box6" class="box"><br />
<h3>Alternative Splicing</h3><br />
<h4>Original Design</h4><br />
<p>In our project, we attempt to use intron as a switch. However, according to previous research, there are two splicing forms of SRC1 intron: one is complete splicing (5’S) while the other can leave 4bp at its 5’ end (5’L). The remaining 4-bp causes frame-shift and a stop codon in the region of adaptor, thus the following exon will not be translated. However, this kind of switch is not complete – the ratio of 5’L and 5’S varied between 40-60 and 85-15.</p><br />
<p>In order to make a complete switch, we abandoned the remaining 4bp of SRC1 intron to avoid producing 5’L mRNA. So the result comes out to be: when Hub1p are expressed, they bind to spliceosome to modify it, resulting to easy recognizing 5’S splice site and its splicing; when inhibit the expression of HUB1 by CRISPRi system, spliceosome can hardly recognize 5’S splice site, therefore no intron can be spliced. </p><br />
<h4>Further Plans</h4><br />
<p>However, things didn’t go as we expected. Our experiment showed that this 4bp plays important role in SRC1 intron splicing. So, to achieve our goal, we redesigned another three versions of SRC1 intron:<br />
</p><br />
<p>1. Intron with GG at its 5’ end</p><br />
<p>2. Intron with CGG at its 5’ end</p><br />
<p>3. Intron with its original 6bp at both end</p><br />
<p>Due to the lack of time, we are still constructing these parts to test devices.</p><br />
<br />
</div><br />
<br />
</div></div>BGI K2http://2013.igem.org/File:Capture.jpgFile:Capture.jpg2013-09-28T01:15:19Z<p>BGI K2: </p>
<hr />
<div></div>BGI K2http://2013.igem.org/File:09172_bright.jpgFile:09172 bright.jpg2013-09-28T01:12:50Z<p>BGI K2: uploaded a new version of &quot;File:09172 bright.jpg&quot;</p>
<hr />
<div></div>BGI K2http://2013.igem.org/File:09172_bright.jpgFile:09172 bright.jpg2013-09-28T01:11:28Z<p>BGI K2: uploaded a new version of &quot;File:09172 bright.jpg&quot;: Reverted to version as of 01:09, 28 September 2013</p>
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<div></div>BGI K2http://2013.igem.org/File:09172_bright.jpgFile:09172 bright.jpg2013-09-28T01:10:10Z<p>BGI K2: uploaded a new version of &quot;File:09172 bright.jpg&quot;</p>
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<div></div>BGI K2http://2013.igem.org/File:09172_bright.jpgFile:09172 bright.jpg2013-09-28T01:09:37Z<p>BGI K2: uploaded a new version of &quot;File:09172 bright.jpg&quot;</p>
<hr />
<div></div>BGI K2http://2013.igem.org/File:09172_bright.jpgFile:09172 bright.jpg2013-09-28T01:09:14Z<p>BGI K2: uploaded a new version of &quot;File:09172 bright.jpg&quot;</p>
<hr />
<div></div>BGI K2http://2013.igem.org/File:09172_bright.jpgFile:09172 bright.jpg2013-09-28T01:09:00Z<p>BGI K2: uploaded a new version of &quot;File:09172 bright.jpg&quot;</p>
<hr />
<div></div>BGI K2http://2013.igem.org/Team:Shenzhen_BGIC_ATCG/resultsTeam:Shenzhen BGIC ATCG/results2013-09-28T01:07:17Z<p>BGI K2: </p>
<hr />
<div><!----------------------<br />
<br />
<br />
Title: Results<br />
Team: Shenzhen_BGIC_ATCG<br />
Author: BGI_K2<br />
Twitter: @BGI_K2<br />
Email: im.ss.kk@gmail.com<br />
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<div class="navleftholder"><br />
<ul class="navleft"><br />
<li><a id="navleftsub1" class="navleftsub" href="#board1">The Magic</a></li><br />
<li><a id="navleftsub2" class="navleftsub" href="#board2">Promoter Verification</a></li><br />
<li><a id="navleftsub3" class="navleftsub" href="#board3">Reporter Locating</a></li><br />
<li><a id="navleftsub4" class="navleftsub" href="#board4">Degradation Rate</a></li><br />
<li><a id="navleftsub5" class="navleftsub" href="#board5">Cell Synchronization</a></li><br />
<li><a id="navleftsub6" class="navleftsub" href="#board6">Alternative Splicing</a></li><br />
</ul></div><br />
<br />
<div id="board1" class="board"></div><br />
<div class="media"><br />
<br />
<ul id="myGallery1"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/></li><br />
<li><img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" title="Degradation Tags" description="Degradation tags testing device for E. coli."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" title="Microfluidics" description="Microfluidics device for degradation test."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/2/23/Sync.jpg" title="Synchronization" description="All cells are synchronized to G1 phase by using the microfluidics method."/></li><br />
</ul></div><br />
<div id="box1" class="box"><br />
<h3>The Magic</h3><br />
<p>Our project “Cell Magic", a complex work has been completed partly,due to the time limitation. The results we have gotten are listed below:</p><br />
<p>We verified some of the cyclin promoters. The clb6 promoter, which is supposed to be expressed in G1 phase, has been verified perfectly.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." width=50%/><p>Figure. clb6 promoter for G1 phase has been verified</p><br />
<p>Targeting peptides to three locations have been verified. Others are still being constructed and tested.</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. " width=50%/><p>Figure. Test for targeting peptides to mitochondria, nucleus and vacuolar. </P><br />
<p>We got good data for degradation tags for <i>E. coli</i>. Construction of degradation devices for yeast have been finished and being tested. Degradation rate calculated from both methods mates well.</p><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" width=50%/><p>Figure. Pictures for degradation tag testing devices with positive control.</p><br />
<p>Microfluidics device has been successfully developed for measurement and cell synchronization.</p><br />
<img src="https://static.igem.org/mediawiki/2013/f/fc/Microfluidic.gif" /><p>Figure. Cells being captured by microfluidic chip.</p><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" width=50%/><br />
<p>Figure. Degradation tags were tested in Microfluidic</p><br />
<p>Cell synchronization has been successful by changing the method to microfluidics based one.</p><br />
<img src="https://static.igem.org/mediawiki/2013/2/23/Sync.jpg" title="Synchronization" description="All cells are synchronized to G1 phase by using the microfluidics method." width=50%/><br />
<p>Figure. All cells are synchronized to G1 phase by using the microfluidics method.</p><br />
<p>Alternative splicing device are still in construction, related results will be updated later.</p><br />
<br />
</div><br />
<br />
<div id="board2" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery2"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/1/19/Flu.jpg" title="S Phase Promoter" description="cln3 promoter for S phase has been verified using flow cytometry." /></li><br />
</ul></div><br />
<div id="box2" class="box"><br />
<h3>Promoter Verification</h3><br />
<h4>Achievements</h4><br />
<p> Using the GFP as reporter and morphological alteration as cell cycle representation, we verified the Clb6 can be activated in G1 phase in the yeast.</P><br />
<p> As shown in the picture in normal lights, there are some yeast were budding. These budded yeast cells and small size budding yeasts (within the red circles) are assumed in the G1 phase of cell cycle. And the next picture was taken under activation light, thus the green lights can representing the Clb6 promoter expressing. The two pictures indicated the Clb6 were expressed in the G1 phase as we expected when designing the experiments </p><br />
<p> PS: we modified the contrast ratio to lower the lights of neighbor cells, thus our results looking better.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" /><p>Figure. clb6 promoter for G1 phase has been verified.</p><br />
<p>We also captured fluorescence on cln3 promoter (S phase) test device, for the fluorescence is quite weak, we utilize micro plate reader and flow cytometry to verify the promoter.</p><br />
<img src="https://static.igem.org/mediawiki/2013/1/19/Flu.jpg" /><p>Figure. Flow cytometry to verify S phase promoter cln3.</p><br />
<h4>Problems</h4><br />
<h5>Yeast Version</h5><br />
<p>While the promoters are natural ones without optimization, and our shuttle plasmid is pSR416 which is a single copied one, fluorescence is too weak to be captured by microfluidics device. That is a main reason for why we currently cannot show you a perfect cell magic movie. We are now constructing all devices with high copy number vectors, and try to optimize the junction near kozak sequence to make a better magic.</p><br />
<h5><i>E. coli</i> Version</h5><br />
<p>Promoters for <i>E. coli</i> seems not to be successful yet. In our first version, original 5' utr of promoter's downstream gene is included in BioBrick's sequences. The wild-type promoter and RBS may be too weak, or the junction of RFC[23] may have negative effect to the RBS. So in the present version, original 5' utr has been removed and strong RBS B0030 is used. We will update related results later.</p><br />
</div><br />
<br />
<div id="board3" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery3"><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/0/0d/Reporter.jpg" title="Modified Reporters" description="Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose."/></li><br />
</ul></div><br />
<div id="box3" class="box"><br />
<h3>Reporter Locating</h3><br />
<h4>Reporter Modification</h4><br />
<p>Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose.</p><br />
<img src="https://static.igem.org/mediawiki/2013/0/0d/Reporter.jpg" title="Modified Reporters" description="Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose."/><p>Figure. Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose</p><br />
<h4>Targeting Peptides</h4><br />
<p> The four pictures shows the reporters can rightly locate to Mitochondria, Nucleus and Vacuole,respectively. Picture A is the negative control, all yeast cells are lighted with GFP. And figure B is the reporter to the mitochondria, we can saw there are several light spots in one cells. Figure C is the reporter located in nucleus, the green spots are small and there is only one in a yeast cell.The last picture shows the reporter of Vacuolar membrane, the green lights were discrete in cells which was as expected.</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/><p>Figure. Test for targeting peptides to mitochondria, nucleus and vacuolar.</p><br />
</div><br />
<br />
<div id="board4" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery4"><br />
<li><img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" title="E. coli Version" description="Degradation tags testing device for E. coli"/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" title="IPTG Repressed" description="Microfluidics device for half life test with IPTG input (promoter of RFP repressed)."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/e9/Degladder.jpg" title="RFP Ladder" description="RFP ladder with different degradation tags)."/></li><br />
<br />
</ul></div><br />
<div id="box4" class="box"><br />
<h3>Degradation Rate</h3><br />
<p>In every curcuits measurement, we firstly test the growth curve through detecting the absorbance. Taken small amount of bateria, inoculate into 400ul LB medium, ensuring the initial concentration between 0.02-0.05 (OD600). Cultured in the room temperature, detect the OD600 value every 20 minutes. Y axis represents the logarithm of bacteria number, X axis represents the growth time. </p><br />
<img src="https://static.igem.org/mediawiki/2013/6/68/Growth-Curve.jpg" /><p>Figure. Growth curves have been drawn for all constructed devices.</p><br />
<p>In E.coli, the adaptor SspB tethers ssrA tagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. Which means, a variation of the WT SsrA tag sequence (K1051206, K1051207 and K1051208) will accelerate the degradation of proteins when fused to their C-terminal. Thus the degradation rates are dependent on concentration of proteases and binding mediators.</p><br />
<p>We constructed the measurement pathway of each tag (K1051257, K1051258 and K1051259) to test the rates of degradation of tagged proteins respectively. J04450 was used as positive control because of the same promoter and fluorescent protein. </p><br />
<h4>Achievements</h4><br />
<img src="https://static.igem.org/mediawiki/2013/e/e9/Degladder.jpg" /><br />
<p>Figure. Fluorescence ladder for degradation test devices. From left, the negative control,BBa_K1051257, BBa_K1051258, BBa_K1051259, J04450 as Positive Control. </p><br />
<p>As the pictures showed, the lights of RFP within three degradation tags are decreasing. </p><br />
<!----------------------<br />
<img src="https://static.igem.org/mediawiki/2013/a/a6/IMG_0804.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/c/ca/IMG_0817.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/M0050bright.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/0/06/M0050strong.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/8/89/M0050weak.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/6/63/Flo.jpg" /><br />
<br />
--------------------><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" /><br />
<p>Figure. The test results for K1051258 and positive control. A. K1051258 in bright field; B. K1051258 in exciting lights; C. Positive control in bright field; D. K1051258 in exciting lights.</p><br />
<p>Images are also available for K1051257 and K1051259. While in simulation, we obtained the degradation rate by calculating with [P1_P]/[P2_P]:</p><br />
<img src="https://static.igem.org/mediawiki/2013/b/ba/Simulation_result.png"> (Calculated from microscope data)<br />
<p>Enzyme - labelled meter detect the fluorescent protein intensity</p><br />
<p>First of all, take a certain amount of bacteria liquid, recovery to around OD0.6(600), ensuring the bacteria in the logarithmic growth phase. Diluted and then the bacteria transferred to 96 well plates, measured their fluorescence intensity. Red fluorescent protein using an excitation wavelength of 584nm, and its emission wavelength is 607nm.When measuring, we first detect the OD600 of each strain, removing the factor of bacteria number difference. Thus the fluorescence intensity cannot be altered by bacteria quantity. Then the measurement mode switching for measurement of fluorescence, fluorescence intensity.</p><br />
<!----------------------<br />
<img src="https://static.igem.org/mediawiki/2013/8/83/0917_t00_white_60mm.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/7/72/0917_t15_white_60mm.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/4/4e/0917_t30_white_60mm.jpg" /><br />
--------------------><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg"/><br />
<p>Figure. The test results of BBa_K1051258 in chip. A,LB medium,O minuts; B, IPTG medium,9minutes; C, IPTG medium, 15 minutes</p><br />
<img src="https://static.igem.org/mediawiki/parts/6/6a/DT.png" title="Fluorescence Intensity" description="Fluorescence intensity of positive control and three tested degradation tags." /><p>Figure. Fluorescence intensity calculated by ImageJ positive control and three tested degradation tags.</p><br />
<p>The degradation efficiency of three degradation tags are obvious.</p><br />
<br />
<p>Using the IPTG medium, the new RFP expression was stopped and we can regard the lights as degradation tags' efficiency. Obviously, the lights are decreasing along with the time. Thus, it indicates that the degradation tags work effectively.</p><br />
<img src="https://static.igem.org/mediawiki/parts/1/1e/Average_flurescence_intensity_of_K1051258_measurement.jpg" title="Half Life" description="RFP with degradation tag's half life tested by microfluidics." /><br />
<p>Figure. RFP with degradation tag's half life tested by microfluidics and fit by modeling.</p><br />
<p>Calculated by ImageJ and from experimental fitting curve we got its half-life of our protein 6.88 min. So the degradation rate should be: </p><br />
<img src="https://static.igem.org/mediawiki/2013/9/94/Hl-rate.png"> (Calculated from microfluidics data)<br />
<p>Simulation results calculated and fit from both microscope and microfluidics mates well, which has verified our experimental results.</p><br />
<br />
<h4>Problems</h4><br />
<h5>Yeast Version</h5><br />
<p>The test for yeast degradation tags has not been fully successful. Fluorescence has been captured in some occasions but not stable. It may caused by the same problem with promoter test devices - plasmid copy number. </p><br />
<h5><i>E. coli</i> Version</h5><br />
<p>Data collected from microscope for different degradation tags did not fully meet performance expectation. Reasons may be multiple to increase systematic errors.</p><br />
<p>- Fluorescence is too weak for calculating</p><br />
<p>- Quenching of fluorescence, especially in RFP</p><br />
<p>- Sample size is small for ImageJ calculating</p><br />
<p>We will have more data detected by microfluidics and flow cytometry. Results will be updated later.</p><br />
</div><br />
<br />
<div id="board5" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery5"><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/e4/09172_bright.jpg" title="E. coli captured" description="E. coli cells captured by microfluidics device."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/3/3f/Sc_bright_20.jpg" title="E. coli captured" description="Yeast cells captured by microfluidics device."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/2/23/Sync.jpg" title="Cell Synchronized" description="All cells are synchronized to G1 phase by using the microfluidics method."/></li><br />
</ul></div><br />
<div id="box5" class="box"><br />
<h3>Cell Synchronization</h3><br />
<h4>Microfluidics Device</h4><br />
<p>We made the chip as a platform for watching and synchronize the cells. First step is capture the cells by the chip. As showed in the two figures, both E.coli and budding yeast can be captured by the chip successfully.</p><br />
<img src="https://static.igem.org/mediawiki/2013/e/e4/09172_bright.jpg" /><br />
<p>Figure. <i>E. coli</i> and yeast cells have been captured by the chip successfully.</p><br />
<p>As Sic1 method has not been successful, we tried another way by changing medium by microfluidics. Protocols can be found in Notes page.</p><br />
<img src="https://static.igem.org/mediawiki/2013/2/23/Sync.jpg" title="Cell Synchronized" description="All cells are synchronized to G1 phase by using the microfluidics method."/><br />
<p>Figure. All cells are synchronized to G1 phase by using the microfluidics method.</p><br />
<p>All yeast cells in microfluidics chip are in G1 phase, after budding.</p><br />
<br />
<h4>Sic1 Synchronization</h4><br />
<p>Seven phosphorylation sites are mutated by PCR to make Sic1 mutant. However, it has been taking us too long time that we have been still working on right clones. Experiments results will be updated later. <br />
<br />
</div><br />
<br />
<div id="board6" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery6"><br />
<li><img src="https://static.igem.org/mediawiki/2013/b/b9/Blank-01.png" /><li><br />
</ul></div><br />
<div id="box6" class="box"><br />
<h3>Alternative Splicing</h3><br />
<h4>Original Design</h4><br />
<p>In our project, we attempt to use intron as a switch. However, according to previous research, there are two splicing forms of SRC1 intron: one is complete splicing (5’S) while the other can leave 4bp at its 5’ end (5’L). The remaining 4-bp causes frame-shift and a stop codon in the region of adaptor, thus the following exon will not be translated. However, this kind of switch is not complete – the ratio of 5’L and 5’S varied between 40-60 and 85-15.</p><br />
<p>In order to make a complete switch, we abandoned the remaining 4bp of SRC1 intron to avoid producing 5’L mRNA. So the result comes out to be: when Hub1p are expressed, they bind to spliceosome to modify it, resulting to easy recognizing 5’S splice site and its splicing; when inhibit the expression of HUB1 by CRISPRi system, spliceosome can hardly recognize 5’S splice site, therefore no intron can be spliced. </p><br />
<h4>Further Plans</h4><br />
<p>However, things didn’t go as we expected. Our experiment showed that this 4bp plays important role in SRC1 intron splicing. So, to achieve our goal, we redesigned another three versions of SRC1 intron:<br />
</p><br />
<p>1. Intron with GG at its 5’ end</p><br />
<p>2. Intron with CGG at its 5’ end</p><br />
<p>3. Intron with its original 6bp at both end</p><br />
<p>Due to the lack of time, we are still constructing these parts to test devices.</p><br />
<br />
</div><br />
<br />
</div></div>BGI K2http://2013.igem.org/Team:Shenzhen_BGIC_ATCG/resultsTeam:Shenzhen BGIC ATCG/results2013-09-28T01:06:01Z<p>BGI K2: </p>
<hr />
<div><!----------------------<br />
<br />
<br />
Title: Results<br />
Team: Shenzhen_BGIC_ATCG<br />
Author: BGI_K2<br />
Twitter: @BGI_K2<br />
Email: im.ss.kk@gmail.com<br />
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<div class="navleftholder"><br />
<ul class="navleft"><br />
<li><a id="navleftsub1" class="navleftsub" href="#board1">The Magic</a></li><br />
<li><a id="navleftsub2" class="navleftsub" href="#board2">Promoter Verification</a></li><br />
<li><a id="navleftsub3" class="navleftsub" href="#board3">Reporter Locating</a></li><br />
<li><a id="navleftsub4" class="navleftsub" href="#board4">Degradation Rate</a></li><br />
<li><a id="navleftsub5" class="navleftsub" href="#board5">Cell Synchronization</a></li><br />
<li><a id="navleftsub6" class="navleftsub" href="#board6">Alternative Splicing</a></li><br />
</ul></div><br />
<br />
<div id="board1" class="board"></div><br />
<div class="media"><br />
<br />
<ul id="myGallery1"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/></li><br />
<li><img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" title="Degradation Tags" description="Degradation tags testing device for E. coli."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" title="Microfluidics" description="Microfluidics device for degradation test."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/2/23/Sync.jpg" title="Cell Synchronized" description="All cells are synchronized to G1 phase by using the microfluidics method."/></li><br />
</ul></div><br />
<div id="box1" class="box"><br />
<h3>The Magic</h3><br />
<p>Our project “Cell Magic", a complex work has been completed partly,due to the time limitation. The results we have gotten are listed below:</p><br />
<p>We verified some of the cyclin promoters. The clb6 promoter, which is supposed to be expressed in G1 phase, has been verified perfectly.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." width=50%/><p>Figure. clb6 promoter for G1 phase has been verified</p><br />
<p>Targeting peptides to three locations have been verified. Others are still being constructed and tested.</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. " width=50%/><p>Figure. Test for targeting peptides to mitochondria, nucleus and vacuolar. </P><br />
<p>We got good data for degradation tags for <i>E. coli</i>. Construction of degradation devices for yeast have been finished and being tested. Degradation rate calculated from both methods mates well.</p><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" width=50%/><p>Figure. Pictures for degradation tag testing devices with positive control.</p><br />
<p>Microfluidics device has been successfully developed for measurement and cell synchronization.</p><br />
<img src="https://static.igem.org/mediawiki/2013/f/fc/Microfluidic.gif" /><p>Figure. Cells being captured by microfluidic chip.</p><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" width=50%/><br />
<p>Figure. Degradation tags were tested in Microfluidic</p><br />
<p>Cell synchronization has been successful by changing the method to microfluidics based one.</p><br />
<img src="https://static.igem.org/mediawiki/2013/2/23/Sync.jpg" title="Cell Synchronized" description="All cells are synchronized to G1 phase by using the microfluidics method." width=50%/><br />
<p>Figure. All cells are synchronized to G1 phase by using the microfluidics method.</p><br />
<p>Alternative splicing device are still in construction, related results will be updated later.</p><br />
<br />
</div><br />
<br />
<div id="board2" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery2"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/1/19/Flu.jpg" title="S Phase Promoter" description="cln3 promoter for S phase has been verified using flow cytometry." /></li><br />
</ul></div><br />
<div id="box2" class="box"><br />
<h3>Promoter Verification</h3><br />
<h4>Achievements</h4><br />
<p> Using the GFP as reporter and morphological alteration as cell cycle representation, we verified the Clb6 can be activated in G1 phase in the yeast.</P><br />
<p> As shown in the picture in normal lights, there are some yeast were budding. These budded yeast cells and small size budding yeasts (within the red circles) are assumed in the G1 phase of cell cycle. And the next picture was taken under activation light, thus the green lights can representing the Clb6 promoter expressing. The two pictures indicated the Clb6 were expressed in the G1 phase as we expected when designing the experiments </p><br />
<p> PS: we modified the contrast ratio to lower the lights of neighbor cells, thus our results looking better.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" /><p>Figure. clb6 promoter for G1 phase has been verified.</p><br />
<p>We also captured fluorescence on cln3 promoter (S phase) test device, for the fluorescence is quite weak, we utilize micro plate reader and flow cytometry to verify the promoter.</p><br />
<img src="https://static.igem.org/mediawiki/2013/1/19/Flu.jpg" /><p>Figure. Flow cytometry to verify S phase promoter cln3.</p><br />
<h4>Problems</h4><br />
<h5>Yeast Version</h5><br />
<p>While the promoters are natural ones without optimization, and our shuttle plasmid is pSR416 which is a single copied one, fluorescence is too weak to be captured by microfluidics device. That is a main reason for why we currently cannot show you a perfect cell magic movie. We are now constructing all devices with high copy number vectors, and try to optimize the junction near kozak sequence to make a better magic.</p><br />
<h5><i>E. coli</i> Version</h5><br />
<p>Promoters for <i>E. coli</i> seems not to be successful yet. In our first version, original 5' utr of promoter's downstream gene is included in BioBrick's sequences. The wild-type promoter and RBS may be too weak, or the junction of RFC[23] may have negative effect to the RBS. So in the present version, original 5' utr has been removed and strong RBS B0030 is used. We will update related results later.</p><br />
</div><br />
<br />
<div id="board3" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery3"><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/0/0d/Reporter.jpg" title="Modified Reporters" description="Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose."/></li><br />
</ul></div><br />
<div id="box3" class="box"><br />
<h3>Reporter Locating</h3><br />
<h4>Reporter Modification</h4><br />
<p>Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose.</p><br />
<img src="https://static.igem.org/mediawiki/2013/0/0d/Reporter.jpg" title="Modified Reporters" description="Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose."/><p>Figure. Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose</p><br />
<h4>Targeting Peptides</h4><br />
<p> The four pictures shows the reporters can rightly locate to Mitochondria, Nucleus and Vacuole,respectively. Picture A is the negative control, all yeast cells are lighted with GFP. And figure B is the reporter to the mitochondria, we can saw there are several light spots in one cells. Figure C is the reporter located in nucleus, the green spots are small and there is only one in a yeast cell.The last picture shows the reporter of Vacuolar membrane, the green lights were discrete in cells which was as expected.</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/><p>Figure. Test for targeting peptides to mitochondria, nucleus and vacuolar.</p><br />
</div><br />
<br />
<div id="board4" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery4"><br />
<li><img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" title="E. coli Version" description="Degradation tags testing device for E. coli"/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" title="IPTG Repressed" description="Microfluidics device for half life test with IPTG input (promoter of RFP repressed)."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/e9/Degladder.jpg" title="RFP Ladder" description="RFP ladder with different degradation tags)."/></li><br />
<br />
</ul></div><br />
<div id="box4" class="box"><br />
<h3>Degradation Rate</h3><br />
<p>In every curcuits measurement, we firstly test the growth curve through detecting the absorbance. Taken small amount of bateria, inoculate into 400ul LB medium, ensuring the initial concentration between 0.02-0.05 (OD600). Cultured in the room temperature, detect the OD600 value every 20 minutes. Y axis represents the logarithm of bacteria number, X axis represents the growth time. </p><br />
<img src="https://static.igem.org/mediawiki/2013/6/68/Growth-Curve.jpg" /><p>Figure. Growth curves have been drawn for all constructed devices.</p><br />
<p>In E.coli, the adaptor SspB tethers ssrA tagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. Which means, a variation of the WT SsrA tag sequence (K1051206, K1051207 and K1051208) will accelerate the degradation of proteins when fused to their C-terminal. Thus the degradation rates are dependent on concentration of proteases and binding mediators.</p><br />
<p>We constructed the measurement pathway of each tag (K1051257, K1051258 and K1051259) to test the rates of degradation of tagged proteins respectively. J04450 was used as positive control because of the same promoter and fluorescent protein. </p><br />
<h4>Achievements</h4><br />
<img src="https://static.igem.org/mediawiki/2013/e/e9/Degladder.jpg" /><br />
<p>Figure. Fluorescence ladder for degradation test devices. From left, the negative control,BBa_K1051257, BBa_K1051258, BBa_K1051259, J04450 as Positive Control. </p><br />
<p>As the pictures showed, the lights of RFP within three degradation tags are decreasing. </p><br />
<!----------------------<br />
<img src="https://static.igem.org/mediawiki/2013/a/a6/IMG_0804.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/c/ca/IMG_0817.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/M0050bright.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/0/06/M0050strong.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/8/89/M0050weak.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/6/63/Flo.jpg" /><br />
<br />
--------------------><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" /><br />
<p>Figure. The test results for K1051258 and positive control. A. K1051258 in bright field; B. K1051258 in exciting lights; C. Positive control in bright field; D. K1051258 in exciting lights.</p><br />
<p>Images are also available for K1051257 and K1051259. While in simulation, we obtained the degradation rate by calculating with [P1_P]/[P2_P]:</p><br />
<img src="https://static.igem.org/mediawiki/2013/b/ba/Simulation_result.png"> (Calculated from microscope data)<br />
<p>Enzyme - labelled meter detect the fluorescent protein intensity</p><br />
<p>First of all, take a certain amount of bacteria liquid, recovery to around OD0.6(600), ensuring the bacteria in the logarithmic growth phase. Diluted and then the bacteria transferred to 96 well plates, measured their fluorescence intensity. Red fluorescent protein using an excitation wavelength of 584nm, and its emission wavelength is 607nm.When measuring, we first detect the OD600 of each strain, removing the factor of bacteria number difference. Thus the fluorescence intensity cannot be altered by bacteria quantity. Then the measurement mode switching for measurement of fluorescence, fluorescence intensity.</p><br />
<!----------------------<br />
<img src="https://static.igem.org/mediawiki/2013/8/83/0917_t00_white_60mm.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/7/72/0917_t15_white_60mm.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/4/4e/0917_t30_white_60mm.jpg" /><br />
--------------------><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg"/><br />
<p>Figure. The test results of BBa_K1051258 in chip. A,LB medium,O minuts; B, IPTG medium,9minutes; C, IPTG medium, 15 minutes</p><br />
<img src="https://static.igem.org/mediawiki/parts/6/6a/DT.png" title="Fluorescence Intensity" description="Fluorescence intensity of positive control and three tested degradation tags." /><p>Figure. Fluorescence intensity calculated by ImageJ positive control and three tested degradation tags.</p><br />
<p>The degradation efficiency of three degradation tags are obvious.</p><br />
<br />
<p>Using the IPTG medium, the new RFP expression was stopped and we can regard the lights as degradation tags' efficiency. Obviously, the lights are decreasing along with the time. Thus, it indicates that the degradation tags work effectively.</p><br />
<img src="https://static.igem.org/mediawiki/parts/1/1e/Average_flurescence_intensity_of_K1051258_measurement.jpg" title="Half Life" description="RFP with degradation tag's half life tested by microfluidics." /><br />
<p>Figure. RFP with degradation tag's half life tested by microfluidics and fit by modeling.</p><br />
<p>Calculated by ImageJ and from experimental fitting curve we got its half-life of our protein 6.88 min. So the degradation rate should be: </p><br />
<img src="https://static.igem.org/mediawiki/2013/9/94/Hl-rate.png"> (Calculated from microfluidics data)<br />
<p>Simulation results calculated and fit from both microscope and microfluidics mates well, which has verified our experimental results.</p><br />
<br />
<h4>Problems</h4><br />
<h5>Yeast Version</h5><br />
<p>The test for yeast degradation tags has not been fully successful. Fluorescence has been captured in some occasions but not stable. It may caused by the same problem with promoter test devices - plasmid copy number. </p><br />
<h5><i>E. coli</i> Version</h5><br />
<p>Data collected from microscope for different degradation tags did not fully meet performance expectation. Reasons may be multiple to increase systematic errors.</p><br />
<p>- Fluorescence is too weak for calculating</p><br />
<p>- Quenching of fluorescence, especially in RFP</p><br />
<p>- Sample size is small for ImageJ calculating</p><br />
<p>We will have more data detected by microfluidics and flow cytometry. Results will be updated later.</p><br />
</div><br />
<br />
<div id="board5" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery5"><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/e4/09172_bright.jpg" title="E. coli captured" description="E. coli cells captured by microfluidics device."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/3/3f/Sc_bright_20.jpg" title="E. coli captured" description="Yeast cells captured by microfluidics device."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/2/23/Sync.jpg" title="Cell Synchronized" description="All cells are synchronized to G1 phase by using the microfluidics method."/></li><br />
</ul></div><br />
<div id="box5" class="box"><br />
<h3>Cell Synchronization</h3><br />
<h4>Microfluidics Device</h4><br />
<p>We made the chip as a platform for watching and synchronize the cells. First step is capture the cells by the chip. As showed in the two figures, both E.coli and budding yeast can be captured by the chip successfully.</p><br />
<img src="https://static.igem.org/mediawiki/2013/e/e4/09172_bright.jpg" /><br />
<p>Figure. <i>E. coli</i> and yeast cells have been captured by the chip successfully.</p><br />
<p>As Sic1 method has not been successful, we tried another way by changing medium by microfluidics. Protocols can be found in Notes page.</p><br />
<img src="https://static.igem.org/mediawiki/2013/2/23/Sync.jpg" title="Cell Synchronized" description="All cells are synchronized to G1 phase by using the microfluidics method."/><br />
<p>Figure. All cells are synchronized to G1 phase by using the microfluidics method.</p><br />
<p>All yeast cells in microfluidics chip are in G1 phase, after budding.</p><br />
<br />
<h4>Sic1 Synchronization</h4><br />
<p>Seven phosphorylation sites are mutated by PCR to make Sic1 mutant. However, it has been taking us too long time that we have been still working on right clones. Experiments results will be updated later. <br />
<br />
</div><br />
<br />
<div id="board6" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery6"><br />
<li><img src="https://static.igem.org/mediawiki/2013/b/b9/Blank-01.png" /><li><br />
</ul></div><br />
<div id="box6" class="box"><br />
<h3>Alternative Splicing</h3><br />
<h4>Original Design</h4><br />
<p>In our project, we attempt to use intron as a switch. However, according to previous research, there are two splicing forms of SRC1 intron: one is complete splicing (5’S) while the other can leave 4bp at its 5’ end (5’L). The remaining 4-bp causes frame-shift and a stop codon in the region of adaptor, thus the following exon will not be translated. However, this kind of switch is not complete – the ratio of 5’L and 5’S varied between 40-60 and 85-15.</p><br />
<p>In order to make a complete switch, we abandoned the remaining 4bp of SRC1 intron to avoid producing 5’L mRNA. So the result comes out to be: when Hub1p are expressed, they bind to spliceosome to modify it, resulting to easy recognizing 5’S splice site and its splicing; when inhibit the expression of HUB1 by CRISPRi system, spliceosome can hardly recognize 5’S splice site, therefore no intron can be spliced. </p><br />
<h4>Further Plans</h4><br />
<p>However, things didn’t go as we expected. Our experiment showed that this 4bp plays important role in SRC1 intron splicing. So, to achieve our goal, we redesigned another three versions of SRC1 intron:<br />
</p><br />
<p>1. Intron with GG at its 5’ end</p><br />
<p>2. Intron with CGG at its 5’ end</p><br />
<p>3. Intron with its original 6bp at both end</p><br />
<p>Due to the lack of time, we are still constructing these parts to test devices.</p><br />
<br />
</div><br />
<br />
</div></div>BGI K2http://2013.igem.org/Team:Shenzhen_BGIC_ATCG/resultsTeam:Shenzhen BGIC ATCG/results2013-09-28T01:03:36Z<p>BGI K2: </p>
<hr />
<div><!----------------------<br />
<br />
<br />
Title: Results<br />
Team: Shenzhen_BGIC_ATCG<br />
Author: BGI_K2<br />
Twitter: @BGI_K2<br />
Email: im.ss.kk@gmail.com<br />
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<div class="navleftholder"><br />
<ul class="navleft"><br />
<li><a id="navleftsub1" class="navleftsub" href="#board1">The Magic</a></li><br />
<li><a id="navleftsub2" class="navleftsub" href="#board2">Promoter Verification</a></li><br />
<li><a id="navleftsub3" class="navleftsub" href="#board3">Reporter Locating</a></li><br />
<li><a id="navleftsub4" class="navleftsub" href="#board4">Degradation Rate</a></li><br />
<li><a id="navleftsub5" class="navleftsub" href="#board5">Cell Synchronization</a></li><br />
<li><a id="navleftsub6" class="navleftsub" href="#board6">Alternative Splicing</a></li><br />
</ul></div><br />
<br />
<div id="board1" class="board"></div><br />
<div class="media"><br />
<br />
<ul id="myGallery1"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/></li><br />
<li><img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" title="Degradation Tags" description="Degradation tags testing device for E. coli."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" title="Microfluidics" description="Microfluidics device for degradation test."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/2/23/Sync.jpg" title="Cell Synchronized" description="All cells are synchronized to G1 phase by using the microfluidics method."/></li><br />
</ul></div><br />
<div id="box1" class="box"><br />
<h3>The Magic</h3><br />
<p>Our project “Cell Magic", a complex work has been completed partly,due to the time limitation. The results we have gotten are listed below:</p><br />
<p>We verified some of the cyclin promoters. The clb6 promoter, which is supposed to be expressed in G1 phase, has been verified perfectly.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." width=50%/><p>Figure. clb6 promoter for G1 phase has been verified</p><br />
<p>Targeting peptides to three locations have been verified. Others are still being constructed and tested.</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. " width=50%/><p>Figure. Test for targeting peptides to mitochondria, nucleus and vacuolar. </P><br />
<p>We got good data for degradation tags for <i>E. coli</i>. Construction of degradation devices for yeast have been finished and being tested.</p><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" width=50%/><p>Figure. Good data for degradation tags</p><br />
<p>Microfluidics device has been successfully developed for measurement and cell synchronization.</p><br />
<img src="https://static.igem.org/mediawiki/2013/f/fc/Microfluidic.gif" /><p>Figure. Cells being captured by microfluidic chip.</p><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" width=50%/><br />
<p>Figure. Degradation tags were tested in Microfluidic</p><br />
<p>Cell synchronization has been successful by changing the method to microfluidics based one.</p><br />
<img src="https://static.igem.org/mediawiki/2013/2/23/Sync.jpg" title="Cell Synchronized" description="All cells are synchronized to G1 phase by using the microfluidics method."/><br />
<p>Figure. All cells are synchronized to G1 phase by using the microfluidics method.</p><br />
<p>Alternative splicing device are still in construction, related results will be updated later.</p><br />
<br />
</div><br />
<br />
<div id="board2" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery2"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/1/19/Flu.jpg" title="S Phase Promoter" description="cln3 promoter for S phase has been verified using flow cytometry." /></li><br />
</ul></div><br />
<div id="box2" class="box"><br />
<h3>Promoter Verification</h3><br />
<h4>Achievements</h4><br />
<p> Using the GFP as reporter and morphological alteration as cell cycle representation, we verified the Clb6 can be activated in G1 phase in the yeast.</P><br />
<p> As shown in the picture in normal lights, there are some yeast were budding. These budded yeast cells and small size budding yeasts (within the red circles) are assumed in the G1 phase of cell cycle. And the next picture was taken under activation light, thus the green lights can representing the Clb6 promoter expressing. The two pictures indicated the Clb6 were expressed in the G1 phase as we expected when designing the experiments </p><br />
<p> PS: we modified the contrast ratio to lower the lights of neighbor cells, thus our results looking better.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" /><p>Figure. clb6 promoter for G1 phase has been verified.</p><br />
<p>We also captured fluorescence on cln3 promoter (S phase) test device, for the fluorescence is quite weak, we utilize micro plate reader and flow cytometry to verify the promoter.</p><br />
<img src="https://static.igem.org/mediawiki/2013/1/19/Flu.jpg" /><p>Figure. Flow cytometry to verify S phase promoter cln3.</p><br />
<h4>Problems</h4><br />
<h5>Yeast Version</h5><br />
<p>While the promoters are natural ones without optimization, and our shuttle plasmid is pSR416 which is a single copied one, fluorescence is too weak to be captured by microfluidics device. That is a main reason for why we currently cannot show you a perfect cell magic movie. We are now constructing all devices with high copy number vectors, and try to optimize the junction near kozak sequence to make a better magic.</p><br />
<h5><i>E. coli</i> Version</h5><br />
<p>Promoters for <i>E. coli</i> seems not to be successful yet. In our first version, original 5' utr of promoter's downstream gene is included in BioBrick's sequences. The wild-type promoter and RBS may be too weak, or the junction of RFC[23] may have negative effect to the RBS. So in the present version, original 5' utr has been removed and strong RBS B0030 is used. We will update related results later.</p><br />
</div><br />
<br />
<div id="board3" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery3"><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/0/0d/Reporter.jpg" title="Modified Reporters" description="Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose."/></li><br />
</ul></div><br />
<div id="box3" class="box"><br />
<h3>Reporter Locating</h3><br />
<h4>Reporter Modification</h4><br />
<p>Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose.</p><br />
<img src="https://static.igem.org/mediawiki/2013/0/0d/Reporter.jpg" title="Modified Reporters" description="Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose."/><p>Figure. Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose</p><br />
<h4>Targeting Peptides</h4><br />
<p> The four pictures shows the reporters can rightly locate to Mitochondria, Nucleus and Vacuole,respectively. Picture A is the negative control, all yeast cells are lighted with GFP. And figure B is the reporter to the mitochondria, we can saw there are several light spots in one cells. Figure C is the reporter located in nucleus, the green spots are small and there is only one in a yeast cell.The last picture shows the reporter of Vacuolar membrane, the green lights were discrete in cells which was as expected.</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/><p>Figure. Test for targeting peptides to mitochondria, nucleus and vacuolar.</p><br />
</div><br />
<br />
<div id="board4" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery4"><br />
<li><img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" title="E. coli Version" description="Degradation tags testing device for E. coli"/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" title="IPTG Repressed" description="Microfluidics device for half life test with IPTG input (promoter of RFP repressed)."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/e9/Degladder.jpg" title="RFP Ladder" description="RFP ladder with different degradation tags)."/></li><br />
<br />
</ul></div><br />
<div id="box4" class="box"><br />
<h3>Degradation Rate</h3><br />
<p>In every curcuits measurement, we firstly test the growth curve through detecting the absorbance. Taken small amount of bateria, inoculate into 400ul LB medium, ensuring the initial concentration between 0.02-0.05 (OD600). Cultured in the room temperature, detect the OD600 value every 20 minutes. Y axis represents the logarithm of bacteria number, X axis represents the growth time. </p><br />
<img src="https://static.igem.org/mediawiki/2013/6/68/Growth-Curve.jpg" /><p>Figure. Growth curves have been drawn for all constructed devices.</p><br />
<p>In E.coli, the adaptor SspB tethers ssrA tagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. Which means, a variation of the WT SsrA tag sequence (K1051206, K1051207 and K1051208) will accelerate the degradation of proteins when fused to their C-terminal. Thus the degradation rates are dependent on concentration of proteases and binding mediators.</p><br />
<p>We constructed the measurement pathway of each tag (K1051257, K1051258 and K1051259) to test the rates of degradation of tagged proteins respectively. J04450 was used as positive control because of the same promoter and fluorescent protein. </p><br />
<h4>Achievements</h4><br />
<img src="https://static.igem.org/mediawiki/2013/e/e9/Degladder.jpg" /><br />
<p>Figure. Fluorescence ladder for degradation test devices. From left, the negative control,BBa_K1051257, BBa_K1051258, BBa_K1051259, J04450 as Positive Control. </p><br />
<p>As the pictures showed, the lights of RFP within three degradation tags are decreasing. </p><br />
<!----------------------<br />
<img src="https://static.igem.org/mediawiki/2013/a/a6/IMG_0804.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/c/ca/IMG_0817.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/M0050bright.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/0/06/M0050strong.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/8/89/M0050weak.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/6/63/Flo.jpg" /><br />
<br />
--------------------><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" /><br />
<p>Figure. The test results for K1051258 and positive control. A. K1051258 in bright field; B. K1051258 in exciting lights; C. Positive control in bright field; D. K1051258 in exciting lights.</p><br />
<p>Images are also available for K1051257 and K1051259. While in simulation, we obtained the degradation rate by calculating with [P1_P]/[P2_P]:</p><br />
<img src="https://static.igem.org/mediawiki/2013/b/ba/Simulation_result.png"> (Calculated from microscope data)<br />
<p>Enzyme - labelled meter detect the fluorescent protein intensity</p><br />
<p>First of all, take a certain amount of bacteria liquid, recovery to around OD0.6(600), ensuring the bacteria in the logarithmic growth phase. Diluted and then the bacteria transferred to 96 well plates, measured their fluorescence intensity. Red fluorescent protein using an excitation wavelength of 584nm, and its emission wavelength is 607nm.When measuring, we first detect the OD600 of each strain, removing the factor of bacteria number difference. Thus the fluorescence intensity cannot be altered by bacteria quantity. Then the measurement mode switching for measurement of fluorescence, fluorescence intensity.</p><br />
<!----------------------<br />
<img src="https://static.igem.org/mediawiki/2013/8/83/0917_t00_white_60mm.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/7/72/0917_t15_white_60mm.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/4/4e/0917_t30_white_60mm.jpg" /><br />
--------------------><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg"/><br />
<p>Figure. The test results of BBa_K1051258 in chip. A,LB medium,O minuts; B, IPTG medium,9minutes; C, IPTG medium, 15 minutes</p><br />
<img src="https://static.igem.org/mediawiki/parts/6/6a/DT.png" title="Fluorescence Intensity" description="Fluorescence intensity of positive control and three tested degradation tags." /><p>Figure. Fluorescence intensity calculated by ImageJ positive control and three tested degradation tags.</p><br />
<p>The degradation efficiency of three degradation tags are obvious.</p><br />
<br />
<p>Using the IPTG medium, the new RFP expression was stopped and we can regard the lights as degradation tags' efficiency. Obviously, the lights are decreasing along with the time. Thus, it indicates that the degradation tags work effectively.</p><br />
<img src="https://static.igem.org/mediawiki/parts/1/1e/Average_flurescence_intensity_of_K1051258_measurement.jpg" title="Half Life" description="RFP with degradation tag's half life tested by microfluidics." /><br />
<p>Figure. RFP with degradation tag's half life tested by microfluidics and fit by modeling.</p><br />
<p>Calculated by ImageJ and from experimental fitting curve we got its half-life of our protein 6.88 min. So the degradation rate should be: </p><br />
<img src="https://static.igem.org/mediawiki/2013/9/94/Hl-rate.png"> (Calculated from microfluidics data)<br />
<p>Simulation results calculated and fit from both microscope and microfluidics mates well, which has verified our experimental results.</p><br />
<br />
<h4>Problems</h4><br />
<h5>Yeast Version</h5><br />
<p>The test for yeast degradation tags has not been fully successful. Fluorescence has been captured in some occasions but not stable. It may caused by the same problem with promoter test devices - plasmid copy number. </p><br />
<h5><i>E. coli</i> Version</h5><br />
<p>Data collected from microscope for different degradation tags did not fully meet performance expectation. Reasons may be multiple to increase systematic errors.</p><br />
<p>- Fluorescence is too weak for calculating</p><br />
<p>- Quenching of fluorescence, especially in RFP</p><br />
<p>- Sample size is small for ImageJ calculating</p><br />
<p>We will have more data detected by microfluidics and flow cytometry. Results will be updated later.</p><br />
</div><br />
<br />
<div id="board5" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery5"><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/e4/09172_bright.jpg" title="E. coli captured" description="E. coli cells captured by microfluidics device."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/3/3f/Sc_bright_20.jpg" title="E. coli captured" description="Yeast cells captured by microfluidics device."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/2/23/Sync.jpg" title="Cell Synchronized" description="All cells are synchronized to G1 phase by using the microfluidics method."/></li><br />
</ul></div><br />
<div id="box5" class="box"><br />
<h3>Cell Synchronization</h3><br />
<h4>Microfluidics Device</h4><br />
<p>We made the chip as a platform for watching and synchronize the cells. First step is capture the cells by the chip. As showed in the two figures, both E.coli and budding yeast can be captured by the chip successfully.</p><br />
<img src="https://static.igem.org/mediawiki/2013/e/e4/09172_bright.jpg" /><br />
<p>Figure. <i>E. coli</i> and yeast cells have been captured by the chip successfully.</p><br />
<p>As Sic1 method has not been successful, we tried another way by changing medium by microfluidics. Protocols can be found in Notes page.</p><br />
<img src="https://static.igem.org/mediawiki/2013/2/23/Sync.jpg" title="Cell Synchronized" description="All cells are synchronized to G1 phase by using the microfluidics method."/><br />
<p>Figure. All cells are synchronized to G1 phase by using the microfluidics method.</p><br />
<p>All yeast cells in microfluidics chip are in G1 phase, after budding.</p><br />
<br />
<h4>Sic1 Synchronization</h4><br />
<p>Seven phosphorylation sites are mutated by PCR to make Sic1 mutant. However, it has been taking us too long time that we have been still working on right clones. Experiments results will be updated later. <br />
<br />
</div><br />
<br />
<div id="board6" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery6"><br />
<li><img src="https://static.igem.org/mediawiki/2013/b/b9/Blank-01.png" /><li><br />
</ul></div><br />
<div id="box6" class="box"><br />
<h3>Alternative Splicing</h3><br />
<h4>Original Design</h4><br />
<p>In our project, we attempt to use intron as a switch. However, according to previous research, there are two splicing forms of SRC1 intron: one is complete splicing (5’S) while the other can leave 4bp at its 5’ end (5’L). The remaining 4-bp causes frame-shift and a stop codon in the region of adaptor, thus the following exon will not be translated. However, this kind of switch is not complete – the ratio of 5’L and 5’S varied between 40-60 and 85-15.</p><br />
<p>In order to make a complete switch, we abandoned the remaining 4bp of SRC1 intron to avoid producing 5’L mRNA. So the result comes out to be: when Hub1p are expressed, they bind to spliceosome to modify it, resulting to easy recognizing 5’S splice site and its splicing; when inhibit the expression of HUB1 by CRISPRi system, spliceosome can hardly recognize 5’S splice site, therefore no intron can be spliced. </p><br />
<h4>Further Plans</h4><br />
<p>However, things didn’t go as we expected. Our experiment showed that this 4bp plays important role in SRC1 intron splicing. So, to achieve our goal, we redesigned another three versions of SRC1 intron:<br />
</p><br />
<p>1. Intron with GG at its 5’ end</p><br />
<p>2. Intron with CGG at its 5’ end</p><br />
<p>3. Intron with its original 6bp at both end</p><br />
<p>Due to the lack of time, we are still constructing these parts to test devices.</p><br />
<br />
</div><br />
<br />
</div></div>BGI K2http://2013.igem.org/Team:Shenzhen_BGIC_ATCG/resultsTeam:Shenzhen BGIC ATCG/results2013-09-28T01:02:32Z<p>BGI K2: </p>
<hr />
<div><!----------------------<br />
<br />
<br />
Title: Results<br />
Team: Shenzhen_BGIC_ATCG<br />
Author: BGI_K2<br />
Twitter: @BGI_K2<br />
Email: im.ss.kk@gmail.com<br />
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<div class="navleftholder"><br />
<ul class="navleft"><br />
<li><a id="navleftsub1" class="navleftsub" href="#board1">The Magic</a></li><br />
<li><a id="navleftsub2" class="navleftsub" href="#board2">Promoter Verification</a></li><br />
<li><a id="navleftsub3" class="navleftsub" href="#board3">Reporter Locating</a></li><br />
<li><a id="navleftsub4" class="navleftsub" href="#board4">Degradation Rate</a></li><br />
<li><a id="navleftsub5" class="navleftsub" href="#board5">Cell Synchronization</a></li><br />
<li><a id="navleftsub6" class="navleftsub" href="#board6">Alternative Splicing</a></li><br />
</ul></div><br />
<br />
<div id="board1" class="board"></div><br />
<div class="media"><br />
<br />
<ul id="myGallery1"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/></li><br />
<li><img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" title="Degradation Tags" description="Degradation tags testing device for E. coli."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" title="Microfluidics" description="Microfluidics device for degradation test."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/2/23/Sync.jpg" title="Cell Synchronized" description="All cells are synchronized to G1 phase by using the microfluidics method."/></li><br />
</ul></div><br />
<div id="box1" class="box"><br />
<h3>The Magic</h3><br />
<p>Our project “Cell Magic", a complex work has been completed partly,due to the time limitation. The results we have gotten are listed below:</p><br />
<p>We verified some of the cyclin promoters. The clb6 promoter, which is supposed to be expressed in G1 phase, has been verified perfectly.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." width=50%/><p>Figure. clb6 promoter for G1 phase has been verified</p><br />
<p>Targeting peptides to three locations have been verified. Others are still being constructed and tested.</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. " width=50%/><p>Figure. Test for targeting peptides to mitochondria, nucleus and vacuolar. </P><br />
<p>We got good data for degradation tags for <i>E. coli</i>. Construction of degradation devices for yeast have been finished and being tested.</p><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" width=50%/><p>Figure. Good data for degradation tags</p><br />
<p>Microfluidics device has been successfully developed for measurement and cell synchronization.</p><br />
<img src="https://static.igem.org/mediawiki/2013/f/fc/Microfluidic.gif" /><p>Figure. Microfluidic</p><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" width=50%/><br />
<p>Figure. Degradation tags were tested in Microfluidic</p><br />
<p>Cell synchronization has been successful by changing the method to microfluidics based one.</p><br />
<img src="https://static.igem.org/mediawiki/2013/2/23/Sync.jpg" title="Cell Synchronized" description="All cells are synchronized to G1 phase by using the microfluidics method."/><br />
<p>Figure. All cells are synchronized to G1 phase by using the microfluidics method.</p><br />
<p>Alternative splicing device are still in construction, related results will be updated later.</p><br />
<br />
</div><br />
<br />
<div id="board2" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery2"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/1/19/Flu.jpg" title="S Phase Promoter" description="cln3 promoter for S phase has been verified using flow cytometry." /></li><br />
</ul></div><br />
<div id="box2" class="box"><br />
<h3>Promoter Verification</h3><br />
<h4>Achievements</h4><br />
<p> Using the GFP as reporter and morphological alteration as cell cycle representation, we verified the Clb6 can be activated in G1 phase in the yeast.</P><br />
<p> As shown in the picture in normal lights, there are some yeast were budding. These budded yeast cells and small size budding yeasts (within the red circles) are assumed in the G1 phase of cell cycle. And the next picture was taken under activation light, thus the green lights can representing the Clb6 promoter expressing. The two pictures indicated the Clb6 were expressed in the G1 phase as we expected when designing the experiments </p><br />
<p> PS: we modified the contrast ratio to lower the lights of neighbor cells, thus our results looking better.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" /><p>Figure. clb6 promoter for G1 phase has been verified.</p><br />
<p>We also captured fluorescence on cln3 promoter (S phase) test device, for the fluorescence is quite weak, we utilize micro plate reader and flow cytometry to verify the promoter.</p><br />
<img src="https://static.igem.org/mediawiki/2013/1/19/Flu.jpg" /><p>Figure. Flow cytometry to verify S phase promoter cln3.</p><br />
<h4>Problems</h4><br />
<h5>Yeast Version</h5><br />
<p>While the promoters are natural ones without optimization, and our shuttle plasmid is pSR416 which is a single copied one, fluorescence is too weak to be captured by microfluidics device. That is a main reason for why we currently cannot show you a perfect cell magic movie. We are now constructing all devices with high copy number vectors, and try to optimize the junction near kozak sequence to make a better magic.</p><br />
<h5><i>E. coli</i> Version</h5><br />
<p>Promoters for <i>E. coli</i> seems not to be successful yet. In our first version, original 5' utr of promoter's downstream gene is included in BioBrick's sequences. The wild-type promoter and RBS may be too weak, or the junction of RFC[23] may have negative effect to the RBS. So in the present version, original 5' utr has been removed and strong RBS B0030 is used. We will update related results later.</p><br />
</div><br />
<br />
<div id="board3" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery3"><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/0/0d/Reporter.jpg" title="Modified Reporters" description="Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose."/></li><br />
</ul></div><br />
<div id="box3" class="box"><br />
<h3>Reporter Locating</h3><br />
<h4>Reporter Modification</h4><br />
<p>Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose.</p><br />
<img src="https://static.igem.org/mediawiki/2013/0/0d/Reporter.jpg" title="Modified Reporters" description="Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose."/><p>Figure. Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose</p><br />
<h4>Targeting Peptides</h4><br />
<p> The four pictures shows the reporters can rightly locate to Mitochondria, Nucleus and Vacuole,respectively. Picture A is the negative control, all yeast cells are lighted with GFP. And figure B is the reporter to the mitochondria, we can saw there are several light spots in one cells. Figure C is the reporter located in nucleus, the green spots are small and there is only one in a yeast cell.The last picture shows the reporter of Vacuolar membrane, the green lights were discrete in cells which was as expected.</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/><p>Figure. Test for targeting peptides to mitochondria, nucleus and vacuolar.</p><br />
</div><br />
<br />
<div id="board4" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery4"><br />
<li><img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" title="E. coli Version" description="Degradation tags testing device for E. coli"/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" title="IPTG Repressed" description="Microfluidics device for half life test with IPTG input (promoter of RFP repressed)."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/e9/Degladder.jpg" title="RFP Ladder" description="RFP ladder with different degradation tags)."/></li><br />
<br />
</ul></div><br />
<div id="box4" class="box"><br />
<h3>Degradation Rate</h3><br />
<p>In every curcuits measurement, we firstly test the growth curve through detecting the absorbance. Taken small amount of bateria, inoculate into 400ul LB medium, ensuring the initial concentration between 0.02-0.05 (OD600). Cultured in the room temperature, detect the OD600 value every 20 minutes. Y axis represents the logarithm of bacteria number, X axis represents the growth time. </p><br />
<img src="https://static.igem.org/mediawiki/2013/6/68/Growth-Curve.jpg" /><p>Figure. Growth curves have been drawn for all constructed devices.</p><br />
<p>In E.coli, the adaptor SspB tethers ssrA tagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. Which means, a variation of the WT SsrA tag sequence (K1051206, K1051207 and K1051208) will accelerate the degradation of proteins when fused to their C-terminal. Thus the degradation rates are dependent on concentration of proteases and binding mediators.</p><br />
<p>We constructed the measurement pathway of each tag (K1051257, K1051258 and K1051259) to test the rates of degradation of tagged proteins respectively. J04450 was used as positive control because of the same promoter and fluorescent protein. </p><br />
<h4>Achievements</h4><br />
<img src="https://static.igem.org/mediawiki/2013/e/e9/Degladder.jpg" /><br />
<p>Figure. Fluorescence ladder for degradation test devices. From left, the negative control,BBa_K1051257, BBa_K1051258, BBa_K1051259, J04450 as Positive Control. </p><br />
<p>As the pictures showed, the lights of RFP within three degradation tags are decreasing. </p><br />
<!----------------------<br />
<img src="https://static.igem.org/mediawiki/2013/a/a6/IMG_0804.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/c/ca/IMG_0817.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/M0050bright.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/0/06/M0050strong.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/8/89/M0050weak.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/6/63/Flo.jpg" /><br />
<br />
--------------------><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" /><br />
<p>Figure. The test results for K1051258 and positive control. A. K1051258 in bright field; B. K1051258 in exciting lights; C. Positive control in bright field; D. K1051258 in exciting lights.</p><br />
<p>Images are also available for K1051257 and K1051259. While in simulation, we obtained the degradation rate by calculating with [P1_P]/[P2_P]:</p><br />
<img src="https://static.igem.org/mediawiki/2013/b/ba/Simulation_result.png"> (Calculated from microscope data)<br />
<p>Enzyme - labelled meter detect the fluorescent protein intensity</p><br />
<p>First of all, take a certain amount of bacteria liquid, recovery to around OD0.6(600), ensuring the bacteria in the logarithmic growth phase. Diluted and then the bacteria transferred to 96 well plates, measured their fluorescence intensity. Red fluorescent protein using an excitation wavelength of 584nm, and its emission wavelength is 607nm.When measuring, we first detect the OD600 of each strain, removing the factor of bacteria number difference. Thus the fluorescence intensity cannot be altered by bacteria quantity. Then the measurement mode switching for measurement of fluorescence, fluorescence intensity.</p><br />
<!----------------------<br />
<img src="https://static.igem.org/mediawiki/2013/8/83/0917_t00_white_60mm.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/7/72/0917_t15_white_60mm.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/4/4e/0917_t30_white_60mm.jpg" /><br />
--------------------><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg"/><br />
<p>Figure. The test results of BBa_K1051258 in chip. A,LB medium,O minuts; B, IPTG medium,9minutes; C, IPTG medium, 15 minutes</p><br />
<img src="https://static.igem.org/mediawiki/parts/6/6a/DT.png" title="Fluorescence Intensity" description="Fluorescence intensity of positive control and three tested degradation tags." /><p>Figure. Fluorescence intensity calculated by ImageJ positive control and three tested degradation tags.</p><br />
<p>The degradation efficiency of three degradation tags are obvious.</p><br />
<br />
<p>Using the IPTG medium, the new RFP expression was stopped and we can regard the lights as degradation tags' efficiency. Obviously, the lights are decreasing along with the time. Thus, it indicates that the degradation tags work effectively.</p><br />
<img src="https://static.igem.org/mediawiki/parts/1/1e/Average_flurescence_intensity_of_K1051258_measurement.jpg" title="Half Life" description="RFP with degradation tag's half life tested by microfluidics." /><br />
<p>Figure. RFP with degradation tag's half life tested by microfluidics and fit by modeling.</p><br />
<p>Calculated by ImageJ and from experimental fitting curve we got its half-life of our protein 6.88 min. So the degradation rate should be: </p><br />
<img src="https://static.igem.org/mediawiki/2013/9/94/Hl-rate.png"> (Calculated from microfluidics data)<br />
<p>Simulation results calculated and fit from both microscope and microfluidics mates well, which has verified our experimental results.</p><br />
<br />
<h4>Problems</h4><br />
<h5>Yeast Version</h5><br />
<p>The test for yeast degradation tags has not been fully successful. Fluorescence has been captured in some occasions but not stable. It may caused by the same problem with promoter test devices - plasmid copy number. </p><br />
<h5><i>E. coli</i> Version</h5><br />
<p>Data collected from microscope for different degradation tags did not fully meet performance expectation. Reasons may be multiple to increase systematic errors.</p><br />
<p>- Fluorescence is too weak for calculating</p><br />
<p>- Quenching of fluorescence, especially in RFP</p><br />
<p>- Sample size is small for ImageJ calculating</p><br />
<p>We will have more data detected by microfluidics and flow cytometry. Results will be updated later.</p><br />
</div><br />
<br />
<div id="board5" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery5"><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/e4/09172_bright.jpg" title="E. coli captured" description="E. coli cells captured by microfluidics device."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/3/3f/Sc_bright_20.jpg" title="E. coli captured" description="Yeast cells captured by microfluidics device."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/2/23/Sync.jpg" title="Cell Synchronized" description="All cells are synchronized to G1 phase by using the microfluidics method."/></li><br />
</ul></div><br />
<div id="box5" class="box"><br />
<h3>Cell Synchronization</h3><br />
<h4>Microfluidics Device</h4><br />
<p>We made the chip as a platform for watching and synchronize the cells. First step is capture the cells by the chip. As showed in the two figures, both E.coli and budding yeast can be captured by the chip successfully.</p><br />
<img src="https://static.igem.org/mediawiki/2013/e/e4/09172_bright.jpg" /><br />
<p>Figure. <i>E. coli</i> and yeast cells have been captured by the chip successfully.</p><br />
<p>As Sic1 method has not been successful, we tried another way by changing medium by microfluidics. Protocols can be found in Notes page.</p><br />
<img src="https://static.igem.org/mediawiki/2013/2/23/Sync.jpg" title="Cell Synchronized" description="All cells are synchronized to G1 phase by using the microfluidics method."/><br />
<p>Figure. All cells are synchronized to G1 phase by using the microfluidics method.</p><br />
<p>All yeast cells in microfluidics chip are in G1 phase, after budding.</p><br />
<br />
<h4>Sic1 Synchronization</h4><br />
<p>Seven phosphorylation sites are mutated by PCR to make Sic1 mutant. However, it has been taking us too long time that we have been still working on right clones. Experiments results will be updated later. <br />
<br />
</div><br />
<br />
<div id="board6" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery6"><br />
<li><img src="https://static.igem.org/mediawiki/2013/b/b9/Blank-01.png" /><li><br />
</ul></div><br />
<div id="box6" class="box"><br />
<h3>Alternative Splicing</h3><br />
<h4>Original Design</h4><br />
<p>In our project, we attempt to use intron as a switch. However, according to previous research, there are two splicing forms of SRC1 intron: one is complete splicing (5’S) while the other can leave 4bp at its 5’ end (5’L). The remaining 4-bp causes frame-shift and a stop codon in the region of adaptor, thus the following exon will not be translated. However, this kind of switch is not complete – the ratio of 5’L and 5’S varied between 40-60 and 85-15.</p><br />
<p>In order to make a complete switch, we abandoned the remaining 4bp of SRC1 intron to avoid producing 5’L mRNA. So the result comes out to be: when Hub1p are expressed, they bind to spliceosome to modify it, resulting to easy recognizing 5’S splice site and its splicing; when inhibit the expression of HUB1 by CRISPRi system, spliceosome can hardly recognize 5’S splice site, therefore no intron can be spliced. </p><br />
<h4>Further Plans</h4><br />
<p>However, things didn’t go as we expected. Our experiment showed that this 4bp plays important role in SRC1 intron splicing. So, to achieve our goal, we redesigned another three versions of SRC1 intron:<br />
</p><br />
<p>1. Intron with GG at its 5’ end</p><br />
<p>2. Intron with CGG at its 5’ end</p><br />
<p>3. Intron with its original 6bp at both end</p><br />
<p>Due to the lack of time, we are still constructing these parts to test devices.</p><br />
<br />
</div><br />
<br />
</div></div>BGI K2http://2013.igem.org/Team:Shenzhen_BGIC_ATCG/resultsTeam:Shenzhen BGIC ATCG/results2013-09-28T00:54:24Z<p>BGI K2: </p>
<hr />
<div><!----------------------<br />
<br />
<br />
Title: Results<br />
Team: Shenzhen_BGIC_ATCG<br />
Author: BGI_K2<br />
Twitter: @BGI_K2<br />
Email: im.ss.kk@gmail.com<br />
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<div class="navleftholder"><br />
<ul class="navleft"><br />
<li><a id="navleftsub1" class="navleftsub" href="#board1">The Magic</a></li><br />
<li><a id="navleftsub2" class="navleftsub" href="#board2">Promoter Verification</a></li><br />
<li><a id="navleftsub3" class="navleftsub" href="#board3">Reporter Locating</a></li><br />
<li><a id="navleftsub4" class="navleftsub" href="#board4">Degradation Rate</a></li><br />
<li><a id="navleftsub5" class="navleftsub" href="#board5">Cell Synchronization</a></li><br />
<li><a id="navleftsub6" class="navleftsub" href="#board6">Alternative Splicing</a></li><br />
</ul></div><br />
<br />
<div id="board1" class="board"></div><br />
<div class="media"><br />
<br />
<ul id="myGallery1"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/></li><br />
<li><img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" title="Degradation Tags" description="Degradation tags testing device for E. coli."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" title="Microfluidics" description="Microfluidics device for degradation test."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/2/23/Sync.jpg" title="Cell Synchronized" description="All cells are synchronized to G1 phase by using the microfluidics method."/></li><br />
</ul></div><br />
<div id="box1" class="box"><br />
<h3>The Magic</h3><br />
<p>Our project “Cell Magic", a complex work has been completed partly,due to the time limitation. The results we have gotten are listed below:</p><br />
<p>We verified some of the cyclin promoters. The clb6 promoter, which is supposed to be expressed in G1 phase, has been verified perfectly.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." width=50%/><p>Figure. clb6 promoter for G1 phase has been verified</p><br />
<p>Targeting peptides to three locations have been verified. Others are still being constructed and tested.</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. " width=50%/><p>Figure. Test for targeting peptides to mitochondria, nucleus and vacuolar. </P><br />
<p>We got good data for degradation tags for <i>E. coli</i>. Construction of degradation devices for yeast have been finished and being tested.</p><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" width=50%/><p>Figure. Good data for degradation tags</p><br />
<p>Microfluidics device has been successfully developed for measurement and cell synchronization.</p><br />
<img src="https://static.igem.org/mediawiki/2013/f/fc/Microfluidic.gif" /><p>Figure. Microfluidic</p><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" width=50%/><p>Figure. Degradation tags were tested in Microfluidic</p><br />
<p>Cell synchronization device and alternative splicing device are still in construction, related results will be updated later.</p><br />
<br />
</div><br />
<br />
<div id="board2" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery2"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/1/19/Flu.jpg" title="S Phase Promoter" description="cln3 promoter for S phase has been verified using flow cytometry." /></li><br />
</ul></div><br />
<div id="box2" class="box"><br />
<h3>Promoter Verification</h3><br />
<h4>Achievements</h4><br />
<p> Using the GFP as reporter and morphological alteration as cell cycle representation, we verified the Clb6 can be activated in G1 phase in the yeast.</P><br />
<p> As shown in the picture in normal lights, there are some yeast were budding. These budded yeast cells and small size budding yeasts (within the red circles) are assumed in the G1 phase of cell cycle. And the next picture was taken under activation light, thus the green lights can representing the Clb6 promoter expressing. The two pictures indicated the Clb6 were expressed in the G1 phase as we expected when designing the experiments </p><br />
<p> PS: we modified the contrast ratio to lower the lights of neighbor cells, thus our results looking better.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" /><p>Figure. clb6 promoter for G1 phase has been verified</p><br />
<p>We also captured fluorescence on cln3 promoter (S phase) test device, for the fluorescence is quite weak, we utilize micro plate reader and flow cytometry to verify the promoter.</p><br />
<img src="https://static.igem.org/mediawiki/2013/1/19/Flu.jpg" /><p>Figure. Flow cytometry to verify the promoter.</p><br />
<h4>Problems</h4><br />
<h5>Yeast Version</h5><br />
<p>While the promoters are natural ones without optimization, and our shuttle plasmid is pSR416 which is a single copied one, fluorescence is too weak to be captured by microfluidics device. That is a main reason for why we currently cannot show you a perfect cell magic movie. We are now constructing all devices with high copy number vectors, and try to optimize the junction near kozak sequence to make a better magic.</p><br />
<h5><i>E. coli</i> Version</h5><br />
<p>Promoters for <i>E. coli</i> seems not to be successful yet. In our first version, original 5' utr of promoter's downstream gene is included in BioBrick's sequences. The wild-type promoter and RBS may be too weak, or the junction of RFC[23] may have negative effect to the RBS. So in the present version, original 5' utr has been removed and strong RBS B0030 is used. We will update related results later.</p><br />
</div><br />
<br />
<div id="board3" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery3"><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/0/0d/Reporter.jpg" title="Modified Reporters" description="Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose."/></li><br />
</ul></div><br />
<div id="box3" class="box"><br />
<h3>Reporter Locating</h3><br />
<h4>Reporter Modification</h4><br />
<p>Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose.</p><br />
<img src="https://static.igem.org/mediawiki/2013/0/0d/Reporter.jpg" title="Modified Reporters" description="Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose."/><p>Figure. Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose</p><br />
<h4>Targeting Peptides</h4><br />
<p> The four pictures shows the reporters can rightly locate to Mitochondria, Nucleus and Vacuole,respectively. Picture A is the negative control, all yeast cells are lighted with GFP. And figure B is the reporter to the mitochondria, we can saw there are several light spots in one cells. Figure C is the reporter located in nucleus, the green spots are small and there is only one in a yeast cell.The last picture shows the reporter of Vacuolar membrane, the green lights were discrete in cells which was as expected.</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/><p>Figure. Test for targeting peptides to mitochondria, nucleus and vacuolar.</p><br />
</div><br />
<br />
<div id="board4" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery4"><br />
<li><img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" title="E. coli Version" description="Degradation tags testing device for E. coli"/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" title="IPTG Repressed" description="Microfluidics device for half life test with IPTG input (promoter of RFP repressed)."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/e9/Degladder.jpg" title="RFP Ladder" description="RFP ladder with different degradation tags)."/></li><br />
<br />
</ul></div><br />
<div id="box4" class="box"><br />
<h3>Degradation Rate</h3><br />
<p>In every curcuits measurement, we firstly test the growth curve through detecting the absorbance. Taken small amount of bateria, inoculate into 400ul LB medium, ensuring the initial concentration between 0.02-0.05 (OD600). Cultured in the room temperature, detect the OD600 value every 20 minutes. Y axis represents the logarithm of bacteria number, X axis represents the growth time. </p><br />
<img src="https://static.igem.org/mediawiki/2013/6/68/Growth-Curve.jpg" /><p>Figure. Growth Curve</p><br />
<p>In E.coli, the adaptor SspB tethers ssrA tagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. Which means, a variation of the WT SsrA tag sequence (K1051206, K1051207 and K1051208) will accelerate the degradation of proteins when fused to their C-terminal. Thus the degradation rates are dependent on concentration of proteases and binding mediators.</p><br />
<p>We constructed the measurement pathway of each tag (K1051257, K1051258 and K1051259) to test the rates of degradation of tagged proteins respectively. J04450 was used as positive control because of the same promoter and fluorescent protein. </p><br />
<h4>Achievements</h4><br />
<img src="https://static.igem.org/mediawiki/2013/e/e9/Degladder.jpg" /><p>Figure. Degladder</p><br />
<p>From right to left, the negative control,BBa_K1051257, BBa_K1051258, BBa_K1051259, J04450 as Positive Control. As the pictures showed, the lights of RFP within three degradation tags are decreasing. </p><br />
<!----------------------<br />
<img src="https://static.igem.org/mediawiki/2013/a/a6/IMG_0804.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/c/ca/IMG_0817.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/M0050bright.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/0/06/M0050strong.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/8/89/M0050weak.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/6/63/Flo.jpg" /><br />
<br />
--------------------><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" /><br />
<p>Figure. The test results for K1051258 and positive control. A. K1051258 in bright field; B. K1051258 in exciting lights; C. Positive control in bright field; D. K1051258 in exciting lights.</p><br />
<p>Images are also available for K1051257 and K1051259. While in simulation, we obtained the degradation rate by calculating with [P1_P]/[P2_P]:</p><br />
<img src="https://static.igem.org/mediawiki/2013/b/ba/Simulation_result.png"> (Calculated from microscope data)<br />
<p>Enzyme - labelled meter detect the fluorescent protein intensity</p><br />
<p>First of all, take a certain amount of bacteria liquid, recovery to around OD0.6(600), ensuring the bacteria in the logarithmic growth phase. Diluted and then the bacteria transferred to 96 well plates, measured their fluorescence intensity. Red fluorescent protein using an excitation wavelength of 584nm, and its emission wavelength is 607nm.When measuring, we first detect the OD600 of each strain, removing the factor of bacteria number difference. Thus the fluorescence intensity cannot be altered by bacteria quantity. Then the measurement mode switching for measurement of fluorescence, fluorescence intensity.</p><br />
<!----------------------<br />
<img src="https://static.igem.org/mediawiki/2013/8/83/0917_t00_white_60mm.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/7/72/0917_t15_white_60mm.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/4/4e/0917_t30_white_60mm.jpg" /><br />
--------------------><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg"/><br />
<p>Figure. Microfluidics device for degradation test</p><br />
<p>The test results of BBa_K1051258 in chip. A,LB medium,O minuts; B, IPTG medium,9minutes; C, IPTG medium, 15 minutes</p><br />
<img src="https://static.igem.org/mediawiki/parts/6/6a/DT.png" title="Fluorescence Intensity" description="Fluorescence intensity of positive control and three tested degradation tags." /><p>Figure. Fluorescence intensity of positive control and three tested degradation tags.</p><br />
<p>Calculated by .ImageJ, the degradation efficiency of three degradation tags are obvious.</p><br />
<p>First of all, yeast will be measured after shaking to about OD2.0 (600)based on the chip which was washed by plasma water, vacuum pumping. The bacteria liquid was pushed into the chip, letting the cells enter the small triangle. Then use the constant flow pump culture medium into chip (the laboratory constant temperature, can not guarantee the training environment, 37 ° E. coli slower growth). The medium speed is about 200 ul/h. Finally we test the data after yeast filled in the triangles.</p><br />
<p>Using the IPTG medium, the new RFP expression was stopped and we can regard the lights as degradation tags' efficiency. Obviously, the lights are decreasing along with the time. Thus, it indicates that the degradation tags work effectively.</p><br />
<img src="https://static.igem.org/mediawiki/parts/1/1e/Average_flurescence_intensity_of_K1051258_measurement.jpg" title="Half Life" description="RFP with degradation tag's half life tested by microfluidics." /><br />
<p>Figure. RFP with degradation tag's half life tested by microfluidics and fit by modeling.</p><br />
<p>Calculated by ImageJ and from experimental fitting curve we got its half-life of our protein 6.88 min. So the degradation rate should be: </p><br />
<img src="https://static.igem.org/mediawiki/2013/9/94/Hl-rate.png"> (Calculated from microfluidics data)<br />
<p>Simulation results calculated and fit from both microscope and microfluidics mates well, which has verified our experimental results.</p><br />
<br />
<h4>Problems</h4><br />
<h5>Yeast Version</h5><br />
<p>The test for yeast degradation tags has not been fully successful. Fluorescence has been captured in some occasions but not stable. It may caused by the same problem with promoter test devices - plasmid copy number. </p><br />
<h5><i>E. coli</i> Version</h5><br />
<p>Data collected from microscope for different degradation tags did not fully meet performance expectation. Reasons may be multiple to increase systematic errors.</p><br />
<p>- Fluorescence is too weak for calculating</p><br />
<p>- Quenching of fluorescence, especially in RFP</p><br />
<p>- Sample size is small for ImageJ calculating</p><br />
<p>We will have more data detected by microfluidics and flow cytometry. Results will be updated later.</p><br />
</div><br />
<br />
<div id="board5" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery5"><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/e4/09172_bright.jpg" title="E. coli captured" description="E. coli cells captured by microfluidics device."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/3/3f/Sc_bright_20.jpg" title="E. coli captured" description="Yeast cells captured by microfluidics device."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/2/23/Sync.jpg" title="Cell Synchronized" description="All cells are synchronized to G1 phase by using the microfluidics method."/></li><br />
</ul></div><br />
<div id="box5" class="box"><br />
<h3>Cell Synchronization</h3><br />
<h4>Microfluidics Device</h4><br />
<p>We made the chip as a platform for watching and synchronize the cells. First step is capture the cells by the chip. As showed in the two figures, both E.coli and budding yeast can be captured by the chip successfully.</p><br />
<img src="https://static.igem.org/mediawiki/2013/e/e4/09172_bright.jpg" /><br />
<p>Figure. Captured by the chip successfully</p><br />
<p>As Sic1 method has not been successful, we tried another way by changing medium by microfluidics. Protocols can be found in Notes page.</p><br />
<img src="https://static.igem.org/mediawiki/2013/2/23/Sync.jpg" title="Cell Synchronized" description="All cells are synchronized to G1 phase by using the microfluidics method."/><br />
<p>Figure. All cells are synchronized to G1 phase by using the microfluidics method.</p><br />
<p>All yeast cells in microfluidics chip are in G1 phase, after budding.</p><br />
<br />
<h4>Sic1 Synchronization</h4><br />
<p>Seven phosphorylation sites are mutated by PCR to make Sic1 mutant. However, it has been taking us too long time that we have been still working on right clones. Experiments results will be updated later. <br />
<br />
</div><br />
<br />
<div id="board6" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery6"><br />
<li><img src="https://static.igem.org/mediawiki/2013/b/b9/Blank-01.png" /><li><br />
</ul></div><br />
<div id="box6" class="box"><br />
<h3>Alternative Splicing</h3><br />
<h4>Original Design</h4><br />
<p>In our project, we attempt to use intron as a switch. However, according to previous research, there are two splicing forms of SRC1 intron: one is complete splicing (5’S) while the other can leave 4bp at its 5’ end (5’L). The remaining 4-bp causes frame-shift and a stop codon in the region of adaptor, thus the following exon will not be translated. However, this kind of switch is not complete – the ratio of 5’L and 5’S varied between 40-60 and 85-15.</p><br />
<p>In order to make a complete switch, we abandoned the remaining 4bp of SRC1 intron to avoid producing 5’L mRNA. So the result comes out to be: when Hub1p are expressed, they bind to spliceosome to modify it, resulting to easy recognizing 5’S splice site and its splicing; when inhibit the expression of HUB1 by CRISPRi system, spliceosome can hardly recognize 5’S splice site, therefore no intron can be spliced. </p><br />
<h4>Further Plans</h4><br />
<p>However, things didn’t go as we expected. Our experiment showed that this 4bp plays important role in SRC1 intron splicing. So, to achieve our goal, we redesigned another three versions of SRC1 intron:<br />
</p><br />
<p>1. Intron with GG at its 5’ end</p><br />
<p>2. Intron with CGG at its 5’ end</p><br />
<p>3. Intron with its original 6bp at both end</p><br />
<p>Due to the lack of time, we are still constructing these parts to test devices.</p><br />
<br />
</div><br />
<br />
</div></div>BGI K2http://2013.igem.org/Team:Shenzhen_BGIC_ATCG/resultsTeam:Shenzhen BGIC ATCG/results2013-09-28T00:49:58Z<p>BGI K2: </p>
<hr />
<div><!----------------------<br />
<br />
<br />
Title: Results<br />
Team: Shenzhen_BGIC_ATCG<br />
Author: BGI_K2<br />
Twitter: @BGI_K2<br />
Email: im.ss.kk@gmail.com<br />
<br />
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<div class="navleftholder"><br />
<ul class="navleft"><br />
<li><a id="navleftsub1" class="navleftsub" href="#board1">The Magic</a></li><br />
<li><a id="navleftsub2" class="navleftsub" href="#board2">Promoter Verification</a></li><br />
<li><a id="navleftsub3" class="navleftsub" href="#board3">Reporter Locating</a></li><br />
<li><a id="navleftsub4" class="navleftsub" href="#board4">Degradation Rate</a></li><br />
<li><a id="navleftsub5" class="navleftsub" href="#board5">Cell Synchronization</a></li><br />
<li><a id="navleftsub6" class="navleftsub" href="#board6">Alternative Splicing</a></li><br />
</ul></div><br />
<br />
<div id="board1" class="board"></div><br />
<div class="media"><br />
<br />
<ul id="myGallery1"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/></li><br />
<li><img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" title="Degradation Tags" description="Degradation tags testing device for E. coli."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" title="Microfluidics" description="Microfluidics device for degradation test."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/2/23/Sync.jpg" title="Cell Synchronized" description="All cells are synchronized to G1 phase by using the microfluidics method."/></li><br />
</ul></div><br />
<div id="box1" class="box"><br />
<h3>The Magic</h3><br />
<p>Our project “Cell Magic", a complex work has been completed partly,due to the time limitation. The results we have gotten are listed below:</p><br />
<p>We verified some of the cyclin promoters. The clb6 promoter, which is supposed to be expressed in G1 phase, has been verified perfectly.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." width=50%/><p>Figure. clb6 promoter for G1 phase has been verified</p><br />
<p>Targeting peptides to three locations have been verified. Others are still being constructed and tested.</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. " width=50%/><p>Figure. Test for targeting peptides to mitochondria, nucleus and vacuolar. </P><br />
<p>We got good data for degradation tags for <i>E. coli</i>. Construction of degradation devices for yeast have been finished and being tested.</p><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" width=50%/><p>Figure. Good data for degradation tags</p><br />
<p>Microfluidics device has been successfully developed for measurement and cell synchronization.</p><br />
<img src="https://static.igem.org/mediawiki/2013/f/fc/Microfluidic.gif" /><p>Figure. Microfluidic</p><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" width=50%/><p>Figure. Degradation tags were tested in Microfluidic</p><br />
<p>Cell synchronization device and alternative splicing device are still in construction, related results will be updated later.</p><br />
<br />
</div><br />
<br />
<div id="board2" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery2"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/1/19/Flu.jpg" title="S Phase Promoter" description="cln3 promoter for S phase has been verified using flow cytometry." /></li><br />
</ul></div><br />
<div id="box2" class="box"><br />
<h3>Promoter Verification</h3><br />
<h4>Achievements</h4><br />
<p> Using the GFP as reporter and morphological alteration as cell cycle representation, we verified the Clb6 can be activated in G1 phase in the yeast.</P><br />
<p> As shown in the picture in normal lights, there are some yeast were budding. These budded yeast cells and small size budding yeasts (within the red circles) are assumed in the G1 phase of cell cycle. And the next picture was taken under activation light, thus the green lights can representing the Clb6 promoter expressing. The two pictures indicated the Clb6 were expressed in the G1 phase as we expected when designing the experiments </p><br />
<p> PS: we modified the contrast ratio to lower the lights of neighbor cells, thus our results looking better.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" /><p>Figure. clb6 promoter for G1 phase has been verified</p><br />
<p>We also captured fluorescence on cln3 promoter (S phase) test device, for the fluorescence is quite weak, we utilize micro plate reader and flow cytometry to verify the promoter.</p><br />
<img src="https://static.igem.org/mediawiki/2013/1/19/Flu.jpg" /><p>Figure. Flow cytometry to verify the promoter.</p><br />
<h4>Problems</h4><br />
<h5>Yeast Version</h5><br />
<p>While the promoters are natural ones without optimization, and our shuttle plasmid is pSR416 which is a single copied one, fluorescence is too weak to be captured by microfluidics device. That is a main reason for why we currently cannot show you a perfect cell magic movie. We are now constructing all devices with high copy number vectors, and try to optimize the junction near kozak sequence to make a better magic.</p><br />
<h5><i>E. coli</i> Version</h5><br />
<p>Promoters for <i>E. coli</i> seems not to be successful yet. In our first version, original 5' utr of promoter's downstream gene is included in BioBrick's sequences. The wild-type promoter and RBS may be too weak, or the junction of RFC[23] may have negative effect to the RBS. So in the present version, original 5' utr has been removed and strong RBS B0030 is used. We will update related results later.</p><br />
</div><br />
<br />
<div id="board3" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery3"><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/0/0d/Reporter.jpg" title="Modified Reporters" description="Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose."/></li><br />
</ul></div><br />
<div id="box3" class="box"><br />
<h3>Reporter Locating</h3><br />
<h4>Reporter Modification</h4><br />
<p>Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose.</p><br />
<img src="https://static.igem.org/mediawiki/2013/0/0d/Reporter.jpg" title="Modified Reporters" description="Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose."/><p>Figure. Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose</p><br />
<h4>Targeting Peptides</h4><br />
<p> The four pictures shows the reporters can rightly locate to Mitochondria, Nucleus and Vacuole,respectively. Picture A is the negative control, all yeast cells are lighted with GFP. And figure B is the reporter to the mitochondria, we can saw there are several light spots in one cells. Figure C is the reporter located in nucleus, the green spots are small and there is only one in a yeast cell.The last picture shows the reporter of Vacuolar membrane, the green lights were discrete in cells which was as expected.</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/><p>Figure. Test for targeting peptides to mitochondria, nucleus and vacuolar.</p><br />
</div><br />
<br />
<div id="board4" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery4"><br />
<li><img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" title="E. coli Version" description="Degradation tags testing device for E. coli"/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" title="IPTG Repressed" description="Microfluidics device for half life test with IPTG input (promoter of RFP repressed)."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/e9/Degladder.jpg" title="RFP Ladder" description="RFP ladder with different degradation tags)."/></li><br />
<br />
</ul></div><br />
<div id="box4" class="box"><br />
<h3>Degradation Rate</h3><br />
<p>In every curcuits measurement, we firstly test the growth curve through detecting the absorbance. Taken small amount of bateria, inoculate into 400ul LB medium, ensuring the initial concentration between 0.02-0.05 (OD600). Cultured in the room temperature, detect the OD600 value every 20 minutes. Y axis represents the logarithm of bacteria number, X axis represents the growth time. </p><br />
<img src="https://static.igem.org/mediawiki/2013/6/68/Growth-Curve.jpg" /><p>Figure. Growth Curve</p><br />
<p>In E.coli, the adaptor SspB tethers ssrA tagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. Which means, a variation of the WT SsrA tag sequence (K1051206, K1051207 and K1051208) will accelerate the degradation of proteins when fused to their C-terminal. Thus the degradation rates are dependent on concentration of proteases and binding mediators.</p><br />
<p>We constructed the measurement pathway of each tag (K1051257, K1051258 and K1051259) to test the rates of degradation of tagged proteins respectively. J04450 was used as positive control because of the same promoter and fluorescent protein. </p><br />
<h4>Achievements</h4><br />
<img src="https://static.igem.org/mediawiki/2013/e/e9/Degladder.jpg" /><p>Figure. Degladder</p><br />
<p>From right to left, the negative control,BBa_K1051257, BBa_K1051258, BBa_K1051259, J04450 as Positive Control. As the pictures showed, the lights of RFP within three degradation tags are decreasing. </p><br />
<!----------------------<br />
<img src="https://static.igem.org/mediawiki/2013/a/a6/IMG_0804.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/c/ca/IMG_0817.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/M0050bright.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/0/06/M0050strong.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/8/89/M0050weak.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/6/63/Flo.jpg" /><br />
<br />
--------------------><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" /><br />
<p>Figure. Degradation tags</p><br />
<p>The test results for K1051258 and positive control. A. K1051258 in bright field; B. K1051258 in exciting lights; C. Positive control in bright field; D. K1051258 in exciting lights.</p><br />
<p>In picture, there are only obvious lights in the picture B, indicated the degradation rates are working.</p><br />
<p>Enzyme - labelled meter detect the fluorescent protein intensity</p><br />
<p>First of all, take a certain amount of bacteria liquid, recovery to around OD0.6(600), ensuring the bacteria in the logarithmic growth phase. Diluted and then the bacteria transferred to 96 well plates, measured their fluorescence intensity. Red fluorescent protein using an excitation wavelength of 584nm, and its emission wavelength is 607nm.When measuring, we first detect the OD600 of each strain, removing the factor of bacteria number difference. Thus the fluorescence intensity cannot be altered by bacteria quantity. Then the measurement mode switching for measurement of fluorescence, fluorescence intensity.</p><br />
<!----------------------<br />
<img src="https://static.igem.org/mediawiki/2013/8/83/0917_t00_white_60mm.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/7/72/0917_t15_white_60mm.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/4/4e/0917_t30_white_60mm.jpg" /><br />
--------------------><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg"/><br />
<p>Figure. Microfluidics device for degradation test</p><br />
<p>The test results of BBa_K1051258 in chip. A,LB medium,O minuts; B, IPTG medium,9minutes; C, IPTG medium, 15 minutes</p><br />
<img src="https://static.igem.org/mediawiki/parts/6/6a/DT.png" title="Fluorescence Intensity" description="Fluorescence intensity of positive control and three tested degradation tags." /><p>Figure. Fluorescence intensity of positive control and three tested degradation tags.</p><br />
<p>Calculated by .ImageJ, the degradation efficiency of three degradation tags are obvious.</p><br />
<p>First of all, yeast will be measured after shaking to about OD2.0 (600)based on the chip which was washed by plasma water, vacuum pumping. The bacteria liquid was pushed into the chip, letting the cells enter the small triangle. Then use the constant flow pump culture medium into chip (the laboratory constant temperature, can not guarantee the training environment, 37 ° E. coli slower growth). The medium speed is about 200 ul/h. Finally we test the data after yeast filled in the triangles.</p><br />
<p>Using the IPTG medium, the new RFP expression was stopped and we can regard the lights as degradation tags' efficiency. Obviously, the lights are decreasing along with the time. Thus, it indicates that the degradation tags work effectively.</p><br />
<img src="https://static.igem.org/mediawiki/parts/1/1e/Average_flurescence_intensity_of_K1051258_measurement.jpg" title="Half Life" description="RFP with degradation tag's half life tested by microfluidics." /><p>Figure. RFP with degradation tag's half life tested by microfluidics and fit by modeling.</p><br />
<p>Calculated by .ImageJ and from experimental fitting curve we got its half-life of our protein 6.88 min. So the degradation rate should be: </p><br />
<img src="https://static.igem.org/mediawiki/2013/9/94/Hl-rate.png"><br />
<p>While in simulation, we obtained the degradation rate by calculating with [P1_P]/[P2_P]:</p><br />
<img src="https://static.igem.org/mediawiki/2013/b/ba/Simulation_result.png"><br />
<p>Simulation results calculated and fit from both microscope and microfluidics mates well, which has verified our experimental results.</p><br />
<br />
<h4>Problems</h4><br />
<h5>Yeast Version</h5><br />
<p>The test for yeast degradation tags has not been fully successful. Fluorescence has been captured in some occasions but not stable. It may caused by the same problem with promoter test devices - plasmid copy number. </p><br />
<h5><i>E. coli</i> Version</h5><br />
<p>Data collected from microscope for different degradation tags did not fully meet performance expectation. Reasons may be multiple to increase systematic errors.</p><br />
<p>- Fluorescence is too weak for calculating</p><br />
<p>- Quenching of fluorescence, especially in RFP</p><br />
<p>- Sample size is small for ImageJ calculating</p><br />
<p>We will have more data detected by microfluidics and flow cytometry. Results will be updated later.</p><br />
</div><br />
<br />
<div id="board5" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery5"><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/e4/09172_bright.jpg" title="E. coli captured" description="E. coli cells captured by microfluidics device."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/3/3f/Sc_bright_20.jpg" title="E. coli captured" description="Yeast cells captured by microfluidics device."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/2/23/Sync.jpg" title="Cell Synchronized" description="All cells are synchronized to G1 phase by using the microfluidics method."/></li><br />
</ul></div><br />
<div id="box5" class="box"><br />
<h3>Cell Synchronization</h3><br />
<h4>Microfluidics Device</h4><br />
<p>We made the chip as a platform for watching and synchronize the cells. First step is capture the cells by the chip. As showed in the two figures, both E.coli and budding yeast can be captured by the chip successfully.</p><br />
<img src="https://static.igem.org/mediawiki/2013/e/e4/09172_bright.jpg" /><br />
<p>Figure. Captured by the chip successfully</p><br />
<p>As Sic1 method has not been successful, we tried another way by changing medium by microfluidics. Protocols can be found in Notes page.</p><br />
<img src="https://static.igem.org/mediawiki/2013/2/23/Sync.jpg" title="Cell Synchronized" description="All cells are synchronized to G1 phase by using the microfluidics method."/><br />
<p>Figure. All cells are synchronized to G1 phase by using the microfluidics method.</p><br />
<p>All yeast cells in microfluidics chip are in G1 phase, after budding.</p><br />
<br />
<h4>Sic1 Synchronization</h4><br />
<p>Seven phosphorylation sites are mutated by PCR to make Sic1 mutant. However, it has been taking us too long time that we have been still working on right clones. Experiments results will be updated later. <br />
<br />
</div><br />
<br />
<div id="board6" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery6"><br />
<li><img src="https://static.igem.org/mediawiki/2013/b/b9/Blank-01.png" /><li><br />
</ul></div><br />
<div id="box6" class="box"><br />
<h3>Alternative Splicing</h3><br />
<h4>Original Design</h4><br />
<p>In our project, we attempt to use intron as a switch. However, according to previous research, there are two splicing forms of SRC1 intron: one is complete splicing (5’S) while the other can leave 4bp at its 5’ end (5’L). The remaining 4-bp causes frame-shift and a stop codon in the region of adaptor, thus the following exon will not be translated. However, this kind of switch is not complete – the ratio of 5’L and 5’S varied between 40-60 and 85-15.</p><br />
<p>In order to make a complete switch, we abandoned the remaining 4bp of SRC1 intron to avoid producing 5’L mRNA. So the result comes out to be: when Hub1p are expressed, they bind to spliceosome to modify it, resulting to easy recognizing 5’S splice site and its splicing; when inhibit the expression of HUB1 by CRISPRi system, spliceosome can hardly recognize 5’S splice site, therefore no intron can be spliced. </p><br />
<h4>Further Plans</h4><br />
<p>However, things didn’t go as we expected. Our experiment showed that this 4bp plays important role in SRC1 intron splicing. So, to achieve our goal, we redesigned another three versions of SRC1 intron:<br />
</p><br />
<p>1. Intron with GG at its 5’ end</p><br />
<p>2. Intron with CGG at its 5’ end</p><br />
<p>3. Intron with its original 6bp at both end</p><br />
<p>Due to the lack of time, we are still constructing these parts to test devices.</p><br />
<br />
</div><br />
<br />
</div></div>BGI K2http://2013.igem.org/File:09172_bright.jpgFile:09172 bright.jpg2013-09-28T00:37:34Z<p>BGI K2: uploaded a new version of &quot;File:09172 bright.jpg&quot;</p>
<hr />
<div></div>BGI K2http://2013.igem.org/File:09172_bright.jpgFile:09172 bright.jpg2013-09-28T00:37:29Z<p>BGI K2: uploaded a new version of &quot;File:09172 bright.jpg&quot;</p>
<hr />
<div></div>BGI K2http://2013.igem.org/File:09172_bright.jpgFile:09172 bright.jpg2013-09-28T00:37:12Z<p>BGI K2: uploaded a new version of &quot;File:09172 bright.jpg&quot;</p>
<hr />
<div></div>BGI K2http://2013.igem.org/Team:Shenzhen_BGIC_ATCG/resultsTeam:Shenzhen BGIC ATCG/results2013-09-28T00:34:49Z<p>BGI K2: </p>
<hr />
<div><!----------------------<br />
<br />
<br />
Title: Results<br />
Team: Shenzhen_BGIC_ATCG<br />
Author: BGI_K2<br />
Twitter: @BGI_K2<br />
Email: im.ss.kk@gmail.com<br />
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<div class="navleftholder"><br />
<ul class="navleft"><br />
<li><a id="navleftsub1" class="navleftsub" href="#board1">The Magic</a></li><br />
<li><a id="navleftsub2" class="navleftsub" href="#board2">Promoter Verification</a></li><br />
<li><a id="navleftsub3" class="navleftsub" href="#board3">Reporter Locating</a></li><br />
<li><a id="navleftsub4" class="navleftsub" href="#board4">Degradation Rate</a></li><br />
<li><a id="navleftsub5" class="navleftsub" href="#board5">Cell Synchronization</a></li><br />
<li><a id="navleftsub6" class="navleftsub" href="#board6">Alternative Splicing</a></li><br />
</ul></div><br />
<br />
<div id="board1" class="board"></div><br />
<div class="media"><br />
<br />
<ul id="myGallery1"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/></li><br />
<li><img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" title="Degradation Tags" description="Degradation tags testing device for E. coli."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" title="Microfluidics" description="Microfluidics device for degradation test."/></li><br />
<br />
</ul></div><br />
<div id="box1" class="box"><br />
<h3>The Magic</h3><br />
<p>Our project “Cell Magic", a complex work has been completed partly,due to the time limitation. The results we have gotten are listed below:</p><br />
<p>We verified some of the cyclin promoters. The clb6 promoter, which is supposed to be expressed in G1 phase, has been verified perfectly.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." width=50%/><p>Figure. clb6 promoter for G1 phase has been verified</p><br />
<p>Targeting peptides to three locations have been verified. Others are still being constructed and tested.</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. " width=50%/><p>Figure. Test for targeting peptides to mitochondria, nucleus and vacuolar. </P><br />
<p>We got good data for degradation tags for <i>E. coli</i>. Construction of degradation devices for yeast have been finished and being tested.</p><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" width=50%/><p>Figure. Good data for degradation tags</p><br />
<p>Microfluidics device has been successfully developed for measurement and cell synchronization.</p><br />
<img src="https://static.igem.org/mediawiki/2013/f/fc/Microfluidic.gif" /><p>Figure. Microfluidic</p><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" width=50%/><p>Figure. Degradation tags were tested in Microfluidic</p><br />
<p>Cell synchronization device and alternative splicing device are still in construction, related results will be updated later.</p><br />
<br />
</div><br />
<br />
<div id="board2" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery2"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/1/19/Flu.jpg" title="S Phase Promoter" description="cln3 promoter for S phase has been verified using flow cytometry." /></li><br />
</ul></div><br />
<div id="box2" class="box"><br />
<h3>Promoter Verification</h3><br />
<h4>Achievements</h4><br />
<p> Using the GFP as reporter and morphological alteration as cell cycle representation, we verified the Clb6 can be activated in G1 phase in the yeast.</P><br />
<p> As shown in the picture in normal lights, there are some yeast were budding. These budded yeast cells and small size budding yeasts (within the red circles) are assumed in the G1 phase of cell cycle. And the next picture was taken under activation light, thus the green lights can representing the Clb6 promoter expressing. The two pictures indicated the Clb6 were expressed in the G1 phase as we expected when designing the experiments </p><br />
<p> PS: we modified the contrast ratio to lower the lights of neighbor cells, thus our results looking better.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" /><p>Figure. clb6 promoter for G1 phase has been verified</p><br />
<p>We also captured fluorescence on cln3 promoter (S phase) test device, for the fluorescence is quite weak, we utilize micro plate reader and flow cytometry to verify the promoter.</p><br />
<img src="https://static.igem.org/mediawiki/2013/1/19/Flu.jpg" /><p>Figure. Flow cytometry to verify the promoter.</p><br />
<h4>Problems</h4><br />
<h5>Yeast Version</h5><br />
<p>While the promoters are natural ones without optimization, and our shuttle plasmid is pSR416 which is a single copied one, fluorescence is too weak to be captured by microfluidics device. That is a main reason for why we currently cannot show you a perfect cell magic movie. We are now constructing all devices with high copy number vectors, and try to optimize the junction near kozak sequence to make a better magic.</p><br />
<h5><i>E. coli</i> Version</h5><br />
<p>Promoters for <i>E. coli</i> seems not to be successful yet. In our first version, original 5' utr of promoter's downstream gene is included in BioBrick's sequences. The wild-type promoter and RBS may be too weak, or the junction of RFC[23] may have negative effect to the RBS. So in the present version, original 5' utr has been removed and strong RBS B0030 is used. We will update related results later.</p><br />
</div><br />
<br />
<div id="board3" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery3"><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/0/0d/Reporter.jpg" title="Modified Reporters" description="Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose."/></li><br />
</ul></div><br />
<div id="box3" class="box"><br />
<h3>Reporter Locating</h3><br />
<h4>Reporter Modification</h4><br />
<p>Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose.</p><br />
<img src="https://static.igem.org/mediawiki/2013/0/0d/Reporter.jpg" title="Modified Reporters" description="Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose."/><p>Figure. Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose</p><br />
<h4>Targeting Peptides</h4><br />
<p> The four pictures shows the reporters can rightly locate to Mitochondria, Nucleus and Vacuole,respectively. Picture A is the negative control, all yeast cells are lighted with GFP. And figure B is the reporter to the mitochondria, we can saw there are several light spots in one cells. Figure C is the reporter located in nucleus, the green spots are small and there is only one in a yeast cell.The last picture shows the reporter of Vacuolar membrane, the green lights were discrete in cells which was as expected.</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/><p>Figure. Test for targeting peptides to mitochondria, nucleus and vacuolar.</p><br />
</div><br />
<br />
<div id="board4" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery4"><br />
<li><img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" title="E. coli Version" description="Degradation tags testing device for E. coli"/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" title="IPTG Repressed" description="Microfluidics device for half life test with IPTG input (promoter of RFP repressed)."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/e9/Degladder.jpg" title="RFP Ladder" description="RFP ladder with different degradation tags)."/></li><br />
<br />
</ul></div><br />
<div id="box4" class="box"><br />
<h3>Degradation Rate</h3><br />
<p>In every curcuits measurement, we firstly test the growth curve through detecting the absorbance. Taken small amount of bateria, inoculate into 400ul LB medium, ensuring the initial concentration between 0.02-0.05 (OD600). Cultured in the room temperature, detect the OD600 value every 20 minutes. Y axis represents the logarithm of bacteria number, X axis represents the growth time. </p><br />
<img src="https://static.igem.org/mediawiki/2013/6/68/Growth-Curve.jpg" /><p>Figure. Growth Curve</p><br />
<p>In E.coli, the adaptor SspB tethers ssrA tagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. Which means, a variation of the WT SsrA tag sequence (K1051206, K1051207 and K1051208) will accelerate the degradation of proteins when fused to their C-terminal. Thus the degradation rates are dependent on concentration of proteases and binding mediators.</p><br />
<p>We constructed the measurement pathway of each tag (K1051257, K1051258 and K1051259) to test the rates of degradation of tagged proteins respectively. J04450 was used as positive control because of the same promoter and fluorescent protein. </p><br />
<h4>Achievements</h4><br />
<img src="https://static.igem.org/mediawiki/2013/e/e9/Degladder.jpg" /><p>Figure. Degladder</p><br />
<p>From right to left, the negative control,BBa_K1051257, BBa_K1051258, BBa_K1051259, J04450 as Positive Control. As the pictures showed, the lights of RFP within three degradation tags are decreasing. </p><br />
<!----------------------<br />
<img src="https://static.igem.org/mediawiki/2013/a/a6/IMG_0804.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/c/ca/IMG_0817.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/M0050bright.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/0/06/M0050strong.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/8/89/M0050weak.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/6/63/Flo.jpg" /><br />
<br />
--------------------><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" /><br />
<p>Figure. Degradation tags</p><br />
<p>The test results for K1051258 and positive control. A. K1051258 in bright field; B. K1051258 in exciting lights; C. Positive control in bright field; D. K1051258 in exciting lights.</p><br />
<p>In picture, there are only obvious lights in the picture B, indicated the degradation rates are working.</p><br />
<p>Enzyme - labelled meter detect the fluorescent protein intensity</p><br />
<p>First of all, take a certain amount of bacteria liquid, recovery to around OD0.6(600), ensuring the bacteria in the logarithmic growth phase. Diluted and then the bacteria transferred to 96 well plates, measured their fluorescence intensity. Red fluorescent protein using an excitation wavelength of 584nm, and its emission wavelength is 607nm.When measuring, we first detect the OD600 of each strain, removing the factor of bacteria number difference. Thus the fluorescence intensity cannot be altered by bacteria quantity. Then the measurement mode switching for measurement of fluorescence, fluorescence intensity.</p><br />
<!----------------------<br />
<img src="https://static.igem.org/mediawiki/2013/8/83/0917_t00_white_60mm.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/7/72/0917_t15_white_60mm.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/4/4e/0917_t30_white_60mm.jpg" /><br />
--------------------><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg"/><br />
<p>Figure. Microfluidics device for degradation test</p><br />
<p>The test results of BBa_K1051258 in chip. A,LB medium,O minuts; B, IPTG medium,9minutes; C, IPTG medium, 15 minutes</p><br />
<img src="https://static.igem.org/mediawiki/parts/6/6a/DT.png" title="Fluorescence Intensity" description="Fluorescence intensity of positive control and three tested degradation tags." /><p>Figure. Fluorescence intensity of positive control and three tested degradation tags</p><br />
<p>Calculated by .ImageJ, the degradation efficiency of three degradation tags are obvious.</p><br />
<p>First of all, yeast will be measured after shaking to about OD2.0 (600)based on the chip which was washed by plasma water, vacuum pumping. The bacteria liquid was pushed into the chip, letting the cells enter the small triangle. Then use the constant flow pump culture medium into chip (the laboratory constant temperature, can not guarantee the training environment, 37 ° E. coli slower growth). The medium speed is about 200 ul/h. Finally we test the data after yeast filled in the triangles.</p><br />
<p>Using the IPTG medium, the new RFP expression was stopped and we can regard the lights as degradation tags' efficiency. Obviously, the lights are decreasing along with the time. Thus, it indicates that the degradation tags work effectively.</p><br />
<img src="https://static.igem.org/mediawiki/parts/8/80/Average-fluorescence-intensity-of-K1051258-measurement.jpg" title="Half Life" description="RFP with degradation tag's half life tested by microfluidics." /><p>Figure. RFP with degradation tag's half life tested by microfluidics</p><br />
<p>Calculated by .ImageJ, half life of RFP in K1051258 is shown to be less than 9 minutes.</p><br />
<br />
<h4>Problems</h4><br />
<h5>Yeast Version</h5><br />
<p>The test for yeast degradation tags has not been fully successful. Fluorescence has been captured in some occasions but not stable. It may caused by the same problem with promoter test devices - plasmid copy number. </p><br />
<h5><i>E. coli</i> Version</h5><br />
<p>Data collected from microscope for different degradation tags did not fully meet performance expectation. Reasons may be multiple to increase systematic errors.</p><br />
<p>- Fluorescence is too weak for calculating</p><br />
<p>- Quenching of fluorescence, especially in RFP</p><br />
<p>- Sample size is small for ImageJ calculating</p><br />
<p>We will have more data detected by microfluidics and flow cytometry. Results will be updated later.</p><br />
</div><br />
<br />
<div id="board5" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery5"><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/e4/09172_bright.jpg" title="E. coli captured" description="E. coli cells captured by microfluidics device."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/3/3f/Sc_bright_20.jpg" title="E. coli captured" description="Yeast cells captured by microfluidics device."/></li><br />
</ul></div><br />
<div id="box5" class="box"><br />
<h3>Cell Synchronization</h3><br />
<h4>Microfluidics Device</h4><br />
<p>We made the chip as a platform for watching and synchronize the cells. First step is capture the cells by the chip. As showed in the two figures, both E.coli and budding yeast can be captured by the chip successfully.</p><br />
<img src="https://static.igem.org/mediawiki/2013/e/e4/09172_bright.jpg" /><br />
<p>Figure. Captured by the chip successfully</p><br />
<p>As Sic1 method has not been successful, we tried another way by changing medium by microfluidics. Protocols can be found in Notes page.</p><br />
<img src="https://static.igem.org/mediawiki/2013/2/23/Sync.jpg" title="Cell Synchronized" description="All cells are synchronized to G1 phase by using the microfluidics method."/><br />
<p>Figure. All cells are synchronized to G1 phase by using the microfluidics method.</p><br />
<p>All yeast cells in microfluidics chip are in G1 phase, after budding.</p><br />
<br />
<h4>Sic1 Synchronization</h4><br />
<p>Seven phosphorylation sites are mutated by PCR to make Sic1 mutant. However, it has been taking us too long time that we have been still working on right clones. Experiments results will be updated later. <br />
<br />
</div><br />
<br />
<div id="board6" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery6"><br />
<li><img src="https://static.igem.org/mediawiki/2013/b/b9/Blank-01.png" /><li><br />
</ul></div><br />
<div id="box6" class="box"><br />
<h3>Alternative Splicing</h3><br />
<h4>Original Design</h4><br />
<p>In our project, we attempt to use intron as a switch. However, according to previous research, there are two splicing forms of SRC1 intron: one is complete splicing (5’S) while the other can leave 4bp at its 5’ end (5’L). The remaining 4-bp causes frame-shift and a stop codon in the region of adaptor, thus the following exon will not be translated. However, this kind of switch is not complete – the ratio of 5’L and 5’S varied between 40-60 and 85-15.</p><br />
<p>In order to make a complete switch, we abandoned the remaining 4bp of SRC1 intron to avoid producing 5’L mRNA. So the result comes out to be: when Hub1p are expressed, they bind to spliceosome to modify it, resulting to easy recognizing 5’S splice site and its splicing; when inhibit the expression of HUB1 by CRISPRi system, spliceosome can hardly recognize 5’S splice site, therefore no intron can be spliced. </p><br />
<h4>Further Plans</h4><br />
<p>However, things didn’t go as we expected. Our experiment showed that this 4bp plays important role in SRC1 intron splicing. So, to achieve our goal, we redesigned another three versions of SRC1 intron:<br />
</p><br />
<p>1. Intron with GG at its 5’ end</p><br />
<p>2. Intron with CGG at its 5’ end</p><br />
<p>3. Intron with its original 6bp at both end</p><br />
<p>Due to the lack of time, we are still constructing these parts to test devices.</p><br />
<br />
</div><br />
<br />
</div></div>BGI K2http://2013.igem.org/File:Sync.jpgFile:Sync.jpg2013-09-28T00:21:38Z<p>BGI K2: </p>
<hr />
<div></div>BGI K2http://2013.igem.org/Team:Shenzhen_BGIC_ATCG/notesTeam:Shenzhen BGIC ATCG/notes2013-09-28T00:16:54Z<p>BGI K2: </p>
<hr />
<div><!----------------------<br />
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Title: Notes<br />
Team: Shenzhen_BGIC_ATCG<br />
Author: BGI_K2<br />
Twitter: @BGI_K2<br />
Email: im.ss.kk@gmail.com<br />
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<h3>Timeline</h3><br />
<h4>Promoter Group</h4><br />
<p>Week1: research about the previous work of iGEM, especially the work done by the European teams.</p><br />
<p>Week2~week4: project idea discussing and determination, group issue asssigned.</p><br />
<p>Week5~week6: research about the principle of promoter in E.coli and yeast.</p><br />
<p>Week7: Experiments process design, constructs the basic parts and test curcuits. </p><br />
<p>Week8: Design the primers needed to amplification the promotor</p><br />
<p>Week9: Workshop among several schools</p><br />
<p>Week10-week13: constructs the basic parts: BBa_K1051300, BBa_K1051301, BBa_K1051302,BBa_K1051303, BBa_K1051304, BBa_K1051305, BBa_K1051306</p><br />
<p>Week14-week18: Measurement designing</p><br />
<p>Week19-week22: Test measurement circuits and then data analysis</p><br />
<p>Week23-week25: results drawing and made the wiki</p><br />
<br />
<h4>Degradation Group</h4><br />
<p>Week1: previous team project data collection, especially the Latin America teams</p><br />
<p>Week2~week4: project idea decided,group issue divided into E.coli one and yeast one.</p><br />
<p>Week5~week6: research of principle of degradation peptide and design the experiment generally combining with the mechanism of cyclin protein degradation.</p><br />
<p>Week7: Experiments process design, constructs the basic parts and test curcuits. </p><br />
<p>Week8: Design the primers needed to amplification the degradation tags.</p><br />
<p>Week9: Workshop among several schools</p><br />
<p>Week10-week13: constructs the basic parts: K051200/K1051201/K1051202/K1051203/K1051204/K1051205/K1051206/K1051207</p><br />
<p>Week14-week18: Measurement circuits construction</p><br />
<p>Week19-week22: Test measurement circuits and then data analysis</p><br />
<p>Week23-week25: results drawing and made the wiki</p><br />
<br />
<h4>Targeting Group</h4><br />
<p>Week1: research about the previous work of iGEM, especially the work done by the Asia teams.</p><br />
<p>Week2~week4: project idea discussing and determination, group issue assigned.</p><br />
<p>Week5~week6: research about the principle of promoter in E.coli and yeast.</p><br />
<p>Week7: Experiments process design, constructs the basic parts and test circuits. </p><br />
<p>Week8: Design the primers </p><br />
<p>Week9: Workshop among several schools</p><br />
<p>Week10-week13: constructs the basic parts: BBa_K1051100 to BBa_K1051118</p><br />
<p>Week14-week18: Measurement designing</p><br />
<p>Week19-week22: Test measurement circuits and then data analysis</p><br />
<p>Week23-week25: results drawing and made the wiki</p><br />
<br />
<h4>Synchronization Group</h4><br />
<p>Week1: research about the previous work of iGEM, especially the work done by the European teams.</p><br />
<p>Week2~week4: project idea discussing and determination, group issue assigned.</p><br />
<p>Week5~week6: research about the principle of promoter in E.coli and yeast.</p><br />
<p>Week7: Experiments process design, constructs the basic parts and test circuits. </p><br />
<p>Week8: Design the primers needed to amplification the degradation tags.</p><br />
<p>Week9: Workshop among several schools</p><br />
<p>Week10-week13: constructs the basic parts: BBa_K1051500, etc.</p><br />
<p>Week14-week18: Measurement designing</p><br />
<p>Week19-week22: Test measurement circuits and then data analysis</p><br />
<p>Week23-week25: results processing<br />
<br />
<h4>Modeling</h4><br />
<p>Week 1-8 research about the previous best model </p><br />
<p>Week 9 Decided using the budding yeast cell cycle model as basic part</p><br />
<p>Week 10-13 Learning the cell designer,Matlab Simbiology as the modeling software and trying to make some test <p>model.</p><br />
<p>Week 14-16 Understanding the cell cycle model in cell designer and using cell designer drawing reaction network, <p>setting the parameters.</p><br />
<p>Week 17 Learning how to use the Matlab Simbiology part and input results within cell designer</p><br />
<p>Week 18-20 Decided making three modeling: alternative splicing, Sic1 regulation and degradation tags. Do related research.</p><br />
<p>Week 21-23 Made the cell cycle circuits, find parameters</p><br />
<p>Week 24-25 combine with experiments data and made wiki</p><br />
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<h3>Attributions</h3><br />
<h4> Work Design</h4><br />
<p>Gone Jianhui as a team leader and K2 as our instructor draft our project "Cell Magic".</p><br />
<h4>Experiments Conduct</h4><br />
<p> Li Xiang Li,Xu Yanhui,Wu Fanzi, Yu yang from SCU: responsible for targeting peptide,XFP,terminators design and experiments.</p> <br />
<p> Chen Shihong,Gu Chenguang, Lu Yanping, Liang Jiale,from South China University of Technology, are responsible for the alternative splicing Src1 and Mer2 intron design and experiments.</p><br />
<p> Zhu Shuang, Lin Kequan from Wuhan University and Wei Wei, Zheng Bingwei, Yi Lan in HUST work together for the promotors.</p><br />
<p> Guan Rui from SEU and He funan, Wang Rui, Lin Li,Zhang Yaolei from UESTC made their efforts to the degradation parts.</p><br />
<p> Zhou Wanling,Zhang Aiping, Li Dongdong, the undergraduates in AHMU, joined the part one</p><br />
<p> Chen Yichun of SCNU, Zhong Na of JNU work for the microfulidic part.</p><br />
<p> The SCNU student: Chen Chengxuan, Lin Qiongfen, Xie Qiaolin, worked for the cell cycle regulator Sic1</p><br />
<h4> Modeling</h4><br />
<p> Liu Shuang Liu from SEU, Zhou Yang from SCUT, Jinchun Zhang from SCU, Qiu Bitao from BGI</p><br />
<h4>Wiki Construction</h4><br />
<p> Zhang Jinchun and Zhou Yang </p><br />
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<h3>Protocols</h3><br />
<h4>Protocol of microfluidic </h4><br />
<p>1. design the pattern of the biochip. </p><br />
<p>2. print the pattern on the mask.</p><br />
<p>3. prepare the basic materials like clean silicon wafer, photoresist and so on.</p><br />
<p>4. spread the photoresist on the silicon wafer and make sure the thickness is 10 um.</p><br />
<p>5. with the help of the ultraviolet ray, the pattern of the mask can be transferred to the metamorphic photoresist.</p><br />
<p>6. (photographic fixing)Bake the silicon wafer to solidify the photoresist for about 2 hours.</p><br />
<p>7. (develop the pattern)wash off the photoresist which wasn’t exposure to the ultraviolet ray by using the special chemical reagent</p><br />
<p>8. Place the silicon wafer in the culture dish, then prepare the PDMS and pour it into the culture dish.</p><br />
<p>9. When the PDMS freeze, downcut it and fit it on a slide by surface plasma.</p><br />
<p>10. Connect the capillary tube and the chip.</p><br />
<br />
<h4>Protocol of chip-based XFP degradation rate detection in E.coli.</h4><br />
<p>First of all, E.coli will be measured after shaking to about OD2.0(600)in the chip which was washed by plasma water, vacuum pumping. The bacteria liquid was pushed into the chip, letting the cells enter the small triangle. Then use the constant flow pump culture medium into chip (the laboratory constant temperature, can not guarantee the training environment, 37°E. coli slower growth). The medium speed is about 200ul/h. Finally we test the data after yeast fulled in the triangles.Using the IPTG medium, the new RFP expression was stopped and we can regard the lights as degradation tags' efficiency.</p><br />
<br />
<h4>Protocol of Enzyme - labelled meter detecting the fluorescent protein intensity</h4><br />
<p>First of all, take a certain amount of bacteria liquid, recovery to around OD0.6(600), ensuring the bacteria in the logarithmic growth phase. Diluted and then the bacteria transferred to 96 well plates, measured their fluorescence intensity. Red fluorescent protein using an excitation wavelength of 584nm, and its emission wavelength is 607nm.When measuring, we first detect the OD600 of each strain, removing the factor of bacteria number difference. Thus the fluorescence intensity cannot be altered by bacteria quantity. Then the measurement mode switching for measurement of fluorescence, fluorescence intensity.</p><br />
<br />
<h4>Protocol of Fluoresces Intensity Measurement via ImageJ</h4><br />
<p>1. Install and open the ImageJ</p><br />
<p>2. Open the picture:File>open</p><br />
<p>3. Transit to 8bit grey style:Image>Type>8-bit </p><br />
<p>4. Invert white and black:Edit>Invert</p><br />
<p>5. Revise light intensity </p><br />
<p>6. Using Global calibration,revise the lilght intensity </p><br />
<p>7. Set pixel</p> <br />
<p>8. Set Measurements: Area、Integrated density.</p><br />
<p>9. Set the threshold :Image>Adjust>Threshold </p><br />
<p>10. Meaure: Analyze>Measure</p><br />
<p>11. Record the results</p><br />
<br />
<h4>Protocol of Yeast Synchronization with Microfluidics</h4><br />
<p>With the micro-fluidic device described above, and based on the fact that the length of the G1 phase of budding yeast is more sensitive to starvation, we carried out a series of experiments with various modulation schemes of changing medium. The rich medium used in budding yeast modulation experiments was SC -Ura, the poor medium was a mixture of 1.5% SC -Ura and 98.5% PBS (phosphate-buffered saline, pH = 6.0). After a scheme of 60 min period of poor medium alternating with 90 min period of rich medium three times.</p><br />
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<h3>References</h3><br />
<p>Acid, S. A. (2004). Lehninger principles of biochemistry. </p><br />
<p>Atlung, T., Løbner-Olesen, A., & Hansen, F. G. (1987). Overproduction of DnaA protein stimulates initiation of chromosome and minichromosome replication in Escherichia coli. Molecular and General Genetics MGG, 206(1), 51-59. </p><br />
<p>Bi, E., & Lutkenhaus, J. (1990). Interaction between the min locus and ftsZ. Journal of bacteriology, 172(10), 5610-5616. </p><br />
<p>Bi, E., & Lutkenhaus, J. (1991). FtsZ ring structure associated with division in Escherichia coli. </p><br />
<p>Bi, E., & Lutkenhaus, J. (1993). Cell division inhibitors SulA and MinCD prevent formation of the FtsZ ring. Journal of bacteriology, 175(4), 1118-1125. </p><br />
<p>Cho, S. W., Kim, S., Kim, J. M., & Kim, J.-S. (2013). Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol. </p><br />
<p>Conklin, D. S., Culbertson, M. R., & Kung, C. (1994). Interactions between gene products involved in divalent cation transport in Saccharomyces cerevisiae. Molecular and General Genetics MGG, 244(3), 303-311. </p><br />
<p>Deltcheva, E., Chylinski, K., Sharma, C. M., Gonzales, K., Chao, Y., Pirzada, Z. A., . . . Charpentier, E. (2011). CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature, 471(7340), 602-607. </p><br />
<p>Drubin, D. G., Mulholland, J., Zhu, Z., & Botstein, D. (1990). Homology of a yeast actin-binding protein to signal transduction proteins and myosin-I. Nature, 343(6255), 288-290. </p><br />
<p>Engebrecht, J., & Roeder, G. S. (1990). MER1, a yeast gene required for chromosome pairing and genetic recombination, is induced in meiosis. Molecular and cellular biology, 10(5), 2379-2389. </p><br />
<p>Fuller, R. S., & Kornberg, A. (1983). Purified dnaA protein in initiation of replication at the Escherichia coli chromosomal origin of replication. Proceedings of the National Academy of Sciences, 80(19), 5817-5821. </p><br />
<p>Grunau, S., Schliebs, W., Linnepe, R., Neufeld, C., Cizmowski, C., Reinartz, B., . . . Erdmann, R. (2009). Peroxisomal Targeting of PTS2 Pre‐Import Complexes in the Yeast Saccharomyces cerevisiae. Traffic, 10(4), 451-460. </p><br />
<p>Hershko, A. (1997). Roles of ubiquitin-mediated proteolysis in cell cycle control. Current Opinion in Cell Biology, 9(6), 788-799. <br />
Huibregtse, J. M., Scheffner, M., Beaudenon, S., & Howley, P. M. (1995). </p><br />
<p>A family of proteins structurally and functionally related to the E6-AP ubiquitin-protein ligase. Proceedings of the National Academy of Sciences, 92(7), 2563-2567. </p><br />
<p>Khorasanizadeh, S. (2004). The nucleosome: from genomic organization to genomic regulation. Cell, 116(2), 259-272. </p><br />
<p>Luo, C., Zhu, X., Yu, T., Luo, X., Ouyang, Q., Ji, H., & Chen, Y. (2008). A fast cell loading and high‐throughput microfluidic system for long‐term cell culture in zero‐flow environments. Biotechnology and bioengineering, 101(1), 190-195. </p><br />
<p>Miyabe, S., Izawa, S., & Inoue, Y. (2001). The Zrc1 Is Involved in Zinc Transport System between Vacuole and Cytosol in< i> Saccharomyces cerevisiae</i>. Biochemical and Biophysical Research Communications, 282(1), 79-83. </p><br />
<p>Munding, E. M., Igel, A. H., Shiue, L., Dorighi, K. M., Treviño, L. R., & Ares, M. (2010). Integration of a splicing regulatory network within the meiotic gene expression program of Saccharomyces cerevisiae. Genes & Development, 24(23), 2693-2704. </p><br />
<p>Murray, A. W., Solomon, M. J., & Kirschner, M. W. (1989). The role of cyclin synthesis and degradation in the control of maturation promoting factor activity. Nature, 339(6222), 280-286. </p><br />
<p>Nash, P., Tang, X., Orlicky, S., Chen, Q., Gertler, F. B., Mendenhall, M. D., . . . Tyers, M. (2001). Multisite phosphorylation of a CDK inhibitor sets a threshold for the onset of DNA replication. Nature, 414(6863), 514-521. </p><br />
<p>Nugroho, T. T., & Mendenhall, M. D. (1994). An inhibitor of yeast cyclin-dependent protein kinase plays an important role in ensuring the genomic integrity of daughter cells. Molecular and cellular biology, 14(5), 3320-3328. </p><br />
<p>Qiu, Z. R., Shuman, S., & Schwer, B. (2011). An essential role for trimethylguanosine RNA caps in Saccharomyces cerevisiae meiosis and their requirement for splicing of SAE3 and PCH2 meiotic pre-mRNAs. Nucleic Acids Research, 39(13), 5633-5646. </p><br />
<p>Spellman, P. T., Sherlock, G., Zhang, M. Q., Iyer, V. R., Anders, K., Eisen, M. B., . . . Futcher, B. (1998). Comprehensive identification of cell cycle–regulated genes of the yeast Saccharomyces cerevisiae by microarray hybridization. Molecular biology of the cell, 9(12), 3273-3297. </p><br />
<p>Ward, J. E., & Lutkenhaus, J. (1985). Overproduction of FtsZ induces minicell formation in E. coli. Cell, 42(3), 941-949. </p><br />
<p>Wolfsberg, T. G., Gabrielian, A. E., Campbell, M. J., Cho, R. J., Spouge, J. L., & Landsman, D. (1999). Candidate regulatory sequence elements for cell cycle-dependent transcription in Saccharomyces cerevisiae. Genome Res, 9(8), 775-792. </p><br />
<p>Yamano, H., Tsurumi, C., Gannon, J., & Hunt, T. (1998). The role of the destruction box and its neighbouring lysine residues in cyclin B for anaphase ubiquitin-dependent proteolysis in fission yeast: defining the D-box receptor. The EMBO Journal, 17(19), 5670-5678. </p><br />
<p>Belle, A., Tanay, A., Bitincka, L., Shamir, R., & O'Shea, E. K. (2006). Quantification of protein half-lives in the budding yeast proteome. Proc Natl Acad Sci U S A, 103(35), 13004-13009. doi: 10.1073/pnas.0605420103 </p><br />
<p>Belli, G., Gari, E., Aldea, M., & Herrero, E. (2001). Osmotic stress causes a G1 cell cycle delay and downregulation of Cln3/Cdc28 activity in Saccharomyces cerevisiae. Mol Microbiol, 39(4), 1022-1035. </p><br />
<p>Chen, K. C., Calzone, L., Csikasz-Nagy, A., Cross, F. R., Novak, B., & Tyson, J. J. (2004). Integrative analysis of cell cycle control in budding yeast. Mol Biol Cell, 15(8), 3841-3862. doi: 10.1091/mbc.E03-11-0794</p><br />
<p>Gilchrist, M. A., & Wagner, A. (2006). A model of protein translation including codon bias, nonsense errors, and ribosome recycling. J Theor Biol, 239(4), 417-434. doi: 10.1016/j.jtbi.2005.08.007</p><br />
<p>Mason, P. B., & Struhl, K. (2005). Distinction and relationship between elongation rate and processivity of RNA polymerase II in vivo. Mol Cell, 17(6), 831-840. doi: 10.1016/j.molcel.2005.02.017</p><br />
<p>Wang, Y., Liu, C. L., Storey, J. D., Tibshirani, R. J., Herschlag, D., & Brown, P. O. (2002). Precision and functional specificity in mRNA decay. Proc Natl Acad Sci U S A, 99(9), 5860-5865. doi: 10.1073/pnas.092538799</p><br />
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<h3>Acknowledgements</h3><br />
<h4>At the Beginning</h4><br />
<p>The BGI-ATCG iGEM 2013 team is a huge and diversity group consists of undergraduate from ten universities. Our team members’ major is really different, such as biotechnology bioinformatics, physical and even mathematics. Our work is partly assigned into each group made up by the students from the same university, which you can find in the group part in this page. And you can also browse about who and how everyone contributed to the project in this page. We really appreciate all our advisors and instructors that have assisted us throughout this project, without whom the project could not been carried out. We would also like to thank all everyone else who has helped us to achieve our project through put up advice or providing DNA, seeds, or other materials. Their contributions have helped us enormously. For a full list of acknowledgments, please see the bottom of this page.</p><br />
<h4>Financial Support</h4><br />
<p>BGI College provides the totally funding including our team registration fee and competition travel fee.</p><br />
<p>BGI Research Institute covers the wet-lab costs and provides equipments and labs.</p><br />
<h4>General Support</h4><br />
<p>Unit of Synthetic Biology at BGI trains team members about the basic experiment technology and knowledge which was really necessary for us.</p><br />
<p>Cho-Kiu Wong at SMC, BGI Tech Solutions help us much to send and receive the BioBricks.</p><br />
<p>MathWorks provides free software of Matlab and SimBiology.</p><br />
<h4>Material Support</h4><br />
<p>Boeke Lab of John Hopkins Medical Institutions (Boeke Lab @ John Hopkins Medical) provide us pRS413, pRS414, pRS415, pRS416 </p><br />
<p>Dr. Chi-Ming Wong (Dr. Chi-Ming Wong @ HKU) at Hong Kong University provides us the plasmid YEpLac195 YEpLac181</p><br />
<p>Kai Tian and Yong Li at BGI, helping us with the CRIPSR system.</p><br />
<p>SUSTC Biology Department and the BGI Unit of Synthetic Biology help us purchasing some materials.</p><br />
<p>WHU-China has provided us the <i>E. coli</i> version of CRISPRi system.</p><br />
<h4>At Last</h4><br />
<p>We really appreciate </p><br />
<p>1) the BGIC_0101 team for their cooperation, </p><br />
<p>2) the SCSTC help us for borrowing device and booking materials, and </p><br />
<p>3) SYSU, SUSTC borrowing us some experiments equipment.</p><br />
<h4>Lab Support</h4><br />
<p>Unit of Synthetic Biology at BGI supports us with the lab for the most important cell and molecular biology experiments.</p><br />
<p>Dr. Ming Ni, team leader in BGI cancer group, let us use his lab for the microfluidic experiments. </p><br />
<p>Pr. Lingling Shui at South China Normal University provides the microfluidic lab and materials for our experiments.</p><br />
<h4>Cooperation College or University</h4><br />
<p>Huazhong University of Science and Technology</p><br />
<p>Wuhan University</p><br />
<p>China University of Geosciences</p><br />
<p>South China University of Technology</p><br />
<p>South China Normal University</p><br />
<p>Jinan University</p><br />
<p>Sichuan University</p><br />
<p>University of Electronic Science and Technology of China</p><br />
<p>Southeast University</p><br />
<p>Qingdao University</p><br />
<p>University of Chinese Academy of Sciences</p><br />
<br />
</div></div>BGI K2http://2013.igem.org/File:09172_bright.jpgFile:09172 bright.jpg2013-09-28T00:14:29Z<p>BGI K2: uploaded a new version of &quot;File:09172 bright.jpg&quot;</p>
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<div></div>BGI K2http://2013.igem.org/File:09172_bright.jpgFile:09172 bright.jpg2013-09-28T00:14:22Z<p>BGI K2: uploaded a new version of &quot;File:09172 bright.jpg&quot;</p>
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<div></div>BGI K2http://2013.igem.org/File:09172_bright.jpgFile:09172 bright.jpg2013-09-28T00:14:07Z<p>BGI K2: uploaded a new version of &quot;File:09172 bright.jpg&quot;</p>
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<div></div>BGI K2http://2013.igem.org/Team:Shenzhen_BGIC_ATCG/notesTeam:Shenzhen BGIC ATCG/notes2013-09-27T23:44:17Z<p>BGI K2: </p>
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<br />
Title: Notes<br />
Team: Shenzhen_BGIC_ATCG<br />
Author: BGI_K2<br />
Twitter: @BGI_K2<br />
Email: im.ss.kk@gmail.com<br />
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<li><a id="navleftsub3" class="navleftsub" href="#board3">Protocols</a></li><br />
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<h3>Timeline</h3><br />
<h4>Promoter Group</h4><br />
<p>Week1: research about the previous work of iGEM, especially the work done by the European teams.</p><br />
<p>Week2~week4: project idea discussing and determination, group issue asssigned.</p><br />
<p>Week5~week6: research about the principle of promoter in E.coli and yeast.</p><br />
<p>Week7: Experiments process design, constructs the basic parts and test curcuits. </p><br />
<p>Week8: Design the primers needed to amplification the promotor</p><br />
<p>Week9: Workshop among several schools</p><br />
<p>Week10-week13: constructs the basic parts: BBa_K1051300, BBa_K1051301, BBa_K1051302,BBa_K1051303, BBa_K1051304, BBa_K1051305, BBa_K1051306</p><br />
<p>Week14-week18: Measurement designing</p><br />
<p>Week19-week22: Test measurement circuits and then data analysis</p><br />
<p>Week23-week25: results drawing and made the wiki</p><br />
<br />
<h4>Degradation Group</h4><br />
<p>Week1: previous team project data collection, especially the Latin America teams</p><br />
<p>Week2~week4: project idea decided,group issue divided into E.coli one and yeast one.</p><br />
<p>Week5~week6: research of principle of degradation peptide and design the experiment generally combining with the mechanism of cyclin protein degradation.</p><br />
<p>Week7: Experiments process design, constructs the basic parts and test curcuits. </p><br />
<p>Week8: Design the primers needed to amplification the degradation tags.</p><br />
<p>Week9: Workshop among several schools</p><br />
<p>Week10-week13: constructs the basic parts: K051200/K1051201/K1051202/K1051203/K1051204/K1051205/K1051206/K1051207</p><br />
<p>Week14-week18: Measurement circuits construction</p><br />
<p>Week19-week22: Test measurement circuits and then data analysis</p><br />
<p>Week23-week25: results drawing and made the wiki</p><br />
<br />
<h4>Targeting Group</h4><br />
<p>Week1: research about the previous work of iGEM, especially the work done by the Asia teams.</p><br />
<p>Week2~week4: project idea discussing and determination, group issue assigned.</p><br />
<p>Week5~week6: research about the principle of promoter in E.coli and yeast.</p><br />
<p>Week7: Experiments process design, constructs the basic parts and test circuits. </p><br />
<p>Week8: Design the primers </p><br />
<p>Week9: Workshop among several schools</p><br />
<p>Week10-week13: constructs the basic parts: BBa_K1051100 to BBa_K1051118</p><br />
<p>Week14-week18: Measurement designing</p><br />
<p>Week19-week22: Test measurement circuits and then data analysis</p><br />
<p>Week23-week25: results drawing and made the wiki</p><br />
<br />
<h4>Synchronization Group</h4><br />
<p>Week1: research about the previous work of iGEM, especially the work done by the European teams.</p><br />
<p>Week2~week4: project idea discussing and determination, group issue assigned.</p><br />
<p>Week5~week6: research about the principle of promoter in E.coli and yeast.</p><br />
<p>Week7: Experiments process design, constructs the basic parts and test circuits. </p><br />
<p>Week8: Design the primers needed to amplification the degradation tags.</p><br />
<p>Week9: Workshop among several schools</p><br />
<p>Week10-week13: constructs the basic parts: BBa_K1051500, etc.</p><br />
<p>Week14-week18: Measurement designing</p><br />
<p>Week19-week22: Test measurement circuits and then data analysis</p><br />
<p>Week23-week25: results processing<br />
<br />
<h4>Modeling</h4><br />
<p>Week 1-8 research about the previous best model </p><br />
<p>Week 9 Decided using the budding yeast cell cycle model as basic part</p><br />
<p>Week 10-13 Learning the cell designer,Matlab Simbiology as the modeling software and trying to make some test <p>model.</p><br />
<p>Week 14-16 Understanding the cell cycle model in cell designer and using cell designer drawing reaction network, <p>setting the parameters.</p><br />
<p>Week 17 Learning how to use the Matlab Simbiology part and input results within cell designer</p><br />
<p>Week 18-20 Decided making three modeling: alternative splicing, Sic1 regulation and degradation tags. Do related research.</p><br />
<p>Week 21-23 Made the cell cycle circuits, find parameters</p><br />
<p>Week 24-25 combine with experiments data and made wiki</p><br />
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<h3>Attributions</h3><br />
<h4> Work Design</h4><br />
<p>Gone Jianhui as a team leader and K2 as our instructor draft our project "Cell Magic".</p><br />
<h4>Experiments Conduct</h4><br />
<p> Li Xiang Li,Xu Yanhui,Wu Fanzi, Yu yang from SCU: responsible for targeting peptide,XFP,terminators design and experiments.</p> <br />
<p> Chen Shihong,Gu Chenguang, Lu Yanping, Liang Jiale,from South China University of Technology, are responsible for the alternative splicing Src1 and Mer2 intron design and experiments.</p><br />
<p> Zhu Shuang, Lin Kequan from Wuhan University and Wei Wei, Zheng Bingwei, Yi Lan in HUST work together for the promotors.</p><br />
<p> Guan Rui from SEU and He funan, Wang Rui, Lin Li,Zhang Yaolei from UESTC made their efforts to the degradation parts.</p><br />
<p> Zhou Wanling,Zhang Aiping, Li Dongdong, the undergraduates in AHMU, joined the part one</p><br />
<p> Chen Yichun of SCNU, Zhong Na of JNU work for the microfulidic part.</p><br />
<p> The SCNU student: Chen Chengxuan, Lin Qiongfen, Xie Qiaolin, worked for the cell cycle regulator Sic1</p><br />
<h4> Modeling</h4><br />
<p> Liu Shuang Liu from SEU, Zhou Yang from SCUT, Jinchun Zhang from SCU, Qiu Bitao from BGI</p><br />
<h4>Wiki Construction</h4><br />
<p> Zhang Jinchun and Zhou Yang </p><br />
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<h3>Protocols</h3><br />
<h4>Protocol of microfluidic </h4><br />
<p>1. design the pattern of the biochip. </p><br />
<p>2. print the pattern on the mask.</p><br />
<p>3. prepare the basic materials like clean silicon wafer, photoresist and so on.</p><br />
<p>4. spread the photoresist on the silicon wafer and make sure the thickness is 10 um.</p><br />
<p>5. with the help of the ultraviolet ray, the pattern of the mask can be transferred to the metamorphic photoresist.</p><br />
<p>6. (photographic fixing)Bake the silicon wafer to solidify the photoresist for about 2 hours.</p><br />
<p>7. (develop the pattern)wash off the photoresist which wasn’t exposure to the ultraviolet ray by using the special chemical reagent</p><br />
<p>8. Place the silicon wafer in the culture dish, then prepare the PDMS and pour it into the culture dish.</p><br />
<p>9. When the PDMS freeze, downcut it and fit it on a slide by surface plasma.</p><br />
<p>10. Connect the capillary tube and the chip.</p><br />
<br />
<h4>Protocol of chip-based XFP degradation rate detection in E.coli.</h4><br />
<p>First of all, E.coli will be measured after shaking to about OD2.0(600)in the chip which was washed by plasma water, vacuum pumping. The bacteria liquid was pushed into the chip, letting the cells enter the small triangle. Then use the constant flow pump culture medium into chip (the laboratory constant temperature, can not guarantee the training environment, 37°E. coli slower growth). The medium speed is about 200ul/h. Finally we test the data after yeast fulled in the triangles.Using the IPTG medium, the new RFP expression was stopped and we can regard the lights as degradation tags' efficiency.</p><br />
<br />
<h4>Protocol of Enzyme - labelled meter detecting the fluorescent protein intensity</h4><br />
<p>First of all, take a certain amount of bacteria liquid, recovery to around OD0.6(600), ensuring the bacteria in the logarithmic growth phase. Diluted and then the bacteria transferred to 96 well plates, measured their fluorescence intensity. Red fluorescent protein using an excitation wavelength of 584nm, and its emission wavelength is 607nm.When measuring, we first detect the OD600 of each strain, removing the factor of bacteria number difference. Thus the fluorescence intensity cannot be altered by bacteria quantity. Then the measurement mode switching for measurement of fluorescence, fluorescence intensity.</p><br />
<br />
<h4>Protocol of Fluoresces Intensity Measurement via ImageJ</h4><br />
<p>1. Install and open the ImageJ</p><br />
<p>2. Open the picture:File>open</p><br />
<p>3. Transit to 8bit grey style:Image>Type>8-bit </p><br />
<p>4. Invert white and black:Edit>Invert</p><br />
<p>5. Revise light intensity </p><br />
<p>6. Using Global calibration,revise the lilght intensity </p><br />
<p>7. Set pixel</p> <br />
<p>8. Set Measurements: Area、Integrated density.</p><br />
<p>9. Set the threshold :Image>Adjust>Threshold </p><br />
<p>10. Meaure: Analyze>Measure</p><br />
<p>11. Record the results</p><br />
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<h3>References</h3><br />
<p>Acid, S. A. (2004). Lehninger principles of biochemistry. </p><br />
<p>Atlung, T., Løbner-Olesen, A., & Hansen, F. G. (1987). Overproduction of DnaA protein stimulates initiation of chromosome and minichromosome replication in Escherichia coli. Molecular and General Genetics MGG, 206(1), 51-59. </p><br />
<p>Bi, E., & Lutkenhaus, J. (1990). Interaction between the min locus and ftsZ. Journal of bacteriology, 172(10), 5610-5616. </p><br />
<p>Bi, E., & Lutkenhaus, J. (1991). FtsZ ring structure associated with division in Escherichia coli. </p><br />
<p>Bi, E., & Lutkenhaus, J. (1993). Cell division inhibitors SulA and MinCD prevent formation of the FtsZ ring. Journal of bacteriology, 175(4), 1118-1125. </p><br />
<p>Cho, S. W., Kim, S., Kim, J. M., & Kim, J.-S. (2013). Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol. </p><br />
<p>Conklin, D. S., Culbertson, M. R., & Kung, C. (1994). Interactions between gene products involved in divalent cation transport in Saccharomyces cerevisiae. Molecular and General Genetics MGG, 244(3), 303-311. </p><br />
<p>Deltcheva, E., Chylinski, K., Sharma, C. M., Gonzales, K., Chao, Y., Pirzada, Z. A., . . . Charpentier, E. (2011). CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature, 471(7340), 602-607. </p><br />
<p>Drubin, D. G., Mulholland, J., Zhu, Z., & Botstein, D. (1990). Homology of a yeast actin-binding protein to signal transduction proteins and myosin-I. Nature, 343(6255), 288-290. </p><br />
<p>Engebrecht, J., & Roeder, G. S. (1990). MER1, a yeast gene required for chromosome pairing and genetic recombination, is induced in meiosis. Molecular and cellular biology, 10(5), 2379-2389. </p><br />
<p>Fuller, R. S., & Kornberg, A. (1983). Purified dnaA protein in initiation of replication at the Escherichia coli chromosomal origin of replication. Proceedings of the National Academy of Sciences, 80(19), 5817-5821. </p><br />
<p>Grunau, S., Schliebs, W., Linnepe, R., Neufeld, C., Cizmowski, C., Reinartz, B., . . . Erdmann, R. (2009). Peroxisomal Targeting of PTS2 Pre‐Import Complexes in the Yeast Saccharomyces cerevisiae. Traffic, 10(4), 451-460. </p><br />
<p>Hershko, A. (1997). Roles of ubiquitin-mediated proteolysis in cell cycle control. Current Opinion in Cell Biology, 9(6), 788-799. <br />
Huibregtse, J. M., Scheffner, M., Beaudenon, S., & Howley, P. M. (1995). </p><br />
<p>A family of proteins structurally and functionally related to the E6-AP ubiquitin-protein ligase. Proceedings of the National Academy of Sciences, 92(7), 2563-2567. </p><br />
<p>Khorasanizadeh, S. (2004). The nucleosome: from genomic organization to genomic regulation. Cell, 116(2), 259-272. </p><br />
<p>Luo, C., Zhu, X., Yu, T., Luo, X., Ouyang, Q., Ji, H., & Chen, Y. (2008). A fast cell loading and high‐throughput microfluidic system for long‐term cell culture in zero‐flow environments. Biotechnology and bioengineering, 101(1), 190-195. </p><br />
<p>Miyabe, S., Izawa, S., & Inoue, Y. (2001). The Zrc1 Is Involved in Zinc Transport System between Vacuole and Cytosol in< i> Saccharomyces cerevisiae</i>. Biochemical and Biophysical Research Communications, 282(1), 79-83. </p><br />
<p>Munding, E. M., Igel, A. H., Shiue, L., Dorighi, K. M., Treviño, L. R., & Ares, M. (2010). Integration of a splicing regulatory network within the meiotic gene expression program of Saccharomyces cerevisiae. Genes & Development, 24(23), 2693-2704. </p><br />
<p>Murray, A. W., Solomon, M. J., & Kirschner, M. W. (1989). The role of cyclin synthesis and degradation in the control of maturation promoting factor activity. Nature, 339(6222), 280-286. </p><br />
<p>Nash, P., Tang, X., Orlicky, S., Chen, Q., Gertler, F. B., Mendenhall, M. D., . . . Tyers, M. (2001). Multisite phosphorylation of a CDK inhibitor sets a threshold for the onset of DNA replication. Nature, 414(6863), 514-521. </p><br />
<p>Nugroho, T. T., & Mendenhall, M. D. (1994). An inhibitor of yeast cyclin-dependent protein kinase plays an important role in ensuring the genomic integrity of daughter cells. Molecular and cellular biology, 14(5), 3320-3328. </p><br />
<p>Qiu, Z. R., Shuman, S., & Schwer, B. (2011). An essential role for trimethylguanosine RNA caps in Saccharomyces cerevisiae meiosis and their requirement for splicing of SAE3 and PCH2 meiotic pre-mRNAs. Nucleic Acids Research, 39(13), 5633-5646. </p><br />
<p>Spellman, P. T., Sherlock, G., Zhang, M. Q., Iyer, V. R., Anders, K., Eisen, M. B., . . . Futcher, B. (1998). Comprehensive identification of cell cycle–regulated genes of the yeast Saccharomyces cerevisiae by microarray hybridization. Molecular biology of the cell, 9(12), 3273-3297. </p><br />
<p>Ward, J. E., & Lutkenhaus, J. (1985). Overproduction of FtsZ induces minicell formation in E. coli. Cell, 42(3), 941-949. </p><br />
<p>Wolfsberg, T. G., Gabrielian, A. E., Campbell, M. J., Cho, R. J., Spouge, J. L., & Landsman, D. (1999). Candidate regulatory sequence elements for cell cycle-dependent transcription in Saccharomyces cerevisiae. Genome Res, 9(8), 775-792. </p><br />
<p>Yamano, H., Tsurumi, C., Gannon, J., & Hunt, T. (1998). The role of the destruction box and its neighbouring lysine residues in cyclin B for anaphase ubiquitin-dependent proteolysis in fission yeast: defining the D-box receptor. The EMBO Journal, 17(19), 5670-5678. </p><br />
<p>Belle, A., Tanay, A., Bitincka, L., Shamir, R., & O'Shea, E. K. (2006). Quantification of protein half-lives in the budding yeast proteome. Proc Natl Acad Sci U S A, 103(35), 13004-13009. doi: 10.1073/pnas.0605420103 </p><br />
<p>Belli, G., Gari, E., Aldea, M., & Herrero, E. (2001). Osmotic stress causes a G1 cell cycle delay and downregulation of Cln3/Cdc28 activity in Saccharomyces cerevisiae. Mol Microbiol, 39(4), 1022-1035. </p><br />
<p>Chen, K. C., Calzone, L., Csikasz-Nagy, A., Cross, F. R., Novak, B., & Tyson, J. J. (2004). Integrative analysis of cell cycle control in budding yeast. Mol Biol Cell, 15(8), 3841-3862. doi: 10.1091/mbc.E03-11-0794</p><br />
<p>Gilchrist, M. A., & Wagner, A. (2006). A model of protein translation including codon bias, nonsense errors, and ribosome recycling. J Theor Biol, 239(4), 417-434. doi: 10.1016/j.jtbi.2005.08.007</p><br />
<p>Mason, P. B., & Struhl, K. (2005). Distinction and relationship between elongation rate and processivity of RNA polymerase II in vivo. Mol Cell, 17(6), 831-840. doi: 10.1016/j.molcel.2005.02.017</p><br />
<p>Wang, Y., Liu, C. L., Storey, J. D., Tibshirani, R. J., Herschlag, D., & Brown, P. O. (2002). Precision and functional specificity in mRNA decay. Proc Natl Acad Sci U S A, 99(9), 5860-5865. doi: 10.1073/pnas.092538799</p><br />
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<h3>Acknowledgements</h3><br />
<h4>At the Beginning</h4><br />
<p>The BGI-ATCG iGEM 2013 team is a huge and diversity group consists of undergraduate from ten universities. Our team members’ major is really different, such as biotechnology bioinformatics, physical and even mathematics. Our work is partly assigned into each group made up by the students from the same university, which you can find in the group part in this page. And you can also browse about who and how everyone contributed to the project in this page. We really appreciate all our advisors and instructors that have assisted us throughout this project, without whom the project could not been carried out. We would also like to thank all everyone else who has helped us to achieve our project through put up advice or providing DNA, seeds, or other materials. Their contributions have helped us enormously. For a full list of acknowledgments, please see the bottom of this page.</p><br />
<h4>Financial Support</h4><br />
<p>BGI College provides the totally funding including our team registration fee and competition travel fee.</p><br />
<p>BGI Research Institute covers the wet-lab costs and provides equipments and labs.</p><br />
<h4>General Support</h4><br />
<p>Unit of Synthetic Biology at BGI trains team members about the basic experiment technology and knowledge which was really necessary for us.</p><br />
<p>Cho-Kiu Wong at SMC, BGI Tech Solutions help us much to send and receive the BioBricks.</p><br />
<p>MathWorks provides free software of Matlab and SimBiology.</p><br />
<h4>Material Support</h4><br />
<p>Boeke Lab of John Hopkins Medical Institutions (Boeke Lab @ John Hopkins Medical) provide us pRS413, pRS414, pRS415, pRS416 </p><br />
<p>Dr. Chi-Ming Wong (Dr. Chi-Ming Wong @ HKU) at Hong Kong University provides us the plasmid YEpLac195 YEpLac181</p><br />
<p>Kai Tian and Yong Li at BGI, helping us with the CRIPSR system.</p><br />
<p>SUSTC Biology Department and the BGI Unit of Synthetic Biology help us purchasing some materials.</p><br />
<p>WHU-China has provided us the <i>E. coli</i> version of CRISPRi system.</p><br />
<h4>At Last</h4><br />
<p>We really appreciate </p><br />
<p>1) the BGIC_0101 team for their cooperation, </p><br />
<p>2) the SCSTC help us for borrowing device and booking materials, and </p><br />
<p>3) SYSU, SUSTC borrowing us some experiments equipment.</p><br />
<h4>Lab Support</h4><br />
<p>Unit of Synthetic Biology at BGI supports us with the lab for the most important cell and molecular biology experiments.</p><br />
<p>Dr. Ming Ni, team leader in BGI cancer group, let us use his lab for the microfluidic experiments. </p><br />
<p>Pr. Lingling Shui at South China Normal University provides the microfluidic lab and materials for our experiments.</p><br />
<h4>Cooperation College or University</h4><br />
<p>Huazhong University of Science and Technology</p><br />
<p>Wuhan University</p><br />
<p>China University of Geosciences</p><br />
<p>South China University of Technology</p><br />
<p>South China Normal University</p><br />
<p>Jinan University</p><br />
<p>Sichuan University</p><br />
<p>University of Electronic Science and Technology of China</p><br />
<p>Southeast University</p><br />
<p>Qingdao University</p><br />
<p>University of Chinese Academy of Sciences</p><br />
<br />
</div></div>BGI K2http://2013.igem.org/Team:Shenzhen_BGIC_ATCG/notesTeam:Shenzhen BGIC ATCG/notes2013-09-27T23:42:42Z<p>BGI K2: </p>
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Title: Notes<br />
Team: Shenzhen_BGIC_ATCG<br />
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<h3>Timeline</h3><br />
<h4>Promoter Group</h4><br />
<p>Week1: research about the previous work of iGEM, especially the work done by the European teams.</p><br />
<p>Week2~week4: project idea discussing and determination, group issue asssigned.</p><br />
<p>Week5~week6: research about the principle of promoter in E.coli and yeast.</p><br />
<p>Week7: Experiments process design, constructs the basic parts and test curcuits. </p><br />
<p>Week8: Design the primers needed to amplification the promotor</p><br />
<p>Week9: Workshop among several schools</p><br />
<p>Week10-week13: constructs the basic parts: BBa_K1051300, BBa_K1051301, BBa_K1051302,BBa_K1051303, BBa_K1051304, BBa_K1051305, BBa_K1051306</p><br />
<p>Week14-week18: Measurement designing</p><br />
<p>Week19-week22: Test measurement circuits and then data analysis</p><br />
<p>Week23-week25: results drawing and made the wiki</p><br />
<br />
<h4>Degradation Group</h4><br />
<p>Week1: previous team project data collection, especially the Latin America teams</p><br />
<p>Week2~week4: project idea decided,group issue divided into E.coli one and yeast one.</p><br />
<p>Week5~week6: research of principle of degradation peptide and design the experiment generally combining with the mechanism of cyclin protein degradation.</p><br />
<p>Week7: Experiments process design, constructs the basic parts and test curcuits. </p><br />
<p>Week8: Design the primers needed to amplification the degradation tags.</p><br />
<p>Week9: Workshop among several schools</p><br />
<p>Week10-week13: constructs the basic parts: K051200/K1051201/K1051202/K1051203/K1051204/K1051205/K1051206/K1051207</p><br />
<p>Week14-week18: Measurement circuits construction</p><br />
<p>Week19-week22: Test measurement circuits and then data analysis</p><br />
<p>Week23-week25: results drawing and made the wiki</p><br />
<br />
<h4>Targeting Group</h4><br />
<p>Week1: research about the previous work of iGEM, especially the work done by the Asia teams.</p><br />
<p>Week2~week4: project idea discussing and determination, group issue assigned.</p><br />
<p>Week5~week6: research about the principle of promoter in E.coli and yeast.</p><br />
<p>Week7: Experiments process design, constructs the basic parts and test circuits. </p><br />
<p>Week8: Design the primers </p><br />
<p>Week9: Workshop among several schools</p><br />
<p>Week10-week13: constructs the basic parts: BBa_K1051100 to BBa_K1051118</p><br />
<p>Week14-week18: Measurement designing</p><br />
<p>Week19-week22: Test measurement circuits and then data analysis</p><br />
<p>Week23-week25: results drawing and made the wiki</p><br />
<br />
<h4>Synchronization Group</h4><br />
<p>Week1: research about the previous work of iGEM, especially the work done by the European teams.</p><br />
<p>Week2~week4: project idea discussing and determination, group issue assigned.</p><br />
<p>Week5~week6: research about the principle of promoter in E.coli and yeast.</p><br />
<p>Week7: Experiments process design, constructs the basic parts and test circuits. </p><br />
<p>Week8: Design the primers needed to amplification the degradation tags.</p><br />
<p>Week9: Workshop among several schools</p><br />
<p>Week10-week13: constructs the basic parts: BBa_K1051500, etc.</p><br />
<p>Week14-week18: Measurement designing</p><br />
<p>Week19-week22: Test measurement circuits and then data analysis</p><br />
<p>Week23-week25: results processing<br />
<br />
<h4>Modeling</h4><br />
<p>Week 1-8 research about the previous best model </p><br />
<p>Week 9 Decided using the budding yeast cell cycle model as basic part</p><br />
<p>Week 10-13 Learning the cell designer,Matlab Simbiology as the modeling software and trying to make some test <p>model.</p><br />
<p>Week 14-16 Understanding the cell cycle model in cell designer and using cell designer drawing reaction network, <p>setting the parameters.</p><br />
<p>Week 17 Learning how to use the Matlab Simbiology part and input results within cell designer</p><br />
<p>Week 18-20 Decided making three modeling: alternative splicing, Sic1 regulation and degradation tags. Do related research.</p><br />
<p>Week 21-23 Made the cell cycle circuits, find parameters</p><br />
<p>Week 24-25 combine with experiments data and made wiki</p><br />
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<h3>Attributions</h3><br />
<h4> Work Design</h4><br />
<p>Gone Jianhui as a team leader and K2 as our instructor draft our project "Cell Magic".</p><br />
<h4>Experiments Conduct</h4><br />
<p> Li Xiang Li,Xu Yanhui,Wu Fanzi, Yu yang from SCU: responsible for targeting peptide,XFP,terminators design and experiments.</p> <br />
<p> Chen Shihong,Gu Chenguang, Lu Yanping, Liang Jiale,from South China University of Technology, are responsible for the alternative splicing Src1 and Mer2 intron design and experiments.</p><br />
<p> Zhu Shuang, Lin Kequan from Wuhan University and Wei Wei, Zheng Bingwei, Yi Lan in HUST work together for the promotors.</p><br />
<p> Guan Rui from SEU and He funan, Wang Rui, Lin Li,Zhang Yaolei from UESTC made their efforts to the degradation parts.</p><br />
<p> Zhou Wanling,Zhang Aiping, Li Dongdong, the undergraduates in AHMU, joined the part one</p><br />
<p> Chen Yichun of SCNU, Zhong Na of JNU work for the microfulidic part.</p><br />
<p> The SCNU student: Chen Chengxuan, Lin Qiongfen, Xie Qiaolin, worked for the cell cycle regulator Sic1</p><br />
<h4> Modeling</h4><br />
<p> Liu Shuang Liu from SEU, Zhou Yang from SCUT, Jinchun Zhang from SCU, Qiu Bitao from BGI</p><br />
<h4>Wiki Construction</h4><br />
<p> Zhang Jinchun and Zhou Yang </p><br />
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<h3>Protocols</h3><br />
<h4>Protocol of microfluidic </h4><br />
<p>1. design the pattern of the biochip. </p><br />
<p>2. print the pattern on the mask.</p><br />
<p>3. prepare the basic materials like clean silicon wafer, photoresist and so on.</p><br />
<p>4. spread the photoresist on the silicon wafer and make sure the thickness is 10 um.</p><br />
<p>5. with the help of the ultraviolet ray, the pattern of the mask can be transferred to the metamorphic photoresist.</p><br />
<p>6. (photographic fixing)Bake the silicon wafer to solidify the photoresist for about 2 hours.</p><br />
<p>7. (develop the pattern)wash off the photoresist which wasn’t exposure to the ultraviolet ray by using the special chemical reagent</p><br />
<p>8. Place the silicon wafer in the culture dish, then prepare the PDMS and pour it into the culture dish.</p><br />
<p>9. When the PDMS freeze, downcut it and fit it on a slide by surface plasma.</p><br />
<p>10. Connect the capillary tube and the chip.</p><br />
<br />
<h4>Protocol of chip-based XFP degradation rate detection in E.coli.</h4><br />
<p>First of all, E.coli will be measured after shaking to about OD2.0(600)in the chip which was washed by plasma water, vacuum pumping. The bacteria liquid was pushed into the chip, letting the cells enter the small triangle. Then use the constant flow pump culture medium into chip (the laboratory constant temperature, can not guarantee the training environment, 37°E. coli slower growth). The medium speed is about 200ul/h. Finally we test the data after yeast fulled in the triangles.Using the IPTG medium, the new RFP expression was stopped and we can regard the lights as degradation tags' efficiency.</p><br />
<br />
<h4>Protocol of Enzyme - labelled meter detecting the fluorescent protein intensity</h4><br />
<p>First of all, take a certain amount of bacteria liquid, recovery to around OD0.6(600), ensuring the bacteria in the logarithmic growth phase. Diluted and then the bacteria transferred to 96 well plates, measured their fluorescence intensity. Red fluorescent protein using an excitation wavelength of 584nm, and its emission wavelength is 607nm.When measuring, we first detect the OD600 of each strain, removing the factor of bacteria number difference. Thus the fluorescence intensity cannot be altered by bacteria quantity. Then the measurement mode switching for measurement of fluorescence, fluorescence intensity.</p><br />
<br />
<h4>Protocol of Fluoresces Intensity Measurement via ImageJ</h4><br />
<p>1. Install and open the ImageJ</p><br />
<p>2. Open the picture:File>open</p><br />
<p>3. Transit to 8bit grey style:Image>Type>8-bit </p><br />
<p>4. Invert white and black:Edit>Invert</p><br />
<p>5. Revise light intensity </p><br />
<p>6. Using Global calibration,revise the lilght intensity </p><br />
<p>7. Set pixel</p> <br />
<p>8. Set Measurements: Area、Integrated density.</p><br />
<p>9. Set the threshold :Image>Adjust>Threshold </p><br />
<p>10. Meaure: Analyze>Measure</p><br />
<p>11. Record the results</p><br />
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<h3>References</h3><br />
<p>Acid, S. A. (2004). Lehninger principles of biochemistry. </p><br />
<p>Atlung, T., Løbner-Olesen, A., & Hansen, F. G. (1987). Overproduction of DnaA protein stimulates initiation of chromosome and minichromosome replication in Escherichia coli. Molecular and General Genetics MGG, 206(1), 51-59. </p><br />
<p>Bi, E., & Lutkenhaus, J. (1990). Interaction between the min locus and ftsZ. Journal of bacteriology, 172(10), 5610-5616. </p><br />
<p>Bi, E., & Lutkenhaus, J. (1991). FtsZ ring structure associated with division in Escherichia coli. </p><br />
<p>Bi, E., & Lutkenhaus, J. (1993). Cell division inhibitors SulA and MinCD prevent formation of the FtsZ ring. Journal of bacteriology, 175(4), 1118-1125. </p><br />
<p>Cho, S. W., Kim, S., Kim, J. M., & Kim, J.-S. (2013). Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol. </p><br />
<p>Conklin, D. S., Culbertson, M. R., & Kung, C. (1994). Interactions between gene products involved in divalent cation transport in Saccharomyces cerevisiae. Molecular and General Genetics MGG, 244(3), 303-311. </p><br />
<p>Deltcheva, E., Chylinski, K., Sharma, C. M., Gonzales, K., Chao, Y., Pirzada, Z. A., . . . Charpentier, E. (2011). CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature, 471(7340), 602-607. </p><br />
<p>Drubin, D. G., Mulholland, J., Zhu, Z., & Botstein, D. (1990). Homology of a yeast actin-binding protein to signal transduction proteins and myosin-I. Nature, 343(6255), 288-290. </p><br />
<p>Engebrecht, J., & Roeder, G. S. (1990). MER1, a yeast gene required for chromosome pairing and genetic recombination, is induced in meiosis. Molecular and cellular biology, 10(5), 2379-2389. </p><br />
<p>Fuller, R. S., & Kornberg, A. (1983). Purified dnaA protein in initiation of replication at the Escherichia coli chromosomal origin of replication. Proceedings of the National Academy of Sciences, 80(19), 5817-5821. </p><br />
<p>Grunau, S., Schliebs, W., Linnepe, R., Neufeld, C., Cizmowski, C., Reinartz, B., . . . Erdmann, R. (2009). Peroxisomal Targeting of PTS2 Pre‐Import Complexes in the Yeast Saccharomyces cerevisiae. Traffic, 10(4), 451-460. </p><br />
<p>Hershko, A. (1997). Roles of ubiquitin-mediated proteolysis in cell cycle control. Current Opinion in Cell Biology, 9(6), 788-799. <br />
Huibregtse, J. M., Scheffner, M., Beaudenon, S., & Howley, P. M. (1995). </p><br />
<p>A family of proteins structurally and functionally related to the E6-AP ubiquitin-protein ligase. Proceedings of the National Academy of Sciences, 92(7), 2563-2567. </p><br />
<p>Khorasanizadeh, S. (2004). The nucleosome: from genomic organization to genomic regulation. Cell, 116(2), 259-272. </p><br />
<p>Luo, C., Zhu, X., Yu, T., Luo, X., Ouyang, Q., Ji, H., & Chen, Y. (2008). A fast cell loading and high‐throughput microfluidic system for long‐term cell culture in zero‐flow environments. Biotechnology and bioengineering, 101(1), 190-195. </p><br />
<p>Miyabe, S., Izawa, S., & Inoue, Y. (2001). The Zrc1 Is Involved in Zinc Transport System between Vacuole and Cytosol in< i> Saccharomyces cerevisiae</i>. Biochemical and Biophysical Research Communications, 282(1), 79-83. </p><br />
<p>Munding, E. M., Igel, A. H., Shiue, L., Dorighi, K. M., Treviño, L. R., & Ares, M. (2010). Integration of a splicing regulatory network within the meiotic gene expression program of Saccharomyces cerevisiae. Genes & Development, 24(23), 2693-2704. </p><br />
<p>Murray, A. W., Solomon, M. J., & Kirschner, M. W. (1989). The role of cyclin synthesis and degradation in the control of maturation promoting factor activity. Nature, 339(6222), 280-286. </p><br />
<p>Nash, P., Tang, X., Orlicky, S., Chen, Q., Gertler, F. B., Mendenhall, M. D., . . . Tyers, M. (2001). Multisite phosphorylation of a CDK inhibitor sets a threshold for the onset of DNA replication. Nature, 414(6863), 514-521. </p><br />
<p>Nugroho, T. T., & Mendenhall, M. D. (1994). An inhibitor of yeast cyclin-dependent protein kinase plays an important role in ensuring the genomic integrity of daughter cells. Molecular and cellular biology, 14(5), 3320-3328. </p><br />
<p>Qiu, Z. R., Shuman, S., & Schwer, B. (2011). An essential role for trimethylguanosine RNA caps in Saccharomyces cerevisiae meiosis and their requirement for splicing of SAE3 and PCH2 meiotic pre-mRNAs. Nucleic Acids Research, 39(13), 5633-5646. </p><br />
<p>Spellman, P. T., Sherlock, G., Zhang, M. Q., Iyer, V. R., Anders, K., Eisen, M. B., . . . Futcher, B. (1998). Comprehensive identification of cell cycle–regulated genes of the yeast Saccharomyces cerevisiae by microarray hybridization. Molecular biology of the cell, 9(12), 3273-3297. </p><br />
<p>Ward, J. E., & Lutkenhaus, J. (1985). Overproduction of FtsZ induces minicell formation in E. coli. Cell, 42(3), 941-949. </p><br />
<p>Wolfsberg, T. G., Gabrielian, A. E., Campbell, M. J., Cho, R. J., Spouge, J. L., & Landsman, D. (1999). Candidate regulatory sequence elements for cell cycle-dependent transcription in Saccharomyces cerevisiae. Genome Res, 9(8), 775-792. </p><br />
<p>Yamano, H., Tsurumi, C., Gannon, J., & Hunt, T. (1998). The role of the destruction box and its neighbouring lysine residues in cyclin B for anaphase ubiquitin-dependent proteolysis in fission yeast: defining the D-box receptor. The EMBO Journal, 17(19), 5670-5678. </p><br />
<p>Belle, A., Tanay, A., Bitincka, L., Shamir, R., & O'Shea, E. K. (2006). Quantification of protein half-lives in the budding yeast proteome. Proc Natl Acad Sci U S A, 103(35), 13004-13009. doi: 10.1073/pnas.0605420103 </p><br />
<p>Belli, G., Gari, E., Aldea, M., & Herrero, E. (2001). Osmotic stress causes a G1 cell cycle delay and downregulation of Cln3/Cdc28 activity in Saccharomyces cerevisiae. Mol Microbiol, 39(4), 1022-1035. </p><br />
<p>Chen, K. C., Calzone, L., Csikasz-Nagy, A., Cross, F. R., Novak, B., & Tyson, J. J. (2004). Integrative analysis of cell cycle control in budding yeast. Mol Biol Cell, 15(8), 3841-3862. doi: 10.1091/mbc.E03-11-0794</p><br />
<p>Gilchrist, M. A., & Wagner, A. (2006). A model of protein translation including codon bias, nonsense errors, and ribosome recycling. J Theor Biol, 239(4), 417-434. doi: 10.1016/j.jtbi.2005.08.007</p><br />
<p>Mason, P. B., & Struhl, K. (2005). Distinction and relationship between elongation rate and processivity of RNA polymerase II in vivo. Mol Cell, 17(6), 831-840. doi: 10.1016/j.molcel.2005.02.017</p><br />
<p>Wang, Y., Liu, C. L., Storey, J. D., Tibshirani, R. J., Herschlag, D., & Brown, P. O. (2002). Precision and functional specificity in mRNA decay. Proc Natl Acad Sci U S A, 99(9), 5860-5865. doi: 10.1073/pnas.092538799</p><br />
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<h3>Acknowledgements</h3><br />
<h4>At the Beginning</h4><br />
<p>The BGI-ATCG iGEM 2013 team is a huge and diversity group consists of undergraduate from ten universities. Our team members’ major is really different, such as biotechnology bioinformatics, physical and even mathematics. Our work is partly assigned into each group made up by the students from the same university, which you can find in the group part in this page. And you can also browse about who and how everyone contributed to the project in this page. We really appreciate all our advisors and instructors that have assisted us throughout this project, without whom the project could not been carried out. We would also like to thank all everyone else who has helped us to achieve our project through put up advice or providing DNA, seeds, or other materials. Their contributions have helped us enormously. For a full list of acknowledgments, please see the bottom of this page.</p><br />
<h4>Financial Support</h4><br />
<p>BGI College provides the totally funding including our team registration fee and competition travel fee.</p><br />
<p>BGI Research Institute covers the wet-lab costs and provides equipments and labs.</p><br />
<h4>General Support</h4><br />
<p>Unit of Synthetic Biology at BGI trains team members about the basic experiment technology and knowledge which was really necessary for us.</p><br />
<p>Cho-Kiu Wong at SMC, BGI Tech Solutions help us much to send and receive the BioBricks.</p><br />
<p>MathWorks provides free software of Matlab and SimBiology.</p><br />
<h4>Material Support</h4><br />
<p>Boeke Lab of John Hopkins Medical Institutions (Boeke Lab @ John Hopkins Medical) provide us pRS413, pRS414, pRS415, pRS416 </p><br />
<p>Dr. Chi-Ming Wong (Dr. Chi-Ming Wong @ HKU) at Hong Kong University provides us the plasmid YEpLac195 YEpLac181</p><br />
<p>Kai Tian and Yong Li at BGI, helping us with the CRIPSR system.</p><br />
<p>SUSTC Biology Department and the BGI Unit of Synthetic Biology help us purchasing some materials.</p><br />
<p>WHU-China has provided us the <i>E. coli</i> version of CRISPRi system.</p><br />
<h4>At Last</h4><br />
<p>We really appreciate 1) the BGIC_0101 team for their cooperation, 2) the SCSTC help us for borrowing device and booking materials,<br />
and 3) SYSU, SUSTC borrowing us some experiments equipment.</p><br />
<h4>Lab Support</h4><br />
<p>Unit of Synthetic Biology at BGI supports us with the lab for the most important cell and molecular biology experiments.</p><br />
<p>Dr. Ming Ni, team leader in BGI cancer group, let us use his lab for the microfluidic experiments. </p><br />
<p>Pr. Lingling Shui at South China Normal University provides the microfluidic lab and materials for our experiments.</p><br />
<h4>Cooperation College or University</h4><br />
<p>Huazhong University of Science and Technology</p><br />
<p>Wuhan University</p><br />
<p>China University of Geosciences</p><br />
<p>South China University of Technology</p><br />
<p>South China Normal University</p><br />
<p>Jinan University</p><br />
<p>Sichuan University</p><br />
<p>University of Electronic Science and Technology of China</p><br />
<p>Southeast University</p><br />
<p>Qingdao University</p><br />
<p>University of Chinese Academy of Sciences</p><br />
<br />
</div></div>BGI K2http://2013.igem.org/Team:Shenzhen_BGIC_ATCG/storiesTeam:Shenzhen BGIC ATCG/stories2013-09-27T23:36:34Z<p>BGI K2: </p>
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<br />
Title: Stories<br />
Team: Shenzhen_BGIC_ATCG<br />
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<div class="navleftholder"><br />
<ul class="navleft"><br />
<li><a id="navleftsub1" class="navleftsub" href="#board1">The Magic</a></li><br />
<li><a id="navleftsub2" class="navleftsub" href="#board2">Cyclin Promoters</a></li><br />
<li><a id="navleftsub3" class="navleftsub" href="#board3">Reporter Modification</a></li><br />
<li><a id="navleftsub4" class="navleftsub" href="#board4">Degradation Tags</a></li><br />
<li><a id="navleftsub5" class="navleftsub" href="#board5">Targeting Peptides</a></li><br />
<li><a id="navleftsub6" class="navleftsub" href="#board6">Cell Synchronization</a></li><br />
<li><a id="navleftsub7" class="navleftsub" href="#board7">Alternative Splicing Device</a></li><br />
<li><a id="navleftsub8" class="navleftsub" href="#board8">Microfluidic Device</a></li><br />
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<li><img src="https://static.igem.org/mediawiki/2013/5/58/P%2BD%2BT%2BS.png" title="Genetic circuit of cell cycle version #4" description="To make the magic more fantasy, alternative splicing device acting synchronously with the synchronization device." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/1/10/Chip.gif" title="Microfuidics Device" description="Cell magic in microfuidics device." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/e4/Yeast.gif" title="DT X TP X CCR X" description="Version #1, cell magic without degradation tags, targeting peptides and cell cycle regulator."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/7/79/Yeast_D.gif" title="DT √ TP X CCR X" description="Version #2, cell magic with degradation tags, without targeting peptides and cell cycle regulator." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/e6/Yeast_DT.gif" title="DT √ TP √ CCR X" description="Version #3, cell magic with degradation tags and targeting peptides, without cell cycle regulator." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/c/c1/Yeast_DTR.gif" title="DT √ TP √ CCR √" description="Version #4, cell magic with degradation tag, targeting peptides and cell <li>cycle regulator." /></li><br />
</ul></div><br />
<div id="box1" class="box"><br />
<h3>The Magic</h3><br />
<p>Life, the most brilliant magic in the universe, celebrated with the ability of reproduction and revolution. And the magic, is based on a sophisticated mechanism, called cell cycle. Most genetic reactions in a cell are regulated, directly or indirectly, by cell cycle. As we wish to build artificial lives, it is worthwhile to learn from the exquisite design of creature, to make use of cell cycle. As a pioneer study, we try to performing a "Cell Magic", by capturing cell cycle with fantastic reporters. By grasping the usage of cell cycle tools, we are promised to direct refined actions in a cell. For instance, when producing Paclitaxel (harmful to centrosome in S phase) with a cell factory, we may able to transport it out before it comes detrimental. </p><br />
<p>So this is our project, the "Cell Magic", to engineer two versions of cell cycle based magic in both budding yeast and <i>E. coli</i>. We will present the blueprint of our stories by the yeast version. </p><br />
<h4>Version #1, Degradation Tag X, Targeting Peptides X, Cell Cycle Regulator X</h4><br />
<p>Promoters of cyclins, which can initial transcription in specific phase in a cell cycle, are selected to produce different fluorescence proteins in G1/SG2/M phases respectably. However, in our first version of story, with low natural degradation rate, fluorescence proteins would remain in cell after their promoters stop working. So colors for each phase cannot be distinguished, as the figure shows.</p><br />
<img src="https://static.igem.org/mediawiki/2013/e/e4/Yeast.gif" title="DT X TP X CCR X" description="Version #1, cell magic without degradation tags, targeting peptides and cell cycle regulator." width=47%/><br />
<img src="https://static.igem.org/mediawiki/2013/3/36/P.png" width=50% ><br />
<p>Figure. Version #1, cell magic without degradation tags, targeting peptides and cell cycle regulator. Animation and genetic circuits.</p><br />
<br />
<h4>Version #2, Degradation Tag √, Targeting Peptides X, Cell Cycle Regulator X</h4><br />
<p>In the second version, degradation tags are added to the reporters to solve this problem. All fluorescence proteins are thought to be degraded in 15 minutes which is far less than the period of a cell cycle.</p><br />
<img src="https://static.igem.org/mediawiki/2013/7/79/Yeast_D.gif" title="DT √ TP X CCR X" description="Version #2, cell magic with degradation tags, without targeting peptides and cell cycle regulator." width=47%/><br />
<img src="https://static.igem.org/mediawiki/2013/d/df/Gc.P%2BD.png" width=50%><br />
<p>Figure. Version #2, cell magic with degradation tags, without targeting peptides and cell cycle regulator. Animation and genetic circuits.</p><br />
<br />
<h4>Version #3, Degradation Tag √, Targeting Peptides √, Cell Cycle Regulator X</h4><br />
<p>While we are not sure if degradation tags can work as expected, we try to turn the time magic into a time-space one. Reporters are located to different cell structures by targeting peptides. Different phase with different color shines in different organelle, that is the third version.</p><br />
<img src="https://static.igem.org/mediawiki/2013/e/e6/Yeast_DT.gif" title="DT √ TP √ CCR X" description="Version #3, cell magic with degradation tags and targeting peptides, without cell cycle regulator." width=47%/><br />
<br />
<img src="https://static.igem.org/mediawiki/2013/8/8d/P%2BD%2BT.png" width=50%><br />
<p>Figure. Version #3, cell magic with degradation tags and targeting peptides, without cell cycle regulator. Animation and genetic circuits.</p><br />
<br />
<h4>Version #4, Degradation Tag √, Targeting Peptides √, Cell Cycle Regulator √</h4><br />
<p>To capture the magic macroscopically, we developed a microfluidic device to synchronize all cells into the same phase (G1). So synchronization devices was added to the forth version. </p><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Chip.gif" title="Microfuidics Device" description="Cell magic in microfuidics device." width=35% /><br />
<p>Figure. Cell magic in microfuidics device.</p><br />
<br />
<p>To make the magic more fantasy, alternative splicing device acting synchronously with the synchronization device (a translational unit of a cyclin regulator), will splice the targeting peptide tail when cell are being synchronized to G1 phase, so all cells turn green; while synchronization device not working, targeting peptide remains in the reporter so only mitochondria turn green. In this way, the cell phase and statues can be reported by fluorescences.</p><br />
<img src="https://static.igem.org/mediawiki/2013/c/c1/Yeast_DTR.gif" title="DT √ TP √ CCR √" description="Version #4, cell magic with degradation tag, targeting peptides and cell cycle regulator." width=47%/><br />
<p>Figure. Version #4, cell magic with degradation tag, targeting peptides and cell cycle regulator. Animation.</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2013/5/58/P%2BD%2BT%2BS.png" /><br />
<p>Figure. Genetic circuits for version #4, in glucose medium, not synchronizing.</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2013/d/d9/P%2BD%2BT%2BS%2BI%2BC.png" /><br />
<p>Figure. Genetic circuits for version #4, in galactose medium, synchronizing.</p><br />
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<h3>Cyclin Promoters</h3><br />
<h4>Yeast Version</h4><br />
<p> As we all know, different proteins are planned to be expressed along the cell cycle. And their mRNAs are transcribed through the specific promoters. These promoters locate at their upstream sequences and can be recognized by the RNA polymerase, which can initiate such process. There are databases containing the upstream 600 amino acid sequence in the upstream of DNA, which can be transcript in the G1, S, G2 and M phase, respectively</p> <br />
<!----- [http://www.genome.jp/kegg-bin/show_pathway?org_name=sce&mapno=04111&mapscale=&show_description=hide]. ----><br />
<p>Previous study (Wolfsberg et al., 1999) demonstrated that cell cycle specific promoters posses their conserved five to six base pairs. Thus, we found the high-confidence 600 promoter-containing sequences in database that harbor the paper-mentioned 5/6 bp sequence. Consequently, we pick up the upstream 600bp sequence of cln2, cln3, clb2, clb5 and clb6.</p><br />
<p>The Clb2 gene is highly expressed in G2 phase, and the genes are very strongly induced by GAL-CLB2, whereas GAL-CLN3 appears somewhat repressive Spellman et al., 1998)</p><br />
<img src="https://static.igem.org/mediawiki/2013/3/31/Sce04111.png" title="Cell Cycle KEGG" description="KEGG diagram for core cell cycle." width=98% /><br />
<p>Figure. KEGG diagram for core cell cycle in yeast.</p><br />
<img src="https://static.igem.org/mediawiki/2013/f/fa/Selected.png" title="Selected Promoters" description="Selected promoters from multiple database and reviews." /><br />
<p>Figure. Selected promoters from multiple database and reviews.</p><br />
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<h4><i>E. coli</i> Version</h4><br />
<p> Cell cycle is a complex process and can be separated into G0, G1, S, G2, M phase. In each phase, distinct transcription factors help the phase-specific gene express through recognizing their promoters. Thus, these promoters are phase-specific too and can be fused with other genes in order to express such gene in a defined cell cycle phase.The well-regulated cell cycle in E.coli consists of three key events: DNA replication, nucleon segregation and the initiation of cell division. The replication initiator is DnaA protein; the second step relates to MukB protein and the cell division activator is the FtsZ protein. In our project, we take full aadvantage of the two initiators DnaA and FtsZ - using their promoter to control the transcript of specific genes binding behind them in the first step or last step of <i>E. coli</i> cell cycle.</p><br />
<h5>DnaA protein</h5><br />
<p>As mutation strain demonstrated, dnaA gene is absolutely required for initiation at oriC. When the concentration of DnaA increases, there is increased initiation?, indicating that DnaA protein is the switch for initiation (Atlung, Løbner-Olesen, & Hansen, 1987). Biochemical studies have shown that, with the help of accessory proteins, DnaA protein binds to five sites within oriC, therefore, leading to strand opening of a region containing three 13-mer repeats. This opening results in the begin of bidirectional replication (Atlung, Løbner-Olesen, & Hansen, 1987).</p><br />
<img src="https://static.igem.org/mediawiki/2013/3/3b/DnaAp.png" title="dnaA Promoter" description="dnaA promoter selected for S1 phase." /><br />
<p>Figure. dnaA promoter selected for S1 phase.</p><br />
<img src="https://static.igem.org/mediawiki/2013/2/2a/GyrBp.png" title="gyrB Promoter" description="gyrB promoter selected for S2 phase." /><br />
<p>Figure. gyrB promoter selected for S2 phase.</p><br />
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<h5>FtsZ</h5><br />
<p>The cell itself seems to regard FtsZ gene expression as the commitment to division, because of the fact that endogenous division inhibitors have FtsZ as target(Bi & Lutkenhaus, 1990). Increasing FtsZ concentration resulted in excess cell division, with the cell size decreasing (Ward & Lutkenhaus, 1985), vice verse. Thus the critical role of FtsZ in division is similar to that of DnaA in replication initiation. FtsZ protein exists randomly throughout cytoplasm during cell elongation, while polymerizes into a membrane-associated ring at the precise site of cell division (Bi & Lutkenhaus, 1991). Just before the appearance of a visible constriction. The ring decreasing along with the septation and FtsZ is dispersed again at completion. Such ring formation is presumed to be essential for cell division, since it cannot be observed in the presence of the cell division inhibitors (Bi & Lutkenhaus, 1993) but is always present when and where septa are formed.</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/6a/FtsQp.png" title="ftsQ Promoter" description="ftsQ promoter selected for S2 phase." /><br />
<p>Figure. ftsQ promoter selected for S2 phase.</p><br />
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<h3>Reporter Modification</h3><br />
<p> The XFP, as reporter in our project has been modified through</p> <br />
<p>1) removed the stop codon in them, 2) modified with 23# prefix and suffix, and then 3) added the terminator within stop codon to them. </p><br />
<p> Furthermore, all BioBricks were added with 23# prefix and suffix, which ensures it to be standard.<br />
</div><br />
<br />
<div id="board4" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery4"><br />
<li><img src="http://upload.wikimedia.org/wikipedia/commons/thumb/5/5b/Ubiquitylation.svg/800px-Ubiquitylation.svg.png" title="Ubiquitin" description="Function of fusion ubiquitin."></li><br />
<li><img src="http://mol-biol4masters.masters.grkraj.org/html/Cell_Cycle_And_Its_Regulation_files/image022.jpg" title="D-box" description="Function of destruction box." /></li><br />
<br />
</ul></div><br />
<div id="box4" class="box"><br />
<h3>Degradation Tags</h3><br />
<h4>Yeast Version</h4><br />
<p>In yeast cell, protein degraded through several mechanisms, a major one is the ubquitin- pathway. In detail the signal in substrates can be recognized by the enzyme E3, which transport the ubiqutin from E2 to such protein. And through such way, the ubiquited protein would finally be degraded by the proteasome. </p><br />
<br />
<h5>PEST</h5><br />
<p>There are two types of cyclins in the budding yeast. One kind contains PEST sequence at C-terminal: Cln1, Cln2, Cln3, Pcl1, Pcl2. Another kind is Clb1~6 which posses 9 conserved amino acid sequence in N-terminal named D-box (destruction box). The D-box are necessary for later kind cyclins degradation in M phase and can function in same way when combined with other proteins. In our study, we utilized the D-box, PEST sequence and ubiquitin-mediated pathway for fluorescent proteins degradation.</p><br />
<br />
<h5>Poly-ubiquitin</h5><br />
<br />
<p>Ubiquitin, a highly conserved 76-residue protein, was involved in the ubiquitin–proteasome pathway of protein proteolysis, which is a fundamental way for protein turnover, signal transduction and cell cycle control. For example, the degradation of CDC34 through such way is necessary for S phase start; another instance is that APC\C(anaphase-promoting complex\cyclosome) is degraded through ubiquitination, which is the foundation of M phase beginning. And both activated by Cdc28-cyclins</p><br />
<p>The mechanism of ubiquitin-mediated protein degradation can be separated into three continuous enzyme catalyze steps. First 1) a ubiquitin is activated by the E1 (ubiquitin-activating enzyme) through a thioester linkage,and then 2)added into the a small ubiquitin-carrier E2. Finally,3) through the E3 ubiquitin protein ligase, this ubiquitin complex was conjugate to the ε-amino group of lysine residues in substrate proteins, forming a glycyllysine isopeptide bond (Hershko, 1997).In some conditions, even E3 enzymes themselves carry ubiquitin as a thioester (Huibregtse et al., 1995).</p><br />
<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/5/5b/Ubiquitylation.svg/800px-Ubiquitylation.svg.png" title="Ubiquitin" description="Function of fusion ubiquitin."><br />
<p>Figure. Function of fusion ubiquitin.</p><br />
<br />
<p>In our project, we design a poly-ubiquitin(5 ubiquitin combined) BioBricks which can directly degrade the proteins fused with it.</p><br />
<br />
<h5>D-box</h5><br />
<p>The best studied substrates of ubiquitin- and APC/C-mediated proteolysis are the mitotic cyclins (Murray, Solomon, & Kirschner, 1989) Mitosis-exit inducer, a cyclin B, are highly dependent on its 90 residues for ubiquitin-mediated proteolysis. Analysis of its N-terminal region demonstrated the sequence essential for cyclin proteolysis, called ‘destruction box’(D-box). Meanwhile, when cyclin destruction started, substrates containing D-boxes were rapidly poly-ubiquitinated. The consensus motif in B-type cyclins is RXALGXIXN. For much of the cell cycle, the D-box may not be recognized with high affinity, and when it is recognized, the ‘bait’ construct becomes highly unstable(Yamano, Tsurumi, Gannon, & Hunt, 1998). </p><br />
<img src="http://mol-biol4masters.masters.grkraj.org/html/Cell_Cycle_And_Its_Regulation_files/image022.jpg" title="D-box" description="Function of destruction box." /><br />
<p>Figure. Function of destruction box.</p><br />
<br />
<p>When the D-box exists in the protein N-terminal, this protein is recognized more easily by the E1 and then captured into the ubiquitin-mediated degradation pathway. Thus, we combined it to the fluorescent proteins’ N-terminal to fasten their degradation and avoid veiling the following fluorescence.</p><br />
<h4><i>E. coli</i> Version</h4><br />
<p>In E.coli, the adaptor SspB tethers ssrAtagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. Which means, a variation of the WT SsrA tag sequence (K1051206, K1051207 and K1051208) will accelerate the degradation of proteins when fused to their C-terminal. Thus the degradation rates are dependent on concentration of proteases and binding mediators. In order to fuse degradation tags freely on the C-terminal of protein, we add TAATAA to the tail of tags.</p><br />
<br />
</div><br />
<br />
<div id="board5" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery5"><br />
<li><img src="https://static.igem.org/mediawiki/2013/1/1f/Figure1.protein-import_pathways_for_mitochondrial_proteins.png" title="Import pathway" description="Mitochrondira import pathway." /></li><br />
<br />
<li><img src="https://static.igem.org/mediawiki/igem.org/7/73/Figure2._Schematical_model_of_the_early_steps_of_PTS2-driven_import.png" title=”Import pathway“ description=”Peroxisome import pathway.“/></li><br />
</ul></div><br />
<div id="box5" class="box"><br />
<h3>Targeting Peptides</h3><br />
<p>A target peptide is a short (3-70 amino acids long) peptide chain that directs the transport of a protein to a specific region in cell, including nucleus, mitochondria, endoplasmic reticulums (ERs), chloroplasts, apoplasts, peroxisomes and plasma membrane. Targeting peptide can exists in both N-terminal, C-terminal and internal sequence of a precursor protein. And after transported, some target peptides are cleaved by signal peptidases.</p><br />
<p>In our project we utilized 19 peptides target to 9 sub-locations in yeast cells, and when combined with fluorescent proteins, such region can be marked by different colors.</p><br />
<br />
<h5>Mitochondria</h5><br />
<p>Though it accounts a small ratio in the cell space, mitochondria possess about 10% to 15% proteins encoded by nuclear genes in eukaryotic organisms. These proteins are synthesized in cytosol and then recognized by the membrane receptors of mitochondria. Translocases in the outer and inner membrane of mitochondria mediate the import and intra-mitochondrial sorting of these proteins. ATP is used as an energy source; Chaperones and auxiliary factors assist in folding and assembly of mitochondrial proteins into their native, three-dimensional structures. </p><br />
<br />
<img src="https://static.igem.org/mediawiki/2013/1/1f/Figure1.protein-import_pathways_for_mitochondrial_proteins.png" title="Import pathway" description="Mitochrondira import pathway." /><br />
<p>Figure. Mitochrondria import pathway.</p><br />
<br />
<p>As shown in the figure above, beta-barrel outer-membrane proteins (dark green), precursor proteins (brown) with positively charged amino-terminal presequences and multispanning inner-membrane proteins (blue) with internal targeting signals are recognized by specific receptors of the outer mitochondrial membrane (TOM) translocases Tom20, Tom22 and/or Tom70. The precursor proteins are then translocated through a small Tom proteins of the TOM complex, Tom40 pore, which the TOM complex contains two or three.</p><br />
<br />
<h5>Peroxisomes </h5><br />
<p>The import of post-translational matrix protein into peroxisomes depends on either of the two peroxisomal targeting signals (PTS), PTS1 and PTS2. PTS2-driven import is facilitated by a complex in the membrane. Under oleic acid-inducing growth conditions, there is a ternary core complex of approximate 150 kDa in the cytosol, which consists of Fox3p,Pex7p and Pex18p. Fox3p is imported as a dimer, while other two are bind in monomeric forms.</p><br />
<br />
<img src="https://static.igem.org/mediawiki/igem.org/7/73/Figure2._Schematical_model_of_the_early_steps_of_PTS2-driven_import.png" title=”Import pathway“ description=”Peroxisome import pathway.“/><br />
<p>Figure. Peroxisome import pathway.</p><br />
<br />
<br />
<p>As study mentioned there are four steps involved in PTS2-driven import. The first step is the recognition of dimerized Fox3p by Pex7p through its PTS2 in the cytosol. In a second step, the Pex7p–Fox3p complex interacts with Pex18p, which targets the PTS2 pre-import complex to the peroxisomal membrane. The third step is the docking process, involving the interaction between Pex7p and the integral membrane protein Pex13p. As a final step of these early steps in the PTS2 import cascade, PTS2 receptor dissociation takes place during or after its assembly into large oligomeric complexes containing Pex14p and Pex13p. Pex18p remains at the peroxisomal membrane in the form of a large-molecular-weight complex in conjunction with Pex14p and/or Pex13p, from where it might be released to the cytosol(Grunau et al., 2009).</p><br />
<br />
<h5>Actin </h5><br />
<p>In yeast, the cortical actin cytoskeleton seems to specify sites of growth of the cell surface. Because the actin-binding protein ABP1p is associated with the cortical cytoskeleton of Saccharomyces cerevisiae, it might be involved in the spatial organization of cell surface growth. ABP1p is localized to the cortical cytoskeleton and its overproduction causes assembly of the cortical actin cytoskeleton at inappropriate sites on the cell surface, resulting in delocalized surface growth. ABP1p is a novel protein with a 50 amino-acid C-terminal domain, which is very similar to the SH3 domain in the non-catalytic region of nonreceptor tyrosine kinases (including those encoded by the proto-oncogenes c-src and c-abl), in phospholipase C gamma and in alpha-spectrin. They also identified an SH3-related motif in the actin-binding tail domain of myosin-I. The identification of SH3 domains in a family of otherwise unrelated proteins that associate with the membrane cytoskeleton indicates that this domain might serve to bring together signal transduction proteins and their targets or regulators, or both, in the membrane cytoskeleton (Drubin, Mulholland, Zhu, & Botstein, 1990; Khorasanizadeh, 2004).</p><br />
<br />
<h5>Nucler</h5><br />
<p>Histones are nuclear proteins package DNA into nucleosomes, and they are responsible for maintaining the shape and structure of a nucleosome. One chromatin molecule is composed of at least one of each core histones per 100 base pairs of DNA.[The Nucleosome: From Genomic Organization to Genomic Regulation.] There are five families of histones known to date, termed H1/H5, H2A, H2B, H3, and H4. H2A is considered a core histone, along with H2B, H3 and H4. Core formation first occurs through the interaction of two H2A molecules(Acid, 2004). Then, H2A forms a dimer with H2B; the core molecule is complete when H3-H4 also attaches to form a tetramer.</p><br />
<br />
<h5>Vacuolar Membrane</h5><br />
<p>The ZRC1 gene encodes a multicopy suppressor of zinc toxicity in Saccharomyces cerevisiae; however, previously it was reported that the expression of ZRC1 was induced when the intracellular zinc level was decreased. The COT1 and ZRC1 genes of Saccharomyces cerevisiae are structurally related dosage-dependent suppressors of metal toxicity. COT1 confers increased tolerance to high levels of cobalt; ZRC1 confers increased tolerance(Conklin, Culbertson, & Kung, 1994).Zrc1 has six putative trans-membrane domains, and Zrc1-GFP fusion protein was localized to the vacuolar membrane. Zrc1 function as a mechanism to maintain the zinc homeostasis in yeast(Miyabe, Izawa, & Inoue, 2001).</p><br />
</div><br />
<br />
<div id="board6" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery6"><br />
<li><img src="http://upload.wikimedia.org/wikipedia/commons/0/06/Sic1_David_Morgan10-5.jpg" title="Principle of Sic1" description="Over expression of Sic1 can stop all yeast cells to G1 phase."></li><br />
<li><img src="https://static.igem.org/mediawiki/parts/8/8b/Sic1-mutate.png" title="Phosphorylation Sites" description="N-terminal phosphorylation sites to be mutated."></li><br />
</ul></div><br />
<div id="box6" class="box"><br />
<h3>Cell Synchronization</h3><br />
<p>As we known, the yeast cell cycle contains a huge and complex regulatory network in the transcription level, translation level as well as protein level. In our project we utilize Sic1 as a regulator to help elongate G1 phase in yeast cell.</p><br />
<p>The activation of B-type cyclin (Clb5/6)+Cdc28 kinases is a necessary step for initiation of DNA replication in vivo. One of its inhibitor Sic1 can be phosphorylated by the activated Cln+Cdc28, thereby targeted for degradation. Over expression of the gene encoding p40, SIC1, produces cells with an elongated bud morphology(figure1)(Nugroho & Mendenhall, 1994). SIC1 deletion is viable and causes increasing frequencies of chromosomes broken and lost. The deletion strain segregates out many dead cells, which are primarily arrested at the G2 checkpoint in an asymmetric fashion. Therefore, it has an important role in ensuring genomic integrity, and that this role has a pronounced mother-daughter asymmetry.</p><br />
<img src="http://upload.wikimedia.org/wikipedia/commons/0/06/Sic1_David_Morgan10-5.jpg" title="Principle of Sic1" description="Over expression of Sic1 can stop all yeast cells to G1 phase."><br />
<p>Figure. Over expression of Sic1 can stop all yeast cells to G1 phase.</p><br />
<br />
<p>After phosphorylation, phospho-Sic1 is specifically recognized by the F-box protein Cdc4, which leads Sic1 being ubiquitinated by the Cdc34±SCF complex (E3). The recognizing and binding by Cdc4 is based on the Sic1’s 9 Cdc4 phospo-degrons (CPDs, figure. a). Several phosphorylation sites contributed to Sic1 instability, with an order of Thr45, Ser76, Thr5,Thr33 and other less significant sites. The immunocipients after selectively phosphorylation as figure e showed suggested that at least six sites phosphorylation is necessary for the Cdc4 recognizing and binding with Sic1. Furthermore, the culture of GAL1-SIC1 constructs strain showed less than 6 sites are phosphorylated is not sufficient for SIC1 degradation in vitro(Nash et al., 2001).</p><br />
<img src="https://static.igem.org/mediawiki/parts/8/8b/Sic1-mutate.png" title="Phosphorylation Sites" description="N-terminal phosphorylation sites to be mutated."><br />
<p>Figure. N-terminal phosphorylation sites to be mutated.</p><br />
<br />
</div><br />
<br />
<div id="board7" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery7"><br />
<li><img src="https://static.igem.org/mediawiki/parts/9/96/SICI_principle.jpg" title="Src1 intron principle" description="Alternative splicing of Src1" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/7/74/Figure4._The_structure_of_sgRNA.png" title="sgRNA" description="sgRNA structure" /></li><br />
<li><img src="https://static.igem.org/mediawiki/parts/1/13/CRISPRi_principle.png" title="Principle of dCAS9" description="Principle of the function of dCAS9 and guide RNA." /></li><br />
</ul></div><br />
<div id="box7" class="box"><br />
<h3>Alternative Splicing Device</h3><br />
<p>In yeast cells, alternative splicing is a common process in before gene transcription. The splicing sites are located in 5'UTR of introns and can by recognized and spliced by some splicesome. Alternative splicing can produce two isoforms from one gene, thus can be used in our project to monitor whether the Sic1 system work efficiently. Here we find a intron with alternative splicing sites in yeast cells named SRCI intron.</p><br />
<br />
<h4>Src1 Intron and Hub1</h4><br />
<p> SRC1 intron has two 5' splicing site whose efficiency is regulated by protein hub1. Originally, the existence of the intron would produce two mature mRNA in proportion. After engineering, the existence of protein hub1 regulates to the preservation of intron precisely, which would make the following mRNA be expressed normally or not. As for the silencing of wild type gene HUB1, we choose CRISPRi that is comparably easy to use reversibly.</p><br />
<p>Alternative splicing substantially increases the gene product diversity and is a major source of cell type differentiation. A good example is the alternative splicing of Saccharomyces cerevisiae SRC1 pre-mRNA, which is promoted by the conserved ubiquitin-like protein Hub1. It can function through binding non-covalently to a conserved element termed HIND in the spliceosomal protein Snu66. Such binding makes the splicesome target sites change and moderately alters spliceosomal interactions. </p><br />
<p>Hub1 is a ubiquitin-like modifier (UBL) that covalent modify the proteins. Interest enough, it harbors several different to other UBLs in which it possesses a C-terminal double tyrosine motif while others having a GG motif. The Snu66, a tri-snRNP in yeast spliceosome, possesses with two N-terminal HINDs (Hub1-INteraction Domain). The Hub1–HIND interaction comprises a strong salt bridge accompanied by several hydrophobic contacts and high affinity. Such binding modifies the spliceosome rather than modulating the properties of an individual binding partner. Hub1-controlled splicing occurs universally in eukaryotes. SR proteins and hnRNPs involved in spliceosome targeting do not seem to exist in S. Cerevisiae , and thus the Hub1-dependent mechanism may be evolutionarily older. </p> <br />
<p>Scr1 is a protein in yeast having alternative splicing sites in its intron. The characteristic differential Hub1 dependence of SRC1 alternative splicing requires the tandem arrangement of overlapping 5’ splice sites. The Hub1 binding spliceosome can splice the intron from both downstream 5’ sites as well as the upstream 5’ sites with preference to the former one.</p><br />
<br />
<img src="https://static.igem.org/mediawiki/parts/9/96/SICI_principle.jpg" title="Src1 intron principle" description="Alternative splicing of Src1" /><br />
<p>Figure. Principle for alternative splicing of Src1.</p><br />
<br />
<p>Showed in Figure.above When cutting in the upstream splice sites, the exons flanking around it would be translated as a fusion protein Scr-S. However, when cutting at the down stream one, the left 4bp in intron would result in a frame shift, thus only the forward exon can express named Scr-L.</p><br />
<br />
<h4>dCas9 CRISPR interface system</h4><br />
<p>CRISPR shorts for Clustered Regularly Interspaced Palindromaic Repeats system, which can be targeted to DNA using RNA, enabling genetic editing of any region of the genome in many organisms.(Cho, Kim, Kim, & Kim, 2013). In the type II CRISPR/Cas system, a ribonucleoprotein complex formed from a single protein (Cas9), a crRNA, and a trans-acting CRISPR RNA (tracrRNA) can carry out efficient crRNA-directed recognition and site-specific cleavage of foreign DNA(Deltcheva et al., 2011). After mutated the endonuclease domains of the Cas9 protein, it creates a programmable RNA-dependent DNA-binding protein. The sgRNA consists of three domains: a 20 nt complementary region for specific DNA binding, a 42 nt hairpin for Cas9 binding (Cas9 handle), and a 40 nt transcription terminator derived from S. Pyogenes. After translation, the Cas9 binds to sgRNA to form a protein-RNA complex, which can recognize target sites in the genome sequence and bind to it. Then, it could block RNA polymerase and transciption elongation.</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2013/7/74/Figure4._The_structure_of_sgRNA.png" title="sgRNA" description="sgRNA structure" /><br />
<p>Figure. sgRNA structure.</p><br />
<br />
<img src="https://static.igem.org/mediawiki/parts/1/13/CRISPRi_principle.png" title="Principle of dCAS9" description="Principle of the function of dCAS9 and guide RNA." /><br />
<p>Figure. Principle of the function of dCAS9 and guide RNA.</p><br />
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</div><br />
<br />
<div id="board8" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery8"><br />
<li><img src="https://static.igem.org/mediawiki/igem.org/e/ec/Figure6._Masks_for_PDMS_microfluidic_device_fabrication..png" title="Microfluidic" description="The CAD design of microfluidics"></li><br />
<br />
</ul></div><br />
<div id="box8" class="box"><br />
<h3>Microfluidic Device</h3><br />
<p>Based physically on the chip, microfluidics can automatically control the biology process. In our project, the microfluidics plays a role as the movie theater, for cell cycle control. We designed a channel which can capture the yeast cell and then alternatively inject medium of different concentrations. Therefore, the yeast cell cycle can be synchronized. Furthermore, due to its transparent attribution, the chip can project the fluorescent lights from the yeast cell sub-locations. Consequently, the magic film can be shown to us. </p><br />
<p>Microfluidic devices possess many advantages such as large-scale integration, fast analyses, and considerably reduced reagent consumption (Comparative study and improvement of current cell micro-patterning techniques). Nowadays many designs are based on cell loading and culturing in a microfluidic channel with particular trapping structures such as micro-well arrays, and micro-cup arrays and micro-chambers. In our project, we design a cell loading technique based on gas absorption of degassed polydimethylsiloxane (PDMS). Because the balance concentration of gas dissolved in PDMS is proportional to the partial pressure of the gas around it, so that one can degas a piece of PDMS by placing it in vacuum. </p><br />
<p>Our cell loading device is a one level PDMS with an array of triangle cavities connected to one or both sides of the main fluidic channel. After degassed, yeast cells in the buffer solutions can be introduced into the device’s triangle cavities by gas absorption therein. Through changing the cell density in the buffer, we can control the number of the yeast cells loaded into each triangle cavity. Statistically, the ratio of cavities with a single cell can reach 40%. Consequently it is convenient to monitor the cell number and cell phenotypic variations because the yeast cells can grow in the micro-triangle-cavities in one layer.</p><br />
<img src="https://static.igem.org/mediawiki/igem.org/e/ec/Figure6._Masks_for_PDMS_microfluidic_device_fabrication..png" title="Microfluidic" description="the structure of Microfluidics"><br />
<p>Figure. Masks for PDMS microfluidic device fabrication.a : Three micro-triangle-cavities at one side of the main channel. b : One single channel for cell culture (100 microcavities). c : Seven parallel channels for different cell culture environments(Luo et al., 2008).</p><br />
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</div><br />
</div></div>BGI K2http://2013.igem.org/Team:Shenzhen_BGIC_ATCG/storiesTeam:Shenzhen BGIC ATCG/stories2013-09-27T23:35:00Z<p>BGI K2: </p>
<hr />
<div><!----------------------<br />
<br />
<br />
Title: Stories<br />
Team: Shenzhen_BGIC_ATCG<br />
Author: BGI_K2<br />
Twitter: @BGI_K2<br />
Email: im.ss.kk@gmail.com<br />
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<div class="navleftholder"><br />
<ul class="navleft"><br />
<li><a id="navleftsub1" class="navleftsub" href="#board1">The Magic</a></li><br />
<li><a id="navleftsub2" class="navleftsub" href="#board2">Cyclin Promoters</a></li><br />
<li><a id="navleftsub3" class="navleftsub" href="#board3">Reporter Modification</a></li><br />
<li><a id="navleftsub4" class="navleftsub" href="#board4">Degradation Tags</a></li><br />
<li><a id="navleftsub5" class="navleftsub" href="#board5">Targeting Peptides</a></li><br />
<li><a id="navleftsub6" class="navleftsub" href="#board6">Cell Synchronization</a></li><br />
<li><a id="navleftsub7" class="navleftsub" href="#board7">Alternative Splicing Device</a></li><br />
<li><a id="navleftsub8" class="navleftsub" href="#board8">Microfluidic Device</a></li><br />
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<li><img src="https://static.igem.org/mediawiki/2013/5/58/P%2BD%2BT%2BS.png" title="Genetic circuit of cell cycle version #4" description="To make the magic more fantasy, alternative splicing device acting synchronously with the synchronization device." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/1/10/Chip.gif" title="Microfuidics Device" description="Cell magic in microfuidics device." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/e4/Yeast.gif" title="DT X TP X CCR X" description="Version #1, cell magic without degradation tags, targeting peptides and cell cycle regulator."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/7/79/Yeast_D.gif" title="DT √ TP X CCR X" description="Version #2, cell magic with degradation tags, without targeting peptides and cell cycle regulator." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/e6/Yeast_DT.gif" title="DT √ TP √ CCR X" description="Version #3, cell magic with degradation tags and targeting peptides, without cell cycle regulator." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/c/c1/Yeast_DTR.gif" title="DT √ TP √ CCR √" description="Version #4, cell magic with degradation tag, targeting peptides and cell <li>cycle regulator." /></li><br />
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<h3>The Magic</h3><br />
<p>Life, the most brilliant magic in the universe, celebrated with the ability of reproduction and revolution. And the magic, is based on a sophisticated mechanism, called cell cycle. Most genetic reactions in a cell are regulated, directly or indirectly, by cell cycle. As we wish to build artificial lives, it is worthwhile to learn from the exquisite design of creature, to make use of cell cycle. As a pioneer study, we try to performing a "Cell Magic", by capturing cell cycle with fantastic reporters. By grasping the usage of cell cycle tools, we are promised to direct refined actions in a cell. For instance, when producing Paclitaxel (harmful to centrosome in S phase) with a cell factory, we may able to transport it out before it comes detrimental. </p><br />
<p>So this is our project, the "Cell Magic", to engineer two versions of cell cycle based magic in both budding yeast and <i>E. coli</i>. We will present the blueprint of our stories by the yeast version. </p><br />
<h4>Version #1, Degradation Tag X, Targeting Peptides X, Cell Cycle Regulator X</h4><br />
<p>Promoters of cyclins, which can initial transcription in specific phase in a cell cycle, are selected to produce different fluorescence proteins in G1/SG2/M phases respectably. However, in our first version of story, with low natural degradation rate, fluorescence proteins would remain in cell after their promoters stop working. So colors for each phase cannot be distinguished, as the figure shows.</p><br />
<img src="https://static.igem.org/mediawiki/2013/e/e4/Yeast.gif" title="DT X TP X CCR X" description="Version #1, cell magic without degradation tags, targeting peptides and cell cycle regulator." width=47%/><br />
<img src="https://static.igem.org/mediawiki/2013/3/36/P.png" width=50% ><br />
<p>Figure. Version #1, cell magic without degradation tags, targeting peptides and cell cycle regulator. Animation and genetic circuits.</p><br />
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<h4>Version #2, Degradation Tag √, Targeting Peptides X, Cell Cycle Regulator X</h4><br />
<p>In the second version, degradation tags are added to the reporters to solve this problem. All fluorescence proteins are thought to be degraded in 15 minutes which is far less than the period of a cell cycle.</p><br />
<img src="https://static.igem.org/mediawiki/2013/7/79/Yeast_D.gif" title="DT √ TP X CCR X" description="Version #2, cell magic with degradation tags, without targeting peptides and cell cycle regulator." width=47%/><br />
<img src="https://static.igem.org/mediawiki/2013/d/df/Gc.P%2BD.png" width=50%><br />
<p>Figure. Version #2, cell magic with degradation tags, without targeting peptides and cell cycle regulator. Animation and genetic circuits.</p><br />
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<h4>Version #3, Degradation Tag √, Targeting Peptides √, Cell Cycle Regulator X</h4><br />
<p>While we are not sure if degradation tags can work as expected, we try to turn the time magic into a time-space one. Reporters are located to different cell structures by targeting peptides. Different phase with different color shines in different organelle, that is the third version.</p><br />
<img src="https://static.igem.org/mediawiki/2013/e/e6/Yeast_DT.gif" title="DT √ TP √ CCR X" description="Version #3, cell magic with degradation tags and targeting peptides, without cell cycle regulator." width=47%/><br />
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<img src="https://static.igem.org/mediawiki/2013/8/8d/P%2BD%2BT.png" width=50%><br />
<p>Figure. Version #3, cell magic with degradation tags and targeting peptides, without cell cycle regulator. Animation and genetic circuits.</p><br />
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<h4>Version #4, Degradation Tag √, Targeting Peptides √, Cell Cycle Regulator √</h4><br />
<p>To capture the magic macroscopically, we developed a microfluidic device to synchronize all cells into the same phase (G1). So synchronization devices was added to the forth version. </p><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Chip.gif" title="Microfuidics Device" description="Cell magic in microfuidics device." width=35% /><br />
<p>Figure. Cell magic in microfuidics device.</p><br />
<br />
<p>To make the magic more fantasy, alternative splicing device acting synchronously with the synchronization device (a translational unit of a cyclin regulator), will splice the targeting peptide tail when cell are being synchronized to G1 phase, so all cells turn green; while synchronization device not working, targeting peptide remains in the reporter so only mitochondria turn green. In this way, the cell phase and statues can be reported by fluorescences.</p><br />
<img src="https://static.igem.org/mediawiki/2013/c/c1/Yeast_DTR.gif" title="DT √ TP √ CCR √" description="Version #4, cell magic with degradation tag, targeting peptides and cell cycle regulator." width=47%/><br />
<p>Figure. Version #4, cell magic with degradation tag, targeting peptides and cell cycle regulator. Animation.</p><br />
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<img src="https://static.igem.org/mediawiki/2013/5/58/P%2BD%2BT%2BS.png" /><br />
<p>Figure. Genetic circuits for version #4, in glucose medium, not synchronizing.</p><br />
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<img src="https://static.igem.org/mediawiki/2013/d/d9/P%2BD%2BT%2BS%2BI%2BC.png" /><br />
<p>Figure. Genetic circuits for version #4, in galactose medium, synchronizing.</p><br />
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<h3>Cyclin Promoters</h3><br />
<h4>Yeast Version</h4><br />
<p> As we all know, different proteins are planned to be expressed along the cell cycle. And their mRNAs are transcribed through the specific promoters. These promoters locate at their upstream sequences and can be recognized by the RNA polymerase, which can initiate such process. There are databases containing the upstream 600 amino acid sequence in the upstream of DNA, which can be transcript in the G1, S, G2 and M phase, respectively</p> <br />
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<p>Previous study (Wolfsberg et al., 1999) demonstrated that cell cycle specific promoters posses their conserved five to six base pairs. Thus, we found the high-confidence 600 promoter-containing sequences in database that harbor the paper-mentioned 5/6 bp sequence. Consequently, we pick up the upstream 600bp sequence of cln2, cln3, clb2, clb5 and clb6.</p><br />
<p>The Clb2 gene is highly expressed in G2 phase, and the genes are very strongly induced by GAL-CLB2, whereas GAL-CLN3 appears somewhat repressive Spellman et al., 1998)</p><br />
<img src="https://static.igem.org/mediawiki/2013/3/31/Sce04111.png" title="Cell Cycle KEGG" description="KEGG diagram for core cell cycle." width=98% /><br />
<p>Figure. KEGG diagram for core cell cycle in yeast.</p><br />
<img src="https://static.igem.org/mediawiki/2013/f/fa/Selected.png" title="Selected Promoters" description="Selected promoters from multiple database and reviews." /><br />
<p>Figure. Selected promoters from multiple database and reviews.</p><br />
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<h4><i>E. coli</i> Version</h4><br />
<p> Cell cycle is a complex process and can be separated into G0, G1, S, G2, M phase. In each phase, distinct transcription factors help the phase-specific gene express through recognizing their promoters. Thus, these promoters are phase-specific too and can be fused with other genes in order to express such gene in a defined cell cycle phase.The well-regulated cell cycle in E.coli consists of three key events: DNA replication, nucleon segregation and the initiation of cell division. The replication initiator is DnaA protein; the second step relates to MukB protein and the cell division activator is the FtsZ protein. In our project, we take full aadvantage of the two initiators DnaA and FtsZ - using their promoter to control the transcript of specific genes binding behind them in the first step or last step of <i>E. coli</i> cell cycle.</p><br />
<h5>DnaA protein</h5><br />
<p>As mutation strain demonstrated, dnaA gene is absolutely required for initiation at oriC. When the concentration of DnaA increases, there is increased initiation?, indicating that DnaA protein is the switch for initiation (Atlung, Løbner-Olesen, & Hansen, 1987). Biochemical studies have shown that, with the help of accessory proteins, DnaA protein binds to five sites within oriC, therefore, leading to strand opening of a region containing three 13-mer repeats. This opening results in the begin of bidirectional replication (Atlung, Løbner-Olesen, & Hansen, 1987).</p><br />
<img src="https://static.igem.org/mediawiki/2013/3/3b/DnaAp.png" title="dnaA Promoter" description="dnaA promoter selected for S1 phase." /><br />
<p>Figure. dnaA promoter selected for S1 phase.</p><br />
<img src="https://static.igem.org/mediawiki/2013/2/2a/GyrBp.png" title="gyrB Promoter" description="gyrB promoter selected for S2 phase." /><br />
<p>Figure. gyrB promoter selected for S2 phase.</p><br />
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<h5>FtsZ</h5><br />
<p>The cell itself seems to regard FtsZ gene expression as the commitment to division, because of the fact that endogenous division inhibitors have FtsZ as target(Bi & Lutkenhaus, 1990). Increasing FtsZ concentration resulted in excess cell division, with the cell size decreasing (Ward & Lutkenhaus, 1985), vice verse. Thus the critical role of FtsZ in division is similar to that of DnaA in replication initiation. FtsZ protein exists randomly throughout cytoplasm during cell elongation, while polymerizes into a membrane-associated ring at the precise site of cell division (Bi & Lutkenhaus, 1991). Just before the appearance of a visible constriction. The ring decreasing along with the septation and FtsZ is dispersed again at completion. Such ring formation is presumed to be essential for cell division, since it cannot be observed in the presence of the cell division inhibitors (Bi & Lutkenhaus, 1993) but is always present when and where septa are formed.</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/6a/FtsQp.png" title="ftsQ Promoter" description="ftsQ promoter selected for S2 phase." /><br />
<p>Figure. ftsQ promoter selected for S2 phase.</p><br />
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<h3>Reporter Modification</h3><br />
<p> The XFP, as reporter in our project has been modified through</p> <br />
<p>1) removed the stop codon in them, 2) modified with 23# prefix and suffix, and then 3) added the terminator within stop codon to them. </p><br />
<p> Furthermore, all BioBricks were added with 23# prefix and suffix, which ensures it to be standard.<br />
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<li><img src="http://upload.wikimedia.org/wikipedia/commons/thumb/5/5b/Ubiquitylation.svg/800px-Ubiquitylation.svg.png" title="Ubiquitin" description="Function of fusion ubiquitin."></li><br />
<li><img src="http://mol-biol4masters.masters.grkraj.org/html/Cell_Cycle_And_Its_Regulation_files/image022.jpg" title="D-box" description="Function of destruction box." /></li><br />
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<h3>Degradation Tags</h3><br />
<h4>Yeast Version</h4><br />
<p>In yeast cell, protein degraded through several mechanisms, a major one is the ubquitin- pathway. In detail the signal in substrates can be recognized by the enzyme E3, which transport the ubiqutin from E2 to such protein. And through such way, the ubiquited protein would finally be degraded by the proteasome. </p><br />
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<h5>PEST</h5><br />
<p>There are two types of cyclins in the budding yeast. One kind contains PEST sequence at C-terminal: Cln1, Cln2, Cln3, Pcl1, Pcl2. Another kind is Clb1~6 which posses 9 conserved amino acid sequence in N-terminal named D-box (destruction box). The D-box are necessary for later kind cyclins degradation in M phase and can function in same way when combined with other proteins. In our study, we utilized the D-box, PEST sequence and ubiquitin-mediated pathway for fluorescent proteins degradation.</p><br />
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<h5>Poly-ubiquitin</h5><br />
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<p>Ubiquitin, a highly conserved 76-residue protein, was involved in the ubiquitin–proteasome pathway of protein proteolysis, which is a fundamental way for protein turnover, signal transduction and cell cycle control. For example, the degradation of CDC34 through such way is necessary for S phase start; another instance is that APC\C(anaphase-promoting complex\cyclosome) is degraded through ubiquitination, which is the foundation of M phase beginning. And both activated by Cdc28-cyclins</p><br />
<p>The mechanism of ubiquitin-mediated protein degradation can be separated into three continuous enzyme catalyze steps. First 1) a ubiquitin is activated by the E1 (ubiquitin-activating enzyme) through a thioester linkage,and then 2)added into the a small ubiquitin-carrier E2. Finally,3) through the E3 ubiquitin protein ligase, this ubiquitin complex was conjugate to the ε-amino group of lysine residues in substrate proteins, forming a glycyllysine isopeptide bond (Hershko, 1997).In some conditions, even E3 enzymes themselves carry ubiquitin as a thioester (Huibregtse et al., 1995).</p><br />
<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/5/5b/Ubiquitylation.svg/800px-Ubiquitylation.svg.png" title="Ubiquitin" description="Function of fusion ubiquitin."><br />
<p>Figure. Function of fusion ubiquitin.</p><br />
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<p>In our project, we design a poly-ubiquitin(5 ubiquitin combined) BioBricks which can directly degrade the proteins fused with it.</p><br />
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<h5>D-box</h5><br />
<p>The best studied substrates of ubiquitin- and APC/C-mediated proteolysis are the mitotic cyclins (Murray, Solomon, & Kirschner, 1989) Mitosis-exit inducer, a cyclin B, are highly dependent on its 90 residues for ubiquitin-mediated proteolysis. Analysis of its N-terminal region demonstrated the sequence essential for cyclin proteolysis, called ‘destruction box’(D-box). Meanwhile, when cyclin destruction started, substrates containing D-boxes were rapidly poly-ubiquitinated. The consensus motif in B-type cyclins is RXALGXIXN. For much of the cell cycle, the D-box may not be recognized with high affinity, and when it is recognized, the ‘bait’ construct becomes highly unstable(Yamano, Tsurumi, Gannon, & Hunt, 1998). </p><br />
<img src="http://mol-biol4masters.masters.grkraj.org/html/Cell_Cycle_And_Its_Regulation_files/image022.jpg" title="D-box" description="Function of destruction box." /><br />
<p>Figure. Function of destruction box.</p><br />
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<p>When the D-box exists in the protein N-terminal, this protein is recognized more easily by the E1 and then captured into the ubiquitin-mediated degradation pathway. Thus, we combined it to the fluorescent proteins’ N-terminal to fasten their degradation and avoid veiling the following fluorescence.</p><br />
<h4><i>E. coli</i> Version</h4><br />
<p>In E.coli, the adaptor SspB tethers ssrAtagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. Which means, a variation of the WT SsrA tag sequence (K1051206, K1051207 and K1051208) will accelerate the degradation of proteins when fused to their C-terminal. Thus the degradation rates are dependent on concentration of proteases and binding mediators. In order to fuse degradation tags freely on the C-terminal of protein, we add TAATAA to the tail of tags.</p><br />
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<li><img src="https://static.igem.org/mediawiki/igem.org/7/73/Figure2._Schematical_model_of_the_early_steps_of_PTS2-driven_import.png" title=”Import pathway“ description=”Peroxisome import pathway.“/></li><br />
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<h3>Targeting Peptides</h3><br />
<p>A target peptide is a short (3-70 amino acids long) peptide chain that directs the transport of a protein to a specific region in cell, including nucleus, mitochondria, endoplasmic reticulums (ERs), chloroplasts, apoplasts, peroxisomes and plasma membrane. Targeting peptide can exists in both N-terminal, C-terminal and internal sequence of a precursor protein. And after transported, some target peptides are cleaved by signal peptidases.</p><br />
<p>In our project we utilized 19 peptides target to 9 sub-locations in yeast cells, and when combined with fluorescent proteins, such region can be marked by different colors.</p><br />
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<h5>Mitochondria</h5><br />
<p>Though it accounts a small ratio in the cell space, mitochondria possess about 10% to 15% proteins encoded by nuclear genes in eukaryotic organisms. These proteins are synthesized in cytosol and then recognized by the membrane receptors of mitochondria. Translocases in the outer and inner membrane of mitochondria mediate the import and intra-mitochondrial sorting of these proteins. ATP is used as an energy source; Chaperones and auxiliary factors assist in folding and assembly of mitochondrial proteins into their native, three-dimensional structures. </p><br />
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<img src="https://static.igem.org/mediawiki/2013/1/1f/Figure1.protein-import_pathways_for_mitochondrial_proteins.png" title="Import pathway" description="Mitochrondira import pathway." /><br />
<p>Figure. Mitochrondria import pathway.</p><br />
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<p>As shown in the figure above, beta-barrel outer-membrane proteins (dark green), precursor proteins (brown) with positively charged amino-terminal presequences and multispanning inner-membrane proteins (blue) with internal targeting signals are recognized by specific receptors of the outer mitochondrial membrane (TOM) translocases Tom20, Tom22 and/or Tom70. The precursor proteins are then translocated through a small Tom proteins of the TOM complex, Tom40 pore, which the TOM complex contains two or three.</p><br />
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<h5>Peroxisomes </h5><br />
<p>The import of post-translational matrix protein into peroxisomes depends on either of the two peroxisomal targeting signals (PTS), PTS1 and PTS2. PTS2-driven import is facilitated by a complex in the membrane. Under oleic acid-inducing growth conditions, there is a ternary core complex of approximate 150 kDa in the cytosol, which consists of Fox3p,Pex7p and Pex18p. Fox3p is imported as a dimer, while other two are bind in monomeric forms.</p><br />
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<img src="https://static.igem.org/mediawiki/igem.org/7/73/Figure2._Schematical_model_of_the_early_steps_of_PTS2-driven_import.png" title=”Import pathway“ description=”Peroxisome import pathway.“/><br />
<p>Figure. Peroxisome import pathway.</p><br />
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<p>As study mentioned there are four steps involved in PTS2-driven import. The first step is the recognition of dimerized Fox3p by Pex7p through its PTS2 in the cytosol. In a second step, the Pex7p–Fox3p complex interacts with Pex18p, which targets the PTS2 pre-import complex to the peroxisomal membrane. The third step is the docking process, involving the interaction between Pex7p and the integral membrane protein Pex13p. As a final step of these early steps in the PTS2 import cascade, PTS2 receptor dissociation takes place during or after its assembly into large oligomeric complexes containing Pex14p and Pex13p. Pex18p remains at the peroxisomal membrane in the form of a large-molecular-weight complex in conjunction with Pex14p and/or Pex13p, from where it might be released to the cytosol(Grunau et al., 2009).</p><br />
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<h5>Actin </h5><br />
<p>In yeast, the cortical actin cytoskeleton seems to specify sites of growth of the cell surface. Because the actin-binding protein ABP1p is associated with the cortical cytoskeleton of Saccharomyces cerevisiae, it might be involved in the spatial organization of cell surface growth. ABP1p is localized to the cortical cytoskeleton and its overproduction causes assembly of the cortical actin cytoskeleton at inappropriate sites on the cell surface, resulting in delocalized surface growth. ABP1p is a novel protein with a 50 amino-acid C-terminal domain, which is very similar to the SH3 domain in the non-catalytic region of nonreceptor tyrosine kinases (including those encoded by the proto-oncogenes c-src and c-abl), in phospholipase C gamma and in alpha-spectrin. They also identified an SH3-related motif in the actin-binding tail domain of myosin-I. The identification of SH3 domains in a family of otherwise unrelated proteins that associate with the membrane cytoskeleton indicates that this domain might serve to bring together signal transduction proteins and their targets or regulators, or both, in the membrane cytoskeleton (Drubin, Mulholland, Zhu, & Botstein, 1990; Khorasanizadeh, 2004).</p><br />
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<h5>Nucler</h5><br />
<p>Histones are nuclear proteins package DNA into nucleosomes, and they are responsible for maintaining the shape and structure of a nucleosome. One chromatin molecule is composed of at least one of each core histones per 100 base pairs of DNA.[The Nucleosome: From Genomic Organization to Genomic Regulation.] There are five families of histones known to date, termed H1/H5, H2A, H2B, H3, and H4. H2A is considered a core histone, along with H2B, H3 and H4. Core formation first occurs through the interaction of two H2A molecules(Acid, 2004). Then, H2A forms a dimer with H2B; the core molecule is complete when H3-H4 also attaches to form a tetramer.</p><br />
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<h5>Vacuolar Membrane</h5><br />
<p>The ZRC1 gene encodes a multicopy suppressor of zinc toxicity in Saccharomyces cerevisiae; however, previously it was reported that the expression of ZRC1 was induced when the intracellular zinc level was decreased. The COT1 and ZRC1 genes of Saccharomyces cerevisiae are structurally related dosage-dependent suppressors of metal toxicity. COT1 confers increased tolerance to high levels of cobalt; ZRC1 confers increased tolerance(Conklin, Culbertson, & Kung, 1994).Zrc1 has six putative trans-membrane domains, and Zrc1-GFP fusion protein was localized to the vacuolar membrane. Zrc1 function as a mechanism to maintain the zinc homeostasis in yeast(Miyabe, Izawa, & Inoue, 2001).</p><br />
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<li><img src="http://upload.wikimedia.org/wikipedia/commons/0/06/Sic1_David_Morgan10-5.jpg" title="Principle of Sic1" description="Over expression of Sic1 can stop all yeast cells to G1 phase."></li><br />
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<h3>Cell Synchronization</h3><br />
<p>As we known, the yeast cell cycle contains a huge and complex regulatory network in the transcription level, translation level as well as protein level. In our project we utilize Sic1 as a regulator to help elongate G1 phase in yeast cell.</p><br />
<p>The activation of B-type cyclin (Clb5/6)+Cdc28 kinases is a necessary step for initiation of DNA replication in vivo. One of its inhibitor Sic1 can be phosphorylated by the activated Cln+Cdc28, thereby targeted for degradation. Over expression of the gene encoding p40, SIC1, produces cells with an elongated bud morphology(figure1)(Nugroho & Mendenhall, 1994). SIC1 deletion is viable and causes increasing frequencies of chromosomes broken and lost. The deletion strain segregates out many dead cells, which are primarily arrested at the G2 checkpoint in an asymmetric fashion. Therefore, it has an important role in ensuring genomic integrity, and that this role has a pronounced mother-daughter asymmetry.</p><br />
<img src="http://upload.wikimedia.org/wikipedia/commons/0/06/Sic1_David_Morgan10-5.jpg" title="Principle of Sic1" description="Over expression of Sic1 can stop all yeast cells to G1 phase."><br />
<p>Figure. Over expression of Sic1 can stop all yeast cells to G1 phase.</p><br />
<br />
<p>After phosphorylation, phospho-Sic1 is specifically recognized by the F-box protein Cdc4, which leads Sic1 being ubiquitinated by the Cdc34±SCF complex (E3). The recognizing and binding by Cdc4 is based on the Sic1’s 9 Cdc4 phospo-degrons (CPDs, figure. a). Several phosphorylation sites contributed to Sic1 instability, with an order of Thr45, Ser76, Thr5,Thr33 and other less significant sites. The immunocipients after selectively phosphorylation as figure e showed suggested that at least six sites phosphorylation is necessary for the Cdc4 recognizing and binding with Sic1. Furthermore, the culture of GAL1-SIC1 constructs strain showed less than 6 sites are phosphorylated is not sufficient for SIC1 degradation in vitro(Nash et al., 2001).</p><br />
<img src="https://static.igem.org/mediawiki/parts/8/8b/Sic1-mutate.png" title="Phosphorylation Sites" description="N-terminal phosphorylation sites to be mutated."><br />
<p>Figure. N-terminal phosphorylation sites to be mutated.</p><br />
<br />
</div><br />
<br />
<div id="board7" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery7"><br />
<li><img src="https://static.igem.org/mediawiki/parts/9/96/SICI_principle.jpg" title="Src1 intron principle" description="Alternative splicing of Src1" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/7/74/Figure4._The_structure_of_sgRNA.png" title="sgRNA" description="sgRNA structure" /></li><br />
<li><img src="https://static.igem.org/mediawiki/parts/1/13/CRISPRi_principle.png" title="Principle of dCAS9" description="Principle of the function of dCAS9 and guide RNA." /></li><br />
</ul></div><br />
<div id="box7" class="box"><br />
<h3>Alternative Splicing Device</h3><br />
<p>In yeast cells, alternative splicing is a common process in before gene transcription. The splicing sites are located in 5'UTR of introns and can by recognized and spliced by some splicesome. Alternative splicing can produce two isoforms from one gene, thus can be used in our project to monitor whether the Sic1 system work efficiently. Here we find a intron with alternative splicing sites in yeast cells named SRCI intron.</p><br />
<br />
<h4>Src1 Intron and Hub1</h4><br />
<p> SRC1 intron has two 5' splicing site whose efficiency is regulated by protein hub1. Originally, the existence of the intron would produce two mature mRNA in proportion. After engineering, the existence of protein hub1 regulates to the preservation of intron precisely, which would make the following mRNA be expressed normally or not. As for the silencing of wild type gene HUB1, we choose CRISPRi that is comparably easy to use reversibly.</p><br />
<p>Alternative splicing substantially increases the gene product diversity and is a major source of cell type differentiation. A good example is the alternative splicing of Saccharomyces cerevisiae SRC1 pre-mRNA, which is promoted by the conserved ubiquitin-like protein Hub1. It can function through binding non-covalently to a conserved element termed HIND in the spliceosomal protein Snu66. Such binding makes the splicesome target sites change and moderately alters spliceosomal interactions. </p><br />
<p>Hub1 is a ubiquitin-like modifier (UBL) that covalent modify the proteins. Interest enough, it harbors several different to other UBLs in which it possesses a C-terminal double tyrosine motif while others having a GG motif. The Snu66, a tri-snRNP in yeast spliceosome, possesses with two N-terminal HINDs (Hub1-INteraction Domain). The Hub1–HIND interaction comprises a strong salt bridge accompanied by several hydrophobic contacts and high affinity. Such binding modifies the spliceosome rather than modulating the properties of an individual binding partner. Hub1-controlled splicing occurs universally in eukaryotes. SR proteins and hnRNPs involved in spliceosome targeting do not seem to exist in S. Cerevisiae , and thus the Hub1-dependent mechanism may be evolutionarily older. </p> <br />
<p>Scr1 is a protein in yeast having alternative splicing sites in its intron. The characteristic differential Hub1 dependence of SRC1 alternative splicing requires the tandem arrangement of overlapping 5’ splice sites. The Hub1 binding spliceosome can splice the intron from both downstream 5’ sites as well as the upstream 5’ sites with preference to the former one.</p><br />
<br />
<img src="https://static.igem.org/mediawiki/parts/9/96/SICI_principle.jpg" title="Src1 intron principle" description="Alternative splicing of Src1" /><br />
<p>Figure. Principle for alternative splicing of Src1.</p><br />
<br />
<p>Showed in Figure.above When cutting in the upstream splice sites, the exons flanking around it would be translated as a fusion protein Scr-S. However, when cutting at the down stream one, the left 4bp in intron would result in a frame shift, thus only the forward exon can express named Scr-L.</p><br />
<br />
<h4>dCas9 CRISPR interface system</h4><br />
<p>CRISPR shorts for Clustered Regularly Interspaced Palindromaic Repeats system, which can be targeted to DNA using RNA, enabling genetic editing of any region of the genome in many organisms.(Cho, Kim, Kim, & Kim, 2013). In the type II CRISPR/Cas system, a ribonucleoprotein complex formed from a single protein (Cas9), a crRNA, and a trans-acting CRISPR RNA (tracrRNA) can carry out efficient crRNA-directed recognition and site-specific cleavage of foreign DNA(Deltcheva et al., 2011). After mutated the endonuclease domains of the Cas9 protein, it creates a programmable RNA-dependent DNA-binding protein. The sgRNA consists of three domains: a 20 nt complementary region for specific DNA binding, a 42 nt hairpin for Cas9 binding (Cas9 handle), and a 40 nt transcription terminator derived from S. Pyogenes. After translation, the Cas9 binds to sgRNA to form a protein-RNA complex, which can recognize target sites in the genome sequence and bind to it. Then, it could block RNA polymerase and transciption elongation.</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2013/7/74/Figure4._The_structure_of_sgRNA.png" title="sgRNA" description="sgRNA structure" /><br />
<p>Figure. sgRNA structure.</p><br />
<br />
<img src="https://static.igem.org/mediawiki/parts/1/13/CRISPRi_principle.png" title="Principle of dCAS9" description="Principle of the function of dCAS9 and guide RNA." /><br />
<p>Figure. Principle of the function of dCAS9 and guide RNA.</p><br />
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</div><br />
<br />
<div id="board8" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery8"><br />
<li><img src="https://static.igem.org/mediawiki/2012/b/b5/Yao.result.jpg" /><br />
</ul></div><br />
<div id="box8" class="box"><br />
<h3>Microfluidic Device</h3><br />
<p>Based physically on the chip, microfluidics can automatically control the biology process. In our project, the microfluidics plays a role as the movie theater, for cell cycle control. We designed a channel which can capture the yeast cell and then alternatively inject medium of different concentrations. Therefore, the yeast cell cycle can be synchronized. Furthermore, due to its transparent attribution, the chip can project the fluorescent lights from the yeast cell sub-locations. Consequently, the magic film can be shown to us. </p><br />
<p>Microfluidic devices possess many advantages such as large-scale integration, fast analyses, and considerably reduced reagent consumption (Comparative study and improvement of current cell micro-patterning techniques). Nowadays many designs are based on cell loading and culturing in a microfluidic channel with particular trapping structures such as micro-well arrays, and micro-cup arrays and micro-chambers. In our project, we design a cell loading technique based on gas absorption of degassed polydimethylsiloxane (PDMS). Because the balance concentration of gas dissolved in PDMS is proportional to the partial pressure of the gas around it, so that one can degas a piece of PDMS by placing it in vacuum. </p><br />
<p>Our cell loading device is a one level PDMS with an array of triangle cavities connected to one or both sides of the main fluidic channel. After degassed, yeast cells in the buffer solutions can be introduced into the device’s triangle cavities by gas absorption therein. Through changing the cell density in the buffer, we can control the number of the yeast cells loaded into each triangle cavity. Statistically, the ratio of cavities with a single cell can reach 40%. Consequently it is convenient to monitor the cell number and cell phenotypic variations because the yeast cells can grow in the micro-triangle-cavities in one layer.</p><br />
<img src="https://static.igem.org/mediawiki/igem.org/e/ec/Figure6._Masks_for_PDMS_microfluidic_device_fabrication..png" title="Microfluidic" description="the structure of Microfluidics"><br />
<p>Figure. Masks for PDMS microfluidic device fabrication.a : Three micro-triangle-cavities at one side of the main channel. b : One single channel for cell culture (100 microcavities). c : Seven parallel channels for different cell culture environments(Luo et al., 2008).</p><br />
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</div><br />
</div></div>BGI K2http://2013.igem.org/File:Selected.pngFile:Selected.png2013-09-27T23:31:29Z<p>BGI K2: </p>
<hr />
<div></div>BGI K2http://2013.igem.org/Team:Shenzhen_BGIC_ATCG/resultsTeam:Shenzhen BGIC ATCG/results2013-09-27T23:03:39Z<p>BGI K2: </p>
<hr />
<div><!----------------------<br />
<br />
<br />
Title: Results<br />
Team: Shenzhen_BGIC_ATCG<br />
Author: BGI_K2<br />
Twitter: @BGI_K2<br />
Email: im.ss.kk@gmail.com<br />
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<div class="navleftholder"><br />
<ul class="navleft"><br />
<li><a id="navleftsub1" class="navleftsub" href="#board1">The Magic</a></li><br />
<li><a id="navleftsub2" class="navleftsub" href="#board2">Promoter Verification</a></li><br />
<li><a id="navleftsub3" class="navleftsub" href="#board3">Reporter Locating</a></li><br />
<li><a id="navleftsub4" class="navleftsub" href="#board4">Degradation Rate</a></li><br />
<li><a id="navleftsub5" class="navleftsub" href="#board5">Cell Synchronization</a></li><br />
<li><a id="navleftsub6" class="navleftsub" href="#board6">Alternative Splicing</a></li><br />
</ul></div><br />
<br />
<div id="board1" class="board"></div><br />
<div class="media"><br />
<br />
<ul id="myGallery1"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/></li><br />
<li><img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" title="Degradation Tags" description="Degradation tags testing device for E. coli."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" title="Microfluidics" description="Microfluidics device for degradation test."/></li><br />
<br />
</ul></div><br />
<div id="box1" class="box"><br />
<h3>The Magic</h3><br />
<p>Our project “Cell Magic", a complex work has been completed partly,due to the time limitation. The results we have gotten are listed below:</p><br />
<p>We verified some of the cyclin promoters. The clb6 promoter, which is supposed to be expressed in G1 phase, has been verified perfectly.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." width=50%/><br />
<p>Targeting peptides to three locations have been verified. Others are still being constructed and tested.</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. " width=50%/><br />
<p>We got good data for degradation tags for <i>E. coli</i>. Construction of degradation devices for yeast have been finished and being tested.</p><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" width=50%/><br />
<p>Microfluidics device has been successfully developed for measurement and cell synchronization.</p><br />
<img src="https://static.igem.org/mediawiki/2013/f/fc/Microfluidic.gif" /><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" width=50%/><br />
<p>Cell synchronization device and alternative splicing device are still in construction, related results will be updated later.</p><br />
<br />
</div><br />
<br />
<div id="board2" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery2"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/1/19/Flu.jpg" title="S Phase Promoter" description="cln3 promoter for S phase has been verified using flow cytometry." /></li><br />
</ul></div><br />
<div id="box2" class="box"><br />
<h3>Promoter Verification</h3><br />
<h4>Achievements</h4><br />
<p> Using the GFP as reporter and morphological alteration as cell cycle representation, we verified the Clb6 can be activated in G1 phase in the yeast.</P><br />
<p> As shown in the picture in normal lights, there are some yeast were budding. These budded yeast cells and small size budding yeasts (within the red circles) are assumed in the G1 phase of cell cycle. And the next picture was taken under activation light, thus the green lights can representing the Clb6 promoter expressing. The two pictures indicated the Clb6 were expressed in the G1 phase as we expected when designing the experiments </p><br />
<p> PS: we modified the contrast ratio to lower the lights of neighbor cells, thus our results looking better.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" /><br />
<p>We also captured fluorescence on cln3 promoter (S phase) test device, for the fluorescence is quite weak, we utilize micro plate reader and flow cytometry to verify the promoter.</p><br />
<img src="https://static.igem.org/mediawiki/2013/1/19/Flu.jpg" /><br />
<h4>Problems</h4><br />
<h5>Yeast Version</h5><br />
<p>While the promoters are natural ones without optimization, and our shuttle plasmid is pSR416 which is a single copied one, fluorescence is too weak to be captured by microfluidics device. That is a main reason for why we currently cannot show you a perfect cell magic movie. We are now constructing all devices with high copy number vectors, and try to optimize the junction near kozak sequence to make a better magic.</p><br />
<h5><i>E. coli</i> Version</h5><br />
<p>Promoters for <i>E. coli</i> seems not to be successful yet. In our first version, original 5' utr of promoter's downstream gene is included in BioBrick's sequences. The wild-type promoter and RBS may be too weak, or the junction of RFC[23] may have negative effect to the RBS. So in the present version, original 5' utr has been removed and strong RBS B0030 is used. We will update related results later.</p><br />
</div><br />
<br />
<div id="board3" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery3"><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/0/0d/Reporter.jpg" title="Modified Reporters" description="Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose."/></li><br />
</ul></div><br />
<div id="box3" class="box"><br />
<h3>Reporter Locating</h3><br />
<h4>Reporter Modification</h4><br />
<p>Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose.</p><br />
<img src="https://static.igem.org/mediawiki/2013/0/0d/Reporter.jpg" title="Modified Reporters" description="Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose."/><br />
<h4>Targeting Peptides</h4><br />
<p> The four pictures shows the reporters can rightly locate to Mitochondria, Nucleus and Vacuole,respectively. Picture A is the negative control, all yeast cells are lighted with GFP. And figure B is the reporter to the mitochondria, we can saw there are several light spots in one cells. Figure C is the reporter located in nucleus, the green spots are small and there is only one in a yeast cell.The last picture shows the reporter of Vacuolar membrane, the green lights were discrete in cells which was as expected.</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/><br />
</div><br />
<br />
<div id="board4" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery4"><br />
<li><img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" title="E. coli Version" description="Degradation tags testing device for E. coli"/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" title="IPTG Repressed" description="Microfluidics device for half life test with IPTG input (promoter of RFP repressed)."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/e9/Degladder.jpg" title="RFP Ladder" description="RFP ladder with different degradation tags)."/></li><br />
<br />
</ul></div><br />
<div id="box4" class="box"><br />
<h3>Degradation Rate</h3><br />
<p>In every curcuits measurement, we firstly test the growth curve through detecting the absorbance. Taken small amount of bateria, inoculate into 400ul LB medium, ensuring the initial concentration between 0.02-0.05 (OD600). Cultured in the room temperature, detect the OD600 value every 20 minutes. Y axis represents the logarithm of bacteria number, X axis represents the growth time. </p><br />
<img src="https://static.igem.org/mediawiki/2013/6/68/Growth-Curve.jpg" /><br />
<p>In E.coli, the adaptor SspB tethers ssrA tagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. Which means, a variation of the WT SsrA tag sequence (K1051206, K1051207 and K1051208) will accelerate the degradation of proteins when fused to their C-terminal. Thus the degradation rates are dependent on concentration of proteases and binding mediators.</p><br />
<p>We constructed the measurement pathway of each tag (K1051257, K1051258 and K1051259) to test the rates of degradation of tagged proteins respectively. J04450 was used as positive control because of the same promoter and fluorescent protein. </p><br />
<h4>Achievements</h4><br />
<img src="https://static.igem.org/mediawiki/2013/e/e9/Degladder.jpg" /><br />
<p>From right to left, the negative control,BBa_K1051257, BBa_K1051258, BBa_K1051259, J04450 as Positive Control. As the pictures showed, the lights of RFP within three degradation tags are decreasing. </p><br />
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<img src="https://static.igem.org/mediawiki/2013/c/ca/IMG_0817.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/M0050bright.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/0/06/M0050strong.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/8/89/M0050weak.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/6/63/Flo.jpg" /><br />
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<img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" /><br />
<p>The test results for K1051258 and positive control. A. K1051258 in bright field; B. K1051258 in exciting lights; C. Positive control in bright field; D. K1051258 in exciting lights.</p><br />
<p>In picture, there are only obvious lights in the picture B, indicated the degradation rates are working.</p><br />
<p>Enzyme - labelled meter detect the fluorescent protein intensity</p><br />
<p>First of all, take a certain amount of bacteria liquid, recovery to around OD0.6(600), ensuring the bacteria in the logarithmic growth phase. Diluted and then the bacteria transferred to 96 well plates, measured their fluorescence intensity. Red fluorescent protein using an excitation wavelength of 584nm, and its emission wavelength is 607nm.When measuring, we first detect the OD600 of each strain, removing the factor of bacteria number difference. Thus the fluorescence intensity cannot be altered by bacteria quantity. Then the measurement mode switching for measurement of fluorescence, fluorescence intensity.</p><br />
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<img src="https://static.igem.org/mediawiki/2013/8/83/0917_t00_white_60mm.jpg" /><br />
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<img src="https://static.igem.org/mediawiki/2013/4/4e/0917_t30_white_60mm.jpg" /><br />
--------------------><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg"/><br />
<p>The test results of BBa_K1051258 in chip. A,LB medium,O minuts; B, IPTG medium,9minutes; C, IPTG medium, 15 minutes</p><br />
<img src="https://static.igem.org/mediawiki/parts/6/6a/DT.png" title="Fluorescence Intensity" description="Fluorescence intensity of positive control and three tested degradation tags." /><br />
<p>Calculated by .ImageJ, the degradation efficiency of three degradation tags are obvious.</p><br />
<p>First of all, yeast will be measured after shaking to about OD2.0 (600)based on the chip which was washed by plasma water, vacuum pumping. The bacteria liquid was pushed into the chip, letting the cells enter the small triangle. Then use the constant flow pump culture medium into chip (the laboratory constant temperature, can not guarantee the training environment, 37 ° E. coli slower growth). The medium speed is about 200 ul/h. Finally we test the data after yeast filled in the triangles.</p><br />
<p>Using the IPTG medium, the new RFP expression was stopped and we can regard the lights as degradation tags' efficiency. Obviously, the lights are decreasing along with the time. Thus, it indicates that the degradation tags work effectively.</p><br />
<img src="https://static.igem.org/mediawiki/parts/8/80/Average-fluorescence-intensity-of-K1051258-measurement.jpg" title="Half Life" description="RFP with degradation tag's half life tested by microfluidics." /><br />
<p>Calculated by .ImageJ, half life of RFP in K1051258 is shown to be less than 9 minutes.</p><br />
<br />
<h4>Problems</h4><br />
<h5>Yeast Version</h5><br />
<p>The test for yeast degradation tags has not been fully successful. Fluorescence has been captured in some occasions but not stable. It may caused by the same problem with promoter test devices - plasmid copy number. </p><br />
<h5><i>E. coli</i> Version</h5><br />
<p>Data collected from microscope for different degradation tags did not fully meet performance expectation. Reasons may be multiple to increase systematic errors.</p><br />
<p>- Fluorescence is too weak for calculating</p><br />
<p>- Quenching of fluorescence, especially in RFP</p><br />
<p>- Sample size is small for ImageJ calculating</p><br />
<p>We will have more data detected by microfluidics and flow cytometry. Results will be updated later.</p><br />
</div><br />
<br />
<div id="board5" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery5"><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/e4/09172_bright.jpg" title="E. coli captured" description="E. coli cells captured by microfluidics device."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/3/3f/Sc_bright_20.jpg" title="E. coli captured" description="Yeast cells captured by microfluidics device."/></li><br />
</ul></div><br />
<div id="box5" class="box"><br />
<h3>Cell Synchronization</h3><br />
<h4>Microfluidics Device</h4><br />
<p>We made the chip as a platform for watching and synchronize the cells. First step is capture the cells by the chip. As showed in the two figures, both E.coli and budding yeast can be captured by the chip successfully.</p><br />
<img src="https://static.igem.org/mediawiki/2013/e/e4/09172_bright.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/3/3f/Sc_bright_20.jpg" /><br />
<h4>Sic1 Synchronization</h4><br />
<p>Seven phosphorylation sites are mutated by PCR to make Sic1 mutant. However, it has been taking us too long time that we have been still working on right clones. Experiments results will be updated later.</p><br />
</div><br />
<br />
<div id="board6" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery6"><br />
<li><img src="https://static.igem.org/mediawiki/2013/b/b9/Blank-01.png" /><li><br />
</ul></div><br />
<div id="box6" class="box"><br />
<h3>Alternative Splicing</h3><br />
<h4>Original Design</h4><br />
<p>In our project, we attempt to use intron as a switch. However, according to previous research, there are two splicing forms of SRC1 intron: one is complete splicing (5’S) while the other can leave 4bp at its 5’ end (5’L). The remaining 4-bp causes frame-shift and a stop codon in the region of adaptor, thus the following exon will not be translated. However, this kind of switch is not complete – the ratio of 5’L and 5’S varied between 40-60 and 85-15.</p><br />
<p>In order to make a complete switch, we abandoned the remaining 4bp of SRC1 intron to avoid producing 5’L mRNA. So the result comes out to be: when Hub1p are expressed, they bind to spliceosome to modify it, resulting to easy recognizing 5’S splice site and its splicing; when inhibit the expression of HUB1 by CRISPRi system, spliceosome can hardly recognize 5’S splice site, therefore no intron can be spliced. </p><br />
<h4>Further Plans</h4><br />
<p>However, things didn’t go as we expected. Our experiment showed that this 4bp plays important role in SRC1 intron splicing. So, to achieve our goal, we redesigned another three versions of SRC1 intron:<br />
</p><br />
<p>1. Intron with GG at its 5’ end</p><br />
<p>2. Intron with CGG at its 5’ end</p><br />
<p>3. Intron with its original 6bp at both end</p><br />
<p>Due to the lack of time, we are still constructing these parts to test devices.</p><br />
<br />
</div><br />
<br />
</div></div>BGI K2http://2013.igem.org/Team:Shenzhen_BGIC_ATCG/resultsTeam:Shenzhen BGIC ATCG/results2013-09-27T22:59:39Z<p>BGI K2: </p>
<hr />
<div><!----------------------<br />
<br />
<br />
Title: Results<br />
Team: Shenzhen_BGIC_ATCG<br />
Author: BGI_K2<br />
Twitter: @BGI_K2<br />
Email: im.ss.kk@gmail.com<br />
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<div class="navleftholder"><br />
<ul class="navleft"><br />
<li><a id="navleftsub1" class="navleftsub" href="#board1">The Magic</a></li><br />
<li><a id="navleftsub2" class="navleftsub" href="#board2">Promoter Verification</a></li><br />
<li><a id="navleftsub3" class="navleftsub" href="#board3">Reporter Locating</a></li><br />
<li><a id="navleftsub4" class="navleftsub" href="#board4">Degradation Rate</a></li><br />
<li><a id="navleftsub5" class="navleftsub" href="#board5">Cell Synchronization</a></li><br />
<li><a id="navleftsub6" class="navleftsub" href="#board6">Alternative Splicing</a></li><br />
</ul></div><br />
<br />
<div id="board1" class="board"></div><br />
<div class="media"><br />
<br />
<ul id="myGallery1"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/></li><br />
<li><img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" title="Degradation Tags" description="Degradation tags testing device for E. coli."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" title="Microfluidics" description="Microfluidics device for degradation test."/></li><br />
<br />
</ul></div><br />
<div id="box1" class="box"><br />
<h3>The Magic</h3><br />
<p>Our project “Cell Magic", a complex work has been completed partly,due to the time limitation. The results we have gotten are listed below:</p><br />
<p>We verified some of the cyclin promoters. The clb6 promoter, which is supposed to be expressed in G1 phase, has been verified perfectly.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." width=50%/><br />
<p>Targeting peptides to three locations have been verified. Others are still being constructed and tested.</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. " width=50%/><br />
<p>We got good data for degradation tags for <i>E. coli</i>. Construction of degradation devices for yeast have been finished and being tested.</p><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" width=50%/><br />
<p>Microfluidics device has been successfully developed for measurement and cell synchronization.</p><br />
<img src="https://static.igem.org/mediawiki/2013/f/fc/Microfluidic.gif" /><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" width=50%/><br />
<p>Cell synchronization device and alternative splicing device are still in construction, related results will be updated later.</p><br />
<br />
</div><br />
<br />
<div id="board2" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery2"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/1/19/Flu.jpg" title="S Phase Promoter" description="cln3 promoter for S phase has been verified using flow cytometry." /></li><br />
</ul></div><br />
<div id="box2" class="box"><br />
<h3>Promoter Verification</h3><br />
<h4>Achievements</h4><br />
<p> Using the GFP as reporter and morphological alteration as cell cycle representation, we verified the Clb6 can be activated in G1 phase in the yeast.</P><br />
<p> As shown in the picture in normal lights, there are some yeast were budding. These budded yeast cells and small size budding yeasts (within the red circles) are assumed in the G1 phase of cell cycle. And the next picture was taken under activation light, thus the green lights can representing the Clb6 promoter expressing. The two pictures indicated the Clb6 were expressed in the G1 phase as we expected when designing the experiments </p><br />
<p> PS: we modified the contrast ratio to lower the lights of neighbor cells, thus our results looking better.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" /><br />
<p>We also captured fluorescence on cln3 promoter (S phase) test device, for the fluorescence is quite weak, we utilize micro plate reader and flow cytometry to verify the promoter.</p><br />
<img src="https://static.igem.org/mediawiki/2013/1/19/Flu.jpg" /><br />
<h4>Problems</h4><br />
<h5>Yeast Version</h5><br />
<p>While the promoters are natural ones without optimization, and our shuttle plasmid is pSR416 which is a single copied one, fluorescence is too weak to be captured by microfluidics device. That is a main reason for why we currently cannot show you a perfect cell magic movie. We are now constructing all devices with high copy number vectors, and try to optimize the junction near kozak sequence to make a better magic.</p><br />
<h5><i>E. coli</i> Version</h5><br />
<p>Promoters for <i>E. coli</i> seems not to be successful yet. In our first version, original 5' utr of promoter's downstream gene is included in BioBrick's sequences. The wild-type promoter and RBS may be too weak, or the junction of RFC[23] may have negative effect to the RBS. So in the present version, original 5' utr has been removed and strong RBS B0030 is used. We will update related results later.</p><br />
</div><br />
<br />
<div id="board3" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery3"><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/0/0d/Reporter.jpg" title="Modified Reporters" description="Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose."/></li><br />
</ul></div><br />
<div id="box3" class="box"><br />
<h3>Reporter Locating</h3><br />
<h4>Reporter Modification</h4><br />
<p>Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose.</p><br />
<img src="https://static.igem.org/mediawiki/2013/0/0d/Reporter.jpg" title="Modified Reporters" description="Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose."/><br />
<h4>Targeting Peptides</h4><br />
<p> The four pictures shows the reporters can rightly locate to Mitochondria, Nucleus and Vacuole,respectively. Picture A is the negative control, all yeast cells are lighted with GFP. And figure B is the reporter to the mitochondria, we can saw there are several light spots in one cells. Figure C is the reporter located in nucleus, the green spots are small and there is only one in a yeast cell.The last picture shows the reporter of Vacuolar membrane, the green lights were discrete in cells which was as expected.</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/><br />
</div><br />
<br />
<div id="board4" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery4"><br />
<li><img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" title="E. coli Version" description="Degradation tags testing device for E. coli"/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" title="IPTG Repressed" description="Microfluidics device for half life test with IPTG input (promoter of RFP repressed)."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/e9/Degladder.jpg" title="RFP Ladder" description="RFP ladder with different degradation tags)."/></li><br />
<br />
</ul></div><br />
<div id="box4" class="box"><br />
<h3>Degradation Rate</h3><br />
<p>In every curcuits measurement, we firstly test the growth curve through detecting the absorbance. Taken small amount of bateria, inoculate into 400ul LB medium, ensuring the initial concentration between 0.02-0.05 (OD600). Cultured in the room temperature, detect the OD600 value every 20 minutes. Y axis represents the logarithm of bacteria number, X axis represents the growth time. </p><br />
<img src="https://static.igem.org/mediawiki/2013/6/68/Growth-Curve.jpg" /><br />
<p>In E.coli, the adaptor SspB tethers ssrA tagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. Which means, a variation of the WT SsrA tag sequence (K1051206, K1051207 and K1051208) will accelerate the degradation of proteins when fused to their C-terminal. Thus the degradation rates are dependent on concentration of proteases and binding mediators.</p><br />
<p>We constructed the measurement pathway of each tag (K1051257, K1051258 and K1051259) to test the rates of degradation of tagged proteins respectively. J04450 was used as positive control because of the same promoter and fluorescent protein. </p><br />
<h4>Achievements</h4><br />
<img src="https://static.igem.org/mediawiki/2013/e/e9/Degladder.jpg" /><br />
<p>From right to left, the negative control,BBa_K1051257, BBa_K1051258, BBa_K1051259, J04450 as Positive Control. As the pictures showed, the lights of RFP within three degradation tags are decreasing. </p><br />
<!----------------------<br />
<img src="https://static.igem.org/mediawiki/2013/a/a6/IMG_0804.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/c/ca/IMG_0817.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/M0050bright.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/0/06/M0050strong.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/8/89/M0050weak.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/6/63/Flo.jpg" /><br />
--------------------><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" /><br />
<p>The test results for K1051258 and positive control. A. K1051258 in bright field; B. K1051258 in exciting lights; C. Positive control in bright field; D. K1051258 in exciting lights.</p><br />
<p>In picture, there are only obvious lights in the picture B, indicated the degradation rates are working.</p><br />
<p>预留酶标仪结果图和柱状图,降解效率换算</p><br />
<p>Enzyme - labelled meter detect the fluorescent protein intensity</p><br />
<p>First of all, take a certain amount of bacteria liquid, recovery to around OD0.6(600), ensuring the bacteria in the logarithmic growth phase. Diluted and then the bacteria transferred to 96 well plates, measured their fluorescence intensity. Red fluorescent protein using an excitation wavelength of 584nm, and its emission wavelength is 607nm.When measuring, we first detect the OD600 of each strain, removing the factor of bacteria number difference. Thus the fluorescence intensity cannot be altered by bacteria quantity. Then the measurement mode switching for measurement of fluorescence, fluorescence intensity.</p><br />
<!----------------------<br />
<img src="https://static.igem.org/mediawiki/2013/8/83/0917_t00_white_60mm.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/7/72/0917_t15_white_60mm.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/4/4e/0917_t30_white_60mm.jpg" /><br />
--------------------><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg"/><br />
<p>The test results of BBa_K1051258 in chip. A,LB medium,O minuts; B, IPTG medium,9minutes; C, IPTG medium, 15 minutes</p><br />
<img src="https://static.igem.org/mediawiki/parts/6/6a/DT.png" title="Fluorescence Intensity" description="Fluorescence intensity of positive control and three tested degradation tags." /><br />
<p>Calculated by .ImageJ, the degradation efficiency of three degradation tags are obvious.</p><br />
<p>First of all, yeast will be measured after shaking to about OD2.0 (600)based on the chip which was washed by plasma water, vacuum pumping. The bacteria liquid was pushed into the chip, letting the cells enter the small triangle. Then use the constant flow pump culture medium into chip (the laboratory constant temperature, can not guarantee the training environment, 37 ° E. coli slower growth). The medium speed is about 200 ul/h. Finally we test the data after yeast filled in the triangles.</p><br />
<p>Using the IPTG medium, the new RFP expression was stopped and we can regard the lights as degradation tags' efficiency. Obviously, the lights are decreasing along with the time. Thus, it indicates that the degradation tags work effectively.</p><br />
<img scr="https://static.igem.org/mediawiki/parts/8/80/Average-fluorescence-intensity-of-K1051258-measurement.jpg" title="Half Life" description="RFP with degradation tag's half life tested by microfluidics." /><br />
<p>Calculated by .ImageJ, half life of RFP in K1051258 is shown to be less than 9 minutes.</p><br />
<br />
<h4>Problems</h4><br />
<h5>Yeast Version</h5><br />
<p>The test for yeast degradation tags has not been fully successful. Fluorescence has been captured in some occasions but not stable. It may caused by the same problem with promoter test devices - plasmid copy number. </p><br />
<h5><i>E. coli</i> Version</h5><br />
<p>Data collected from microscope for different degradation tags did not fully meet performance expectation. Reasons may be multiple to increase systematic errors.</p><br />
<p>- Fluorescence is too weak for calculating</p><br />
<p>- Quenching of fluorescence, especially in RFP</p><br />
<p>- Sample size is small for ImageJ calculating</p><br />
<p>We will have more data detected by microfluidics and flow cytometry. Results will be updated later.</p><br />
</div><br />
<br />
<div id="board5" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery5"><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/e4/09172_bright.jpg" title="E. coli captured" description="E. coli cells captured by microfluidics device."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/3/3f/Sc_bright_20.jpg" title="E. coli captured" description="Yeast cells captured by microfluidics device."/></li><br />
</ul></div><br />
<div id="box5" class="box"><br />
<h3>Cell Synchronization</h3><br />
<h4>Microfluidics Device</h4><br />
<p>We made the chip as a platform for watching and synchronize the cells. First step is capture the cells by the chip. As showed in the two figures, both E.coli and budding yeast can be captured by the chip successfully.</p><br />
<img src="https://static.igem.org/mediawiki/2013/e/e4/09172_bright.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/3/3f/Sc_bright_20.jpg" /><br />
<h4>Sic1 Synchronization</h4><br />
<p>Seven phosphorylation sites are mutated by PCR to make Sic1 mutant. However, it has been taking us too long time that we have been still working on right clones. Experiments results will be updated later.</p><br />
</div><br />
<br />
<div id="board6" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery6"><br />
<li><img src="https://static.igem.org/mediawiki/2013/b/b9/Blank-01.png" /><li><br />
</ul></div><br />
<div id="box6" class="box"><br />
<h3>Alternative Splicing</h3><br />
<h4>Original Design</h4><br />
<p>In our project, we attempt to use intron as a switch. However, according to previous research, there are two splicing forms of SRC1 intron: one is complete splicing (5’S) while the other can leave 4bp at its 5’ end (5’L). The remaining 4-bp causes frame-shift and a stop codon in the region of adaptor, thus the following exon will not be translated. However, this kind of switch is not complete – the ratio of 5’L and 5’S varied between 40-60 and 85-15.</p><br />
<p>In order to make a complete switch, we abandoned the remaining 4bp of SRC1 intron to avoid producing 5’L mRNA. So the result comes out to be: when Hub1p are expressed, they bind to spliceosome to modify it, resulting to easy recognizing 5’S splice site and its splicing; when inhibit the expression of HUB1 by CRISPRi system, spliceosome can hardly recognize 5’S splice site, therefore no intron can be spliced. </p><br />
<h4>Further Plans</h4><br />
<p>However, things didn’t go as we expected. Our experiment showed that this 4bp plays important role in SRC1 intron splicing. So, to achieve our goal, we redesigned another three versions of SRC1 intron:<br />
</p><br />
<p>1. Intron with GG at its 5’ end</p><br />
<p>2. Intron with CGG at its 5’ end</p><br />
<p>3. Intron with its original 6bp at both end</p><br />
<p>Due to the lack of time, we are still constructing these parts to test devices.</p><br />
<br />
</div><br />
<br />
</div></div>BGI K2http://2013.igem.org/Team:Shenzhen_BGIC_ATCG/storiesTeam:Shenzhen BGIC ATCG/stories2013-09-27T22:45:33Z<p>BGI K2: </p>
<hr />
<div><!----------------------<br />
<br />
<br />
Title: Stories<br />
Team: Shenzhen_BGIC_ATCG<br />
Author: BGI_K2<br />
Twitter: @BGI_K2<br />
Email: im.ss.kk@gmail.com<br />
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<div class="navleftholder"><br />
<ul class="navleft"><br />
<li><a id="navleftsub1" class="navleftsub" href="#board1">The Magic</a></li><br />
<li><a id="navleftsub2" class="navleftsub" href="#board2">Cyclin Promoters</a></li><br />
<li><a id="navleftsub3" class="navleftsub" href="#board3">Reporter Modification</a></li><br />
<li><a id="navleftsub4" class="navleftsub" href="#board4">Degradation Tags</a></li><br />
<li><a id="navleftsub5" class="navleftsub" href="#board5">Targeting Peptides</a></li><br />
<li><a id="navleftsub6" class="navleftsub" href="#board6">Cell Synchronization</a></li><br />
<li><a id="navleftsub7" class="navleftsub" href="#board7">Alternative Splicing Device</a></li><br />
<li><a id="navleftsub8" class="navleftsub" href="#board8">Microfluidic Device</a></li><br />
</ul></div><br />
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<div id="board1" class="board"></div><br />
<div class="media"><br />
<br />
<ul id="myGallery1"><br />
<li><img src="https://static.igem.org/mediawiki/2013/5/58/P%2BD%2BT%2BS.png" title="Genetic circuit of cell cycle version #4" description="To make the magic more fantasy, alternative splicing device acting synchronously with the synchronization device." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/1/10/Chip.gif" title="Microfuidics Device" description="Cell magic in microfuidics device." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/e4/Yeast.gif" title="DT X TP X CCR X" description="Version #1, cell magic without degradation tags, targeting peptides and cell cycle regulator."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/7/79/Yeast_D.gif" title="DT √ TP X CCR X" description="Version #2, cell magic with degradation tags, without targeting peptides and cell cycle regulator." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/e6/Yeast_DT.gif" title="DT √ TP √ CCR X" description="Version #3, cell magic with degradation tags and targeting peptides, without cell cycle regulator." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/c/c1/Yeast_DTR.gif" title="DT √ TP √ CCR √" description="Version #4, cell magic with degradation tag, targeting peptides and cell <li>cycle regulator." /></li><br />
</ul></div><br />
<div id="box1" class="box"><br />
<h3>The Magic</h3><br />
<p>Life, the most brilliant magic in the universe, celebrated with the ability of reproduction and revolution. And the magic, is based on a sophisticated mechanism, called cell cycle. Most genetic reactions in a cell are regulated, directly or indirectly, by cell cycle. As we wish to build artificial lives, it is worthwhile to learn from the exquisite design of creature, to make use of cell cycle. As a pioneer study, we try to performing a "Cell Magic", by capturing cell cycle with fantastic reporters. By grasping the usage of cell cycle tools, we are promised to direct refined actions in a cell. For instance, when producing Paclitaxel (harmful to centrosome in S phase) with a cell factory, we may able to transport it out before it comes detrimental. </p><br />
<p>So this is our project, the "Cell Magic", to engineer two versions of cell cycle based magic in both budding yeast and <i>E. coli</i>. We will present the blueprint of our stories by the yeast version. </p><br />
<h4>Version #1, Degradation Tag X, Targeting Peptides X, Cell Cycle Regulator X</h4><br />
<p>Promoters of cyclins, which can initial transcription in specific phase in a cell cycle, are selected to produce different fluorescence proteins in G1/SG2/M phases respectably. However, in our first version of story, with low natural degradation rate, fluorescence proteins would remain in cell after their promoters stop working. So colors for each phase cannot be distinguished, as the figure shows.</p><br />
<img src="https://static.igem.org/mediawiki/2013/e/e4/Yeast.gif" title="DT X TP X CCR X" description="Version #1, cell magic without degradation tags, targeting peptides and cell cycle regulator." width=47%/><br />
<img src="https://static.igem.org/mediawiki/2013/3/36/P.png" width=50% ><br />
<p>Figure. Version #1, cell magic without degradation tags, targeting peptides and cell cycle regulator. Animation and genetic circuits.</p><br />
<br />
<h4>Version #2, Degradation Tag √, Targeting Peptides X, Cell Cycle Regulator X</h4><br />
<p>In the second version, degradation tags are added to the reporters to solve this problem. All fluorescence proteins are thought to be degraded in 15 minutes which is far less than the period of a cell cycle.</p><br />
<img src="https://static.igem.org/mediawiki/2013/7/79/Yeast_D.gif" title="DT √ TP X CCR X" description="Version #2, cell magic with degradation tags, without targeting peptides and cell cycle regulator." width=47%/><br />
<img src="https://static.igem.org/mediawiki/2013/d/df/Gc.P%2BD.png" width=50%><br />
<p>Figure. Version #2, cell magic with degradation tags, without targeting peptides and cell cycle regulator. Animation and genetic circuits.</p><br />
<br />
<h4>Version #3, Degradation Tag √, Targeting Peptides √, Cell Cycle Regulator X</h4><br />
<p>While we are not sure if degradation tags can work as expected, we try to turn the time magic into a time-space one. Reporters are located to different cell structures by targeting peptides. Different phase with different color shines in different organelle, that is the third version.</p><br />
<img src="https://static.igem.org/mediawiki/2013/e/e6/Yeast_DT.gif" title="DT √ TP √ CCR X" description="Version #3, cell magic with degradation tags and targeting peptides, without cell cycle regulator." width=47%/><br />
<br />
<img src="https://static.igem.org/mediawiki/2013/8/8d/P%2BD%2BT.png" width=50%><br />
<p>Figure. Version #3, cell magic with degradation tags and targeting peptides, without cell cycle regulator. Animation and genetic circuits.</p><br />
<br />
<h4>Version #4, Degradation Tag √, Targeting Peptides √, Cell Cycle Regulator √</h4><br />
<p>To capture the magic macroscopically, we developed a microfluidic device to synchronize all cells into the same phase (G1). So synchronization devices was added to the forth version. </p><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Chip.gif" title="Microfuidics Device" description="Cell magic in microfuidics device." width=35% /><br />
<p>Figure. Cell magic in microfuidics device.</p><br />
<br />
<p>To make the magic more fantasy, alternative splicing device acting synchronously with the synchronization device (a translational unit of a cyclin regulator), will splice the targeting peptide tail when cell are being synchronized to G1 phase, so all cells turn green; while synchronization device not working, targeting peptide remains in the reporter so only mitochondria turn green. In this way, the cell phase and statues can be reported by fluorescences.</p><br />
<img src="https://static.igem.org/mediawiki/2013/c/c1/Yeast_DTR.gif" title="DT √ TP √ CCR √" description="Version #4, cell magic with degradation tag, targeting peptides and cell cycle regulator." width=47%/><br />
<p>Figure. Version #4, cell magic with degradation tag, targeting peptides and cell cycle regulator. Animation.</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2013/5/58/P%2BD%2BT%2BS.png" /><br />
<p>Figure. Genetic circuits for version #4, in glucose medium, not synchronizing.</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2013/d/d9/P%2BD%2BT%2BS%2BI%2BC.png" /><br />
<p>Figure. Genetic circuits for version #4, in galactose medium, synchronizing.</p><br />
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<div id="box2" class="box"><br />
<h3>Cyclin Promoters</h3><br />
<h4>Yeast Version</h4><br />
<p> As we all know, different proteins are planned to be expressed along the cell cycle. And their mRNAs are transcribed through the specific promoters. These promoters locate at their upstream sequences and can be recognized by the RNA polymerase, which can initiate such process. There are databases containing the upstream 600 amino acid sequence in the upstream of DNA, which can be transcript in the G1, S, G2 and M phase, respectively</p> <br />
<!----- [http://www.genome.jp/kegg-bin/show_pathway?org_name=sce&mapno=04111&mapscale=&show_description=hide]. ----><br />
<p>Previous study (Wolfsberg et al., 1999) demonstrated that cell cycle specific promoters posses their conserved five to six base pairs. Thus, we found the high-confidence 600 promoter-containing sequences in database that harbor the paper-mentioned 5/6 bp sequence. Consequently, we pick up the upstream 600bp sequence of cln2, cln3, clb2, clb5 and clb6.</p><br />
<p>The Clb2 gene is highly expressed in G2 phase, and the genes are very strongly induced by GAL-CLB2, whereas GAL-CLN3 appears somewhat repressive Spellman et al., 1998)</p><br />
<img src="https://static.igem.org/mediawiki/2013/3/31/Sce04111.png" title="Cell Cycle KEGG" description="KEGG diagram for core cell cycle." width=98% /><br />
<p>Figure. KEGG diagram for core cell cycle in yeast.</p><br />
<br />
<h4><i>E. coli</i> Version</h4><br />
<p> Cell cycle is a complex process and can be separated into G0, G1, S, G2, M phase. In each phase, distinct transcription factors help the phase-specific gene express through recognizing their promoters. Thus, these promoters are phase-specific too and can be fused with other genes in order to express such gene in a defined cell cycle phase.The well-regulated cell cycle in E.coli consists of three key events: DNA replication, nucleon segregation and the initiation of cell division. The replication initiator is DnaA protein; the second step relates to MukB protein and the cell division activator is the FtsZ protein. In our project, we take full aadvantage of the two initiators DnaA and FtsZ - using their promoter to control the transcript of specific genes binding behind them in the first step or last step of <i>E. coli</i> cell cycle.</p><br />
<h5>DnaA protein</h5><br />
<p>As mutation strain demonstrated, dnaA gene is absolutely required for initiation at oriC. When the concentration of DnaA increases, there is increased initiation?, indicating that DnaA protein is the switch for initiation (Atlung, Løbner-Olesen, & Hansen, 1987). Biochemical studies have shown that, with the help of accessory proteins, DnaA protein binds to five sites within oriC, therefore, leading to strand opening of a region containing three 13-mer repeats. This opening results in the begin of bidirectional replication (Atlung, Løbner-Olesen, & Hansen, 1987).</p><br />
<img src="https://static.igem.org/mediawiki/2013/3/3b/DnaAp.png" title="dnaA Promoter" description="dnaA promoter selected for S1 phase." /><br />
<p>Figure. dnaA promoter selected for S1 phase.</p><br />
<img src="https://static.igem.org/mediawiki/2013/2/2a/GyrBp.png" title="gyrB Promoter" description="gyrB promoter selected for S2 phase." /><br />
<p>Figure. gyrB promoter selected for S2 phase.</p><br />
<br />
<br />
<h5>FtsZ</h5><br />
<p>The cell itself seems to regard FtsZ gene expression as the commitment to division, because of the fact that endogenous division inhibitors have FtsZ as target(Bi & Lutkenhaus, 1990). Increasing FtsZ concentration resulted in excess cell division, with the cell size decreasing (Ward & Lutkenhaus, 1985), vice verse. Thus the critical role of FtsZ in division is similar to that of DnaA in replication initiation. FtsZ protein exists randomly throughout cytoplasm during cell elongation, while polymerizes into a membrane-associated ring at the precise site of cell division (Bi & Lutkenhaus, 1991). Just before the appearance of a visible constriction. The ring decreasing along with the septation and FtsZ is dispersed again at completion. Such ring formation is presumed to be essential for cell division, since it cannot be observed in the presence of the cell division inhibitors (Bi & Lutkenhaus, 1993) but is always present when and where septa are formed.</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/6a/FtsQp.png" title="ftsQ Promoter" description="ftsQ promoter selected for S2 phase." /><br />
<p>Figure. ftsQ promoter selected for S2 phase.</p><br />
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<div class="media"><br />
<ul id="myGallery3"><br />
<li><img src="https://static.igem.org/mediawiki/2013/b/b9/Blank-01.png" /><li><br />
</ul></div><br />
<div id="box3" class="box"><br />
<h3>Reporter Modification</h3><br />
<p> The XFP, as reporter in our project has been modified through</p> <br />
<p>1) removed the stop codon in them, 2) modified with 23# prefix and suffix, and then 3) added the terminator within stop codon to them. </p><br />
<p> Furthermore, all BioBricks were added with 23# prefix and suffix, which ensures it to be standard.<br />
</div><br />
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<div id="board4" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery4"><br />
<li><img src="http://upload.wikimedia.org/wikipedia/commons/thumb/5/5b/Ubiquitylation.svg/800px-Ubiquitylation.svg.png" title="Ubiquitin" description="Function of fusion ubiquitin."></li><br />
<li><img src="http://mol-biol4masters.masters.grkraj.org/html/Cell_Cycle_And_Its_Regulation_files/image022.jpg" title="D-box" description="Function of destruction box." /></li><br />
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</ul></div><br />
<div id="box4" class="box"><br />
<h3>Degradation Tags</h3><br />
<h4>Yeast Version</h4><br />
<p>In yeast cell, protein degraded through several mechanisms, a major one is the ubquitin- pathway. In detail the signal in substrates can be recognized by the enzyme E3, which transport the ubiqutin from E2 to such protein. And through such way, the ubiquited protein would finally be degraded by the proteasome. </p><br />
<br />
<h5>PEST</h5><br />
<p>There are two types of cyclins in the budding yeast. One kind contains PEST sequence at C-terminal: Cln1, Cln2, Cln3, Pcl1, Pcl2. Another kind is Clb1~6 which posses 9 conserved amino acid sequence in N-terminal named D-box (destruction box). The D-box are necessary for later kind cyclins degradation in M phase and can function in same way when combined with other proteins. In our study, we utilized the D-box, PEST sequence and ubiquitin-mediated pathway for fluorescent proteins degradation.</p><br />
<br />
<h5>Poly-ubiquitin</h5><br />
<br />
<p>Ubiquitin, a highly conserved 76-residue protein, was involved in the ubiquitin–proteasome pathway of protein proteolysis, which is a fundamental way for protein turnover, signal transduction and cell cycle control. For example, the degradation of CDC34 through such way is necessary for S phase start; another instance is that APC\C(anaphase-promoting complex\cyclosome) is degraded through ubiquitination, which is the foundation of M phase beginning. And both activated by Cdc28-cyclins</p><br />
<p>The mechanism of ubiquitin-mediated protein degradation can be separated into three continuous enzyme catalyze steps. First 1) a ubiquitin is activated by the E1 (ubiquitin-activating enzyme) through a thioester linkage,and then 2)added into the a small ubiquitin-carrier E2. Finally,3) through the E3 ubiquitin protein ligase, this ubiquitin complex was conjugate to the ε-amino group of lysine residues in substrate proteins, forming a glycyllysine isopeptide bond (Hershko, 1997).In some conditions, even E3 enzymes themselves carry ubiquitin as a thioester (Huibregtse et al., 1995).</p><br />
<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/5/5b/Ubiquitylation.svg/800px-Ubiquitylation.svg.png" title="Ubiquitin" description="Function of fusion ubiquitin."><br />
<p>Figure. Function of fusion ubiquitin.</p><br />
<br />
<p>In our project, we design a poly-ubiquitin(5 ubiquitin combined) BioBricks which can directly degrade the proteins fused with it.</p><br />
<br />
<h5>D-box</h5><br />
<p>The best studied substrates of ubiquitin- and APC/C-mediated proteolysis are the mitotic cyclins (Murray, Solomon, & Kirschner, 1989) Mitosis-exit inducer, a cyclin B, are highly dependent on its 90 residues for ubiquitin-mediated proteolysis. Analysis of its N-terminal region demonstrated the sequence essential for cyclin proteolysis, called ‘destruction box’(D-box). Meanwhile, when cyclin destruction started, substrates containing D-boxes were rapidly poly-ubiquitinated. The consensus motif in B-type cyclins is RXALGXIXN. For much of the cell cycle, the D-box may not be recognized with high affinity, and when it is recognized, the ‘bait’ construct becomes highly unstable(Yamano, Tsurumi, Gannon, & Hunt, 1998). </p><br />
<img src="http://mol-biol4masters.masters.grkraj.org/html/Cell_Cycle_And_Its_Regulation_files/image022.jpg" title="D-box" description="Function of destruction box." /><br />
<p>Figure. Function of destruction box.</p><br />
<br />
<p>When the D-box exists in the protein N-terminal, this protein is recognized more easily by the E1 and then captured into the ubiquitin-mediated degradation pathway. Thus, we combined it to the fluorescent proteins’ N-terminal to fasten their degradation and avoid veiling the following fluorescence.</p><br />
<h4><i>E. coli</i> Version</h4><br />
<p>In E.coli, the adaptor SspB tethers ssrAtagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. Which means, a variation of the WT SsrA tag sequence (K1051206, K1051207 and K1051208) will accelerate the degradation of proteins when fused to their C-terminal. Thus the degradation rates are dependent on concentration of proteases and binding mediators. In order to fuse degradation tags freely on the C-terminal of protein, we add TAATAA to the tail of tags.</p><br />
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<li><img src="https://static.igem.org/mediawiki/2013/1/1f/Figure1.protein-import_pathways_for_mitochondrial_proteins.png" title="Import pathway" description="Mitochrondira import pathway." /></li><br />
<br />
<li><img src="https://static.igem.org/mediawiki/igem.org/7/73/Figure2._Schematical_model_of_the_early_steps_of_PTS2-driven_import.png" title=”Import pathway“ description=”Peroxisome import pathway.“/></li><br />
</ul></div><br />
<div id="box5" class="box"><br />
<h3>Targeting Peptides</h3><br />
<p>A target peptide is a short (3-70 amino acids long) peptide chain that directs the transport of a protein to a specific region in cell, including nucleus, mitochondria, endoplasmic reticulums (ERs), chloroplasts, apoplasts, peroxisomes and plasma membrane. Targeting peptide can exists in both N-terminal, C-terminal and internal sequence of a precursor protein. And after transported, some target peptides are cleaved by signal peptidases.</p><br />
<p>In our project we utilized 19 peptides target to 9 sub-locations in yeast cells, and when combined with fluorescent proteins, such region can be marked by different colors.</p><br />
<br />
<h5>Mitochondria</h5><br />
<p>Though it accounts a small ratio in the cell space, mitochondria possess about 10% to 15% proteins encoded by nuclear genes in eukaryotic organisms. These proteins are synthesized in cytosol and then recognized by the membrane receptors of mitochondria. Translocases in the outer and inner membrane of mitochondria mediate the import and intra-mitochondrial sorting of these proteins. ATP is used as an energy source; Chaperones and auxiliary factors assist in folding and assembly of mitochondrial proteins into their native, three-dimensional structures. </p><br />
<br />
<img src="https://static.igem.org/mediawiki/2013/1/1f/Figure1.protein-import_pathways_for_mitochondrial_proteins.png" title="Import pathway" description="Mitochrondira import pathway." /><br />
<p>Figure. Mitochrondria import pathway.</p><br />
<br />
<p>As shown in the figure above, beta-barrel outer-membrane proteins (dark green), precursor proteins (brown) with positively charged amino-terminal presequences and multispanning inner-membrane proteins (blue) with internal targeting signals are recognized by specific receptors of the outer mitochondrial membrane (TOM) translocases Tom20, Tom22 and/or Tom70. The precursor proteins are then translocated through a small Tom proteins of the TOM complex, Tom40 pore, which the TOM complex contains two or three.</p><br />
<br />
<h5>Peroxisomes </h5><br />
<p>The import of post-translational matrix protein into peroxisomes depends on either of the two peroxisomal targeting signals (PTS), PTS1 and PTS2. PTS2-driven import is facilitated by a complex in the membrane. Under oleic acid-inducing growth conditions, there is a ternary core complex of approximate 150 kDa in the cytosol, which consists of Fox3p,Pex7p and Pex18p. Fox3p is imported as a dimer, while other two are bind in monomeric forms.</p><br />
<br />
<img src="https://static.igem.org/mediawiki/igem.org/7/73/Figure2._Schematical_model_of_the_early_steps_of_PTS2-driven_import.png" title=”Import pathway“ description=”Peroxisome import pathway.“/><br />
<p>Figure. Peroxisome import pathway.</p><br />
<br />
<br />
<p>As study mentioned there are four steps involved in PTS2-driven import. The first step is the recognition of dimerized Fox3p by Pex7p through its PTS2 in the cytosol. In a second step, the Pex7p–Fox3p complex interacts with Pex18p, which targets the PTS2 pre-import complex to the peroxisomal membrane. The third step is the docking process, involving the interaction between Pex7p and the integral membrane protein Pex13p. As a final step of these early steps in the PTS2 import cascade, PTS2 receptor dissociation takes place during or after its assembly into large oligomeric complexes containing Pex14p and Pex13p. Pex18p remains at the peroxisomal membrane in the form of a large-molecular-weight complex in conjunction with Pex14p and/or Pex13p, from where it might be released to the cytosol(Grunau et al., 2009).</p><br />
<br />
<h5>Actin </h5><br />
<p>In yeast, the cortical actin cytoskeleton seems to specify sites of growth of the cell surface. Because the actin-binding protein ABP1p is associated with the cortical cytoskeleton of Saccharomyces cerevisiae, it might be involved in the spatial organization of cell surface growth. ABP1p is localized to the cortical cytoskeleton and its overproduction causes assembly of the cortical actin cytoskeleton at inappropriate sites on the cell surface, resulting in delocalized surface growth. ABP1p is a novel protein with a 50 amino-acid C-terminal domain, which is very similar to the SH3 domain in the non-catalytic region of nonreceptor tyrosine kinases (including those encoded by the proto-oncogenes c-src and c-abl), in phospholipase C gamma and in alpha-spectrin. They also identified an SH3-related motif in the actin-binding tail domain of myosin-I. The identification of SH3 domains in a family of otherwise unrelated proteins that associate with the membrane cytoskeleton indicates that this domain might serve to bring together signal transduction proteins and their targets or regulators, or both, in the membrane cytoskeleton (Drubin, Mulholland, Zhu, & Botstein, 1990; Khorasanizadeh, 2004).</p><br />
<br />
<h5>Nucler</h5><br />
<p>Histones are nuclear proteins package DNA into nucleosomes, and they are responsible for maintaining the shape and structure of a nucleosome. One chromatin molecule is composed of at least one of each core histones per 100 base pairs of DNA.[The Nucleosome: From Genomic Organization to Genomic Regulation.] There are five families of histones known to date, termed H1/H5, H2A, H2B, H3, and H4. H2A is considered a core histone, along with H2B, H3 and H4. Core formation first occurs through the interaction of two H2A molecules(Acid, 2004). Then, H2A forms a dimer with H2B; the core molecule is complete when H3-H4 also attaches to form a tetramer.</p><br />
<br />
<h5>Vacuolar Membrane</h5><br />
<p>The ZRC1 gene encodes a multicopy suppressor of zinc toxicity in Saccharomyces cerevisiae; however, previously it was reported that the expression of ZRC1 was induced when the intracellular zinc level was decreased. The COT1 and ZRC1 genes of Saccharomyces cerevisiae are structurally related dosage-dependent suppressors of metal toxicity. COT1 confers increased tolerance to high levels of cobalt; ZRC1 confers increased tolerance(Conklin, Culbertson, & Kung, 1994).Zrc1 has six putative trans-membrane domains, and Zrc1-GFP fusion protein was localized to the vacuolar membrane. Zrc1 function as a mechanism to maintain the zinc homeostasis in yeast(Miyabe, Izawa, & Inoue, 2001).</p><br />
</div><br />
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<div id="board6" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery6"><br />
<li><img src="http://upload.wikimedia.org/wikipedia/commons/0/06/Sic1_David_Morgan10-5.jpg" title="Principle of Sic1" description="Over expression of Sic1 can stop all yeast cells to G1 phase."></li><br />
<li><img src="https://static.igem.org/mediawiki/parts/8/8b/Sic1-mutate.png" title="Phosphorylation Sites" description="N-terminal phosphorylation sites to be mutated."></li><br />
</ul></div><br />
<div id="box6" class="box"><br />
<h3>Cell Synchronization</h3><br />
<p>As we known, the yeast cell cycle contains a huge and complex regulatory network in the transcription level, translation level as well as protein level. In our project we utilize Sic1 as a regulator to help elongate G1 phase in yeast cell.</p><br />
<p>The activation of B-type cyclin (Clb5/6)+Cdc28 kinases is a necessary step for initiation of DNA replication in vivo. One of its inhibitor Sic1 can be phosphorylated by the activated Cln+Cdc28, thereby targeted for degradation. Over expression of the gene encoding p40, SIC1, produces cells with an elongated bud morphology(figure1)(Nugroho & Mendenhall, 1994). SIC1 deletion is viable and causes increasing frequencies of chromosomes broken and lost. The deletion strain segregates out many dead cells, which are primarily arrested at the G2 checkpoint in an asymmetric fashion. Therefore, it has an important role in ensuring genomic integrity, and that this role has a pronounced mother-daughter asymmetry.</p><br />
<img src="http://upload.wikimedia.org/wikipedia/commons/0/06/Sic1_David_Morgan10-5.jpg" title="Principle of Sic1" description="Over expression of Sic1 can stop all yeast cells to G1 phase."><br />
<p>Figure. Over expression of Sic1 can stop all yeast cells to G1 phase.</p><br />
<br />
<p>After phosphorylation, phospho-Sic1 is specifically recognized by the F-box protein Cdc4, which leads Sic1 being ubiquitinated by the Cdc34±SCF complex (E3). The recognizing and binding by Cdc4 is based on the Sic1’s 9 Cdc4 phospo-degrons (CPDs, figure. a). Several phosphorylation sites contributed to Sic1 instability, with an order of Thr45, Ser76, Thr5,Thr33 and other less significant sites. The immunocipients after selectively phosphorylation as figure e showed suggested that at least six sites phosphorylation is necessary for the Cdc4 recognizing and binding with Sic1. Furthermore, the culture of GAL1-SIC1 constructs strain showed less than 6 sites are phosphorylated is not sufficient for SIC1 degradation in vitro(Nash et al., 2001).</p><br />
<img src="https://static.igem.org/mediawiki/parts/8/8b/Sic1-mutate.png" title="Phosphorylation Sites" description="N-terminal phosphorylation sites to be mutated."><br />
<p>Figure. N-terminal phosphorylation sites to be mutated.</p><br />
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</div><br />
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<div id="board7" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery7"><br />
<li><img src="https://static.igem.org/mediawiki/parts/9/96/SICI_principle.jpg" title="Src1 intron principle" description="Alternative splicing of Src1" /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/7/74/Figure4._The_structure_of_sgRNA.png" title="sgRNA" description="sgRNA structure" /></li><br />
<li><img src="https://static.igem.org/mediawiki/parts/1/13/CRISPRi_principle.png" title="Principle of dCAS9" description="Principle of the function of dCAS9 and guide RNA." /></li><br />
</ul></div><br />
<div id="box7" class="box"><br />
<h3>Alternative Splicing Device</h3><br />
<p>In yeast cells, alternative splicing is a common process in before gene transcription. The splicing sites are located in 5'UTR of introns and can by recognized and spliced by some splicesome. Alternative splicing can produce two isoforms from one gene, thus can be used in our project to monitor whether the Sic1 system work efficiently. Here we find a intron with alternative splicing sites in yeast cells named SRCI intron.</p><br />
<br />
<h4>Src1 Intron and Hub1</h4><br />
<p> SRC1 intron has two 5' splicing site whose efficiency is regulated by protein hub1. Originally, the existence of the intron would produce two mature mRNA in proportion. After engineering, the existence of protein hub1 regulates to the preservation of intron precisely, which would make the following mRNA be expressed normally or not. As for the silencing of wild type gene HUB1, we choose CRISPRi that is comparably easy to use reversibly.</p><br />
<p>Alternative splicing substantially increases the gene product diversity and is a major source of cell type differentiation. A good example is the alternative splicing of Saccharomyces cerevisiae SRC1 pre-mRNA, which is promoted by the conserved ubiquitin-like protein Hub1. It can function through binding non-covalently to a conserved element termed HIND in the spliceosomal protein Snu66. Such binding makes the splicesome target sites change and moderately alters spliceosomal interactions. </p><br />
<p>Hub1 is a ubiquitin-like modifier (UBL) that covalent modify the proteins. Interest enough, it harbors several different to other UBLs in which it possesses a C-terminal double tyrosine motif while others having a GG motif. The Snu66, a tri-snRNP in yeast spliceosome, possesses with two N-terminal HINDs (Hub1-INteraction Domain). The Hub1–HIND interaction comprises a strong salt bridge accompanied by several hydrophobic contacts and high affinity. Such binding modifies the spliceosome rather than modulating the properties of an individual binding partner. Hub1-controlled splicing occurs universally in eukaryotes. SR proteins and hnRNPs involved in spliceosome targeting do not seem to exist in S. Cerevisiae , and thus the Hub1-dependent mechanism may be evolutionarily older. </p> <br />
<p>Scr1 is a protein in yeast having alternative splicing sites in its intron. The characteristic differential Hub1 dependence of SRC1 alternative splicing requires the tandem arrangement of overlapping 5’ splice sites. The Hub1 binding spliceosome can splice the intron from both downstream 5’ sites as well as the upstream 5’ sites with preference to the former one.</p><br />
<br />
<img src="https://static.igem.org/mediawiki/parts/9/96/SICI_principle.jpg" title="Src1 intron principle" description="Alternative splicing of Src1" /><br />
<p>Figure. Principle for alternative splicing of Src1.</p><br />
<br />
<p>Showed in Figure.above When cutting in the upstream splice sites, the exons flanking around it would be translated as a fusion protein Scr-S. However, when cutting at the down stream one, the left 4bp in intron would result in a frame shift, thus only the forward exon can express named Scr-L.</p><br />
<br />
<h4>dCas9 CRISPR interface system</h4><br />
<p>CRISPR shorts for Clustered Regularly Interspaced Palindromaic Repeats system, which can be targeted to DNA using RNA, enabling genetic editing of any region of the genome in many organisms.(Cho, Kim, Kim, & Kim, 2013). In the type II CRISPR/Cas system, a ribonucleoprotein complex formed from a single protein (Cas9), a crRNA, and a trans-acting CRISPR RNA (tracrRNA) can carry out efficient crRNA-directed recognition and site-specific cleavage of foreign DNA(Deltcheva et al., 2011). After mutated the endonuclease domains of the Cas9 protein, it creates a programmable RNA-dependent DNA-binding protein. The sgRNA consists of three domains: a 20 nt complementary region for specific DNA binding, a 42 nt hairpin for Cas9 binding (Cas9 handle), and a 40 nt transcription terminator derived from S. Pyogenes. After translation, the Cas9 binds to sgRNA to form a protein-RNA complex, which can recognize target sites in the genome sequence and bind to it. Then, it could block RNA polymerase and transciption elongation.</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2013/7/74/Figure4._The_structure_of_sgRNA.png" title="sgRNA" description="sgRNA structure" /><br />
<p>Figure. sgRNA structure.</p><br />
<br />
<img src="https://static.igem.org/mediawiki/parts/1/13/CRISPRi_principle.png" title="Principle of dCAS9" description="Principle of the function of dCAS9 and guide RNA." /><br />
<p>Figure. Principle of the function of dCAS9 and guide RNA.</p><br />
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</div><br />
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<div id="board8" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery8"><br />
<li><img src="https://static.igem.org/mediawiki/2012/b/b5/Yao.result.jpg" /><br />
</ul></div><br />
<div id="box8" class="box"><br />
<h3>Microfluidic Device</h3><br />
<p>Based physically on the chip, microfluidics can automatically control the biology process. In our project, the microfluidics plays a role as the movie theater, for cell cycle control. We designed a channel which can capture the yeast cell and then alternatively inject medium of different concentrations. Therefore, the yeast cell cycle can be synchronized. Furthermore, due to its transparent attribution, the chip can project the fluorescent lights from the yeast cell sub-locations. Consequently, the magic film can be shown to us. </p><br />
<p>Microfluidic devices possess many advantages such as large-scale integration, fast analyses, and considerably reduced reagent consumption (Comparative study and improvement of current cell micro-patterning techniques). Nowadays many designs are based on cell loading and culturing in a microfluidic channel with particular trapping structures such as micro-well arrays, and micro-cup arrays and micro-chambers. In our project, we design a cell loading technique based on gas absorption of degassed polydimethylsiloxane (PDMS). Because the balance concentration of gas dissolved in PDMS is proportional to the partial pressure of the gas around it, so that one can degas a piece of PDMS by placing it in vacuum. </p><br />
<p>Our cell loading device is a one level PDMS with an array of triangle cavities connected to one or both sides of the main fluidic channel. After degassed, yeast cells in the buffer solutions can be introduced into the device’s triangle cavities by gas absorption therein. Through changing the cell density in the buffer, we can control the number of the yeast cells loaded into each triangle cavity. Statistically, the ratio of cavities with a single cell can reach 40%. Consequently it is convenient to monitor the cell number and cell phenotypic variations because the yeast cells can grow in the micro-triangle-cavities in one layer.</p><br />
<img src="https://static.igem.org/mediawiki/igem.org/e/ec/Figure6._Masks_for_PDMS_microfluidic_device_fabrication..png" title="Microfluidic" description="the structure of Microfluidics"><br />
<p>Figure. Masks for PDMS microfluidic device fabrication.a : Three micro-triangle-cavities at one side of the main channel. b : One single channel for cell culture (100 microcavities). c : Seven parallel channels for different cell culture environments(Luo et al., 2008).</p><br />
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</div><br />
</div></div>BGI K2http://2013.igem.org/Team:Shenzhen_BGIC_ATCG/storiesTeam:Shenzhen BGIC ATCG/stories2013-09-27T22:35:37Z<p>BGI K2: </p>
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Title: Stories<br />
Team: Shenzhen_BGIC_ATCG<br />
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<li><a id="navleftsub1" class="navleftsub" href="#board1">The Magic</a></li><br />
<li><a id="navleftsub2" class="navleftsub" href="#board2">Cyclin Promoters</a></li><br />
<li><a id="navleftsub3" class="navleftsub" href="#board3">Reporter Modification</a></li><br />
<li><a id="navleftsub4" class="navleftsub" href="#board4">Degradation Tags</a></li><br />
<li><a id="navleftsub5" class="navleftsub" href="#board5">Targeting Peptides</a></li><br />
<li><a id="navleftsub6" class="navleftsub" href="#board6">Cell Synchronization</a></li><br />
<li><a id="navleftsub7" class="navleftsub" href="#board7">Alternative Splicing Device</a></li><br />
<li><a id="navleftsub8" class="navleftsub" href="#board8">Microfluidic Device</a></li><br />
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<li><img src="https://static.igem.org/mediawiki/2013/5/58/P%2BD%2BT%2BS.png" title="Genetic circuit of cell cycle version #4" description="To make the magic more fantasy, alternative splicing device acting synchronously with the synchronization device." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/1/10/Chip.gif" title="Microfuidics Device" description="Cell magic in microfuidics device." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/e4/Yeast.gif" title="DT X TP X CCR X" description="Version #1, cell magic without degradation tags, targeting peptides and cell cycle regulator."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/7/79/Yeast_D.gif" title="DT √ TP X CCR X" description="Version #2, cell magic with degradation tags, without targeting peptides and cell cycle regulator." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/e6/Yeast_DT.gif" title="DT √ TP √ CCR X" description="Version #3, cell magic with degradation tags and targeting peptides, without cell cycle regulator." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/c/c1/Yeast_DTR.gif" title="DT √ TP √ CCR √" description="Version #4, cell magic with degradation tag, targeting peptides and cell <li>cycle regulator." /></li><br />
</ul></div><br />
<div id="box1" class="box"><br />
<h3>The Magic</h3><br />
<p>Life, the most brilliant magic in the universe, celebrated with the ability of reproduction and revolution. And the magic, is based on a sophisticated mechanism, called cell cycle. Most genetic reactions in a cell are regulated, directly or indirectly, by cell cycle. As we wish to build artificial lives, it is worthwhile to learn from the exquisite design of creature, to make use of cell cycle. As a pioneer study, we try to performing a "Cell Magic", by capturing cell cycle with fantastic reporters. By grasping the usage of cell cycle tools, we are promised to direct refined actions in a cell. For instance, when producing Paclitaxel (harmful to centrosome in S phase) with a cell factory, we may able to transport it out before it comes detrimental. </p><br />
<p>So this is our project, the "Cell Magic", to engineer two versions of cell cycle based magic in both budding yeast and <i>E. coli</i>. We will present the blueprint of our stories by the yeast version. </p><br />
<h4>Version #1, Degradation Tag X, Targeting Peptides X, Cell Cycle Regulator X</h4><br />
<p>Promoters of cyclins, which can initial transcription in specific phase in a cell cycle, are selected to produce different fluorescence proteins in G1/SG2/M phases respectably. However, in our first version of story, with low natural degradation rate, fluorescence proteins would remain in cell after their promoters stop working. So colors for each phase cannot be distinguished, as the figure shows.</p><br />
<img src="https://static.igem.org/mediawiki/2013/e/e4/Yeast.gif" title="DT X TP X CCR X" description="Version #1, cell magic without degradation tags, targeting peptides and cell cycle regulator." width=47%/><br />
<img src="https://static.igem.org/mediawiki/2013/3/36/P.png" width=50% ><br />
<center><p>Figure. Version #1, cell magic without degradation tags, targeting peptides and cell cycle regulator. Animation and genetic circuits.</p></center><br />
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<h4>Version #2, Degradation Tag √, Targeting Peptides X, Cell Cycle Regulator X</h4><br />
<p>In the second version, degradation tags are added to the reporters to solve this problem. All fluorescence proteins are thought to be degraded in 15 minutes which is far less than the period of a cell cycle.</p><br />
<img src="https://static.igem.org/mediawiki/2013/7/79/Yeast_D.gif" title="DT √ TP X CCR X" description="Version #2, cell magic with degradation tags, without targeting peptides and cell cycle regulator." width=47%/><br />
<img src="https://static.igem.org/mediawiki/2013/d/df/Gc.P%2BD.png" width=50%><br />
<center><p>Figure. Version #2, cell magic with degradation tags, without targeting peptides and cell cycle regulator. Animation and genetic circuits.</p></center><br />
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<h4>Version #3, Degradation Tag √, Targeting Peptides √, Cell Cycle Regulator X</h4><br />
<p>While we are not sure if degradation tags can work as expected, we try to turn the time magic into a time-space one. Reporters are located to different cell structures by targeting peptides. Different phase with different color shines in different organelle, that is the third version.</p><br />
<img src="https://static.igem.org/mediawiki/2013/e/e6/Yeast_DT.gif" title="DT √ TP √ CCR X" description="Version #3, cell magic with degradation tags and targeting peptides, without cell cycle regulator." width=47%/><br />
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<img src="https://static.igem.org/mediawiki/2013/8/8d/P%2BD%2BT.png" width=50%><br />
<center><p>Figure. Version #3, cell magic with degradation tags and targeting peptides, without cell cycle regulator. Animation and genetic circuits.</p></center><br />
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<h4>Version #4, Degradation Tag √, Targeting Peptides √, Cell Cycle Regulator √</h4><br />
<p>To capture the magic macroscopically, we developed a microfluidic device to synchronize all cells into the same phase (G1). So synchronization devices was added to the forth version. </p><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Chip.gif" title="Microfuidics Device" description="Cell magic in microfuidics device." width=35% /><br />
<center><p>Figure. Cell magic in microfuidics device.</p></center><br />
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<p>To make the magic more fantasy, alternative splicing device acting synchronously with the synchronization device (a translational unit of a cyclin regulator), will splice the targeting peptide tail when cell are being synchronized to G1 phase, so all cells turn green; while synchronization device not working, targeting peptide remains in the reporter so only mitochondria turn green. In this way, the cell phase and statues can be reported by fluorescences.</p><br />
<img src="https://static.igem.org/mediawiki/2013/c/c1/Yeast_DTR.gif" title="DT √ TP √ CCR √" description="Version #4, cell magic with degradation tag, targeting peptides and cell cycle regulator." width=47%/><br />
<center><p>Figure. Version #4, cell magic with degradation tag, targeting peptides and cell cycle regulator. Animation.</p></center><br />
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<img src="https://static.igem.org/mediawiki/2013/5/58/P%2BD%2BT%2BS.png" width=50% /><br />
<center><p>Figure. Genetic circuits for version #4, in glucose medium, not synchronizing.</p></center><br />
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<img src="https://static.igem.org/mediawiki/2013/d/d9/P%2BD%2BT%2BS%2BI%2BC.png" /><br />
<center><p>Figure. Genetic circuits for version #4, in galactose medium, synchronizing.</p></center><br />
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<h3>Cyclin Promoters</h3><br />
<h4>Yeast Version</h4><br />
<p> As we all know, different proteins are planned to be expressed along the cell cycle. And their mRNAs are transcribed through the specific promoters. These promoters locate at their upstream sequences and can be recognized by the RNA polymerase, which can initiate such process. There are databases containing the upstream 600 amino acid sequence in the upstream of DNA, which can be transcript in the G1, S, G2 and M phase, respectively</p> <br />
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<p>Previous study (Wolfsberg et al., 1999) demonstrated that cell cycle specific promoters posses their conserved five to six base pairs. Thus, we found the high-confidence 600 promoter-containing sequences in database that harbor the paper-mentioned 5/6 bp sequence. Consequently, we pick up the upstream 600bp sequence of cln2, cln3, clb2, clb5 and clb6.</p><br />
<p>The Clb2 gene is highly expressed in G2 phase, and the genes are very strongly induced by GAL-CLB2, whereas GAL-CLN3 appears somewhat repressive Spellman et al., 1998)</p><br />
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<h4><i>E. coli</i> Version</h4><br />
<p> Cell cycle is a complex process and can be separated into G0, G1, S, G2, M phase. In each phase, distinct transcription factors help the phase-specific gene express through recognizing their promoters. Thus, these promoters are phase-specific too and can be fused with other genes in order to express such gene in a defined cell cycle phase.The well-regulated cell cycle in E.coli consists of three key events: DNA replication, nucleon segregation and the initiation of cell division. The replication initiator is DnaA protein; the second step relates to MukB protein and the cell division activator is the FtsZ protein. In our project, we take full aadvantage of the two initiators DnaA and FtsZ - using their promoter to control the transcript of specific genes binding behind them in the first step or last step of <i>E. coli</i> cell cycle.</p><br />
<h5>DnaA protein</h5><br />
<p>As mutation strain demonstrated, dnaA gene is absolutely required for initiation at oriC. When the concentration of DnaA increases, there is increased initiation?, indicating that DnaA protein is the switch for initiation (Atlung, Løbner-Olesen, & Hansen, 1987). Biochemical studies have shown that, with the help of accessory proteins, DnaA protein binds to five sites within oriC, therefore, leading to strand opening of a region containing three 13-mer repeats. This opening results in the begin of bidirectional replication (Atlung, Løbner-Olesen, & Hansen, 1987).</p><br />
<img src="https://static.igem.org/mediawiki/2013/3/3b/DnaAp.png" title="dnaA Promoter" description="dnaA promoter selected for S1 phase." /><br />
<img src="https://static.igem.org/mediawiki/2013/2/2a/GyrBp.png" title="gyrB Promoter" description="gyrB promoter selected for S2 phase." /><br />
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<h5>FtsZ</h5><br />
<p>The cell itself seems to regard FtsZ gene expression as the commitment to division, because of the fact that endogenous division inhibitors have FtsZ as target(Bi & Lutkenhaus, 1990). Increasing FtsZ concentration resulted in excess cell division, with the cell size decreasing (Ward & Lutkenhaus, 1985), vice verse. Thus the critical role of FtsZ in division is similar to that of DnaA in replication initiation. FtsZ protein exists randomly throughout cytoplasm during cell elongation, while polymerizes into a membrane-associated ring at the precise site of cell division (Bi & Lutkenhaus, 1991). Just before the appearance of a visible constriction. The ring decreasing along with the septation and FtsZ is dispersed again at completion. Such ring formation is presumed to be essential for cell division, since it cannot be observed in the presence of the cell division inhibitors (Bi & Lutkenhaus, 1993) but is always present when and where septa are formed.</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/6a/FtsQp.png" title="ftsQ Promoter" description="ftsQ promoter selected for S2 phase." /><br />
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<h3>Reporter Modification</h3><br />
<p> The XFP, as reporter in our project has been modified through</p> <br />
<p>1) removed the stop codon in them, 2) modified with 23# prefix and suffix, and then 3) added the terminator within stop codon to them. </p><br />
<p> Furthermore, all BioBricks were added with 23# prefix and suffix, which ensures it to be standard.<br />
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<h3>Degradation Tags</h3><br />
<h4>Yeast Version</h4><br />
<p>In yeast cell, protein degraded through several mechanisms, a major one is the ubquitin- pathway. In detail the signal in substrates can be recognized by the enzyme E3, which transport the ubiqutin from E2 to such protein. And through such way, the ubiquited protein would finally be degraded by the proteasome. </p><br />
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<h5>PEST</h5><br />
<p>There are two types of cyclins in the budding yeast. One kind contains PEST sequence at C-terminal: Cln1, Cln2, Cln3, Pcl1, Pcl2. Another kind is Clb1~6 which posses 9 conserved amino acid sequence in N-terminal named D-box (destruction box). The D-box are necessary for later kind cyclins degradation in M phase and can function in same way when combined with other proteins. In our study, we utilized the D-box, PEST sequence and ubiquitin-mediated pathway for fluorescent proteins degradation.</p><br />
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<h5>Poly-ubiquitin</h5><br />
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<p>Ubiquitin, a highly conserved 76-residue protein, was involved in the ubiquitin–proteasome pathway of protein proteolysis, which is a fundamental way for protein turnover, signal transduction and cell cycle control. For example, the degradation of CDC34 through such way is necessary for S phase start; another instance is that APC\C(anaphase-promoting complex\cyclosome) is degraded through ubiquitination, which is the foundation of M phase beginning. And both activated by Cdc28-cyclins</p><br />
<p>The mechanism of ubiquitin-mediated protein degradation can be separated into three continuous enzyme catalyze steps. First 1) a ubiquitin is activated by the E1 (ubiquitin-activating enzyme) through a thioester linkage,and then 2)added into the a small ubiquitin-carrier E2. Finally,3) through the E3 ubiquitin protein ligase, this ubiquitin complex was conjugate to the ε-amino group of lysine residues in substrate proteins, forming a glycyllysine isopeptide bond (Hershko, 1997).In some conditions, even E3 enzymes themselves carry ubiquitin as a thioester (Huibregtse et al., 1995).</p><br />
<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/5/5b/Ubiquitylation.svg/800px-Ubiquitylation.svg.png" title="Ubiquitin" description="Function of fusion ubiquitin."><br />
<p>In our project, we design a poly-ubiquitin(5 ubiquitin combined) BioBricks which can directly degrade the proteins fused with it.</p><br />
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<h5>D-box</h5><br />
<p>The best studied substrates of ubiquitin- and APC/C-mediated proteolysis are the mitotic cyclins (Murray, Solomon, & Kirschner, 1989) Mitosis-exit inducer, a cyclin B, are highly dependent on its 90 residues for ubiquitin-mediated proteolysis. Analysis of its N-terminal region demonstrated the sequence essential for cyclin proteolysis, called ‘destruction box’(D-box). Meanwhile, when cyclin destruction started, substrates containing D-boxes were rapidly poly-ubiquitinated. The consensus motif in B-type cyclins is RXALGXIXN. For much of the cell cycle, the D-box may not be recognized with high affinity, and when it is recognized, the ‘bait’ construct becomes highly unstable(Yamano, Tsurumi, Gannon, & Hunt, 1998). </p><br />
<img src="http://mol-biol4masters.masters.grkraj.org/html/Cell_Cycle_And_Its_Regulation_files/image022.jpg" title="D-box" description="Function of destruction box." /><br />
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<p>When the D-box exists in the protein N-terminal, this protein is recognized more easily by the E1 and then captured into the ubiquitin-mediated degradation pathway. Thus, we combined it to the fluorescent proteins’ N-terminal to fasten their degradation and avoid veiling the following fluorescence.</p><br />
<h4><i>E. coli</i> Version</h4><br />
<p>In E.coli, the adaptor SspB tethers ssrAtagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. Which means, a variation of the WT SsrA tag sequence (K1051206, K1051207 and K1051208) will accelerate the degradation of proteins when fused to their C-terminal. Thus the degradation rates are dependent on concentration of proteases and binding mediators. In order to fuse degradation tags freely on the C-terminal of protein, we add TAATAA to the tail of tags.</p><br />
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<h3>Targeting Peptides</h3><br />
<p>A target peptide is a short (3-70 amino acids long) peptide chain that directs the transport of a protein to a specific region in cell, including nucleus, mitochondria, endoplasmic reticulums (ERs), chloroplasts, apoplasts, peroxisomes and plasma membrane. Targeting peptide can exists in both N-terminal, C-terminal and internal sequence of a precursor protein. And after transported, some target peptides are cleaved by signal peptidases.</p><br />
<p>In our project we utilized 19 peptides target to 9 sub-locations in yeast cells, and when combined with fluorescent proteins, such region can be marked by different colors.</p><br />
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<h5>Mitochondria</h5><br />
<p>Though it accounts a small ratio in the cell space, mitochondria possess about 10% to 15% proteins encoded by nuclear genes in eukaryotic organisms. These proteins are synthesized in cytosol and then recognized by the membrane receptors of mitochondria. Translocases in the outer and inner membrane of mitochondria mediate the import and intra-mitochondrial sorting of these proteins. ATP is used as an energy source; Chaperones and auxiliary factors assist in folding and assembly of mitochondrial proteins into their native, three-dimensional structures. </p><br />
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<p>As shown in the figure above, beta-barrel outer-membrane proteins (dark green), precursor proteins (brown) with positively charged amino-terminal presequences and multispanning inner-membrane proteins (blue) with internal targeting signals are recognized by specific receptors of the outer mitochondrial membrane (TOM) translocases Tom20, Tom22 and/or Tom70. The precursor proteins are then translocated through a small Tom proteins of the TOM complex, Tom40 pore, which the TOM complex contains two or three.</p><br />
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<h5>Peroxisomes </h5><br />
<p>The import of post-translational matrix protein into peroxisomes depends on either of the two peroxisomal targeting signals (PTS), PTS1 and PTS2. PTS2-driven import is facilitated by a complex in the membrane. Under oleic acid-inducing growth conditions, there is a ternary core complex of approximate 150 kDa in the cytosol, which consists of Fox3p,Pex7p and Pex18p. Fox3p is imported as a dimer, while other two are bind in monomeric forms.</p><br />
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<img src="https://static.igem.org/mediawiki/igem.org/7/73/Figure2._Schematical_model_of_the_early_steps_of_PTS2-driven_import.png" title=”Import pathway“ description=”Peroxisome import pathway“/><br />
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<p>As study mentioned there are four steps involved in PTS2-driven import. The first step is the recognition of dimerized Fox3p by Pex7p through its PTS2 in the cytosol. In a second step, the Pex7p–Fox3p complex interacts with Pex18p, which targets the PTS2 pre-import complex to the peroxisomal membrane. The third step is the docking process, involving the interaction between Pex7p and the integral membrane protein Pex13p. As a final step of these early steps in the PTS2 import cascade, PTS2 receptor dissociation takes place during or after its assembly into large oligomeric complexes containing Pex14p and Pex13p. Pex18p remains at the peroxisomal membrane in the form of a large-molecular-weight complex in conjunction with Pex14p and/or Pex13p, from where it might be released to the cytosol(Grunau et al., 2009).</p><br />
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<h5>Actin </h5><br />
<p>In yeast, the cortical actin cytoskeleton seems to specify sites of growth of the cell surface. Because the actin-binding protein ABP1p is associated with the cortical cytoskeleton of Saccharomyces cerevisiae, it might be involved in the spatial organization of cell surface growth. ABP1p is localized to the cortical cytoskeleton and its overproduction causes assembly of the cortical actin cytoskeleton at inappropriate sites on the cell surface, resulting in delocalized surface growth. ABP1p is a novel protein with a 50 amino-acid C-terminal domain, which is very similar to the SH3 domain in the non-catalytic region of nonreceptor tyrosine kinases (including those encoded by the proto-oncogenes c-src and c-abl), in phospholipase C gamma and in alpha-spectrin. They also identified an SH3-related motif in the actin-binding tail domain of myosin-I. The identification of SH3 domains in a family of otherwise unrelated proteins that associate with the membrane cytoskeleton indicates that this domain might serve to bring together signal transduction proteins and their targets or regulators, or both, in the membrane cytoskeleton (Drubin, Mulholland, Zhu, & Botstein, 1990; Khorasanizadeh, 2004).</p><br />
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<h5>Nucler</h5><br />
<p>Histones are nuclear proteins package DNA into nucleosomes, and they are responsible for maintaining the shape and structure of a nucleosome. One chromatin molecule is composed of at least one of each core histones per 100 base pairs of DNA.[The Nucleosome: From Genomic Organization to Genomic Regulation.] There are five families of histones known to date, termed H1/H5, H2A, H2B, H3, and H4. H2A is considered a core histone, along with H2B, H3 and H4. Core formation first occurs through the interaction of two H2A molecules(Acid, 2004). Then, H2A forms a dimer with H2B; the core molecule is complete when H3-H4 also attaches to form a tetramer.</p><br />
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<h5>Vacuolar Membrane</h5><br />
<p>The ZRC1 gene encodes a multicopy suppressor of zinc toxicity in Saccharomyces cerevisiae; however, previously it was reported that the expression of ZRC1 was induced when the intracellular zinc level was decreased. The COT1 and ZRC1 genes of Saccharomyces cerevisiae are structurally related dosage-dependent suppressors of metal toxicity. COT1 confers increased tolerance to high levels of cobalt; ZRC1 confers increased tolerance(Conklin, Culbertson, & Kung, 1994).Zrc1 has six putative trans-membrane domains, and Zrc1-GFP fusion protein was localized to the vacuolar membrane. Zrc1 function as a mechanism to maintain the zinc homeostasis in yeast(Miyabe, Izawa, & Inoue, 2001).</p><br />
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<h3>Cell Synchronization</h3><br />
<p>As we known, the yeast cell cycle contains a huge and complex regulatory network in the transcription level, translation level as well as protein level. In our project we utilize Sic1 as a regulator to help elongate G1 phase in yeast cell.</p><br />
<p>The activation of B-type cyclin (Clb5/6)+Cdc28 kinases is a necessary step for initiation of DNA replication in vivo. One of its inhibitor Sic1 can be phosphorylated by the activated Cln+Cdc28, thereby targeted for degradation. Over expression of the gene encoding p40, SIC1, produces cells with an elongated bud morphology(figure1)(Nugroho & Mendenhall, 1994). SIC1 deletion is viable and causes increasing frequencies of chromosomes broken and lost. The deletion strain segregates out many dead cells, which are primarily arrested at the G2 checkpoint in an asymmetric fashion. Therefore, it has an important role in ensuring genomic integrity, and that this role has a pronounced mother-daughter asymmetry.</p><br />
<img src="http://upload.wikimedia.org/wikipedia/commons/0/06/Sic1_David_Morgan10-5.jpg" title="Principle of Sic1" description="Over expression of Sic1 can stop all yeast cells to G1 phase."><br />
<p>After phosphorylation, phospho-Sic1 is specifically recognized by the F-box protein Cdc4, which leads Sic1 being ubiquitinated by the Cdc34±SCF complex (E3). The recognizing and binding by Cdc4 is based on the Sic1’s 9 Cdc4 phospo-degrons (CPDs, figure. a). Several phosphorylation sites contributed to Sic1 instability, with an order of Thr45, Ser76, Thr5,Thr33 and other less significant sites. The immunocipients after selectively phosphorylation as figure e showed suggested that at least six sites phosphorylation is necessary for the Cdc4 recognizing and binding with Sic1. Furthermore, the culture of GAL1-SIC1 constructs strain showed less than 6 sites are phosphorylated is not sufficient for SIC1 degradation in vitro(Nash et al., 2001).</p><br />
<img src="https://static.igem.org/mediawiki/parts/8/8b/Sic1-mutate.png" title="Phosphorylation Sites" description="N-terminal phosphorylation sites to be mutated."><br />
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<h3>Alternative Splicing Device</h3><br />
<p>In yeast cells, alternative splicing is a common process in before gene transcription. The splicing sites are located in 5'UTR of introns and can by recognized and spliced by some splicesome. Alternative splicing can produce two isoforms from one gene, thus can be used in our project to monitor whether the Sic1 system work efficiently. Here we find a intron with alternative splicing sites in yeast cells named SRCI intron.</p><br />
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<h4>Src1 Intron and Hub1</h4><br />
<p> SRC1 intron has two 5' splicing site whose efficiency is regulated by protein hub1. Originally, the existence of the intron would produce two mature mRNA in proportion. After engineering, the existence of protein hub1 regulates to the preservation of intron precisely, which would make the following mRNA be expressed normally or not. As for the silencing of wild type gene HUB1, we choose CRISPRi that is comparably easy to use reversibly.</p><br />
<p>Alternative splicing substantially increases the gene product diversity and is a major source of cell type differentiation. A good example is the alternative splicing of Saccharomyces cerevisiae SRC1 pre-mRNA, which is promoted by the conserved ubiquitin-like protein Hub1. It can function through binding non-covalently to a conserved element termed HIND in the spliceosomal protein Snu66. Such binding makes the splicesome target sites change and moderately alters spliceosomal interactions. </p><br />
<p>Hub1 is a ubiquitin-like modifier (UBL) that covalent modify the proteins. Interest enough, it harbors several different to other UBLs in which it possesses a C-terminal double tyrosine motif while others having a GG motif. The Snu66, a tri-snRNP in yeast spliceosome, possesses with two N-terminal HINDs (Hub1-INteraction Domain). The Hub1–HIND interaction comprises a strong salt bridge accompanied by several hydrophobic contacts and high affinity. Such binding modifies the spliceosome rather than modulating the properties of an individual binding partner. Hub1-controlled splicing occurs universally in eukaryotes. SR proteins and hnRNPs involved in spliceosome targeting do not seem to exist in S. Cerevisiae , and thus the Hub1-dependent mechanism may be evolutionarily older. </p> <br />
<p>Scr1 is a protein in yeast having alternative splicing sites in its intron. The characteristic differential Hub1 dependence of SRC1 alternative splicing requires the tandem arrangement of overlapping 5’ splice sites. The Hub1 binding spliceosome can splice the intron from both downstream 5’ sites as well as the upstream 5’ sites with preference to the former one.</p><br />
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<p>Showed in Figure.above When cutting in the upstream splice sites, the exons flanking around it would be translated as a fusion protein Scr-S. However, when cutting at the down stream one, the left 4bp in intron would result in a frame shift, thus only the forward exon can express named Scr-L.</p><br />
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<h4>dCas9 CRISPR interface system</h4><br />
<p>CRISPR shorts for Clustered Regularly Interspaced Palindromaic Repeats system, which can be targeted to DNA using RNA, enabling genetic editing of any region of the genome in many organisms.(Cho, Kim, Kim, & Kim, 2013). In the type II CRISPR/Cas system, a ribonucleoprotein complex formed from a single protein (Cas9), a crRNA, and a trans-acting CRISPR RNA (tracrRNA) can carry out efficient crRNA-directed recognition and site-specific cleavage of foreign DNA(Deltcheva et al., 2011). After mutated the endonuclease domains of the Cas9 protein, it creates a programmable RNA-dependent DNA-binding protein. The sgRNA consists of three domains: a 20 nt complementary region for specific DNA binding, a 42 nt hairpin for Cas9 binding (Cas9 handle), and a 40 nt transcription terminator derived from S. Pyogenes. After translation, the Cas9 binds to sgRNA to form a protein-RNA complex, which can recognize target sites in the genome sequence and bind to it. Then, it could block RNA polymerase and transciption elongation.</p><br />
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<img src="https://static.igem.org/mediawiki/2013/7/74/Figure4._The_structure_of_sgRNA.png" title="sgRNA" description="sgRNA structure" /><br />
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<h3>Microfluidic Device</h3><br />
<p>Based physically on the chip, microfluidics can automatically control the biology process. In our project, the microfluidics plays a role as the movie theater, for cell cycle control. We designed a channel which can capture the yeast cell and then alternatively inject medium of different concentrations. Therefore, the yeast cell cycle can be synchronized. Furthermore, due to its transparent attribution, the chip can project the fluorescent lights from the yeast cell sub-locations. Consequently, the magic film can be shown to us. </p><br />
<p>Microfluidic devices possess many advantages such as large-scale integration, fast analyses, and considerably reduced reagent consumption (Comparative study and improvement of current cell micro-patterning techniques). Nowadays many designs are based on cell loading and culturing in a microfluidic channel with particular trapping structures such as micro-well arrays, and micro-cup arrays and micro-chambers. In our project, we design a cell loading technique based on gas absorption of degassed polydimethylsiloxane (PDMS). Because the balance concentration of gas dissolved in PDMS is proportional to the partial pressure of the gas around it, so that one can degas a piece of PDMS by placing it in vacuum. </p><br />
<p>Our cell loading device is a one level PDMS with an array of triangle cavities connected to one or both sides of the main fluidic channel. After degassed, yeast cells in the buffer solutions can be introduced into the device’s triangle cavities by gas absorption therein. Through changing the cell density in the buffer, we can control the number of the yeast cells loaded into each triangle cavity. Statistically, the ratio of cavities with a single cell can reach 40%. Consequently it is convenient to monitor the cell number and cell phenotypic variations because the yeast cells can grow in the micro-triangle-cavities in one layer.</p><br />
<img src="https://static.igem.org/mediawiki/igem.org/e/ec/Figure6._Masks_for_PDMS_microfluidic_device_fabrication..png" title="Microfluidic" description="the structure of Microfluidics"><br />
<p>Figure 1. Masks for PDMS microfluidic device fabrication.a : Three micro-triangle-cavities at one side of the main channel. b : One single channel for cell culture (100 microcavities). c : Seven parallel channels for different cell culture environments(Luo et al., 2008).</p><br />
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</div><br />
</div></div>BGI K2http://2013.igem.org/Team:Shenzhen_BGIC_ATCG/storiesTeam:Shenzhen BGIC ATCG/stories2013-09-27T22:31:55Z<p>BGI K2: </p>
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<div><!----------------------<br />
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<br />
Title: Stories<br />
Team: Shenzhen_BGIC_ATCG<br />
Author: BGI_K2<br />
Twitter: @BGI_K2<br />
Email: im.ss.kk@gmail.com<br />
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<div class="navleftholder"><br />
<ul class="navleft"><br />
<li><a id="navleftsub1" class="navleftsub" href="#board1">The Magic</a></li><br />
<li><a id="navleftsub2" class="navleftsub" href="#board2">Cyclin Promoters</a></li><br />
<li><a id="navleftsub3" class="navleftsub" href="#board3">Reporter Modification</a></li><br />
<li><a id="navleftsub4" class="navleftsub" href="#board4">Degradation Tags</a></li><br />
<li><a id="navleftsub5" class="navleftsub" href="#board5">Targeting Peptides</a></li><br />
<li><a id="navleftsub6" class="navleftsub" href="#board6">Cell Synchronization</a></li><br />
<li><a id="navleftsub7" class="navleftsub" href="#board7">Alternative Splicing Device</a></li><br />
<li><a id="navleftsub8" class="navleftsub" href="#board8">Microfluidic Device</a></li><br />
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<div id="board1" class="board"></div><br />
<div class="media"><br />
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<ul id="myGallery1"><br />
<li><img src="https://static.igem.org/mediawiki/2013/5/58/P%2BD%2BT%2BS.png" title="Genetic circuit of cell cycle version #4" description="To make the magic more fantasy, alternative splicing device acting synchronously with the synchronization device." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/1/10/Chip.gif" title="Microfuidics Device" description="Cell magic in microfuidics device." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/e4/Yeast.gif" title="DT X TP X CCR X" description="Version #1, cell magic without degradation tags, targeting peptides and cell cycle regulator."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/7/79/Yeast_D.gif" title="DT √ TP X CCR X" description="Version #2, cell magic with degradation tags, without targeting peptides and cell cycle regulator." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/e6/Yeast_DT.gif" title="DT √ TP √ CCR X" description="Version #3, cell magic with degradation tags and targeting peptides, without cell cycle regulator." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/c/c1/Yeast_DTR.gif" title="DT √ TP √ CCR √" description="Version #4, cell magic with degradation tag, targeting peptides and cell <li>cycle regulator." /></li><br />
</ul></div><br />
<div id="box1" class="box"><br />
<h3>The Magic</h3><br />
<p>Life, the most brilliant magic in the universe, celebrated with the ability of reproduction and revolution. And the magic, is based on a sophisticated mechanism, called cell cycle. Most genetic reactions in a cell are regulated, directly or indirectly, by cell cycle. As we wish to build artificial lives, it is worthwhile to learn from the exquisite design of creature, to make use of cell cycle. As a pioneer study, we try to performing a "Cell Magic", by capturing cell cycle with fantastic reporters. By grasping the usage of cell cycle tools, we are promised to direct refined actions in a cell. For instance, when producing Paclitaxel (harmful to centrosome in S phase) with a cell factory, we may able to transport it out before it comes detrimental. </p><br />
<p>So this is our project, the "Cell Magic", to engineer two versions of cell cycle based magic in both budding yeast and <i>E. coli</i>. We will present the blueprint of our stories by the yeast version. </p><br />
<h4>Version #1, Degradation Tag X, Targeting Peptides X, Cell Cycle Regulator X</h4><br />
<p>Promoters of cyclins, which can initial transcription in specific phase in a cell cycle, are selected to produce different fluorescence proteins in G1/SG2/M phases respectably. However, in our first version of story, with low natural degradation rate, fluorescence proteins would remain in cell after their promoters stop working. So colors for each phase cannot be distinguished, as the figure shows.</p><br />
<img src="https://static.igem.org/mediawiki/2013/e/e4/Yeast.gif" title="DT X TP X CCR X" description="Version #1, cell magic without degradation tags, targeting peptides and cell cycle regulator." width=47%/><br />
<img src="https://static.igem.org/mediawiki/2013/3/36/P.png" width=50% ><br />
<center><p>Figure. Version #1, cell magic without degradation tags, targeting peptides and cell cycle regulator. Animation and genetic circuits.</p></center><br />
<br />
<h4>Version #2, Degradation Tag √, Targeting Peptides X, Cell Cycle Regulator X</h4><br />
<p>In the second version, degradation tags are added to the reporters to solve this problem. All fluorescence proteins are thought to be degraded in 15 minutes which is far less than the period of a cell cycle.</p><br />
<img src="https://static.igem.org/mediawiki/2013/7/79/Yeast_D.gif" title="DT √ TP X CCR X" description="Version #2, cell magic with degradation tags, without targeting peptides and cell cycle regulator." width=47%/><br />
<img src="https://static.igem.org/mediawiki/2013/d/df/Gc.P%2BD.png" width=50%><br />
<center><p>Figure. Version #2, cell magic with degradation tags, without targeting peptides and cell cycle regulator. Animation and genetic circuits.</p></center><br />
<br />
<h4>Version #3, Degradation Tag √, Targeting Peptides √, Cell Cycle Regulator X</h4><br />
<p>While we are not sure if degradation tags can work as expected, we try to turn the time magic into a time-space one. Reporters are located to different cell structures by targeting peptides. Different phase with different color shines in different organelle, that is the third version.</p><br />
<img src="https://static.igem.org/mediawiki/2013/e/e6/Yeast_DT.gif" title="DT √ TP √ CCR X" description="Version #3, cell magic with degradation tags and targeting peptides, without cell cycle regulator." width=47%/><br />
<center><p>Figure. Version #3, cell magic with degradation tags and targeting peptides, without cell cycle regulator. Animation and genetic circuits.</p></center><br />
<br />
<img src="https://static.igem.org/mediawiki/2013/8/8d/P%2BD%2BT.png" width=50%><br />
<h4>Version #4, Degradation Tag √, Targeting Peptides √, Cell Cycle Regulator √</h4><br />
<p>To capture the magic macroscopically, we developed a microfluidic device to synchronize all cells into the same phase (G1). So synchronization devices was added to the forth version. </p><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Chip.gif" title="Microfuidics Device" description="Cell magic in microfuidics device." width=35% /><br />
<img src="https://static.igem.org/mediawiki/2013/5/58/P%2BD%2BT%2BS.png" width=62% /><br />
<center><p>Figure. Cell magic in microfuidics device.</p></center><br />
<br />
<p>To make the magic more fantasy, alternative splicing device acting synchronously with the synchronization device (a translational unit of a cyclin regulator), will splice the targeting peptide tail when cell are being synchronized to G1 phase, so all cells turn green; while synchronization device not working, targeting peptide remains in the reporter so only mitochondria turn green. In this way, the cell phase and statues can be reported by fluorescences.</p><br />
<img src="https://static.igem.org/mediawiki/2013/c/c1/Yeast_DTR.gif" title="DT √ TP √ CCR √" description="Version #4, cell magic with degradation tag, targeting peptides and cell cycle regulator." width=47%/><br />
<img src="https://static.igem.org/mediawiki/2013/d/d9/P%2BD%2BT%2BS%2BI%2BC.png" /><br />
<center><p>Figure. Version #4, cell magic with degradation tag, targeting peptides and cell cycle regulator. Animation and genetic circuits.</p></center><br />
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<h3>Cyclin Promoters</h3><br />
<h4>Yeast Version</h4><br />
<p> As we all know, different proteins are planned to be expressed along the cell cycle. And their mRNAs are transcribed through the specific promoters. These promoters locate at their upstream sequences and can be recognized by the RNA polymerase, which can initiate such process. There are databases containing the upstream 600 amino acid sequence in the upstream of DNA, which can be transcript in the G1, S, G2 and M phase, respectively</p> <br />
<!----- [http://www.genome.jp/kegg-bin/show_pathway?org_name=sce&mapno=04111&mapscale=&show_description=hide]. ----><br />
<p>Previous study (Wolfsberg et al., 1999) demonstrated that cell cycle specific promoters posses their conserved five to six base pairs. Thus, we found the high-confidence 600 promoter-containing sequences in database that harbor the paper-mentioned 5/6 bp sequence. Consequently, we pick up the upstream 600bp sequence of cln2, cln3, clb2, clb5 and clb6.</p><br />
<p>The Clb2 gene is highly expressed in G2 phase, and the genes are very strongly induced by GAL-CLB2, whereas GAL-CLN3 appears somewhat repressive Spellman et al., 1998)</p><br />
<img src="https://static.igem.org/mediawiki/2013/3/31/Sce04111.png" title="Cell Cycle KEGG" description="KEGG diagram for core cell cycle." width=98% /><br />
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<h4><i>E. coli</i> Version</h4><br />
<p> Cell cycle is a complex process and can be separated into G0, G1, S, G2, M phase. In each phase, distinct transcription factors help the phase-specific gene express through recognizing their promoters. Thus, these promoters are phase-specific too and can be fused with other genes in order to express such gene in a defined cell cycle phase.The well-regulated cell cycle in E.coli consists of three key events: DNA replication, nucleon segregation and the initiation of cell division. The replication initiator is DnaA protein; the second step relates to MukB protein and the cell division activator is the FtsZ protein. In our project, we take full aadvantage of the two initiators DnaA and FtsZ - using their promoter to control the transcript of specific genes binding behind them in the first step or last step of <i>E. coli</i> cell cycle.</p><br />
<h5>DnaA protein</h5><br />
<p>As mutation strain demonstrated, dnaA gene is absolutely required for initiation at oriC. When the concentration of DnaA increases, there is increased initiation?, indicating that DnaA protein is the switch for initiation (Atlung, Løbner-Olesen, & Hansen, 1987). Biochemical studies have shown that, with the help of accessory proteins, DnaA protein binds to five sites within oriC, therefore, leading to strand opening of a region containing three 13-mer repeats. This opening results in the begin of bidirectional replication (Atlung, Løbner-Olesen, & Hansen, 1987).</p><br />
<img src="https://static.igem.org/mediawiki/2013/3/3b/DnaAp.png" title="dnaA Promoter" description="dnaA promoter selected for S1 phase." /><br />
<img src="https://static.igem.org/mediawiki/2013/2/2a/GyrBp.png" title="gyrB Promoter" description="gyrB promoter selected for S2 phase." /><br />
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<h5>FtsZ</h5><br />
<p>The cell itself seems to regard FtsZ gene expression as the commitment to division, because of the fact that endogenous division inhibitors have FtsZ as target(Bi & Lutkenhaus, 1990). Increasing FtsZ concentration resulted in excess cell division, with the cell size decreasing (Ward & Lutkenhaus, 1985), vice verse. Thus the critical role of FtsZ in division is similar to that of DnaA in replication initiation. FtsZ protein exists randomly throughout cytoplasm during cell elongation, while polymerizes into a membrane-associated ring at the precise site of cell division (Bi & Lutkenhaus, 1991). Just before the appearance of a visible constriction. The ring decreasing along with the septation and FtsZ is dispersed again at completion. Such ring formation is presumed to be essential for cell division, since it cannot be observed in the presence of the cell division inhibitors (Bi & Lutkenhaus, 1993) but is always present when and where septa are formed.</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/6a/FtsQp.png" title="ftsQ Promoter" description="ftsQ promoter selected for S2 phase." /><br />
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<h3>Reporter Modification</h3><br />
<p> The XFP, as reporter in our project has been modified through</p> <br />
<p>1) removed the stop codon in them, 2) modified with 23# prefix and suffix, and then 3) added the terminator within stop codon to them. </p><br />
<p> Furthermore, all BioBricks were added with 23# prefix and suffix, which ensures it to be standard.<br />
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<li><img src="https://static.igem.org/mediawiki/2012/b/b5/Yao.result.jpg"/><br />
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<h3>Degradation Tags</h3><br />
<h4>Yeast Version</h4><br />
<p>In yeast cell, protein degraded through several mechanisms, a major one is the ubquitin- pathway. In detail the signal in substrates can be recognized by the enzyme E3, which transport the ubiqutin from E2 to such protein. And through such way, the ubiquited protein would finally be degraded by the proteasome. </p><br />
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<h5>PEST</h5><br />
<p>There are two types of cyclins in the budding yeast. One kind contains PEST sequence at C-terminal: Cln1, Cln2, Cln3, Pcl1, Pcl2. Another kind is Clb1~6 which posses 9 conserved amino acid sequence in N-terminal named D-box (destruction box). The D-box are necessary for later kind cyclins degradation in M phase and can function in same way when combined with other proteins. In our study, we utilized the D-box, PEST sequence and ubiquitin-mediated pathway for fluorescent proteins degradation.</p><br />
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<h5>Poly-ubiquitin</h5><br />
<br />
<p>Ubiquitin, a highly conserved 76-residue protein, was involved in the ubiquitin–proteasome pathway of protein proteolysis, which is a fundamental way for protein turnover, signal transduction and cell cycle control. For example, the degradation of CDC34 through such way is necessary for S phase start; another instance is that APC\C(anaphase-promoting complex\cyclosome) is degraded through ubiquitination, which is the foundation of M phase beginning. And both activated by Cdc28-cyclins</p><br />
<p>The mechanism of ubiquitin-mediated protein degradation can be separated into three continuous enzyme catalyze steps. First 1) a ubiquitin is activated by the E1 (ubiquitin-activating enzyme) through a thioester linkage,and then 2)added into the a small ubiquitin-carrier E2. Finally,3) through the E3 ubiquitin protein ligase, this ubiquitin complex was conjugate to the ε-amino group of lysine residues in substrate proteins, forming a glycyllysine isopeptide bond (Hershko, 1997).In some conditions, even E3 enzymes themselves carry ubiquitin as a thioester (Huibregtse et al., 1995).</p><br />
<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/5/5b/Ubiquitylation.svg/800px-Ubiquitylation.svg.png" title="Ubiquitin" description="Function of fusion ubiquitin."><br />
<p>In our project, we design a poly-ubiquitin(5 ubiquitin combined) BioBricks which can directly degrade the proteins fused with it.</p><br />
<br />
<h5>D-box</h5><br />
<p>The best studied substrates of ubiquitin- and APC/C-mediated proteolysis are the mitotic cyclins (Murray, Solomon, & Kirschner, 1989) Mitosis-exit inducer, a cyclin B, are highly dependent on its 90 residues for ubiquitin-mediated proteolysis. Analysis of its N-terminal region demonstrated the sequence essential for cyclin proteolysis, called ‘destruction box’(D-box). Meanwhile, when cyclin destruction started, substrates containing D-boxes were rapidly poly-ubiquitinated. The consensus motif in B-type cyclins is RXALGXIXN. For much of the cell cycle, the D-box may not be recognized with high affinity, and when it is recognized, the ‘bait’ construct becomes highly unstable(Yamano, Tsurumi, Gannon, & Hunt, 1998). </p><br />
<img src="http://mol-biol4masters.masters.grkraj.org/html/Cell_Cycle_And_Its_Regulation_files/image022.jpg" title="D-box" description="Function of destruction box." /><br />
<br />
<p>When the D-box exists in the protein N-terminal, this protein is recognized more easily by the E1 and then captured into the ubiquitin-mediated degradation pathway. Thus, we combined it to the fluorescent proteins’ N-terminal to fasten their degradation and avoid veiling the following fluorescence.</p><br />
<h4><i>E. coli</i> Version</h4><br />
<p>In E.coli, the adaptor SspB tethers ssrAtagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. Which means, a variation of the WT SsrA tag sequence (K1051206, K1051207 and K1051208) will accelerate the degradation of proteins when fused to their C-terminal. Thus the degradation rates are dependent on concentration of proteases and binding mediators. In order to fuse degradation tags freely on the C-terminal of protein, we add TAATAA to the tail of tags.</p><br />
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<h3>Targeting Peptides</h3><br />
<p>A target peptide is a short (3-70 amino acids long) peptide chain that directs the transport of a protein to a specific region in cell, including nucleus, mitochondria, endoplasmic reticulums (ERs), chloroplasts, apoplasts, peroxisomes and plasma membrane. Targeting peptide can exists in both N-terminal, C-terminal and internal sequence of a precursor protein. And after transported, some target peptides are cleaved by signal peptidases.</p><br />
<p>In our project we utilized 19 peptides target to 9 sub-locations in yeast cells, and when combined with fluorescent proteins, such region can be marked by different colors.</p><br />
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<h5>Mitochondria</h5><br />
<p>Though it accounts a small ratio in the cell space, mitochondria possess about 10% to 15% proteins encoded by nuclear genes in eukaryotic organisms. These proteins are synthesized in cytosol and then recognized by the membrane receptors of mitochondria. Translocases in the outer and inner membrane of mitochondria mediate the import and intra-mitochondrial sorting of these proteins. ATP is used as an energy source; Chaperones and auxiliary factors assist in folding and assembly of mitochondrial proteins into their native, three-dimensional structures. </p><br />
<br />
<img src="https://static.igem.org/mediawiki/2013/1/1f/Figure1.protein-import_pathways_for_mitochondrial_proteins.png" title="Import pathway" description="Mitochrondira import pathway" /><br />
<br />
<p>As shown in the figure above, beta-barrel outer-membrane proteins (dark green), precursor proteins (brown) with positively charged amino-terminal presequences and multispanning inner-membrane proteins (blue) with internal targeting signals are recognized by specific receptors of the outer mitochondrial membrane (TOM) translocases Tom20, Tom22 and/or Tom70. The precursor proteins are then translocated through a small Tom proteins of the TOM complex, Tom40 pore, which the TOM complex contains two or three.</p><br />
<br />
<h5>Peroxisomes </h5><br />
<p>The import of post-translational matrix protein into peroxisomes depends on either of the two peroxisomal targeting signals (PTS), PTS1 and PTS2. PTS2-driven import is facilitated by a complex in the membrane. Under oleic acid-inducing growth conditions, there is a ternary core complex of approximate 150 kDa in the cytosol, which consists of Fox3p,Pex7p and Pex18p. Fox3p is imported as a dimer, while other two are bind in monomeric forms.</p><br />
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<img src="https://static.igem.org/mediawiki/igem.org/7/73/Figure2._Schematical_model_of_the_early_steps_of_PTS2-driven_import.png" title=”Import pathway“ description=”Peroxisome import pathway“/><br />
<br />
<br />
<p>As study mentioned there are four steps involved in PTS2-driven import. The first step is the recognition of dimerized Fox3p by Pex7p through its PTS2 in the cytosol. In a second step, the Pex7p–Fox3p complex interacts with Pex18p, which targets the PTS2 pre-import complex to the peroxisomal membrane. The third step is the docking process, involving the interaction between Pex7p and the integral membrane protein Pex13p. As a final step of these early steps in the PTS2 import cascade, PTS2 receptor dissociation takes place during or after its assembly into large oligomeric complexes containing Pex14p and Pex13p. Pex18p remains at the peroxisomal membrane in the form of a large-molecular-weight complex in conjunction with Pex14p and/or Pex13p, from where it might be released to the cytosol(Grunau et al., 2009).</p><br />
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<h5>Actin </h5><br />
<p>In yeast, the cortical actin cytoskeleton seems to specify sites of growth of the cell surface. Because the actin-binding protein ABP1p is associated with the cortical cytoskeleton of Saccharomyces cerevisiae, it might be involved in the spatial organization of cell surface growth. ABP1p is localized to the cortical cytoskeleton and its overproduction causes assembly of the cortical actin cytoskeleton at inappropriate sites on the cell surface, resulting in delocalized surface growth. ABP1p is a novel protein with a 50 amino-acid C-terminal domain, which is very similar to the SH3 domain in the non-catalytic region of nonreceptor tyrosine kinases (including those encoded by the proto-oncogenes c-src and c-abl), in phospholipase C gamma and in alpha-spectrin. They also identified an SH3-related motif in the actin-binding tail domain of myosin-I. The identification of SH3 domains in a family of otherwise unrelated proteins that associate with the membrane cytoskeleton indicates that this domain might serve to bring together signal transduction proteins and their targets or regulators, or both, in the membrane cytoskeleton (Drubin, Mulholland, Zhu, & Botstein, 1990; Khorasanizadeh, 2004).</p><br />
<br />
<h5>Nucler</h5><br />
<p>Histones are nuclear proteins package DNA into nucleosomes, and they are responsible for maintaining the shape and structure of a nucleosome. One chromatin molecule is composed of at least one of each core histones per 100 base pairs of DNA.[The Nucleosome: From Genomic Organization to Genomic Regulation.] There are five families of histones known to date, termed H1/H5, H2A, H2B, H3, and H4. H2A is considered a core histone, along with H2B, H3 and H4. Core formation first occurs through the interaction of two H2A molecules(Acid, 2004). Then, H2A forms a dimer with H2B; the core molecule is complete when H3-H4 also attaches to form a tetramer.</p><br />
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<h5>Vacuolar Membrane</h5><br />
<p>The ZRC1 gene encodes a multicopy suppressor of zinc toxicity in Saccharomyces cerevisiae; however, previously it was reported that the expression of ZRC1 was induced when the intracellular zinc level was decreased. The COT1 and ZRC1 genes of Saccharomyces cerevisiae are structurally related dosage-dependent suppressors of metal toxicity. COT1 confers increased tolerance to high levels of cobalt; ZRC1 confers increased tolerance(Conklin, Culbertson, & Kung, 1994).Zrc1 has six putative trans-membrane domains, and Zrc1-GFP fusion protein was localized to the vacuolar membrane. Zrc1 function as a mechanism to maintain the zinc homeostasis in yeast(Miyabe, Izawa, & Inoue, 2001).</p><br />
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<h3>Cell Synchronization</h3><br />
<p>As we known, the yeast cell cycle contains a huge and complex regulatory network in the transcription level, translation level as well as protein level. In our project we utilize Sic1 as a regulator to help elongate G1 phase in yeast cell.</p><br />
<p>The activation of B-type cyclin (Clb5/6)+Cdc28 kinases is a necessary step for initiation of DNA replication in vivo. One of its inhibitor Sic1 can be phosphorylated by the activated Cln+Cdc28, thereby targeted for degradation. Over expression of the gene encoding p40, SIC1, produces cells with an elongated bud morphology(figure1)(Nugroho & Mendenhall, 1994). SIC1 deletion is viable and causes increasing frequencies of chromosomes broken and lost. The deletion strain segregates out many dead cells, which are primarily arrested at the G2 checkpoint in an asymmetric fashion. Therefore, it has an important role in ensuring genomic integrity, and that this role has a pronounced mother-daughter asymmetry.</p><br />
<img src="http://upload.wikimedia.org/wikipedia/commons/0/06/Sic1_David_Morgan10-5.jpg" title="Principle of Sic1" description="Over expression of Sic1 can stop all yeast cells to G1 phase."><br />
<p>After phosphorylation, phospho-Sic1 is specifically recognized by the F-box protein Cdc4, which leads Sic1 being ubiquitinated by the Cdc34±SCF complex (E3). The recognizing and binding by Cdc4 is based on the Sic1’s 9 Cdc4 phospo-degrons (CPDs, figure. a). Several phosphorylation sites contributed to Sic1 instability, with an order of Thr45, Ser76, Thr5,Thr33 and other less significant sites. The immunocipients after selectively phosphorylation as figure e showed suggested that at least six sites phosphorylation is necessary for the Cdc4 recognizing and binding with Sic1. Furthermore, the culture of GAL1-SIC1 constructs strain showed less than 6 sites are phosphorylated is not sufficient for SIC1 degradation in vitro(Nash et al., 2001).</p><br />
<img src="https://static.igem.org/mediawiki/parts/8/8b/Sic1-mutate.png" title="Phosphorylation Sites" description="N-terminal phosphorylation sites to be mutated."><br />
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<h3>Alternative Splicing Device</h3><br />
<p>In yeast cells, alternative splicing is a common process in before gene transcription. The splicing sites are located in 5'UTR of introns and can by recognized and spliced by some splicesome. Alternative splicing can produce two isoforms from one gene, thus can be used in our project to monitor whether the Sic1 system work efficiently. Here we find a intron with alternative splicing sites in yeast cells named SRCI intron.</p><br />
<br />
<h4>Src1 Intron and Hub1</h4><br />
<p> SRC1 intron has two 5' splicing site whose efficiency is regulated by protein hub1. Originally, the existence of the intron would produce two mature mRNA in proportion. After engineering, the existence of protein hub1 regulates to the preservation of intron precisely, which would make the following mRNA be expressed normally or not. As for the silencing of wild type gene HUB1, we choose CRISPRi that is comparably easy to use reversibly.</p><br />
<p>Alternative splicing substantially increases the gene product diversity and is a major source of cell type differentiation. A good example is the alternative splicing of Saccharomyces cerevisiae SRC1 pre-mRNA, which is promoted by the conserved ubiquitin-like protein Hub1. It can function through binding non-covalently to a conserved element termed HIND in the spliceosomal protein Snu66. Such binding makes the splicesome target sites change and moderately alters spliceosomal interactions. </p><br />
<p>Hub1 is a ubiquitin-like modifier (UBL) that covalent modify the proteins. Interest enough, it harbors several different to other UBLs in which it possesses a C-terminal double tyrosine motif while others having a GG motif. The Snu66, a tri-snRNP in yeast spliceosome, possesses with two N-terminal HINDs (Hub1-INteraction Domain). The Hub1–HIND interaction comprises a strong salt bridge accompanied by several hydrophobic contacts and high affinity. Such binding modifies the spliceosome rather than modulating the properties of an individual binding partner. Hub1-controlled splicing occurs universally in eukaryotes. SR proteins and hnRNPs involved in spliceosome targeting do not seem to exist in S. Cerevisiae , and thus the Hub1-dependent mechanism may be evolutionarily older. </p> <br />
<p>Scr1 is a protein in yeast having alternative splicing sites in its intron. The characteristic differential Hub1 dependence of SRC1 alternative splicing requires the tandem arrangement of overlapping 5’ splice sites. The Hub1 binding spliceosome can splice the intron from both downstream 5’ sites as well as the upstream 5’ sites with preference to the former one.</p><br />
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<img src="https://static.igem.org/mediawiki/parts/9/96/SICI_principle.jpg" title="Src1 intron principle" description="Alternative splicing of Src1" /><br />
<br />
<p>Showed in Figure.above When cutting in the upstream splice sites, the exons flanking around it would be translated as a fusion protein Scr-S. However, when cutting at the down stream one, the left 4bp in intron would result in a frame shift, thus only the forward exon can express named Scr-L.</p><br />
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<h4>dCas9 CRISPR interface system</h4><br />
<p>CRISPR shorts for Clustered Regularly Interspaced Palindromaic Repeats system, which can be targeted to DNA using RNA, enabling genetic editing of any region of the genome in many organisms.(Cho, Kim, Kim, & Kim, 2013). In the type II CRISPR/Cas system, a ribonucleoprotein complex formed from a single protein (Cas9), a crRNA, and a trans-acting CRISPR RNA (tracrRNA) can carry out efficient crRNA-directed recognition and site-specific cleavage of foreign DNA(Deltcheva et al., 2011). After mutated the endonuclease domains of the Cas9 protein, it creates a programmable RNA-dependent DNA-binding protein. The sgRNA consists of three domains: a 20 nt complementary region for specific DNA binding, a 42 nt hairpin for Cas9 binding (Cas9 handle), and a 40 nt transcription terminator derived from S. Pyogenes. After translation, the Cas9 binds to sgRNA to form a protein-RNA complex, which can recognize target sites in the genome sequence and bind to it. Then, it could block RNA polymerase and transciption elongation.</p><br />
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<img src="https://static.igem.org/mediawiki/2013/7/74/Figure4._The_structure_of_sgRNA.png" title="sgRNA" description="sgRNA structure" /><br />
<img src="https://static.igem.org/mediawiki/parts/1/13/CRISPRi_principle.png" title="Principle of dCAS9" description="Principle of the function of dCAS9 and guide RNA." /><br />
<!---[[File:Figure4._The_structure_of_sgRNA.png ]]---><br />
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<h3>Microfluidic Device</h3><br />
<p>Based physically on the chip, microfluidics can automatically control the biology process. In our project, the microfluidics plays a role as the movie theater, for cell cycle control. We designed a channel which can capture the yeast cell and then alternatively inject medium of different concentrations. Therefore, the yeast cell cycle can be synchronized. Furthermore, due to its transparent attribution, the chip can project the fluorescent lights from the yeast cell sub-locations. Consequently, the magic film can be shown to us. </p><br />
<p>Microfluidic devices possess many advantages such as large-scale integration, fast analyses, and considerably reduced reagent consumption (Comparative study and improvement of current cell micro-patterning techniques). Nowadays many designs are based on cell loading and culturing in a microfluidic channel with particular trapping structures such as micro-well arrays, and micro-cup arrays and micro-chambers. In our project, we design a cell loading technique based on gas absorption of degassed polydimethylsiloxane (PDMS). Because the balance concentration of gas dissolved in PDMS is proportional to the partial pressure of the gas around it, so that one can degas a piece of PDMS by placing it in vacuum. </p><br />
<p>Our cell loading device is a one level PDMS with an array of triangle cavities connected to one or both sides of the main fluidic channel. After degassed, yeast cells in the buffer solutions can be introduced into the device’s triangle cavities by gas absorption therein. Through changing the cell density in the buffer, we can control the number of the yeast cells loaded into each triangle cavity. Statistically, the ratio of cavities with a single cell can reach 40%. Consequently it is convenient to monitor the cell number and cell phenotypic variations because the yeast cells can grow in the micro-triangle-cavities in one layer.</p><br />
<img src="https://static.igem.org/mediawiki/igem.org/e/ec/Figure6._Masks_for_PDMS_microfluidic_device_fabrication..png" title="Microfluidic" description="the structure of Microfluidics"><br />
<p>Figure 1. Masks for PDMS microfluidic device fabrication.a : Three micro-triangle-cavities at one side of the main channel. b : One single channel for cell culture (100 microcavities). c : Seven parallel channels for different cell culture environments(Luo et al., 2008).</p><br />
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</div><br />
</div></div>BGI K2http://2013.igem.org/Team:Shenzhen_BGIC_ATCG/teamTeam:Shenzhen BGIC ATCG/team2013-09-27T22:25:17Z<p>BGI K2: </p>
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<li><img src="https://static.igem.org/mediawiki/2013/7/7c/Wet2013.jpg" title="Our Consortium" description="All members of team Shenzhen-BGIC-ATCG." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/a/aa/Guangzhou2013.jpg" title="SCUTers and SCNUers" description="Two universities from Guangzhou city. We have three six members from South China University of Technology and five from South China Normal University."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/ee/Wh2013.jpg" title="WHUers & HUSTers & CGUer" description="Three Universities from Wuhan city. Three from Wuhan University, two from Huazhong University of Science and Technology, and one from China University of Geography."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/8/87/Scu22013.jpg" title="SCUers" description="Four guys from Sichuan University." /></li><br />
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<li><img src="https://static.igem.org/mediawiki/2013/0/0d/Seu2013.jpg" title="SEUers" description="Two girls from Southeast University." /></li><br />
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<h3>Consortium</h3><br />
<p>Shenzhen_BGIC_ATCG is a special consortium. Team members are from all over the country. In the 3rd or 4th year in university, some undergraduate students came to Shenzhen and involved in a joint program called Innovation Program in BGI College. After lectures introducing synthetic biology and iGEM given by our instructor K2, 29 students from ten schools have joined the consortium BGIC_ATCG, which include:<br> - Huazhong University of Science and Technology, China<br> - Sichuan University, China<br> - South China University of Technology, China<br> - Wuhan University, China<br> - University of Electronic Science and Technology of China, China<br> - Southeast University, China<br> - South China Normal University, China<br> - Anhui Medical University<br> - China University of Geosciences<br> - Jinan University </p> </div><br />
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<li><img src="https://static.igem.org/mediawiki/2013/7/7d/Promoter2013.jpg" title = "Promoter Group" description="All members of Promotor Group"/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/64/Lanyi2013.jpg" title = "Lan Yi" description=""/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/7/74/Lidongdong2013.jpg" title = "Li Dongdong" description=""/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/c/c3/Linkequan2013.jpg" title = "Lin Kequan" description=""/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/9/99/Liuyang2013.jpg" title = "Liu Yang" description=""/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/8/8a/Zhushuang2013.jpg" title = "Zhu Shuang" description=""/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/7/7b/Weiwei2013.jpg" title = "Wei Wei" description=""/></li><br />
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<h3>Promoter Group</h3><br />
<p> Zhu Shuang, Lin Kequan, Wang Boxiang from Wuhan University, Wei Wei, Zheng Bingwei, Lan Yi in HUST and Liu Yang in CUG work together for the promotors.</p><br />
<p> Zhu Shuang: Studious and very outgoing. Being popular with everyone.</p> <br />
<p> Lin Kequan: A steady fellow ,with serious appearance and hot heart .Like to make friends</p><br />
<p> Wang Boxiang: An easy-going guy. I am a crazy man, Wang said.</p><br />
<p> Wei Wei: Always act as a gentleman, never refute to help others.<br />
<p> Zheng Bingwei: Looks like a common students but really different when you know him well. </p><br />
<p> Lan Yi: Like laughing, a smart and pretty girl</p><br />
<p> Liu Yang: A shy and interesting girl , from the CUG </p><br />
</div><br />
<br />
<div id="board3" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery3"><br />
<li><img src="https://static.igem.org/mediawiki/2013/d/dd/Degradation2013.jpg" title="Degradation Group" description="All member from Degradation Group." /> </li><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/61/Guanrui2013.jpg" title="Guan Rui" description= ""/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/1/19/Hefunan2013.jpg" title="He Funan" description = ""/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/0/03/Lilin2013.jpg" title="Li Lin" description = ""/></li><br />
<li><img src="https://static.igem.org/mediawiki/igem.org/f/f5/Wangrui2013.jpg" title= "Wang Rui" description=""/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/e0/Zhangyaolei2013.jpg" title="Zhang Yaolei" description = ""/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/2/2e/Zhouwanling2013.jpg" title="Zhou Wanling" description = ""/></li> </ul></div><br />
<div id="box3" class="box"><br />
<h3>Degradation Group</h3><br />
<p>Guan Rui from SEU and He funan, Wang Rui, Lin Li, Yaolei Zhang from UESTC made their efforts to the degradation parts.</p><br />
<p>Wang Rui: A smart,pretty and slim girl from UESTC </p><br />
<p>Guan Rui: Cute and excellent girl.Had a nickname, "a little Pokemon". With a strong will in doing what she likes to do.</p><br />
<p>He funan: I'm a senior student from UESTC, major in biotech, I am also a member of team degradation. I'm keen to learn and have passion in life science.</p><br />
<p> Lin Li:A dreaming girl, works hard to be a good "Time Commander".</p><br />
<p> Zhang Yaolei : A somewhat introverted but confident boy,who always believes that "Tomorrow Will Be Brighter".</p><br />
</div><br />
<br />
<div id="board4" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery4"><br />
<li><img src="https://static.igem.org/mediawiki/2013/5/59/Targeting2013.jpg" title = "Targeting Group" description="All members of Targeting Group."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/8/8f/Zhangjinchun2013.jpg" title = "Zhang Jinchun" description=""/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/2/28/Lixiang2013.jpg" title = "Li Xiang" description=""/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/3/31/Xuyanhui2013.jpg" title = "Xu Yanhui" description=""/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/4d/Yuyang2013.jpg" title = "Yu Yang" description=""/></li> </ul></div><br />
<div id="box4" class="box"><br />
<h3>Targeting Group</h3><br />
<p>LiXiang,Xu Yanhui,Wu Fanzi, Yu yang and Zhang Jinchun from SCU: responsible for targeting peptide,XFP,terminators design and experiments.</p><br />
<p>Zhang Jinchun: A creative person, outgoing and friendly.Everyone called her "Aunt Chun" due to her maturation and proficient.</p><br />
<p>Xu Yanhui : Studious Student, always fighting for her dream</p><br />
<p>Li Xiang: A good man.</p><br />
<p>Yu Yang: A Warm-hearted boy, major in biotechnology. Love sports,especially the basketball.</p><br />
</div><br />
<br />
<div id="board5" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery5"><br />
<li><img src="https://static.igem.org/mediawiki/2013/3/3a/Regulator2013.jpg" title = "Synchronization Group" description="All members of Synchronization Group."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/9/9c/Xieqiaoling2013.jpg" title = "Xie Qiaolin" description=""/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/6d/Zhangaiping2013.jpg" title = "Zhang Aiping" description=""/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/5/52/Linqiongfen2013.jpg" title = "Lin Qiongfen" description=""/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/c/c7/Chenchengxuan2013.jpg" title = "Cheng Chengxuan" description=""/></li> </ul></div><br />
<div id="box5" class="box"><br />
<h3>Synchronization Group</h3><br />
<p>Xie Qiaolin, Zhang Aiping, Lin Qiongfen, Cheng Chengxuan working together in the Synchronization Group</p><br />
<p>Xie Qiaolin: In the words of Adam Smith (The Wealth of Nations, 1776): “Science is the great antidote to the poison of enthusiasm and superstition.”So come on,let's work hard</p><br />
<p>Lin Qiongfen: Just doing myself, not for best, but for better.</p><br />
<p>Cheng Chengxuan: Nothing to say, just work and being great.</p><br />
</div><br />
<br />
<div id="board6" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery6"><br />
<li><img src="https://static.igem.org/mediawiki/2013/c/c3/Intron2013.jpg" titile="Splicing Group" description = "All members of Splicing Group"/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/0/01/Gongjianhui2013.jpg" title = "Gong Jianhui" description= ""/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/a/a6/Chenshihong2013.jpg" title = "Chen Shihong" description= ""/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/6d/Guchenguang2013.jpg"title = "Gu Chenegguang" description= ""/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/9/99/Liangjiale2013.jpg"title = "Liang Jiale" description= ""/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/a/a7/Luyanping2013.jpg" title = "Lu Yanping" description= ""/></li> </ul></div><br />
<div id="box6" class="box"><br />
<h3>Splicing Group</h3><br />
<p>Gong Jianhui,Chen Shihong,Gu Chenguang,Lu Yanping Liang Jiale,from South China University of Technology, are responsible for the Src1 and Mer1 intron design and experiments.</p><br />
<p>Gong Jianhui: Our team leader, good at bioinformatics and synthetic biology.</p><br />
<p>Chen Shihong:Due to his cute lips, we call him "little sausage".</p><br />
<p>Gu Chenguang: A slightly fat boy and seems to be shy. Everybody loves that guy.</p><br />
<p>Lu Yanping:......................................</p><br />
<p>Liang Jiale:..........................................</p><br />
</div><br />
<br />
<div id="board7" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery7"><br />
<li><img src="https://static.igem.org/mediawiki/2013/f/f1/Chengyichun2013.jpg" title = "Cheng Yichun" description=""/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/2/24/Zhongna2013.jpg" title = Zhong Na" description=""/></li> </ul></div><br />
<div id="box7" class="box"><br />
<h3>Microfluidic Group</h3><br />
<p>Chen Yichun of SCNU, Zhong Na of JNU work for the microfulidic part.</p><br />
<p>Cheng Yichun: Outgoing and interesting to talking with for his humorous </p><br />
<p> Zhong Na: Good at cooking, we all love the meal she did.</p><br />
</div><br />
<br />
<div id="board8" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery8"><br />
<li><img src="https://static.igem.org/mediawiki/2013/2/21/Modeling2013.jpg" title = "Modeling Group" description="All members of Modeling Group"/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/d/d1/Liushuang2013.jpg"title = "Liu Shuang" description=""/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/0/0e/Qiubitao2013.jpg"title = "Qiu Bitao" description=""/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/d/d4/Zhouyang2013.jpg" title = "Zhou Yang" description=""/></li> </ul></div><br />
<div id="box8" class="box"><br />
<h3>Modeling Group</h3><br />
<p> Liu Shuang from SEU,Zhou Yang from SCUT and Qiu Bitao from BGI working for the modelling</p><br />
<p> Liu Shuang: One of the smartest girl in our team, majoring in Mathematics and Application Mathematics.</p><br />
<p> Zhou Yang: Loves iGEM and working hard in modeling</p><br />
<p> Qiu Bitao: Studious</p><br />
</div><br />
<br />
<div id="board9" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery9"><br />
<li><img src="https://static.igem.org/mediawiki/2013/8/8c/Niming.jpg" title = "Dr. Ni Ming"/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/a/aa/Kangkang2013.jpg" title = "Kang Kang"/></li><br />
</ul></div><br />
<div id="box9" class="box"><br />
<h3>Instructors</h3><br />
<P>Prof. Dr. Yang Huanming: Advisor in safety and bio-ethic issues, iGEM judge.</p><br />
<p>Dr. Ni Ming: Advisor in systems biology and microfluidics.</p><br />
<p>Kang Kang: Team Instructor, project designer, iGEM judge.</p><br />
</div><br />
<br />
</div></div>BGI K2http://2013.igem.org/Team:Shenzhen_BGIC_ATCG/storiesTeam:Shenzhen BGIC ATCG/stories2013-09-27T22:22:58Z<p>BGI K2: </p>
<hr />
<div><!----------------------<br />
<br />
<br />
Title: Stories<br />
Team: Shenzhen_BGIC_ATCG<br />
Author: BGI_K2<br />
Twitter: @BGI_K2<br />
Email: im.ss.kk@gmail.com<br />
<br />
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<div class="navleftholder"><br />
<ul class="navleft"><br />
<li><a id="navleftsub1" class="navleftsub" href="#board1">The Magic</a></li><br />
<li><a id="navleftsub2" class="navleftsub" href="#board2">Cyclin Promoters</a></li><br />
<li><a id="navleftsub3" class="navleftsub" href="#board3">Reporter Modification</a></li><br />
<li><a id="navleftsub4" class="navleftsub" href="#board4">Degradation Tags</a></li><br />
<li><a id="navleftsub5" class="navleftsub" href="#board5">Targeting Peptides</a></li><br />
<li><a id="navleftsub6" class="navleftsub" href="#board6">Cell Synchronization</a></li><br />
<li><a id="navleftsub7" class="navleftsub" href="#board7">Alternative Splicing Device</a></li><br />
<li><a id="navleftsub8" class="navleftsub" href="#board8">Microfluidic Device</a></li><br />
</ul></div><br />
<br />
<div id="board1" class="board"></div><br />
<div class="media"><br />
<br />
<ul id="myGallery1"><br />
<li><img src="https://static.igem.org/mediawiki/2013/5/58/P%2BD%2BT%2BS.png" title="Genetic circuit of cell cycle version #4" description="To make the magic more fantasy, alternative splicing device acting synchronously with the synchronization device." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/1/10/Chip.gif" title="Microfuidics Device" description="Cell magic in microfuidics device." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/e4/Yeast.gif" title="DT X TP X CCR X" description="Version #1, cell magic without degradation tags, targeting peptides and cell cycle regulator."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/7/79/Yeast_D.gif" title="DT √ TP X CCR X" description="Version #2, cell magic with degradation tags, without targeting peptides and cell cycle regulator." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/e6/Yeast_DT.gif" title="DT √ TP √ CCR X" description="Version #3, cell magic with degradation tags and targeting peptides, without cell cycle regulator." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/c/c1/Yeast_DTR.gif" title="DT √ TP √ CCR √" description="Version #4, cell magic with degradation tag, targeting peptides and cell <li>cycle regulator." /></li><br />
</ul></div><br />
<div id="box1" class="box"><br />
<h3>The Magic</h3><br />
<p>Life, the most brilliant magic in the universe, celebrated with the ability of reproduction and revolution. And the magic, is based on a sophisticated mechanism, called cell cycle. Most genetic reactions in a cell are regulated, directly or indirectly, by cell cycle. As we wish to build artificial lives, it is worthwhile to learn from the exquisite design of creature, to make use of cell cycle. As a pioneer study, we try to performing a "Cell Magic", by capturing cell cycle with fantastic reporters. By grasping the usage of cell cycle tools, we are promised to direct refined actions in a cell. For instance, when producing Paclitaxel (harmful to centrosome in S phase) with a cell factory, we may able to transport it out before it comes detrimental. </p><br />
<p>So this is our project, the "Cell Magic", to engineer two versions of cell cycle based magic in both budding yeast and <i>E. coli</i>. We will present the blueprint of our stories by the yeast version. </p><br />
<h4>Version #1, Degradation Tag X, Targeting Peptides X, Cell Cycle Regulator X</h4><br />
<p>Promoters of cyclins, which can initial transcription in specific phase in a cell cycle, are selected to produce different fluorescence proteins in G1/SG2/M phases respectably. However, in our first version of story, with low natural degradation rate, fluorescence proteins would remain in cell after their promoters stop working. So colors for each phase cannot be distinguished, as the figure shows.</p><br />
<img src="https://static.igem.org/mediawiki/2013/e/e4/Yeast.gif" title="DT X TP X CCR X" description="Version #1, cell magic without degradation tags, targeting peptides and cell cycle regulator." width=47%/><br />
<img src="https://static.igem.org/mediawiki/2013/3/36/P.png" width=50% ><br />
<h4>Version #2, Degradation Tag √, Targeting Peptides X, Cell Cycle Regulator X</h4><br />
<p>In the second version, degradation tags are added to the reporters to solve this problem. All fluorescence proteins are thought to be degraded in 15 minutes which is far less than the period of a cell cycle.</p><br />
<img src="https://static.igem.org/mediawiki/2013/7/79/Yeast_D.gif" title="DT √ TP X CCR X" description="Version #2, cell magic with degradation tags, without targeting peptides and cell cycle regulator." width=47%/><br />
<img src="https://static.igem.org/mediawiki/2013/d/df/Gc.P%2BD.png" width=50%><br />
<h4>Version #3, Degradation Tag √, Targeting Peptides √, Cell Cycle Regulator X</h4><br />
<p>While we are not sure if degradation tags can work as expected, we try to turn the time magic into a time-space one. Reporters are located to different cell structures by targeting peptides. Different phase with different color shines in different organelle, that is the third version.</p><br />
<img src="https://static.igem.org/mediawiki/2013/e/e6/Yeast_DT.gif" title="DT √ TP √ CCR X" description="Version #3, cell magic with degradation tags and targeting peptides, without cell cycle regulator." width=47%/><br />
<img src="https://static.igem.org/mediawiki/2013/8/8d/P%2BD%2BT.png" width=50%><br />
<h4>Version #4, Degradation Tag √, Targeting Peptides √, Cell Cycle Regulator √</h4><br />
<p>To capture the magic macroscopically, we developed a microfluidic device to synchronize all cells into the same phase (G1). So synchronization devices was added to the forth version. </p><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Chip.gif" title="Microfuidics Device" description="Cell magic in microfuidics device." width=35% /><br />
<img src="https://static.igem.org/mediawiki/2013/5/58/P%2BD%2BT%2BS.png" width=62% /><br />
<p>To make the magic more fantasy, alternative splicing device acting synchronously with the synchronization device (a translational unit of a cyclin regulator), will splice the targeting peptide tail when cell are being synchronized to G1 phase, so all cells turn green; while synchronization device not working, targeting peptide remains in the reporter so only mitochondria turn green. In this way, the cell phase and statues can be reported by fluorescences.</p><br />
<img src="https://static.igem.org/mediawiki/2013/c/c1/Yeast_DTR.gif" title="DT √ TP √ CCR √" description="Version #4, cell magic with degradation tag, targeting peptides and cell cycle regulator." width=47%/><br />
<img src="https://static.igem.org/mediawiki/2013/d/d9/P%2BD%2BT%2BS%2BI%2BC.png" /><br />
<!----------<br />
<img src="https://static.igem.org/mediawiki/2013/6/6f/Chip.png"/><br />
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<ul id="myGallery2"><br />
<li><img src="https://static.igem.org/mediawiki/2013/3/31/Sce04111.png" title="Cell cycle of yeast"/><br />
</ul></div><br />
<div id="box2" class="box"><br />
<h3>Cyclin Promoters</h3><br />
<h4>Yeast Version</h4><br />
<p> As we all know, different proteins are planned to be expressed along the cell cycle. And their mRNAs are transcribed through the specific promoters. These promoters locate at their upstream sequences and can be recognized by the RNA polymerase, which can initiate such process. There are databases containing the upstream 600 amino acid sequence in the upstream of DNA, which can be transcript in the G1, S, G2 and M phase, respectively</p> <br />
<!----- [http://www.genome.jp/kegg-bin/show_pathway?org_name=sce&mapno=04111&mapscale=&show_description=hide]. ----><br />
<p>Previous study (Wolfsberg et al., 1999) demonstrated that cell cycle specific promoters posses their conserved five to six base pairs. Thus, we found the high-confidence 600 promoter-containing sequences in database that harbor the paper-mentioned 5/6 bp sequence. Consequently, we pick up the upstream 600bp sequence of cln2, cln3, clb2, clb5 and clb6.</p><br />
<p>The Clb2 gene is highly expressed in G2 phase, and the genes are very strongly induced by GAL-CLB2, whereas GAL-CLN3 appears somewhat repressive Spellman et al., 1998)</p><br />
<img src="https://static.igem.org/mediawiki/2013/3/31/Sce04111.png" title="Cell Cycle KEGG" description="KEGG diagram for core cell cycle." width=98% /><br />
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<h4><i>E. coli</i> Version</h4><br />
<p> Cell cycle is a complex process and can be separated into G0, G1, S, G2, M phase. In each phase, distinct transcription factors help the phase-specific gene express through recognizing their promoters. Thus, these promoters are phase-specific too and can be fused with other genes in order to express such gene in a defined cell cycle phase.The well-regulated cell cycle in E.coli consists of three key events: DNA replication, nucleon segregation and the initiation of cell division. The replication initiator is DnaA protein; the second step relates to MukB protein and the cell division activator is the FtsZ protein. In our project, we take full aadvantage of the two initiators DnaA and FtsZ - using their promoter to control the transcript of specific genes binding behind them in the first step or last step of <i>E. coli</i> cell cycle.</p><br />
<h5>DnaA protein</h5><br />
<p>As mutation strain demonstrated, dnaA gene is absolutely required for initiation at oriC. When the concentration of DnaA increases, there is increased initiation?, indicating that DnaA protein is the switch for initiation (Atlung, Løbner-Olesen, & Hansen, 1987). Biochemical studies have shown that, with the help of accessory proteins, DnaA protein binds to five sites within oriC, therefore, leading to strand opening of a region containing three 13-mer repeats. This opening results in the begin of bidirectional replication (Atlung, Løbner-Olesen, & Hansen, 1987).</p><br />
<img src="https://static.igem.org/mediawiki/2013/3/3b/DnaAp.png" title="dnaA Promoter" description="dnaA promoter selected for S1 phase." /><br />
<img src="https://static.igem.org/mediawiki/2013/2/2a/GyrBp.png" title="gyrB Promoter" description="gyrB promoter selected for S2 phase." /><br />
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<h5>FtsZ</h5><br />
<p>The cell itself seems to regard FtsZ gene expression as the commitment to division, because of the fact that endogenous division inhibitors have FtsZ as target(Bi & Lutkenhaus, 1990). Increasing FtsZ concentration resulted in excess cell division, with the cell size decreasing (Ward & Lutkenhaus, 1985), vice verse. Thus the critical role of FtsZ in division is similar to that of DnaA in replication initiation. FtsZ protein exists randomly throughout cytoplasm during cell elongation, while polymerizes into a membrane-associated ring at the precise site of cell division (Bi & Lutkenhaus, 1991). Just before the appearance of a visible constriction. The ring decreasing along with the septation and FtsZ is dispersed again at completion. Such ring formation is presumed to be essential for cell division, since it cannot be observed in the presence of the cell division inhibitors (Bi & Lutkenhaus, 1993) but is always present when and where septa are formed.</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/6a/FtsQp.png" title="ftsQ Promoter" description="ftsQ promoter selected for S2 phase." /><br />
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<h3>Reporter Modification</h3><br />
<p> The XFP, as reporter in our project has been modified through</p> <br />
<p>1) removed the stop codon in them, 2) modified with 23# prefix and suffix, and then 3) added the terminator within stop codon to them. </p><br />
<p> Furthermore, all BioBricks were added with 23# prefix and suffix, which ensures it to be standard.<br />
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<h3>Degradation Tags</h3><br />
<h4>Yeast Version</h4><br />
<p>In yeast cell, protein degraded through several mechanisms, a major one is the ubquitin- pathway. In detail the signal in substrates can be recognized by the enzyme E3, which transport the ubiqutin from E2 to such protein. And through such way, the ubiquited protein would finally be degraded by the proteasome. </p><br />
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<h5>PEST</h5><br />
<p>There are two types of cyclins in the budding yeast. One kind contains PEST sequence at C-terminal: Cln1, Cln2, Cln3, Pcl1, Pcl2. Another kind is Clb1~6 which posses 9 conserved amino acid sequence in N-terminal named D-box (destruction box). The D-box are necessary for later kind cyclins degradation in M phase and can function in same way when combined with other proteins. In our study, we utilized the D-box, PEST sequence and ubiquitin-mediated pathway for fluorescent proteins degradation.</p><br />
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<h5>Poly-ubiquitin</h5><br />
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<p>Ubiquitin, a highly conserved 76-residue protein, was involved in the ubiquitin–proteasome pathway of protein proteolysis, which is a fundamental way for protein turnover, signal transduction and cell cycle control. For example, the degradation of CDC34 through such way is necessary for S phase start; another instance is that APC\C(anaphase-promoting complex\cyclosome) is degraded through ubiquitination, which is the foundation of M phase beginning. And both activated by Cdc28-cyclins</p><br />
<p>The mechanism of ubiquitin-mediated protein degradation can be separated into three continuous enzyme catalyze steps. First 1) a ubiquitin is activated by the E1 (ubiquitin-activating enzyme) through a thioester linkage,and then 2)added into the a small ubiquitin-carrier E2. Finally,3) through the E3 ubiquitin protein ligase, this ubiquitin complex was conjugate to the ε-amino group of lysine residues in substrate proteins, forming a glycyllysine isopeptide bond (Hershko, 1997).In some conditions, even E3 enzymes themselves carry ubiquitin as a thioester (Huibregtse et al., 1995).</p><br />
<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/5/5b/Ubiquitylation.svg/800px-Ubiquitylation.svg.png" title="Ubiquitin" description="Function of fusion ubiquitin."><br />
<p>In our project, we design a poly-ubiquitin(5 ubiquitin combined) BioBricks which can directly degrade the proteins fused with it.</p><br />
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<h5>D-box</h5><br />
<p>The best studied substrates of ubiquitin- and APC/C-mediated proteolysis are the mitotic cyclins (Murray, Solomon, & Kirschner, 1989) Mitosis-exit inducer, a cyclin B, are highly dependent on its 90 residues for ubiquitin-mediated proteolysis. Analysis of its N-terminal region demonstrated the sequence essential for cyclin proteolysis, called ‘destruction box’(D-box). Meanwhile, when cyclin destruction started, substrates containing D-boxes were rapidly poly-ubiquitinated. The consensus motif in B-type cyclins is RXALGXIXN. For much of the cell cycle, the D-box may not be recognized with high affinity, and when it is recognized, the ‘bait’ construct becomes highly unstable(Yamano, Tsurumi, Gannon, & Hunt, 1998). </p><br />
<img src="http://mol-biol4masters.masters.grkraj.org/html/Cell_Cycle_And_Its_Regulation_files/image022.jpg" title="D-box" description="Function of destruction box." /><br />
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<p>When the D-box exists in the protein N-terminal, this protein is recognized more easily by the E1 and then captured into the ubiquitin-mediated degradation pathway. Thus, we combined it to the fluorescent proteins’ N-terminal to fasten their degradation and avoid veiling the following fluorescence.</p><br />
<h4><i>E. coli</i> Version</h4><br />
<p>In E.coli, the adaptor SspB tethers ssrAtagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. Which means, a variation of the WT SsrA tag sequence (K1051206, K1051207 and K1051208) will accelerate the degradation of proteins when fused to their C-terminal. Thus the degradation rates are dependent on concentration of proteases and binding mediators. In order to fuse degradation tags freely on the C-terminal of protein, we add TAATAA to the tail of tags.</p><br />
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<h3>Targeting Peptides</h3><br />
<p>A target peptide is a short (3-70 amino acids long) peptide chain that directs the transport of a protein to a specific region in cell, including nucleus, mitochondria, endoplasmic reticulums (ERs), chloroplasts, apoplasts, peroxisomes and plasma membrane. Targeting peptide can exists in both N-terminal, C-terminal and internal sequence of a precursor protein. And after transported, some target peptides are cleaved by signal peptidases.</p><br />
<p>In our project we utilized 19 peptides target to 9 sub-locations in yeast cells, and when combined with fluorescent proteins, such region can be marked by different colors.</p><br />
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<h5>Mitochondria</h5><br />
<p>Though it accounts a small ratio in the cell space, mitochondria possess about 10% to 15% proteins encoded by nuclear genes in eukaryotic organisms. These proteins are synthesized in cytosol and then recognized by the membrane receptors of mitochondria. Translocases in the outer and inner membrane of mitochondria mediate the import and intra-mitochondrial sorting of these proteins. ATP is used as an energy source; Chaperones and auxiliary factors assist in folding and assembly of mitochondrial proteins into their native, three-dimensional structures. </p><br />
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<img src="https://static.igem.org/mediawiki/2013/1/1f/Figure1.protein-import_pathways_for_mitochondrial_proteins.png" title="Import pathway" description="Mitochrondira import pathway" /><br />
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<p>As shown in the figure above, beta-barrel outer-membrane proteins (dark green), precursor proteins (brown) with positively charged amino-terminal presequences and multispanning inner-membrane proteins (blue) with internal targeting signals are recognized by specific receptors of the outer mitochondrial membrane (TOM) translocases Tom20, Tom22 and/or Tom70. The precursor proteins are then translocated through a small Tom proteins of the TOM complex, Tom40 pore, which the TOM complex contains two or three.</p><br />
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<h5>Peroxisomes </h5><br />
<p>The import of post-translational matrix protein into peroxisomes depends on either of the two peroxisomal targeting signals (PTS), PTS1 and PTS2. PTS2-driven import is facilitated by a complex in the membrane. Under oleic acid-inducing growth conditions, there is a ternary core complex of approximate 150 kDa in the cytosol, which consists of Fox3p,Pex7p and Pex18p. Fox3p is imported as a dimer, while other two are bind in monomeric forms.</p><br />
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<img src="https://static.igem.org/mediawiki/igem.org/7/73/Figure2._Schematical_model_of_the_early_steps_of_PTS2-driven_import.png" title=”Import pathway“ description=”Peroxisome import pathway“/><br />
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<p>As study mentioned there are four steps involved in PTS2-driven import. The first step is the recognition of dimerized Fox3p by Pex7p through its PTS2 in the cytosol. In a second step, the Pex7p–Fox3p complex interacts with Pex18p, which targets the PTS2 pre-import complex to the peroxisomal membrane. The third step is the docking process, involving the interaction between Pex7p and the integral membrane protein Pex13p. As a final step of these early steps in the PTS2 import cascade, PTS2 receptor dissociation takes place during or after its assembly into large oligomeric complexes containing Pex14p and Pex13p. Pex18p remains at the peroxisomal membrane in the form of a large-molecular-weight complex in conjunction with Pex14p and/or Pex13p, from where it might be released to the cytosol(Grunau et al., 2009).</p><br />
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<h5>Actin </h5><br />
<p>In yeast, the cortical actin cytoskeleton seems to specify sites of growth of the cell surface. Because the actin-binding protein ABP1p is associated with the cortical cytoskeleton of Saccharomyces cerevisiae, it might be involved in the spatial organization of cell surface growth. ABP1p is localized to the cortical cytoskeleton and its overproduction causes assembly of the cortical actin cytoskeleton at inappropriate sites on the cell surface, resulting in delocalized surface growth. ABP1p is a novel protein with a 50 amino-acid C-terminal domain, which is very similar to the SH3 domain in the non-catalytic region of nonreceptor tyrosine kinases (including those encoded by the proto-oncogenes c-src and c-abl), in phospholipase C gamma and in alpha-spectrin. They also identified an SH3-related motif in the actin-binding tail domain of myosin-I. The identification of SH3 domains in a family of otherwise unrelated proteins that associate with the membrane cytoskeleton indicates that this domain might serve to bring together signal transduction proteins and their targets or regulators, or both, in the membrane cytoskeleton (Drubin, Mulholland, Zhu, & Botstein, 1990; Khorasanizadeh, 2004).</p><br />
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<h5>Nucler</h5><br />
<p>Histones are nuclear proteins package DNA into nucleosomes, and they are responsible for maintaining the shape and structure of a nucleosome. One chromatin molecule is composed of at least one of each core histones per 100 base pairs of DNA.[The Nucleosome: From Genomic Organization to Genomic Regulation.] There are five families of histones known to date, termed H1/H5, H2A, H2B, H3, and H4. H2A is considered a core histone, along with H2B, H3 and H4. Core formation first occurs through the interaction of two H2A molecules(Acid, 2004). Then, H2A forms a dimer with H2B; the core molecule is complete when H3-H4 also attaches to form a tetramer.</p><br />
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<h5>Vacuolar Membrane</h5><br />
<p>The ZRC1 gene encodes a multicopy suppressor of zinc toxicity in Saccharomyces cerevisiae; however, previously it was reported that the expression of ZRC1 was induced when the intracellular zinc level was decreased. The COT1 and ZRC1 genes of Saccharomyces cerevisiae are structurally related dosage-dependent suppressors of metal toxicity. COT1 confers increased tolerance to high levels of cobalt; ZRC1 confers increased tolerance(Conklin, Culbertson, & Kung, 1994).Zrc1 has six putative trans-membrane domains, and Zrc1-GFP fusion protein was localized to the vacuolar membrane. Zrc1 function as a mechanism to maintain the zinc homeostasis in yeast(Miyabe, Izawa, & Inoue, 2001).</p><br />
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<h3>Cell Synchronization</h3><br />
<p>As we known, the yeast cell cycle contains a huge and complex regulatory network in the transcription level, translation level as well as protein level. In our project we utilize Sic1 as a regulator to help elongate G1 phase in yeast cell.</p><br />
<p>The activation of B-type cyclin (Clb5/6)+Cdc28 kinases is a necessary step for initiation of DNA replication in vivo. One of its inhibitor Sic1 can be phosphorylated by the activated Cln+Cdc28, thereby targeted for degradation. Over expression of the gene encoding p40, SIC1, produces cells with an elongated bud morphology(figure1)(Nugroho & Mendenhall, 1994). SIC1 deletion is viable and causes increasing frequencies of chromosomes broken and lost. The deletion strain segregates out many dead cells, which are primarily arrested at the G2 checkpoint in an asymmetric fashion. Therefore, it has an important role in ensuring genomic integrity, and that this role has a pronounced mother-daughter asymmetry.</p><br />
<img src="http://upload.wikimedia.org/wikipedia/commons/0/06/Sic1_David_Morgan10-5.jpg" title="Principle of Sic1" description="Over expression of Sic1 can stop all yeast cells to G1 phase."><br />
<p>After phosphorylation, phospho-Sic1 is specifically recognized by the F-box protein Cdc4, which leads Sic1 being ubiquitinated by the Cdc34±SCF complex (E3). The recognizing and binding by Cdc4 is based on the Sic1’s 9 Cdc4 phospo-degrons (CPDs, figure. a). Several phosphorylation sites contributed to Sic1 instability, with an order of Thr45, Ser76, Thr5,Thr33 and other less significant sites. The immunocipients after selectively phosphorylation as figure e showed suggested that at least six sites phosphorylation is necessary for the Cdc4 recognizing and binding with Sic1. Furthermore, the culture of GAL1-SIC1 constructs strain showed less than 6 sites are phosphorylated is not sufficient for SIC1 degradation in vitro(Nash et al., 2001).</p><br />
<img src="https://static.igem.org/mediawiki/parts/8/8b/Sic1-mutate.png" title="Phosphorylation Sites" description="N-terminal phosphorylation sites to be mutated."><br />
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<h3>Alternative Splicing Device</h3><br />
<p>In yeast cells, alternative splicing is a common process in before gene transcription. The splicing sites are located in 5'UTR of introns and can by recognized and spliced by some splicesome. Alternative splicing can produce two isoforms from one gene, thus can be used in our project to monitor whether the Sic1 system work efficiently. Here we find a intron with alternative splicing sites in yeast cells named SRCI intron.</p><br />
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<h4>Src1 Intron and Hub1</h4><br />
<p> SRC1 intron has two 5' splicing site whose efficiency is regulated by protein hub1. Originally, the existence of the intron would produce two mature mRNA in proportion. After engineering, the existence of protein hub1 regulates to the preservation of intron precisely, which would make the following mRNA be expressed normally or not. As for the silencing of wild type gene HUB1, we choose CRISPRi that is comparably easy to use reversibly.</p><br />
<p>Alternative splicing substantially increases the gene product diversity and is a major source of cell type differentiation. A good example is the alternative splicing of Saccharomyces cerevisiae SRC1 pre-mRNA, which is promoted by the conserved ubiquitin-like protein Hub1. It can function through binding non-covalently to a conserved element termed HIND in the spliceosomal protein Snu66. Such binding makes the splicesome target sites change and moderately alters spliceosomal interactions. </p><br />
<p>Hub1 is a ubiquitin-like modifier (UBL) that covalent modify the proteins. Interest enough, it harbors several different to other UBLs in which it possesses a C-terminal double tyrosine motif while others having a GG motif. The Snu66, a tri-snRNP in yeast spliceosome, possesses with two N-terminal HINDs (Hub1-INteraction Domain). The Hub1–HIND interaction comprises a strong salt bridge accompanied by several hydrophobic contacts and high affinity. Such binding modifies the spliceosome rather than modulating the properties of an individual binding partner. Hub1-controlled splicing occurs universally in eukaryotes. SR proteins and hnRNPs involved in spliceosome targeting do not seem to exist in S. Cerevisiae , and thus the Hub1-dependent mechanism may be evolutionarily older. </p> <br />
<p>Scr1 is a protein in yeast having alternative splicing sites in its intron. The characteristic differential Hub1 dependence of SRC1 alternative splicing requires the tandem arrangement of overlapping 5’ splice sites. The Hub1 binding spliceosome can splice the intron from both downstream 5’ sites as well as the upstream 5’ sites with preference to the former one.</p><br />
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<img src="https://static.igem.org/mediawiki/parts/9/96/SICI_principle.jpg" title="Src1 intron principle" description="Alternative splicing of Src1" /><br />
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<p>Showed in Figure.above When cutting in the upstream splice sites, the exons flanking around it would be translated as a fusion protein Scr-S. However, when cutting at the down stream one, the left 4bp in intron would result in a frame shift, thus only the forward exon can express named Scr-L.</p><br />
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<h4>dCas9 CRISPR interface system</h4><br />
<p>CRISPR shorts for Clustered Regularly Interspaced Palindromaic Repeats system, which can be targeted to DNA using RNA, enabling genetic editing of any region of the genome in many organisms.(Cho, Kim, Kim, & Kim, 2013). In the type II CRISPR/Cas system, a ribonucleoprotein complex formed from a single protein (Cas9), a crRNA, and a trans-acting CRISPR RNA (tracrRNA) can carry out efficient crRNA-directed recognition and site-specific cleavage of foreign DNA(Deltcheva et al., 2011). After mutated the endonuclease domains of the Cas9 protein, it creates a programmable RNA-dependent DNA-binding protein. The sgRNA consists of three domains: a 20 nt complementary region for specific DNA binding, a 42 nt hairpin for Cas9 binding (Cas9 handle), and a 40 nt transcription terminator derived from S. Pyogenes. After translation, the Cas9 binds to sgRNA to form a protein-RNA complex, which can recognize target sites in the genome sequence and bind to it. Then, it could block RNA polymerase and transciption elongation.</p><br />
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<img src="https://static.igem.org/mediawiki/2013/7/74/Figure4._The_structure_of_sgRNA.png" title="sgRNA" description="sgRNA structure" /><br />
<img src="https://static.igem.org/mediawiki/parts/1/13/CRISPRi_principle.png" title="Principle of dCAS9" description="Principle of the function of dCAS9 and guide RNA." /><br />
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<h3>Microfluidic Device</h3><br />
<p>Based physically on the chip, microfluidics can automatically control the biology process. In our project, the microfluidics plays a role as the movie theater, for cell cycle control. We designed a channel which can capture the yeast cell and then alternatively inject medium of different concentrations. Therefore, the yeast cell cycle can be synchronized. Furthermore, due to its transparent attribution, the chip can project the fluorescent lights from the yeast cell sub-locations. Consequently, the magic film can be shown to us. </p><br />
<p>Microfluidic devices possess many advantages such as large-scale integration, fast analyses, and considerably reduced reagent consumption (Comparative study and improvement of current cell micro-patterning techniques). Nowadays many designs are based on cell loading and culturing in a microfluidic channel with particular trapping structures such as micro-well arrays, and micro-cup arrays and micro-chambers. In our project, we design a cell loading technique based on gas absorption of degassed polydimethylsiloxane (PDMS). Because the balance concentration of gas dissolved in PDMS is proportional to the partial pressure of the gas around it, so that one can degas a piece of PDMS by placing it in vacuum. </p><br />
<p>Our cell loading device is a one level PDMS with an array of triangle cavities connected to one or both sides of the main fluidic channel. After degassed, yeast cells in the buffer solutions can be introduced into the device’s triangle cavities by gas absorption therein. Through changing the cell density in the buffer, we can control the number of the yeast cells loaded into each triangle cavity. Statistically, the ratio of cavities with a single cell can reach 40%. Consequently it is convenient to monitor the cell number and cell phenotypic variations because the yeast cells can grow in the micro-triangle-cavities in one layer.</p><br />
<img src="https://static.igem.org/mediawiki/igem.org/e/ec/Figure6._Masks_for_PDMS_microfluidic_device_fabrication..png" title="Microfluidic" description="the structure of Microfluidics"><br />
<p>Figure 1. Masks for PDMS microfluidic device fabrication.a : Three micro-triangle-cavities at one side of the main channel. b : One single channel for cell culture (100 microcavities). c : Seven parallel channels for different cell culture environments(Luo et al., 2008).</p><br />
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</div></div>BGI K2http://2013.igem.org/Team:Shenzhen_BGIC_ATCG/modelingTeam:Shenzhen BGIC ATCG/modeling2013-09-27T22:21:52Z<p>BGI K2: </p>
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<div><!----------------------<br />
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<br />
Title: Modeling<br />
Team: Shenzhen_BGIC_ATCG<br />
Author: BGI_K2<br />
Twitter: @BGI_K2<br />
Email: im.ss.kk@gmail.com<br />
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<div id="board1" class="board"></div><br />
<div class="media"><br />
<br />
<ul id="myGallery1"><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/e7/Kd%3D0.png" title="Phase Specific Promoter + XFP" description="Different XFPs emerge at different phases with its own degradation rate"/><br />
<li><img src="https://static.igem.org/mediawiki/2013/b/bc/P%2BD.png" title="Phase Specific Promoter + XFP + Degradation Tag" description="Different XFPs emerge at different phases with a faster degradation rate"/><br />
<li><img src="https://static.igem.org/mediawiki/2013/c/c7/3D.png" title="Phase Specific Promoter + XFP + Degradation Tag + Targeting Peptide" description="Different XFPs emerge at different phases with faster degradation rate and are transported to different organelles"/><br />
</ul></div><br />
<div id="box1" class="box"><br />
<h3>Blueprint</h3><br />
<p>Our project based a lot on cell cycle, especially the cyclin-promoters and cyclin-degradation tags. Through modelling Cell cycle is one of the most complex network in biology world. Better understanding of cell cycle and it’s regulation, to some extent, faciliate the fermentation industry because we can easily accelarate or decelarate a cell cycle or even one phase in the cycle which are important for metabolism product synthesis. In order to simulation and predict the experimets of the effeciency of Sic1, alternative splicing and degradation tags in the whole cell cycle, we build tree ordinary differential equation system models.</p><br />
<p>With the use of cyclins' promoters, we got the simulation of period XFP.<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2013/e/e7/Kd%3D0.png"><br />
<center><p>Figure 15. Simulation Result of Phase Specific Promoter + XFP<br />
</p></center><br />
<p>Each XFP will finally merge together so it's hard to tell part. So degradation tags were introduced. Degradation tags were also obtained from cyclins because cyclins should degrade fast enough to avoid binding to cdc28 and delaying its own phase. From our simulation we can find out that transformed proteins can also be degraded at a convenient speed.<br />
</p><br />
<center><p><b>Parameter Table<br />
</b></p></center><br />
<table><br />
<td><p>Parameter</p></td><br />
<td><p>Rate(min<sup>-1</sup>)</p></td><br />
<td><p>Citation</p></td><br />
<tr><br />
<td><p>D(PEST1)</p></td><br />
<td><p>0.12</p></td><br />
<td><p> Chen et al. (2004) </p></td><br />
<tr><br />
<td><p>D(PEST2)</p></td><br />
<td><p>0.12</p></td><br />
<td><p> Chen et al. (2004) </p></td><br />
<tr><br />
<td><p>D(PEST3)</p></td><br />
<td><p>0.14</p></td><br />
<td><p> Belli, Gari, Aldea, & Herrero (2001) </p></td><br />
<tr><br />
<td><p>D(D-box)</p></td><br />
<td><p>Vdb5</p></td><br />
<td><p> Chen et al. (2004) </p></td><br />
</table><br />
<p></p><br />
<img src="https://static.igem.org/mediawiki/2013/b/bc/P%2BD.png"><br />
<center><p>Figure 16. Simulation Result of Phase Specific Promoter + XFP + Degradation Tag<br />
</p></center><br />
<p>As Degradation tags could not fully help tell apart each phase by the light of XFP, we built targeting peptide into model to make a more distinguishable visual result. As shown here, we present a 3D simulation result by adding another axis to specify different organelles.<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2013/c/c7/3D.png"><br />
<center><p>Figure 17. Simulation Result of Phase Specific Promoter + XFP + Degradation Tag + Targeting Peptide<br />
</p></center><br />
<br />
</div><br />
<br />
<div id="board2" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery2"><br />
<li><img src="https://static.igem.org/mediawiki/2013/7/70/Original_Cell_Cycle.png" title="Original Cell Cycle Simulation" description="Simulation of appearance and disappearance of different proteins"/><br />
</ul></div><br />
<div id="box2" class="box"><br />
<h3>Cell Cycle</h3><br />
<p>To make a precise prediction of our project and analyze its feasibility, we build a cell cycle model based on Chen's work Chen et al. (2004). By simulating the periodic cycle, we obtained the promoters we could make use of. <br />
</p><br />
<p>Promoters are selected by observing the appearance and disappearance of proteins shown in cell cycle model. <br />
</p><br />
<img src="https://static.igem.org/mediawiki/2013/7/70/Original_Cell_Cycle.png"><br />
<center><p>Figure 1. Simulation Result of Original Cell Cycle<br />
</p></center><br />
<p>Both the model and related papers show that cln1, 2 and clb1, 2, 5, 6 appear at a periodic rhythm because of the appearance of their transcription factors. There are some other proteins (such as NET1) shown this features, but their mechanisms are also related to some protein-protein interactions.<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2013/b/bd/NET.png"><br />
<center><p>Figure 2. Simulation Result of NET1 and NET1T<br />
</p></center><br />
</div><br />
<br />
<div id="board3" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery3"><br />
<li><img src="https://static.igem.org/mediawiki/2013/8/89/G1_delay.png" title="G1 Phase Delay" description="G1 phase will delay with galactose induced sic1p expression"><br />
<li><img src="https://static.igem.org/mediawiki/2013/9/98/SIC1_Time.png" title="Plateau Stage of sic1p" description="Plateau Stage of sic1p by inducing sic1p expression"><br />
<li><img src="https://static.igem.org/mediawiki/2013/1/1a/Tp_ka_kd.png" title="Influence of Ka and Kd to Tp"><br />
<li><img src="https://static.igem.org/mediawiki/2013/1/1f/Lp_ka_kd.png" title="Influence of Ka and Kd to Lp"><br />
<li><img src="https://static.igem.org/mediawiki/2013/9/9a/Synchronization.png" title="Synchronization" description="Synchronization result of multiple cells"><br />
</ul></div><br />
<div id="box3" class="box"><br />
<h3>Cell Synchronization</h3><br />
<p>Previously study reported the introduction of sic1p could prevent the cell to enter S phase. Based on the sic1 system in yeast, we developed an artificial sic1 system (SIC1_Art). By adding galactose or modifying the phosphorylated sites, we can regulate the synthesis (Ka) and degradation (Kd) rates of the sic1_Art. We are trying to utilize this artificial system to precisely regulate the phase in yeast cell cycle, and our goal is to understand the synchronization behavior in yeast.<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/SIC1.png"><br />
<center><p>Figure 18. SIC1 Model<br />
</p></center><br />
<img src="https://static.igem.org/mediawiki/2013/8/89/G1_delay.png"><br />
<center><p>Figure 3. G1 Phase Delay<br />
</p></center><br />
<h4>SIC1_Art on G1 stage</h4><br />
<h5>G1 length:</h5><br />
<p>To understand the temporal effect of SIC1_Art on the length G1 phase, we performed parameter scan on the amount of time of adding SIC1_Art (DeltaT). By setting Ka=0.12 and Kd=0.016, we estimated the relationship between DeltaT and the length of G1 phase. Our computation simulation showed that, as we added SIC1_Art into the yeast, the amount of SIC1_Art will increase at first, and then it will enter a plateau stage. After the plateau stage, SIC1_Art will decrease gradually, which subsequently followed by the leaving of G1 stage (900 min). Our result suggests a positive correlation between DeltaT and the length of G1 phase in the first 900 min, and we discovered an upper bound at the length of G1 phase.<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2013/3/38/SIC1_G1L.png"><br />
<center><p>Figure 4. Curve Fitting of SIC1_Art and G1 Phase Length<br />
</p></center><br />
<h5>Plateau, the definition:</h5><br />
<p>Based on the relation between DeltaT and the cellular level of SIC1_Art, we defined plateau stage as the time space within which the amount of SIC1_Art is less than the maximum SIC_Art amount during the G1 phase (SIC1_Amax) and greater than (SIC1_Amax - Kd*5min). During this time space, the temporal difference of SIC1_Art degradation is less than 5 min, which we considered the minimum requirement of synchronization.<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2013/9/98/SIC1_Time.png"><br />
<center><p>Figure 5. Plateau Stage of SIC1_Art<br />
</p></center><br />
<img src="https://static.igem.org/mediawiki/2013/9/99/SIC1_Art_Time.png"><br />
<center><p>Figure 6. Plateau Stage of SIC1_Art with Different Galatose Input Time<br />
</p></center><br />
<h5>Ka/Kd, Entering Plateau:</h5><br />
<p>To examine the relation between the synthesis and degradation rate and the timing of entering plateau stage (Tp), we performed parameter scan on Ka and Kd. We found that the Tp is negatively correlated with Ka and positively correlated with Kd.<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2013/1/1a/Tp_ka_kd.png"><br />
<center><p>Figure 7. Influence of Ka and Kd to Tp<br />
</p></center><br />
<h5>Ka/kd, Length of Plateau:</h5><br />
<p>To further explore the optimal Ka/Kd in SIC1_Art system, we investigated the influence of Ka/Kd on the length of plateau domain (Lp).<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2013/1/1f/Lp_ka_kd.png"><br />
<center><p>Figure 8. Influence of Ka and Kd to Lp<br />
</p></center><br />
<h4>Synchronization in yeast cell cycle</h4><br />
<p>Using the result of above analysis, we chose Ka=0.219, Kd=0.048 as the optimal parameters, which give rise to short entering plateau time and long enough plateau stage (Tp = 61 min, Lp=355 min ).<br />
</p><br />
<p>We simulated the multi-cell behavior in yeast cell cycle: we started with cells in different phases, Art_time = 990 min, we added galactose into the systems, which initiated the synthesis of SIC1_Art and subsequently stopped all the cells at G1 phase. At time=1030 min, we stopped adding galactose and subsequently caused fast degradation of SIC1_Art. This process made all the yeasts into the phase and consequently achieved synchronization.<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2013/9/9a/Synchronization.png"><br />
<center><p>Figure 9. Simulation Result of Multiple Cells' Synchronization<br />
</p></center><br />
</div><br />
<br />
<br />
<div id="board4" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery4"><br />
<li><img src="https://static.igem.org/mediawiki/igem.org/0/01/Kass1_eq_kdis1.png" title="Simulation Result of Kass1 and Kdis1" description="Kass1 = Kids1"><br />
<li><img src="https://static.igem.org/mediawiki/igem.org/a/ad/Parameter_scan_of_b_%28gal%29.png" title="Parameter Scan of b with Galactose Input"><br />
</ul></div><br />
<div id="box4" class="box"><br />
<h3>Alternative Splicing by CRISPRi</h3><br />
<p>To predict the alternative outcome, we also made an intron model to show different results due to incubating in different media. In our project, intron can be spliced in two different ways, providing a completely different outcome because of frame-shift, and this result is not a change like 1-0 to 0-1, but somehow more like a change between 0.4-0.6 and 0.8-0.2.<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2013/9/96/DCas9.png"><br />
<center><p>Figure 11. dCas9 Controlled SRC1 Intron Splicing<br />
</p></center><br />
<h4>Reactions:</h4><br />
<img src="https://static.igem.org/mediawiki/2013/a/af/Formula1.png"><br />
<img src="https://static.igem.org/mediawiki/2013/0/03/Formula2.png"><br />
<h4>Parameter Table:</h4><br />
<table><br />
<td><p>Parameter</p></td><br />
<td><p>Explanation</p></td><br />
<tr><br />
<td><p>P(dCas9_m)</p></td><br />
<td><p>dCas9 mRNA transcription rate</p></td><br />
<tr><br />
<td><p>P(sgRNA)</p></td><br />
<td><p>sgRNA transcription rate</p></td><br />
<tr><br />
<td><p>P(dCas9p)</p></td><br />
<td><p>dCas9 protein translation rate</p></td><br />
<tr><br />
<td><p>D(RNA)</p></td><br />
<td><p>Average degradation rate of RNA</p></td><br />
<tr><br />
<td><p>Kass</p></td><br />
<td><p>Association rate of CRISPRi system</p></td><br />
<tr><br />
<td><p>Kass</p></td><br />
<td><p>Association rate of CRISPRi system</p></td><br />
<tr><br />
<td><p>Kdis</p></td><br />
<td><p>Dissociation rate of CRISPRi system</p></td><br />
<tr><br />
<td><p>Kass1</p></td><br />
<td><p>Association rate of modified spliceosome</p></td><br />
<tr><br />
<td><p>Kdis1</p></td><br />
<td><p>Dissociation rate of modified spliceosome</p></td><br />
<tr><br />
<td><p>Kdis1</p></td><br />
<td><p>Dissociation rate of modified spliceosome</p></td><br />
<tr><br />
<td><p>K</p></td><br />
<td><p>Splicing rate</p></td><br />
<tr><br />
<td><p>P(Hub1_m)</p></td><br />
<td><p>Hub1 mRNA transcription rate</p></td><br />
<tr><br />
<td><p>P(Hub1_P)</p></td><br />
<td><p>Hub1 protein translation rate</p></td><br />
<tr><br />
<td><p>P(pre-mRNA)</p></td><br />
<td><p>pre-mRNA transcription rate</p></td><br />
<tr><br />
<td><p>P(ProteinL)</p></td><br />
<td><p>5’L protein translation rate</p></td><br />
<tr><br />
<td><p>P(ProteinS)</p></td><br />
<td><p>5’S protein translation rate</p></td><br />
<tr><br />
<td><p>D(Protein)</p></td><br />
<td><p>Average degradation rate of protein</p></td><br />
<tr><br />
</table><br />
<p>There are 4 parameters that we cannot find during our research, including kass, kdis representing the association and dissociation rate of CRISPRi system, and kass1 and kdis1 representing the association and dissociation rate of Hub1p and spliceosome. <br />
</p><br />
<p></p><br />
<p>We run parameter scan for each system individually, and found out that with CRISPRi system is more efficient with higher kass and lower kdis, as expected.<br />
</p><br />
<p></p><br />
<p>And about the alternative splicing model, we attempted to fit simulation result to experimental one. In Hub1 expressed system, L-mRNA will rise at first but descend to an equilibrium stage while S-mRNA will directly rise to its own equilibrium stage. <br />
</p><br />
<p>kass1 should be larger than kdis1, or L-mRNA will be produced more than S-mRNA instead of a ratio of 40-60. <br />
</p><br />
<img src="https://static.igem.org/mediawiki/igem.org/2/2b/Kass1_lt_kdis1.png"><br />
<center><p>Figure 10. Simulation Result of Two mRNA when Kass1<Kdis1<br />
</p></center><br />
<p>Also, kass1 should not be too smaller than kdis1, or their ratio will be much larger than 40-60.<br />
</p><br />
<img src="https://static.igem.org/mediawiki/igem.org/e/ec/Kass1_gt_kdis1.png"><br />
<center><p>Figure 11. Simulation Result of Two mRNA when Kass1>Kdis1<br />
</p></center><br />
<p>Finally we set down that kass1 = kdis1.<br />
</p><br />
<img src="https://static.igem.org/mediawiki/igem.org/0/01/Kass1_eq_kdis1.png"><br />
<center><p>Figure 12. Simulation Result of Two mRNA when Kass1=Kdis1<br />
</p></center><br />
<p>When inhibiting the expression of HUB1, there is still a background splicing of 5’S site, so we need another parameter b in S-splicing. <br />
</p><br />
<p>Background S-splicing parameter b is mostly related to the ratio of spliceosome and Hub1p-modified spliceosome (Hub1_spliceosome) at equilibrium state. With higher b, L-mRNA and S-mRNA will come closer at equilibrium stat while it influence no-Hub1p situation more than Hub1p situation. <br />
</p><br />
<img src="https://static.igem.org/mediawiki/igem.org/a/ad/Parameter_scan_of_b_%28gal%29.png"><br />
<center><p>Figure 13. Parameter Scan of b with Galactose Input<br />
</p></center><br />
<img src="https://static.igem.org/mediawiki/igem.org/e/ea/Parameter_scan_of_b_%28no_gal%29.png"><br />
<center><p>Figure 14. Parameter Scan of b without Galactose Input<br />
</p></center><br />
</div><br />
<br />
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<div id="box5" class="box"><br />
<h3>Degradation Rate</h3><br />
<br />
</div></div>BGI K2http://2013.igem.org/Team:Shenzhen_BGIC_ATCG/partsTeam:Shenzhen BGIC ATCG/parts2013-09-27T22:19:40Z<p>BGI K2: </p>
<hr />
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<h3>Best BioBricks</h3><br />
<h4>Best BioBrick (Natural/Collection), BBa_K1051303, BBa_K1051300 ~ BBa_K1051306</h4><br />
<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051303"><p>K1051303</p><br />
<img src="https://static.igem.org/mediawiki/2013/0/0c/K1051303.png"></a><br />
<p>K1051300</p><br />
<img src="https://static.igem.org/mediawiki/2013/8/8d/K1051300.png"><br />
<p>K1051301</p><br />
<img src="https://static.igem.org/mediawiki/2013/c/c0/K1051301.png"><br />
<p>K1051302</p><br />
<img src="https://static.igem.org/mediawiki/2013/c/c6/K1051302.png"><br />
<p>K1051304</p><br />
<img src="https://static.igem.org/mediawiki/2013/f/fa/K1051304.png"><br />
<p>K1051305</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/49/K1051305.png"><br />
<p>K1051306</p><br />
<img src="https://static.igem.org/mediawiki/2013/5/5d/K1051306.png"><br />
<h4>Best BioBrick (Engineered), BBa_K1051900</h4><br />
<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051900"><p>K1051900</p><br />
<img src="https://static.igem.org/mediawiki/igem.org/c/ce/K1051900a.png"><br />
<img src="https://static.igem.org/mediawiki/igem.org/f/fa/K1051900b.png"></a><br />
<h4>Best Measurement, BBa_K1051257 ~ BBa_K1051259</h4><br />
<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051257"><p>K1051257</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/43/K1051257.png"></a><br />
<p>K1051258</p><br />
<img src="https://static.igem.org/mediawiki/2013/d/d4/K1051258.png"><br />
<p>K1051259</p><br />
<img src="https://static.igem.org/mediawiki/2013/e/ec/K1051259.png"><br />
<br />
<br />
</div><br />
<br />
<div id="board2" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery2"><br />
<li><img src="https://static.igem.org/mediawiki/2013/b/b9/Blank-01.png" /><li><br />
</ul></div><br />
<div id="box2" class="box"><br />
<h3>Devices List</h3><br />
<h4>Final Constructions</h4><br />
<h5>Final Constructions of <i>E.coli</i></h5><br />
<p>K1051410</p><br />
<img src="https://static.igem.org/mediawiki/2013/f/f5/K1051410.png"><br />
<p>K1051412</p><br />
<img src="https://static.igem.org/mediawiki/2013/8/8d/K1051412.png"><br />
<p>K1051414</p><br />
<img src="https://static.igem.org/mediawiki/2013/f/fa/K1051414.png"><br />
<h5>Final Constructions of Budding Yeast</h5><br />
<p>K1051900a</p><br />
<img src="https://static.igem.org/mediawiki/igem.org/c/ce/K1051900a.png"><br />
<p>K1051900b</p><br />
<img src="https://static.igem.org/mediawiki/igem.org/f/fa/K1051900b.png"><br />
<p>K1051901</p><br />
<img src="https://static.igem.org/mediawiki/2013/e/e3/K1051901.png"><br />
<p>K1051902</p><br />
<img src="https://static.igem.org/mediawiki/2013/2/2c/K1051902.png"><br />
<p>K1051903</p><br />
<img src="https://static.igem.org/mediawiki/2013/1/11/K1051903.png"><br />
<h4>Promoter Verification</h4><br />
<h5>Verification of <i>E.coli</i></h5><br />
<p>K1051245</p><br />
<img src="https://static.igem.org/mediawiki/2013/d/d8/K1051245.png"><br />
<p>K1051246</p><br />
<img src="https://static.igem.org/mediawiki/2013/2/2b/K1051246.png"><br />
<p>K1051247</p><br />
<img src="https://static.igem.org/mediawiki/2013/7/79/K1051247.png"><br />
<h5>Verification of Budding Yeast</h5><br />
<p>K1051307</p><br />
<img src="https://static.igem.org/mediawiki/2013/c/c5/K1051307.png"><br />
<p>K1051355</p><br />
<img src="https://static.igem.org/mediawiki/2013/1/12/K1051355.png"><br />
<p>K1051356</p><br />
<img src="https://static.igem.org/mediawiki/2013/7/74/K1051356.png"><br />
<p>K1051357</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/48/K1051357.png"><br />
<p>K1051358</p><br />
<img src="https://static.igem.org/mediawiki/2013/d/d6/K1051358.png"><br />
<p>K1051359</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/65/K1051359.png"><br />
<p>K1051365</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/40/K1051365.png"><br />
<p>K1051366</p><br />
<img src="https://static.igem.org/mediawiki/2013/3/33/K1051366.png"><br />
<p>K1051367</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/4f/K1051367.png"><br />
<p>K1051368</p><br />
<img src="https://static.igem.org/mediawiki/2013/8/8a/K1051368.png"><br />
<p>K1051369</p><br />
<img src="https://static.igem.org/mediawiki/2013/5/52/K1051369.png"><br />
<h4>Degradation Rate Measurement</h4><br />
<h5>Measurement of <i>E.coli</i></h5><br />
<p>K1051257</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/43/K1051257.png"><br />
<p>K1051258</p><br />
<img src="https://static.igem.org/mediawiki/2013/d/d4/K1051258.png"><br />
<p>K1051259</p><br />
<img src="https://static.igem.org/mediawiki/2013/e/ec/K1051259.png"><br />
<h5>Measurement of Budding Yeast</h5><br />
<p>K1051278</p><br />
<img src="https://static.igem.org/mediawiki/2013/b/b2/K1051278.png"><br />
<p>K1051279</p><br />
<img src="https://static.igem.org/mediawiki/2013/1/1d/K1051279.png"><br />
<p>K1051280</p><br />
<img src="https://static.igem.org/mediawiki/2013/1/1a/K1051280.png"><br />
<p>K1051281</p><br />
<img src="https://static.igem.org/mediawiki/2013/5/5e/K1051281.png"><br />
<p>K1051288</p><br />
<img src="https://static.igem.org/mediawiki/2013/8/88/K1051288.png"><br />
<h4>Targeting Peptides Verification</h4><br />
<p>K1051150</p><br />
<img src="https://static.igem.org/mediawiki/2013/d/db/K1051150.png"><br />
<p>K1051151</p><br />
<img src="https://static.igem.org/mediawiki/2013/9/97/K1051151.png"><br />
<p>K1051152</p><br />
<img src="https://static.igem.org/mediawiki/2013/0/0e/K1051152.png"><br />
<p>K1051153</p><br />
<img src="https://static.igem.org/mediawiki/2013/c/cb/K1051153.png"><br />
<h4>CRISPRi Verification</h4><br />
<p>K1051852</p><br />
<img src="https://static.igem.org/mediawiki/2013/3/33/K1051852.png"><br />
<h4>Alternative Splicing Verification</h4><br />
<p>K1051706</p><br />
<img src="https://static.igem.org/mediawiki/2013/2/2b/K1051706.png"><br />
<br />
</div><br />
<br />
<div id="board3" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery3"><br />
<li><img src="https://static.igem.org/mediawiki/2013/b/b9/Blank-01.png" /><li><br />
</ul></div><br />
<div id="box3" class="box"><br />
<h3>Basic Parts</h3><br />
<h4>Universal Parts</h4><br />
<h5>Reporters</h5><br />
<table><br />
<TH>Name</TH><TH>Type<TH>Description<TH>Designer<TH>Length<TH><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051000'>BBa_K1051000</A></TD><TD>Reporter</TD><TD>Stop codon free RFP in RFC[23] standard</TD><TD >Xiang LI</TD><TD ALIGN='right'>675</TD><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051001'>BBa_K1051001</A></TD><TD>Reporter</TD><TD>Stop codon free ECFP in RFC[23] standard</TD><TD >Xiang LI</TD><TD ALIGN='right'>716</TD><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051002'>BBa_K1051002</A></TD><TD>Reporter</TD><TD>Stop codon free EYFP in RFC[23] standard</TD><TD >Xiang LI</TD><TD ALIGN='right'>717</TD><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051003'>BBa_K1051003</A></TD><TD>Reporter</TD><TD>Stop codon free GFP in RFC[23] standard</TD><TD >Xiang LI</TD><TD ALIGN='right'>714</TD><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051004'>BBa_K1051004</A></TD><TD>Reporter</TD><TD>Stop codon free mOrange in RFC[23] standard</TD><TD >Xiang LI</TD><TD ALIGN='right'>738</TD><br />
</table><br />
<h5>Yeast Terminators</h5><br />
<table><br />
<TH>Name</TH><TH>Type<TH>Description<TH>Designer<TH>Length<TH><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051006'>BBa_K1051006</A></TD><TD>Terminator</TD><TD>Saccharomycescerevisiae TBY-1 terminator with stopcodon</TD><TD >Xiang LI</TD><TD ALIGN='right'>31</TD><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051007'>BBa_K1051007</A></TD><TD>Terminator</TD><TD>S. cerevisiae SSV7 terminator with stopcodon</TD><TD >Xiang LI</TD><TD ALIGN='right'>39</TD><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051008'>BBa_K1051008</A></TD><TD>Terminator</TD><TD>S. cerevisiae CAA terminator with stopcodon</TD><TD >Xiang LI</TD><TD ALIGN='right'>18</TD><br />
</table><br />
<h5><i>E. coli</i> Terminators</h5><br />
<table><br />
<TH>Name</TH><TH>Type<TH>Description<TH>Designer<TH>Length<TH><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051009'>BBa_K1051009</A></TD><TD>Terminator</TD><TD>e.coli terminator ilvGEDA_T with stop codon</TD><TD >Xiang LI</TD><TD ALIGN='right'>92</TD><br />
</table><br />
<h4>Cyclin Promoters</h4><br />
<h5>Yeast</h5><br />
<table><br />
<TH>Name</TH><TH>Type<TH>Description<TH>Designer<TH>Length<TH><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051300'>BBa_K1051300</A></TD><TD>Regulatory</TD><TD> cln2 promoter (G1 phase to S phase)</TD><TD >Kequan Lin</TD><TD ALIGN='right'>1000</TD><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051301'>BBa_K1051301</A></TD><TD>Regulatory</TD><TD> clb2 promoter (during G2 phase)</TD><TD >Kequan Lin</TD><TD ALIGN='right'>1063</TD><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051302'>BBa_K1051302</A></TD><TD>Regulatory</TD><TD> clb5 promoter (during G1 phase)</TD><TD >Kequan Lin</TD><TD ALIGN='right'>648</TD><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051304'>BBa_K1051304</A></TD><TD>Regulatory</TD><TD> cln3 promoter (M phase to G1 phase)</TD><TD >Kequan Lin</TD><TD ALIGN='right'>1075</TD><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051305'>BBa_K1051305</A></TD><TD>Regulatory</TD><TD>met16 promoter (M phase to G1 phase)</TD><TD >Zhu Shuang</TD><TD ALIGN='right'>551</TD><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051306'>BBa_K1051306</A></TD><TD>Regulatory</TD><TD>met28 promoter (S phase)</TD><TD >Zhu Shuang</TD><TD ALIGN='right'>500</TD><br />
</table><br />
<h5><i>E. coli</i></h5><br />
<table><br />
<TH>Name</TH><TH>Type<TH>Description<TH>Designer<TH>Length<TH><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051400'>BBa_K1051400</A></TD><TD>Regulatory</TD><TD>promoter->dnaAp1</TD><TD >Bingwei Zheng</TD><TD ALIGN='right'>359</TD><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051401'>BBa_K1051401</A></TD><TD>Regulatory</TD><TD>promoter->dnaAp1 no RBS</TD><TD >Bingwei Zheng</TD><TD ALIGN='right'>184</TD><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051402'>BBa_K1051402</A></TD><TD>Regulatory</TD><TD>promoter->gyrB</TD><TD >Bingwei Zheng</TD><TD ALIGN='right'>188</TD><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051403'>BBa_K1051403</A></TD><TD>Regulatory</TD><TD>promoter->gyrB no RBS</TD><TD >Bingwei Zheng</TD><TD ALIGN='right'>158</TD><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051404'>BBa_K1051404</A></TD><TD>Regulatory</TD><TD>promoter->ftsQp</TD><TD >Bingwei Zheng</TD><TD ALIGN='right'>485</TD><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051405'>BBa_K1051405</A></TD><TD>Regulatory</TD><TD>promoter->ftsQp no RBS</TD><TD >Bingwei Zheng</TD><TD ALIGN='right'>198</TD><br />
</table><br />
<h4>Degradation Tags</h4><br />
<h5>Yeast</h5><br />
<table><br />
<TH>Name</TH><TH>Type<TH>Description<TH>Designer<TH>Length<TH><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051200'>BBa_K1051200</A></TD><TD>DNA</TD><TD>ubiquitin</TD><TD >wanling ZHOU</TD><TD ALIGN='right'>1146</TD><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051201'>BBa_K1051201</A></TD><TD>DNA</TD><TD>D-BOX</TD><TD >wanling ZHOU</TD><TD ALIGN='right'>39</TD><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051202'>BBa_K1051202</A></TD><TD>DNA</TD><TD>cln1-PEST</TD><TD >wanling ZHOU</TD><TD ALIGN='right'>489</TD><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051203'>BBa_K1051203</A></TD><TD>DNA</TD><TD>Cln2-PEST</TD><TD >wanling ZHOU</TD><TD ALIGN='right'>537</TD><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051204'>BBa_K1051204</A></TD><TD>DNA</TD><TD>cln3-PEST</TD><TD >wanling ZHOU</TD><TD ALIGN='right'>588</TD><br />
</table><br />
<h5><i>E. coli</i></h5><br />
<table><br />
<TH>Name</TH><TH>Type<TH>Description<TH>Designer<TH>Length<TH><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051206'>BBa_K1051206</A></TD><TD>Tag</TD><TD>The degradation tag in E. coil ,M0050 with TAATAA.</TD><TD >Rui Guan</TD><TD ALIGN='right'>39</TD><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051207'>BBa_K1051207</A></TD><TD>Tag</TD><TD>The degradation tag in E. coil ,M0051 with TAATAA.</TD><TD >Rui Guan</TD><TD ALIGN='right'>45</TD><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051208'>BBa_K1051208</A></TD><TD>Tag</TD><TD>The degradation tag in E. coil ,M0052 with TAATAA.</TD><TD >Rui Guan</TD><TD ALIGN='right'>39</TD><br />
</table><br />
<h4>Targeting Peptides</h4><br />
<h5>Mitochondria</h5><br />
<table><br />
<TH>Name</TH><TH>Type<TH>Description<TH>Designer<TH>Length<TH><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051100'>BBa_K1051100</A></TD><TD>Coding</TD><TD>targeting peptide to mitochondria inner membrane</TD><TD >Jinchun Zhang</TD><TD ALIGN='right'>209</TD><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051101'>BBa_K1051101</A></TD><TD>Coding</TD><TD>Cox4 targeting peptide to mitochondria inner membrane</TD><TD >Jinchun Zhang</TD><TD ALIGN='right'>75</TD><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051102'>BBa_K1051102</A></TD><TD>Coding</TD><TD>targeting peptide to mitochondria inner membrane</TD><TD >Jinchun Zhang</TD><TD ALIGN='right'>141</TD><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051103'>BBa_K1051103</A></TD><TD>Coding</TD><TD>TOM22, targeting peptide to mitochondria outer membrane</TD><TD >Jinchun Zhang</TD><TD ALIGN='right'>69</TD><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051104'>BBa_K1051104</A></TD><TD>Coding</TD><TD>VDAC1;targeting peptide to mitochondria outer membrane</TD><TD >Jinchun Zhang</TD><TD ALIGN='right'>93</TD><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051105'>BBa_K1051105</A></TD><TD>Coding</TD><TD>TOM40;targeting peptide to mitochondria outer membrane</TD><TD >Jinchun Zhang</TD><TD ALIGN='right'>147</TD><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051106'>BBa_K1051106</A></TD><TD>Coding</TD><TD>targeting peptide to mitochondria outer membrane</TD><TD >Jinchun Zhang</TD><TD ALIGN='right'>96</TD><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051107'>BBa_K1051107</A></TD><TD>Coding</TD><TD>Tom70;targeting peptide to mitochondria outer membrane</TD><TD >Jinchun Zhang</TD><TD ALIGN='right'>90</TD><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051108'>BBa_K1051108</A></TD><TD>Coding</TD><TD>targeting peptide to mitochondria outer membrane;Tom20</TD><TD >Jinchun Zhang</TD><TD ALIGN='right'>120</TD><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051109'>BBa_K1051109</A></TD><TD>Coding</TD><TD>targeting peptide to mitochondria matrix;MIA40</TD><TD >Jinchun Zhang</TD><TD ALIGN='right'>93</TD><br />
<tr><br />
</table><br />
<h5>Centrosome</h5><br />
<table><br />
<TH>Name</TH><TH>Type<TH>Description<TH>Designer<TH>Length<TH><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051110'>BBa_K1051110</A></TD><TD>Coding</TD><TD>ScSfi1;targeting peptide to centrosome</TD><TD >Jinchun Zhang</TD><TD ALIGN='right'>63</TD><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051111'>BBa_K1051111</A></TD><TD>Coding</TD><TD>ScSfi1E; targeting peptide to centrosome;</TD><TD >Jinchun Zhang</TD><TD ALIGN='right'>63</TD><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051112'>BBa_K1051112</A></TD><TD>Coding</TD><TD>ScKar1;targeting peptide to centrosome</TD><TD >Jinchun Zhang</TD><TD ALIGN='right'>60</TD><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051113'>BBa_K1051113</A></TD><TD>Coding</TD><TD>ScSac3;targeting peptide to centrosome</TD><TD >Jinchun Zhang</TD><TD ALIGN='right'>57</TD><br />
</table><br />
<h5>Nucleus</h5><br />
<table><br />
<TH>Name</TH><TH>Type<TH>Description<TH>Designer<TH>Length<TH><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051114'>BBa_K1051114</A></TD><TD>Coding</TD><TD>H2A2;targeting peptide to nucleus</TD><TD >Jinchun Zhang</TD><TD ALIGN='right'>408</TD><br />
</table><br />
<h5>Vacuolar</h5><br />
<table><br />
<TH>Name</TH><TH>Type<TH>Description<TH>Designer<TH>Length<TH><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051115'>BBa_K1051115</A></TD><TD>Coding</TD><TD>ZRC1;Vacuolar Membrane Targeting Protein</TD><TD >Jinchun Zhang</TD><TD ALIGN='right'>396</TD><br />
</table><br />
<h5>Actin</h5><br />
<table><br />
<TH>Name</TH><TH>Type<TH>Description<TH>Designer<TH>Length<TH><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051116'>BBa_K1051116</A></TD><TD>Coding</TD><TD>Actin Targeting Protein</TD><TD >Jinchun Zhang</TD><TD ALIGN='right'>1779</TD><br />
</table><br />
<h5>Plasma Membrane</h5><br />
<table><br />
<TH>Name</TH><TH>Type<TH>Description<TH>Designer<TH>Length<TH><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051117'>BBa_K1051117</A></TD><TD>Coding</TD><TD>Plasma Membrane Targeting Sequence</TD><TD >Jinchun Zhang</TD><TD ALIGN='right'>12</TD><br />
</table><br />
<h5>Peroxisomes</h5><br />
<table><br />
<TH>Name</TH><TH>Type<TH>Description<TH>Designer<TH>Length<TH><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051118'>BBa_K1051118</A></TD><TD>Coding</TD><TD>ScPEX21p&#65307;baking yeast targeting peptide to peroxisome ;</TD><TD >Jinchun Zhang</TD><TD ALIGN='right'>30</TD><br />
</table><br />
<br />
<h4>CRISPRi Verification</h4><br />
<table><br />
<TH>Name</TH><TH>Type<TH>Description<TH>Designer<TH>Length<TH><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051800'>BBa_K1051800</A></TD><TD>Coding</TD><TD>protein dCas9.Two mutations,D10A and H841A,from Cas9 gene without stop codon.</TD><TD >Shihong Chen</TD><TD ALIGN='right'>4164</TD><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051801'>BBa_K1051801</A></TD><TD>Device</TD><TD>Targeted to HUBI gene ATG downstream position of the 12 bp sgRNA, cooperate to dCas 9 protein, inhib</TD><TD >Shihong Chen</TD><TD ALIGN='right'>388</TD><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051801'>BBa_K1051801</A></TD><TD>Device</TD><TD>Targeted to HUBI gene ATG downstream position of the 12 bp sgRNA, cooperate to dCas 9 protein, inhib</TD><TD >Shihong Chen</TD><TD ALIGN='right'>388</TD><br />
</table><br />
<h4>Alternative Splicing Verification</h4><br />
<table><br />
<TH>Name</TH><TH>Type<TH>Description<TH>Designer<TH>Length<TH><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051700'>BBa_K1051700</A></TD><TD>DNA</TD><TD>SRC1 Intron -4bp at 5'</TD><TD >Yang Zhou</TD><TD ALIGN='right'>129</TD><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051701'>BBa_K1051701</A></TD><TD>DNA</TD><TD>SRC1 Intron+GG</TD><TD >Yang Zhou</TD><TD ALIGN='right'>132</TD><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051702'>BBa_K1051702</A></TD><TD>DNA</TD><TD>SRC1 Intron+CGG</TD><TD >Yang Zhou</TD><TD ALIGN='right'>133</TD><br />
<tr><br />
<TD><A href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K1051703'>BBa_K1051703</A></TD><TD>DNA</TD><TD>SRC1 Intron with additional 6bp at both directions</TD><TD >Yang Zhou</TD><TD ALIGN='right'>142</TD><br />
</table><br />
</div><br />
<br />
<div id="board4" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery4"><br />
<li><img src="https://static.igem.org/mediawiki/2013/8/8d/Sequence_Result.png" title="Sequencing Result" description="An example for part sequencing." ></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/7/74/Alignment.png" title="Alignment Result" description="An example for part sequencing result alignment." ></li><br />
</ul></div><br />
<div id="box4" class="box"><br />
<h3>Parts Sequencing</h3><br />
<p>All of our parts have been sequenced to get verification.<br />
</p><br />
<p>Sequencing example of K1051701<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2013/8/8d/Sequence_Result.png"><br />
<center><p>Figure. Sequencing Result</p></center><br />
<p>Alignment example of K1051701</p><br />
<img src="https://static.igem.org/mediawiki/2013/7/74/Alignment.png"><br />
<center><p>Figure. Alignment Result</p></center><br />
</div></div>BGI K2http://2013.igem.org/Team:Shenzhen_BGIC_ATCG/resultsTeam:Shenzhen BGIC ATCG/results2013-09-27T22:16:09Z<p>BGI K2: </p>
<hr />
<div><!----------------------<br />
<br />
<br />
Title: Results<br />
Team: Shenzhen_BGIC_ATCG<br />
Author: BGI_K2<br />
Twitter: @BGI_K2<br />
Email: im.ss.kk@gmail.com<br />
<br />
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<div class="navleftholder"><br />
<ul class="navleft"><br />
<li><a id="navleftsub1" class="navleftsub" href="#board1">The Magic</a></li><br />
<li><a id="navleftsub2" class="navleftsub" href="#board2">Promoter Verification</a></li><br />
<li><a id="navleftsub3" class="navleftsub" href="#board3">Reporter Locating</a></li><br />
<li><a id="navleftsub4" class="navleftsub" href="#board4">Degradation Rate</a></li><br />
<li><a id="navleftsub5" class="navleftsub" href="#board5">Cell Synchronization</a></li><br />
<li><a id="navleftsub6" class="navleftsub" href="#board6">Alternative Splicing</a></li><br />
</ul></div><br />
<br />
<div id="board1" class="board"></div><br />
<div class="media"><br />
<br />
<ul id="myGallery1"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/></li><br />
<li><img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" title="Degradation Tags" description="Degradation tags testing device for E. coli."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" title="Microfluidics" description="Microfluidics device for degradation test."/></li><br />
<br />
</ul></div><br />
<div id="box1" class="box"><br />
<h3>The Magic</h3><br />
<p>Our project “Cell Magic", a complex work has been completed partly,due to the time limitation. The results we have gotten are listed below:</p><br />
<p>We verified some of the cyclin promoters. The clb6 promoter, which is supposed to be expressed in G1 phase, has been verified perfectly.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." width=50%/><br />
<p>Targeting peptides to three locations have been verified. Others are still being constructed and tested.</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. " width=50%/><br />
<p>We got good data for degradation tags for <i>E. coli</i>. Construction of degradation devices for yeast have been finished and being tested.</p><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" width=50%/><br />
<p>Microfluidics device has been successfully developed for measurement and cell synchronization.</p><br />
<img src="https://static.igem.org/mediawiki/2013/f/fc/Microfluidic.gif" /><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" width=50%/><br />
<p>Cell synchronization device and alternative splicing device are still in construction, related results will be updated later.</p><br />
<br />
</div><br />
<br />
<div id="board2" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery2"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/1/19/Flu.jpg" title="S Phase Promoter" description="cln3 promoter for S phase has been verified using flow cytometry." /></li><br />
</ul></div><br />
<div id="box2" class="box"><br />
<h3>Promoter Verification</h3><br />
<h4>Achievements</h4><br />
<p> Using the GFP as reporter and morphological alteration as cell cycle representation, we verified the Clb6 can be activated in G1 phase in the yeast.</P><br />
<p> As shown in the picture in normal lights, there are some yeast were budding. These budded yeast cells and small size budding yeasts (within the red circles) are assumed in the G1 phase of cell cycle. And the next picture was taken under activation light, thus the green lights can representing the Clb6 promoter expressing. The two pictures indicated the Clb6 were expressed in the G1 phase as we expected when designing the experiments </p><br />
<p> PS: we modified the contrast ratio to lower the lights of neighbor cells, thus our results looking better.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" /><br />
<p>We also captured fluorescence on cln3 promoter (S phase) test device, for the fluorescence is quite weak, we utilize micro plate reader and flow cytometry to verify the promoter.</p><br />
<img src="https://static.igem.org/mediawiki/2013/1/19/Flu.jpg" /><br />
<h4>Problems</h4><br />
<h5>Yeast Version</h5><br />
<p>While the promoters are natural ones without optimization, and our shuttle plasmid is pSR416 which is a single copied one, fluorescence is too weak to be captured by microfluidics device. That is a main reason for why we currently cannot show you a perfect cell magic movie. We are now constructing all devices with high copy number vectors, and try to optimize the junction near kozak sequence to make a better magic.</p><br />
<h5><i>E. coli</i> Version</h5><br />
<p>Promoters for <i>E. coli</i> seems not to be successful yet. In our first version, original 5' utr of promoter's downstream gene is included in BioBrick's sequences. The wild-type promoter and RBS may be too weak, or the junction of RFC[23] may have negative effect to the RBS. So in the present version, original 5' utr has been removed and strong RBS B0030 is used. We will update related results later.</p><br />
</div><br />
<br />
<div id="board3" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery3"><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/0/0d/Reporter.jpg" title="Modified Reporters" description="Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose."/></li><br />
</ul></div><br />
<div id="box3" class="box"><br />
<h3>Reporter Locating</h3><br />
<h4>Reporter Modification</h4><br />
<p>Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose.</p><br />
<img src="https://static.igem.org/mediawiki/2013/0/0d/Reporter.jpg" title="Modified Reporters" description="Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose."/><br />
<h4>Targeting Peptides</h4><br />
<p> The four pictures shows the reporters can rightly locate to Mitochondria, Nucleus and Vacuole,respectively. Picture A is the negative control, all yeast cells are lighted with GFP. And figure B is the reporter to the mitochondria, we can saw there are several light spots in one cells. Figure C is the reporter located in nucleus, the green spots are small and there is only one in a yeast cell.The last picture shows the reporter of Vacuolar membrane, the green lights were discrete in cells which was as expected.</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/><br />
</div><br />
<br />
<div id="board4" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery4"><br />
<li><img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" title="E. coli Version" description="Degradation tags testing device for E. coli"/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" title="IPTG Repressed" description="Microfluidics device for half life test with IPTG input (promoter of RFP repressed)."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/e9/Degladder.jpg" title="RFP Ladder" description="RFP ladder with different degradation tags)."/></li><br />
<br />
</ul></div><br />
<div id="box4" class="box"><br />
<h3>Degradation Rate</h3><br />
<p>In every curcuits measurement, we firstly test the growth curve through detecting the absorbance. Taken small amount of bateria, inoculate into 400ul LB medium, ensuring the initial concentration between 0.02-0.05 (OD600). Cultured in the room temperature, detect the OD600 value every 20 minutes. Y axis represents the logarithm of bacteria number, X axis represents the growth time. </p><br />
<img src="https://static.igem.org/mediawiki/2013/6/68/Growth-Curve.jpg" /><br />
<p>In E.coli, the adaptor SspB tethers ssrA tagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. Which means, a variation of the WT SsrA tag sequence (K1051206, K1051207 and K1051208) will accelerate the degradation of proteins when fused to their C-terminal. Thus the degradation rates are dependent on concentration of proteases and binding mediators.</p><br />
<p>We constructed the measurement pathway of each tag (K1051257, K1051258 and K1051259) to test the rates of degradation of tagged proteins respectively. J04450 was used as positive control because of the same promoter and fluorescent protein. </p><br />
<h4>Achievements</h4><br />
<img src="https://static.igem.org/mediawiki/2013/e/e9/Degladder.jpg" /><br />
<p>From right to left, the negative control,BBa_K1051257, BBa_K1051258, BBa_K1051259, J04450 as Positive Control. As the pictures showed, the lights of RFP within three degradation tags are decreasing. </p><br />
<!----------------------<br />
<img src="https://static.igem.org/mediawiki/2013/a/a6/IMG_0804.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/c/ca/IMG_0817.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/M0050bright.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/0/06/M0050strong.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/8/89/M0050weak.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/6/63/Flo.jpg" /><br />
--------------------><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" /><br />
<p>The test results for K1051258 and positive control. A. K1051258 in bright field; B. K1051258 in exciting lights; C. Positive control in bright field; D. K1051258 in exciting lights.</p><br />
<p>In picture, there are only obvious lights in the picture B, indicated the degradation rates are working.</p><br />
<p>预留酶标仪结果图和柱状图,降解效率换算</p><br />
<p>Enzyme - labelled meter detect the fluorescent protein intensity</p><br />
<p>First of all, take a certain amount of bacteria liquid, recovery to around OD0.6(600), ensuring the bacteria in the logarithmic growth phase. Diluted and then the bacteria transferred to 96 well plates, measured their fluorescence intensity. Red fluorescent protein using an excitation wavelength of 584nm, and its emission wavelength is 607nm.When measuring, we first detect the OD600 of each strain, removing the factor of bacteria number difference. Thus the fluorescence intensity cannot be altered by bacteria quantity. Then the measurement mode switching for measurement of fluorescence, fluorescence intensity.</p><br />
<!----------------------<br />
<img src="https://static.igem.org/mediawiki/2013/8/83/0917_t00_white_60mm.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/7/72/0917_t15_white_60mm.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/4/4e/0917_t30_white_60mm.jpg" /><br />
--------------------><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" /><br />
<p>The test results of BBa_K1051258 in chip. A,LB medium,O minuts; B, IPTG medium,9minutes; C,IPTG medium, 15 minutes</p><br />
<p>First of all, yeast will be measured after shaking to about OD2.0 (600)based on the chip which was washed by plasma water, vacuum pumping. The bacteria liquid was pushed into the chip, letting the cells enter the small triangle. Then use the constant flow pump culture medium into chip (the laboratory constant temperature, can not guarantee the training environment, 37 ° E. coli slower growth). The medium speed is about 200ul/h. Finally we test the data after yeast fulled in the triangles.</p><br />
<p>Using the IPTG medium, the new RFP expression was stopped and we can regard the lights as degradation tags' efficiency. Obviously, the lights are decreasing along with the time. Thus, it indicates that the degradation tags work effectively.</p><br />
<p>预留半衰期换算</p><br />
<h4>Problems</h4><br />
<h5>Yeast Version</h5><br />
<p>The test for yeast degradation tags has not been fully successful. Fluorescence has been captured in some occasions but not stable. It may caused by the same problem with promoter test devices - plasmid copy number. </p><br />
<h5><i>E. coli</i> Version</h5><br />
<p>Data collected from microscope for different degradation tags did not fully meet performance expectation. Reasons may be multiple to increase systematic errors.</p><br />
<p>- Fluorescence is too weak for calculating</p><br />
<p>- Quenching of fluorescence, especially in RFP</p><br />
<p>- Sample size is small for ImageJ calculating</p><br />
<p>We will have more data detected by microfluidics and flow cytometry. Results will be updated later.</p><br />
</div><br />
<br />
<div id="board5" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery5"><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/e4/09172_bright.jpg" title="E. coli captured" description="E. coli cells captured by microfluidics device."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/3/3f/Sc_bright_20.jpg" title="E. coli captured" description="Yeast cells captured by microfluidics device."/></li><br />
</ul></div><br />
<div id="box5" class="box"><br />
<h3>Cell Synchronization</h3><br />
<h4>Microfluidics Device</h4><br />
<p>We made the chip as a platform for watching and synchronize the cells. First step is capture the cells by the chip. As showed in the two figures, both E.coli and budding yeast can be captured by the chip successfully.</p><br />
<img src="https://static.igem.org/mediawiki/2013/e/e4/09172_bright.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/3/3f/Sc_bright_20.jpg" /><br />
<h4>Sic1 Synchronization</h4><br />
<p>Seven phosphorylation sites are mutated by PCR to make Sic1 mutant. However, it has been taking us too long time that we have been still working on right clones. Experiments results will be updated later.</p><br />
</div><br />
<br />
<div id="board6" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery6"><br />
<li><img src="https://static.igem.org/mediawiki/2013/b/b9/Blank-01.png" /><li><br />
</ul></div><br />
<div id="box6" class="box"><br />
<h3>Alternative Splicing</h3><br />
<h4>Original Design</h4><br />
<p>In our project, we attempt to use intron as a switch. However, according to previous research, there are two splicing forms of SRC1 intron: one is complete splicing (5’S) while the other can leave 4bp at its 5’ end (5’L). The remaining 4-bp causes frame-shift and a stop codon in the region of adaptor, thus the following exon will not be translated. However, this kind of switch is not complete – the ratio of 5’L and 5’S varied between 40-60 and 85-15.</p><br />
<p>In order to make a complete switch, we abandoned the remaining 4bp of SRC1 intron to avoid producing 5’L mRNA. So the result comes out to be: when Hub1p are expressed, they bind to spliceosome to modify it, resulting to easy recognizing 5’S splice site and its splicing; when inhibit the expression of HUB1 by CRISPRi system, spliceosome can hardly recognize 5’S splice site, therefore no intron can be spliced. </p><br />
<h4>Further Plans</h4><br />
<p>However, things didn’t go as we expected. Our experiment showed that this 4bp plays important role in SRC1 intron splicing. So, to achieve our goal, we redesigned another three versions of SRC1 intron:<br />
</p><br />
<p>1. Intron with GG at its 5’ end</p><br />
<p>2. Intron with CGG at its 5’ end</p><br />
<p>3. Intron with its original 6bp at both end</p><br />
<p>Due to the lack of time, we are still constructing these parts to test devices.</p><br />
<br />
</div><br />
<br />
</div></div>BGI K2http://2013.igem.org/Team:Shenzhen_BGIC_ATCG/resultsTeam:Shenzhen BGIC ATCG/results2013-09-27T22:14:02Z<p>BGI K2: </p>
<hr />
<div><!----------------------<br />
<br />
<br />
Title: Results<br />
Team: Shenzhen_BGIC_ATCG<br />
Author: BGI_K2<br />
Twitter: @BGI_K2<br />
Email: im.ss.kk@gmail.com<br />
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<div class="navleftholder"><br />
<ul class="navleft"><br />
<li><a id="navleftsub1" class="navleftsub" href="#board1">The Magic</a></li><br />
<li><a id="navleftsub2" class="navleftsub" href="#board2">Promoter Verification</a></li><br />
<li><a id="navleftsub3" class="navleftsub" href="#board3">Reporter Locating</a></li><br />
<li><a id="navleftsub4" class="navleftsub" href="#board4">Degradation Rate</a></li><br />
<li><a id="navleftsub5" class="navleftsub" href="#board5">Cell Synchronization</a></li><br />
<li><a id="navleftsub6" class="navleftsub" href="#board6">Alternative Splicing</a></li><br />
</ul></div><br />
<br />
<div id="board1" class="board"></div><br />
<div class="media"><br />
<br />
<ul id="myGallery1"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/></li><br />
<li><img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" title="Degradation Tags" description="Degradation tags testing device for E. coli."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" title="Microfluidics" description="Microfluidics device for degradation test."/></li><br />
<br />
</ul></div><br />
<div id="box1" class="box"><br />
<h3>The Magic</h3><br />
<p>Our project “Cell Magic", a complex work has been completed partly,due to the time limitation. The results we have gotten are listed below:</p><br />
<p>We verified some of the cyclin promoters. The clb6 promoter, which is supposed to be expressed in G1 phase, has been verified perfectly.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." width=50%/><br />
<p>Targeting peptides to three locations have been verified. Others are still being constructed and tested.</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. " width=50%/><br />
<p>We got good data for degradation tags for <i>E. coli</i>. Construction of degradation devices for yeast have been finished and being tested.</p><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" width=50%/><br />
<p>Microfluidics device has been successfully developed for measurement and cell synchronization.</p><br />
<img src="https://static.igem.org/mediawiki/2013/f/fc/Microfluidic.gif" /><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" width=50%/><br />
<p>Cell synchronization device and alternative splicing device are still in construction, related results will be updated later.</p><br />
<br />
</div><br />
<br />
<div id="board2" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery2"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/1/19/Flu.jpg" title="S Phase Promoter" description="cln3 promoter for S phase has been verified using flow cytometry." /></li><br />
</ul></div><br />
<div id="box2" class="box"><br />
<h3>Promoter Verification</h3><br />
<h4>Achievements</h4><br />
<p> Using the GFP as reporter and morphological alteration as cell cycle representation, we verified the Clb6 can be activated in G1 phase in the yeast.</P><br />
<p> As shown in the picture in normal lights, there are some yeast were budding. These budded yeast cells and small size budding yeasts (within the red circles) are assumed in the G1 phase of cell cycle. And the next picture was taken under activation light, thus the green lights can representing the Clb6 promoter expressing. The two pictures indicated the Clb6 were expressed in the G1 phase as we expected when designing the experiments </p><br />
<p> PS: we modified the contrast ratio to lower the lights of neighbor cells, thus our results looking better.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" /><br />
<p>We also captured fluorescence on cln3 promoter (S phase) test device, for the fluorescence is quite weak, we utilize micro plate reader and flow cytometry to verify the promoter.</p><br />
<img src="https://static.igem.org/mediawiki/2013/1/19/Flu.jpg" /><br />
<h4>Problems</h4><br />
<h5>Yeast Version</h5><br />
<p>While the promoters are natural ones without optimization, and our shuttle plasmid is pSR416 which is a single copied one, fluorescence is too weak to be captured by microfluidics device. That is a main reason for why we currently cannot show you a perfect cell magic movie. We are now constructing all devices with high copy number vectors, and try to optimize the junction near kozak sequence to make a better magic.</p><br />
<h5><i>E. coli</i> Version</h5><br />
<p>Promoters for <i>E. coli</i> seems not to be successful yet. In our first version, original 5' utr of promoter's downstream gene is included in BioBrick's sequences. The wild-type promoter and RBS may be too weak, or the junction of RFC[23] may have negative effect to the RBS. So in the present version, original 5' utr has been removed and strong RBS B0030 is used. We will update related results later.</p><br />
</div><br />
<br />
<div id="board3" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery3"><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/0/0d/Reporter.jpg" title="Modified Reporters" description="Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose."/></li><br />
</ul></div><br />
<div id="box3" class="box"><br />
<h3>Reporter Locating</h3><br />
<h4>Reporter Modification</h4><br />
<p>Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose.</p><br />
<img src="https://static.igem.org/mediawiki/2013/0/0d/Reporter.jpg" title="Modified Reporters" description="Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose."/><br />
<h4>Targeting Peptides</h4><br />
<p> The four pictures shows the reporters can rightly locate to Mitochondria, Nucleus and Vacuole,respectively. Picture A is the negative control, all yeast cells are lighted with GFP. And figure B is the reporter to the mitochondria, we can saw there are several light spots in one cells. Figure C is the reporter located in nucleus, the green spots are small and there is only one in a yeast cell.The last picture shows the reporter of Vacuolar membrane, the green lights were discrete in cells which was as expected.</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/><br />
</div><br />
<br />
<div id="board4" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery4"><br />
<li><img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" title="E. coli Version" description="Degradation tags testing device for E. coli"/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" title="IPTG Repressed" description="Microfluidics device for half life test with IPTG input (promoter of RFP repressed)."/></li><br />
</ul></div><br />
<div id="box4" class="box"><br />
<h3>Degradation Rate</h3><br />
<p>In every curcuits measurement, we firstly test the growth curve through detecting the absorbance. Taken small amount of bateria, inoculate into 400ul LB medium, ensuring the initial concentration between 0.02-0.05 (OD600). Cultured in the room temperature, detect the OD600 value every 20 minutes. Y axis represents the logarithm of bacteria number, X axis represents the growth time. </p><br />
<img src="https://static.igem.org/mediawiki/2013/6/68/Growth-Curve.jpg" /><br />
<p>In E.coli, the adaptor SspB tethers ssrA tagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. Which means, a variation of the WT SsrA tag sequence (K1051206, K1051207 and K1051208) will accelerate the degradation of proteins when fused to their C-terminal. Thus the degradation rates are dependent on concentration of proteases and binding mediators.</p><br />
<p>We constructed the measurement pathway of each tag (K1051257, K1051258 and K1051259) to test the rates of degradation of tagged proteins respectively. J04450 was used as positive control because of the same promoter and fluorescent protein. </p><br />
<h4>Achievements</h4><br />
<img src="https://static.igem.org/mediawiki/2013/e/e9/Degladder.jpg" /><br />
<p>From right to left, the negative control,BBa_K1051257, BBa_K1051258, BBa_K1051259, J04450 as Positive Control. As the pictures showed, the lights of RFP within three degradation tags are decreasing. </p><br />
<!----------------------<br />
<img src="https://static.igem.org/mediawiki/2013/a/a6/IMG_0804.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/c/ca/IMG_0817.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/M0050bright.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/0/06/M0050strong.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/8/89/M0050weak.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/6/63/Flo.jpg" /><br />
--------------------><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" /><br />
<p>The test results for K1051258 and positive control. A. K1051258 in bright field; B. K1051258 in exciting lights; C. Positive control in bright field; D. K1051258 in exciting lights.</p><br />
<p>In picture, there are only obvious lights in the picture B, indicated the degradation rates are working.</p><br />
<p>预留酶标仪结果图和柱状图,降解效率换算</p><br />
<p>Enzyme - labelled meter detect the fluorescent protein intensity</p><br />
<p>First of all, take a certain amount of bacteria liquid, recovery to around OD0.6(600), ensuring the bacteria in the logarithmic growth phase. Diluted and then the bacteria transferred to 96 well plates, measured their fluorescence intensity. Red fluorescent protein using an excitation wavelength of 584nm, and its emission wavelength is 607nm.When measuring, we first detect the OD600 of each strain, removing the factor of bacteria number difference. Thus the fluorescence intensity cannot be altered by bacteria quantity. Then the measurement mode switching for measurement of fluorescence, fluorescence intensity.</p><br />
<!----------------------<br />
<img src="https://static.igem.org/mediawiki/2013/8/83/0917_t00_white_60mm.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/7/72/0917_t15_white_60mm.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/4/4e/0917_t30_white_60mm.jpg" /><br />
--------------------><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" /><br />
<p>The test results of BBa_K1051258 in chip. A,LB medium,O minuts; B, IPTG medium,9minutes; C,IPTG medium, 15 minutes</p><br />
<p>First of all, yeast will be measured after shaking to about OD2.0 (600)based on the chip which was washed by plasma water, vacuum pumping. The bacteria liquid was pushed into the chip, letting the cells enter the small triangle. Then use the constant flow pump culture medium into chip (the laboratory constant temperature, can not guarantee the training environment, 37 ° E. coli slower growth). The medium speed is about 200ul/h. Finally we test the data after yeast fulled in the triangles.</p><br />
<p>Using the IPTG medium, the new RFP expression was stopped and we can regard the lights as degradation tags' efficiency. Obviously, the lights are decreasing along with the time. Thus, it indicates that the degradation tags work effectively.</p><br />
<p>预留半衰期换算</p><br />
<h4>Problems</h4><br />
<h5>Yeast Version</h5><br />
<p>The test for yeast degradation tags has not been fully successful. Fluorescence has been captured in some occasions but not stable. It may caused by the same problem with promoter test devices - plasmid copy number. </p><br />
<h5><i>E. coli</i> Version</h5><br />
<p>Data collected from microscope for different degradation tags did not fully meet performance expectation. Reasons may be multiple to increase systematic errors.</p><br />
<p>- Fluorescence is too weak for calculating</p><br />
<p>- Quenching of fluorescence, especially in RFP</p><br />
<p>- Sample size is small for ImageJ calculating</p><br />
<p>We will have more data detected by microfluidics and flow cytometry. Results will be updated later.</p><br />
</div><br />
<br />
<div id="board5" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery5"><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/e4/09172_bright.jpg" title="E. coli captured" description="E. coli cells captured by microfluidics device."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/3/3f/Sc_bright_20.jpg" title="E. coli captured" description="Yeast cells captured by microfluidics device."/></li><br />
</ul></div><br />
<div id="box5" class="box"><br />
<h3>Cell Synchronization</h3><br />
<h4>Microfluidics Device</h4><br />
<p>We made the chip as a platform for watching and synchronize the cells. First step is capture the cells by the chip. As showed in the two figures, both E.coli and budding yeast can be captured by the chip successfully.</p><br />
<img src="https://static.igem.org/mediawiki/2013/e/e4/09172_bright.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/3/3f/Sc_bright_20.jpg" /><br />
<h4>Sic1 Synchronization</h4><br />
<p>Seven phosphorylation sites are mutated by PCR to make Sic1 mutant. However, it has been taking us too long time that we have been still working on right clones. Experiments results will be updated later.</p><br />
</div><br />
<br />
<div id="board6" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery6"><br />
<li><img src="https://static.igem.org/mediawiki/2013/b/b9/Blank-01.png" /><li><br />
</ul></div><br />
<div id="box6" class="box"><br />
<h3>Alternative Splicing</h3><br />
<h4>Original Design</h4><br />
<p>In our project, we attempt to use intron as a switch. However, according to previous research, there are two splicing forms of SRC1 intron: one is complete splicing (5’S) while the other can leave 4bp at its 5’ end (5’L). The remaining 4-bp causes frame-shift and a stop codon in the region of adaptor, thus the following exon will not be translated. However, this kind of switch is not complete – the ratio of 5’L and 5’S varied between 40-60 and 85-15.</p><br />
<p>In order to make a complete switch, we abandoned the remaining 4bp of SRC1 intron to avoid producing 5’L mRNA. So the result comes out to be: when Hub1p are expressed, they bind to spliceosome to modify it, resulting to easy recognizing 5’S splice site and its splicing; when inhibit the expression of HUB1 by CRISPRi system, spliceosome can hardly recognize 5’S splice site, therefore no intron can be spliced. </p><br />
<h4>Further Plans</h4><br />
<p>However, things didn’t go as we expected. Our experiment showed that this 4bp plays important role in SRC1 intron splicing. So, to achieve our goal, we redesigned another three versions of SRC1 intron:<br />
</p><br />
<p>1. Intron with GG at its 5’ end</p><br />
<p>2. Intron with CGG at its 5’ end</p><br />
<p>3. Intron with its original 6bp at both end</p><br />
<p>Due to the lack of time, we are still constructing these parts to test devices.</p><br />
<br />
</div><br />
<br />
</div></div>BGI K2http://2013.igem.org/Team:Shenzhen_BGIC_ATCG/resultsTeam:Shenzhen BGIC ATCG/results2013-09-27T21:52:49Z<p>BGI K2: </p>
<hr />
<div><!----------------------<br />
<br />
<br />
Title: Results<br />
Team: Shenzhen_BGIC_ATCG<br />
Author: BGI_K2<br />
Twitter: @BGI_K2<br />
Email: im.ss.kk@gmail.com<br />
<br />
Please contact me if you would like to use this script<br />
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<div class="navleftholder"><br />
<ul class="navleft"><br />
<li><a id="navleftsub1" class="navleftsub" href="#board1">The Magic</a></li><br />
<li><a id="navleftsub2" class="navleftsub" href="#board2">Promoter Verification</a></li><br />
<li><a id="navleftsub3" class="navleftsub" href="#board3">Reporter Locating</a></li><br />
<li><a id="navleftsub4" class="navleftsub" href="#board4">Degradation Rate</a></li><br />
<li><a id="navleftsub5" class="navleftsub" href="#board5">Cell Synchronization</a></li><br />
<li><a id="navleftsub6" class="navleftsub" href="#board6">Alternative Splicing</a></li><br />
</ul></div><br />
<br />
<div id="board1" class="board"></div><br />
<div class="media"><br />
<br />
<ul id="myGallery1"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/></li><br />
<li><img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" title="Degradation Tags" description="Degradation tags testing device for E. coli."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" title="Microfluidics" description="Microfluidics device for degradation test."/></li><br />
<br />
</ul></div><br />
<div id="box1" class="box"><br />
<h3>The Magic</h3><br />
<p>Our project “Cell Magic", a complex work has been completed partly,due to the time limitation. The results we have gotten are listed below:</p><br />
<p>We verified some of the cyclin promoters. The clb6 promoter, which is supposed to be expressed in G1 phase, has been verified perfectly.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." width=50%/><br />
<p>Targeting peptides to three locations have been verified. Others are still being constructed and tested.</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. " width=50%/><br />
<p>We got good data for degradation tags for <i>E. coli</i>. Construction of degradation devices for yeast have been finished and being tested.</p><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" width=50%/><br />
<p>Microfluidics device has been successfully developed for measurement and cell synchronization.</p><br />
<img src="https://static.igem.org/mediawiki/2013/f/fc/Microfluidic.gif" /><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" width=50%/><br />
<p>Cell synchronization device and alternative splicing device are still in construction, related results will be updated later.</p><br />
<br />
</div><br />
<br />
<div id="board2" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery2"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/1/19/Flu.jpg" title="S Phase Promoter" description="cln3 promoter for S phase has been verified using flow cytometry." /></li><br />
</ul></div><br />
<div id="box2" class="box"><br />
<h3>Promoter Verification</h3><br />
<h4>Achievements</h4><br />
<p> Using the GFP as reporter and morphological alteration as cell cycle representation, we verified the Clb6 can be activated in G1 phase in the yeast.</P><br />
<p> As shown in the picture in normal lights, there are some yeast were budding. These budded yeast cells and small size budding yeasts (within the red circles) are assumed in the G1 phase of cell cycle. And the next picture was taken under activation light, thus the green lights can representing the Clb6 promoter expressing. The two pictures indicated the Clb6 were expressed in the G1 phase as we expected when designing the experiments </p><br />
<p> PS: we modified the contrast ratio to lower the lights of neighbor cells, thus our results looking better.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" /><br />
<p>We also captured fluorescence on cln3 promoter (S phase) test device, for the fluorescence is quite weak, we utilize micro plate reader and flow cytometry to verify the promoter.</p><br />
<img src="https://static.igem.org/mediawiki/2013/1/19/Flu.jpg" /><br />
<h4>Problems</h4><br />
<p>While the promoters are natural ones without optimization, and our shuttle plasmid is pSR416 which is a single copied one, fluorescence is too weak to be captured by microfluidics device. That is a main reason for why we currently cannot show you a perfect cell magic movie. We are now constructing all devices with high copy number vectors, and try to optimize the junction near kozak sequence to make a better magic.</p><br />
<br />
</div><br />
<br />
<div id="board3" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery3"><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/0/0d/Reporter.jpg" title="Modified Reporters" description="Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose."/></li><br />
</ul></div><br />
<div id="box3" class="box"><br />
<h3>Reporter Locating</h3><br />
<h4>Reporter Modification</h4><br />
<p>Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose.</p><br />
<img src="https://static.igem.org/mediawiki/2013/0/0d/Reporter.jpg" title="Modified Reporters" description="Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose."/><br />
<h4>Targeting Peptides</h4><br />
<p> The four pictures shows the reporters can rightly locate to Mitochondria, Nucleus and Vacuole,respectively. Picture A is the negative control, all yeast cells are lighted with GFP. And figure B is the reporter to the mitochondria, we can saw there are several light spots in one cells. Figure C is the reporter located in nucleus, the green spots are small and there is only one in a yeast cell.The last picture shows the reporter of Vacuolar membrane, the green lights were discrete in cells which was as expected.</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/><br />
</div><br />
<br />
<div id="board4" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery4"><br />
<li><img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" title="E. coli Version" description="Degradation tags testing device for E. coli"/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" title="IPTG Repressed" description="Microfluidics device for half life test with IPTG input (promoter of RFP repressed)."/></li><br />
</ul></div><br />
<div id="box4" class="box"><br />
<h3>Degradation Rate</h3><br />
<p>In every curcuits measurement, we firstly test the growth curve through detecting the absorbance. Taken small amount of bateria, inoculate into 400ul LB medium, ensuring the initial concentration between 0.02-0.05 (OD600). Cultured in the room temperature, detect the OD600 value every 20 minutes. Y axis represents the logarithm of bacteria number, X axis represents the growth time. </p><br />
<img src="https://static.igem.org/mediawiki/2013/6/68/Growth-Curve.jpg" /><br />
<p>In E.coli, the adaptor SspB tethers ssrA tagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. Which means, a variation of the WT SsrA tag sequence (K1051206, K1051207 and K1051208) will accelerate the degradation of proteins when fused to their C-terminal. Thus the degradation rates are dependent on concentration of proteases and binding mediators.</p><br />
<p>We constructed the measurement pathway of each tag (K1051257, K1051258 and K1051259) to test the rates of degradation of tagged proteins respectively. J04450 was used as positive control because of the same promoter and fluorescent protein. </p><br />
<img src="https://static.igem.org/mediawiki/2013/e/e9/Degladder.jpg" /><br />
<p>From right to left, the negative control,BBa_K1051257, BBa_K1051258, BBa_K1051259, J04450 as Positive Control. As the pictures showed, the lights of RFP within three degradation tags are decreasing. </p><br />
<!----------------------<br />
<img src="https://static.igem.org/mediawiki/2013/a/a6/IMG_0804.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/c/ca/IMG_0817.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/M0050bright.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/0/06/M0050strong.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/8/89/M0050weak.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/6/63/Flo.jpg" /><br />
--------------------><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" /><br />
<p>The test results for K1051258 and positive control. A. K1051258 in bright field; B. K1051258 in exciting lights; C. Positive control in bright field; D. K1051258 in exciting lights.</p><br />
<p>In picture, there are only obvious lights in the picture B, indicated the degradation rates are working.</p><br />
<p>预留酶标仪结果图和柱状图,降解效率换算</p><br />
<p>Enzyme - labelled meter detect the fluorescent protein intensity</p><br />
<p>First of all, take a certain amount of bacteria liquid, recovery to around OD0.6(600), ensuring the bacteria in the logarithmic growth phase. Diluted and then the bacteria transferred to 96 well plates, measured their fluorescence intensity. Red fluorescent protein using an excitation wavelength of 584nm, and its emission wavelength is 607nm.When measuring, we first detect the OD600 of each strain, removing the factor of bacteria number difference. Thus the fluorescence intensity cannot be altered by bacteria quantity. Then the measurement mode switching for measurement of fluorescence, fluorescence intensity.</p><br />
<!----------------------<br />
<img src="https://static.igem.org/mediawiki/2013/8/83/0917_t00_white_60mm.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/7/72/0917_t15_white_60mm.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/4/4e/0917_t30_white_60mm.jpg" /><br />
--------------------><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" /><br />
<p>The test results of BBa_K1051258 in chip. A,LB medium,O minuts; B, IPTG medium,9minutes; C,IPTG medium, 15 minutes</p><br />
<p>First of all, yeast will be measured after shaking to about OD2.0 (600)based on the chip which was washed by plasma water, vacuum pumping. The bacteria liquid was pushed into the chip, letting the cells enter the small triangle. Then use the constant flow pump culture medium into chip (the laboratory constant temperature, can not guarantee the training environment, 37 ° E. coli slower growth). The medium speed is about 200ul/h. Finally we test the data after yeast fulled in the triangles.</p><br />
<p>Using the IPTG medium, the new RFP expression was stopped and we can regard the lights as degradation tags' efficiency. Obviously, the lights are decreasing along with the time. Thus, it indicates that the degradation tags work effectively</p><br />
<p>预留半衰期换算</p><br />
<br />
</div><br />
<br />
<div id="board5" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery5"><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/e4/09172_bright.jpg" title="E. coli captured" description="E. coli cells captured by microfluidics device."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/3/3f/Sc_bright_20.jpg" title="E. coli captured" description="Yeast cells captured by microfluidics device."/></li><br />
</ul></div><br />
<div id="box5" class="box"><br />
<h3>Cell Synchronization</h3><br />
<p>We made the chip as a platform for watching and synchronize the cells. First step is capture the cells by the chip. As showed in the two figures, both E.coli and budding yeast can be captured by the chip successfully.</p><br />
<img src="https://static.igem.org/mediawiki/2013/e/e4/09172_bright.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/3/3f/Sc_bright_20.jpg" /><br />
<p>Seven phosphorylation sites are mutated by PCR to make Sic1 mutant. However, it has been taking us too long time that we have been still working on right clones. Experiments results will be updated later.</p><br />
</div><br />
<br />
<div id="board6" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery6"><br />
<li><img src="https://static.igem.org/mediawiki/2013/b/b9/Blank-01.png" /><li><br />
</ul></div><br />
<div id="box6" class="box"><br />
<h3>Alternative Splicing</h3><br />
<p>In our project, we attempt to use intron as a switch. However, according to previous research, there are two splicing forms of SRC1 intron: one is complete splicing (5’S) while the other can leave 4bp at its 5’ end (5’L). The remaining 4-bp causes frame-shift and a stop codon in the region of adaptor, thus the following exon will not be translated. However, this kind of switch is not complete – the ratio of 5’L and 5’S varied between 40-60 and 85-15.<br />
</p><br />
<p></p><br />
<p>In order to make a complete switch, we abandoned the remaining 4bp of SRC1 intron to avoid producing 5’L mRNA. So the result comes out to be: when Hub1p are expressed, they bind to spliceosome to modify it, resulting to easy recognizing 5’S splice site and its splicing; when inhibit the expression of HUB1 by CRISPRi system, spliceosome can hardly recognize 5’S splice site, therefore no intron can be spliced. <br />
</p><br />
<p></p><br />
<p>However, things didn’t go as we expected. Our experiment showed that this 4bp plays important role in SRC1 intron splicing. So, to achieve our goal, we redesigned another three versions of SRC1 intron:<br />
</p><br />
<p>1. Intron with GG at its 5’ end</p><br />
<p>2. Intron with CGG at its 5’ end</p><br />
<p>3. Intron with its original 6bp at both end</p><br />
<p></p><br />
<p>However, due to the lack of time, we could not connect all these parts to our test parts.<br />
</p><br />
<br />
</div><br />
<br />
</div></div>BGI K2http://2013.igem.org/File:Microfluidic.gifFile:Microfluidic.gif2013-09-27T21:40:16Z<p>BGI K2: uploaded a new version of &quot;File:Microfluidic.gif&quot;</p>
<hr />
<div></div>BGI K2http://2013.igem.org/Team:Shenzhen_BGIC_ATCG/resultsTeam:Shenzhen BGIC ATCG/results2013-09-27T21:24:10Z<p>BGI K2: </p>
<hr />
<div><!----------------------<br />
<br />
<br />
Title: Results<br />
Team: Shenzhen_BGIC_ATCG<br />
Author: BGI_K2<br />
Twitter: @BGI_K2<br />
Email: im.ss.kk@gmail.com<br />
<br />
Please contact me if you would like to use this script<br />
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<div class="navleftholder"><br />
<ul class="navleft"><br />
<li><a id="navleftsub1" class="navleftsub" href="#board1">The Magic</a></li><br />
<li><a id="navleftsub2" class="navleftsub" href="#board2">Promoter Verification</a></li><br />
<li><a id="navleftsub3" class="navleftsub" href="#board3">Reporter Locating</a></li><br />
<li><a id="navleftsub4" class="navleftsub" href="#board4">Degradation Rate</a></li><br />
<li><a id="navleftsub5" class="navleftsub" href="#board5">Cell Synchronization</a></li><br />
<li><a id="navleftsub6" class="navleftsub" href="#board6">Alternative Splicing</a></li><br />
</ul></div><br />
<br />
<div id="board1" class="board"></div><br />
<div class="media"><br />
<br />
<ul id="myGallery1"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/></li><br />
<li><img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" title="Degradation Tags" description="Degradation tags testing device for E. coli."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" title="Microfluidics" description="Microfluidics device for degradation test."/></li><br />
<br />
</ul></div><br />
<div id="box1" class="box"><br />
<h3>The Magic</h3><br />
<p>Our project “Cell Magic", a complex work has been completed partly,due to the time limitation. The results we have gotten are listed below:</p><br />
<p>We verified some of the cyclin promoters. The clb6 promoter, which is supposed to be expressed in G1 phase, has been verified perfectly.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." width=50%/><br />
<p>Targeting peptides to three locations have been verified. Others are still being constructed and tested.</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. " width=50%/><br />
<p>We got good data for degradation tags for <i>E. coli</i>. Construction of degradation devices for yeast have been finished and being tested.</p><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" width=50%/><br />
<p>Microfluidics device has been successfully developed for measurement and cell synchronization.</p><br />
<img src="https://static.igem.org/mediawiki/2013/f/fc/Microfluidic.gif" /><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" width=50%/><br />
<p>Cell synchronization device and alternative splicing device are still in construction, related results will be updated later.</p><br />
<br />
</div><br />
<br />
<div id="board2" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery2"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/1/19/Flu.jpg" title="S Phase Promoter" description="cln3 promoter for S phase has been verified using flow cytometry." /></li><br />
</ul></div><br />
<div id="box2" class="box"><br />
<h3>Promoter Verification</h3><br />
<p> Using the GFP as reporter and morphological alteration as cell cycle representation, we verified the Clb6 can be activated in G1 phase in the yeast.</P><br />
<p> As shown in the picture in normal lights, there are some yeast were budding. These budded yeast cells and small size budding yeasts (within the red circles) are assumed in the G1 phase of cell cycle. And the next picture was taken under activation light, thus the green lights can representing the Clb6 promoter expressing. The two pictures indicated the Clb6 were expressed in the G1 phase as we expected when designing the experiments </p><br />
<p> PS: we modified the contrast ratio to lower the lights of neighbor cells, thus our results looking better.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" /><br />
<p>We also captured fluorescence on cln3 promoter (S phase) test device, for the fluorescence is quite weak, we utilize micro plate reader and flow cytometry to verify the promoter.</p><br />
<img src="https://static.igem.org/mediawiki/2013/1/19/Flu.jpg" /><br />
<p>While the promoters are natural ones without optimization, and our shuttle plasmid is pSR416 which is a single copied one, fluorescence is too weak to be captured by microfluidics device. That is a main reason for why we currently cannot show you a perfect cell magic movie. We are now constructing all devices with high copy number vectors, and try to optimize the junction near kozak sequence to make a better magic.</p><br />
<br />
</div><br />
<br />
<div id="board3" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery3"><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/></li><br />
</ul></div><br />
<div id="box3" class="box"><br />
<h3>Reporter Locating</h3><br />
<p>Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose.</p><br />
<img src="https://static.igem.org/mediawiki/2013/0/0d/Reporter.jpg" title="Modified Reporters" description="Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose."/><br />
<p> The four pictures shows the reporters can rightly locate to Mitochondria, Nucleus and Vacuole,respectively. Picture A is the negative control, all yeast cells are lighted with GFP. And figure B is the reporter to the mitochondria, we can saw there are several light spots in one cells. Figure C is the reporter located in nucleus, the green spots are small and there is only one in a yeast cell.The last picture shows the reporter of Vacuolar membrane, the green lights were discrete in cells which was as expected.</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/><br />
</div><br />
<br />
<div id="board4" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery4"><br />
<li><img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" title="E. coli Version" description="Degradation tags testing device for E. coli"/></li><br />
</ul></div><br />
<div id="box4" class="box"><br />
<h3>Degradation Rate</h3><br/><br />
<p>In E.coli, the adaptor SspB tethers ssrAtagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. Which means, a variation of the WT SsrA tag sequence (K1051206, K1051207 and K1051208) will accelerate the degradation of proteins when fused to their C-terminal. Thus the degradation rates are dependent on concentration of proteases and binding mediators.</p><br />
<p>We constructed the measurement pathway of each tag (K1051257, K1051258 and K1051259) to test the rates of degradation of tagged proteins respectively. J04450 was used as positive control because of the same promoter and fluorescent protein. </p><br />
<img src="https://static.igem.org/mediawiki/2013/6/68/Growth-Curve.jpg" /><br />
<p>In every curcuits measurement, we firstly test the growth curve through detecting the absorbance. Taken small amount of bateria, inoculate into 400ul LB medium, ensuring the initial concentration between 0.02-0.05 (OD600). Cultured in the room temperature, detect the OD600 value every 20 minutes. Y axis represents the logarithm of bacteria number, X axis represents the growth time. </p><br />
<br />
<img src="https://static.igem.org/mediawiki/2013/e/e9/Degladder.jpg" /><br />
<p>From right to left, the negative control,BBa_K1051257, BBa_K1051258, BBa_K1051259, J04450 as Positive Control. As the pictures showed, the lights of RFP within three degradation tags are decreasing. </p><br />
<!----------------------<br />
<img src="https://static.igem.org/mediawiki/2013/a/a6/IMG_0804.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/c/ca/IMG_0817.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/M0050bright.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/0/06/M0050strong.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/8/89/M0050weak.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/6/63/Flo.jpg" /><br />
--------------------><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" /><br />
<p>The test results for K1051258 and positive control. A. K1051258 in bright field; B. K1051258 in exciting lights; C. Positive control in bright field; D. K1051258 in exciting lights.</p><br />
<p>In picture, there are only obvious lights in the picture B, indicated the degradation rates are working.</p><br />
<p>预留酶标仪结果图和柱状图,降解效率换算</p><br />
<p>Enzyme - labelled meter detect the fluorescent protein intensity</p><br />
<p>First of all, take a certain amount of bacteria liquid, recovery to around OD0.6(600), ensuring the bacteria in the logarithmic growth phase. Diluted and then the bacteria transferred to 96 well plates, measured their fluorescence intensity. Red fluorescent protein using an excitation wavelength of 584nm, and its emission wavelength is 607nm.When measuring, we first detect the OD600 of each strain, removing the factor of bacteria number difference. Thus the fluorescence intensity cannot be altered by bacteria quantity. Then the measurement mode switching for measurement of fluorescence, fluorescence intensity.</p><br />
<!----------------------<br />
<img src="https://static.igem.org/mediawiki/2013/8/83/0917_t00_white_60mm.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/7/72/0917_t15_white_60mm.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/4/4e/0917_t30_white_60mm.jpg" /><br />
--------------------><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" /><br />
<p>The test results of BBa_K1051258 in chip. A,LB medium,O minuts; B, IPTG medium,9minutes; C,IPTG medium, 15 minutes</p><br />
<p>First of all, yeast will be measured after shaking to about OD2.0 (600)based on the chip which was washed by plasma water, vacuum pumping. The bacteria liquid was pushed into the chip, letting the cells enter the small triangle. Then use the constant flow pump culture medium into chip (the laboratory constant temperature, can not guarantee the training environment, 37 ° E. coli slower growth). The medium speed is about 200ul/h. Finally we test the data after yeast fulled in the triangles.</p><br />
<p>Using the IPTG medium, the new RFP expression was stopped and we can regard the lights as degradation tags'effecience. Obviously, the lights are decreasing along with the time. Thus, it indicates that the degradation tags work effectively</p><br />
<p>预留半衰期换算</p><br />
<br />
</div><br />
<br />
<div id="board5" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery5"><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/e4/09172_bright.jpg" title="E. coli captured" description="E. coli cells captured by microfluidics device."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/3/3f/Sc_bright_20.jpg" title="E. coli captured" description="Yeast cells captured by microfluidics device."/></li><br />
</ul></div><br />
<div id="box5" class="box"><br />
<h3>Cell Synchronization</h3><br />
<p>We made the chip as a platform for watching and synchronize the cells. First step is capture the cells by the chip. As showed in the two figures, both E.coli and budding yeast can be captured by the chip successfully.</p><br />
<img src="https://static.igem.org/mediawiki/2013/e/e4/09172_bright.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/3/3f/Sc_bright_20.jpg" /><br />
<p>Seven phosphorylation sites are mutated by PCR to make Sic1 mutant. However, it has been taking us too long time that we have been still working on right clones. Experiments results will be updated later.</p><br />
</div><br />
<br />
<div id="board6" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery6"><br />
<li><img src="https://static.igem.org/mediawiki/2013/b/b9/Blank-01.png" /><li><br />
</ul></div><br />
<div id="box6" class="box"><br />
<h3>Alternative Splicing</h3><br />
<p>In our project, we attempt to use intron as a switch. However, according to previous research, there are two splicing forms of SRC1 intron: one is complete splicing (5’S) while the other can leave 4bp at its 5’ end (5’L). The remaining 4-bp causes frame-shift and a stop codon in the region of adaptor, thus the following exon will not be translated. However, this kind of switch is not complete – the ratio of 5’L and 5’S varied between 40-60 and 85-15.<br />
</p><br />
<p></p><br />
<p>In order to make a complete switch, we abandoned the remaining 4bp of SRC1 intron to avoid producing 5’L mRNA. So the result comes out to be: when Hub1p are expressed, they bind to spliceosome to modify it, resulting to easy recognizing 5’S splice site and its splicing; when inhibit the expression of HUB1 by CRISPRi system, spliceosome can hardly recognize 5’S splice site, therefore no intron can be spliced. <br />
</p><br />
<p></p><br />
<p>However, things didn’t go as we expected. Our experiment showed that this 4bp plays important role in SRC1 intron splicing. So, to achieve our goal, we redesigned another three versions of SRC1 intron:<br />
</p><br />
<p>1. Intron with GG at its 5’ end</p><br />
<p>2. Intron with CGG at its 5’ end</p><br />
<p>3. Intron with its original 6bp at both end</p><br />
<p></p><br />
<p>However, due to the lack of time, we could not connect all these parts to our test parts.<br />
</p><br />
<br />
</div><br />
<br />
</div></div>BGI K2http://2013.igem.org/Team:Shenzhen_BGIC_ATCG/resultsTeam:Shenzhen BGIC ATCG/results2013-09-27T21:21:02Z<p>BGI K2: </p>
<hr />
<div><!----------------------<br />
<br />
<br />
Title: Results<br />
Team: Shenzhen_BGIC_ATCG<br />
Author: BGI_K2<br />
Twitter: @BGI_K2<br />
Email: im.ss.kk@gmail.com<br />
<br />
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<div class="navleftholder"><br />
<ul class="navleft"><br />
<li><a id="navleftsub1" class="navleftsub" href="#board1">The Magic</a></li><br />
<li><a id="navleftsub2" class="navleftsub" href="#board2">Promoter Verification</a></li><br />
<li><a id="navleftsub3" class="navleftsub" href="#board3">Reporter Locating</a></li><br />
<li><a id="navleftsub4" class="navleftsub" href="#board4">Degradation Rate</a></li><br />
<li><a id="navleftsub5" class="navleftsub" href="#board5">Cell Synchronization</a></li><br />
<li><a id="navleftsub6" class="navleftsub" href="#board6">Alternative Splicing</a></li><br />
</ul></div><br />
<br />
<div id="board1" class="board"></div><br />
<div class="media"><br />
<br />
<ul id="myGallery1"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/></li><br />
<li><img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" title="Degradation Tags" description="Degradation tags testing device for E. coli."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" title="Microfluidics" description="Microfluidics device for degradation test."/></li><br />
<br />
</ul></div><br />
<div id="box1" class="box"><br />
<h3>The Magic</h3><br />
<p>Our project “Cell Magic", a complex work has been completed partly,due to the time limitation. The results we have gotten are listed below:</p><br />
<p>We verified some of the cyclin promoters. The clb6 promoter, which is supposed to be expressed in G1 phase, has been verified perfectly.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." width=50%/><br />
<p>Targeting peptides to three locations have been verified. Others are still being constructed and tested.</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. " width=50%/><br />
<p>We got good data for degradation tags for <i>E. coli</i>. Construction of degradation devices for yeast have been finished and being tested.</p><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" width=50%/><br />
<p>Microfluidics device has been successfully developed for measurement and cell synchronization.</p><br />
<img src="https://2013.igem.org/File:Microfluidic.gif" /><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" width=50%/><br />
<p>Cell synchronization device and alternative splicing device are still in construction, related results will be updated later.</p><br />
<img src="https://2013.igem.org/File:Microfluidic.gif" /><br />
<br />
</div><br />
<br />
<div id="board2" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery2"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/1/19/Flu.jpg" title="S Phase Promoter" description="cln3 promoter for S phase has been verified using flow cytometry." /></li><br />
</ul></div><br />
<div id="box2" class="box"><br />
<h3>Promoter Verification</h3><br />
<p> Using the GFP as reporter and morphological alteration as cell cycle representation, we verified the Clb6 can be activated in G1 phase in the yeast.</P><br />
<p> As shown in the picture in normal lights, there are some yeast were budding. These budded yeast cells and small size budding yeasts (within the red circles) are assumed in the G1 phase of cell cycle. And the next picture was taken under activation light, thus the green lights can representing the Clb6 promoter expressing. The two pictures indicated the Clb6 were expressed in the G1 phase as we expected when designing the experiments </p><br />
<p> PS: we modified the contrast ratio to lower the lights of neighbor cells, thus our results looking better.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" /><br />
<p>We also captured fluorescence on cln3 promoter (S phase) test device, for the fluorescence is quite weak, we utilize micro plate reader and flow cytometry to verify the promoter.</p><br />
<img src="https://static.igem.org/mediawiki/2013/1/19/Flu.jpg" /><br />
<p>While the promoters are natural ones without optimization, and our shuttle plasmid is pSR416 which is a single copied one, fluorescence is too weak to be captured by microfluidics device. That is a main reason for why we currently cannot show you a perfect cell magic movie. We are now constructing all devices with high copy number vectors, and try to optimize the junction near kozak sequence to make a better magic.</p><br />
<br />
</div><br />
<br />
<div id="board3" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery3"><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/></li><br />
</ul></div><br />
<div id="box3" class="box"><br />
<h3>Reporter Locating</h3><br />
<p>Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose.</p><br />
<img src="https://static.igem.org/mediawiki/2013/0/0d/Reporter.jpg" title="Modified Reporters" description="Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose."/><br />
<p> The four pictures shows the reporters can rightly locate to Mitochondria, Nucleus and Vacuole,respectively. Picture A is the negative control, all yeast cells are lighted with GFP. And figure B is the reporter to the mitochondria, we can saw there are several light spots in one cells. Figure C is the reporter located in nucleus, the green spots are small and there is only one in a yeast cell.The last picture shows the reporter of Vacuolar membrane, the green lights were discrete in cells which was as expected.</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/><br />
</div><br />
<br />
<div id="board4" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery4"><br />
<li><img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" title="E. coli Version" description="Degradation tags testing device for E. coli"/></li><br />
</ul></div><br />
<div id="box4" class="box"><br />
<h3>Degradation Rate</h3><br/><br />
<p>In E.coli, the adaptor SspB tethers ssrAtagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. Which means, a variation of the WT SsrA tag sequence (K1051206, K1051207 and K1051208) will accelerate the degradation of proteins when fused to their C-terminal. Thus the degradation rates are dependent on concentration of proteases and binding mediators.</p><br />
<p>We constructed the measurement pathway of each tag (K1051257, K1051258 and K1051259) to test the rates of degradation of tagged proteins respectively. J04450 was used as positive control because of the same promoter and fluorescent protein. </p><br />
<img src="https://static.igem.org/mediawiki/2013/6/68/Growth-Curve.jpg" /><br />
<p>In every curcuits measurement, we firstly test the growth curve through detecting the absorbance. Taken small amount of bateria, inoculate into 400ul LB medium, ensuring the initial concentration between 0.02-0.05 (OD600). Cultured in the room temperature, detect the OD600 value every 20 minutes. Y axis represents the logarithm of bacteria number, X axis represents the growth time. </p><br />
<br />
<img src="https://static.igem.org/mediawiki/2013/e/e9/Degladder.jpg" /><br />
<p>From right to left, the negative control,BBa_K1051257, BBa_K1051258, BBa_K1051259, J04450 as Positive Control. As the pictures showed, the lights of RFP within three degradation tags are decreasing. </p><br />
<!----------------------<br />
<img src="https://static.igem.org/mediawiki/2013/a/a6/IMG_0804.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/c/ca/IMG_0817.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/M0050bright.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/0/06/M0050strong.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/8/89/M0050weak.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/6/63/Flo.jpg" /><br />
--------------------><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" /><br />
<p>The test results for K1051258 and positive control. A. K1051258 in bright field; B. K1051258 in exciting lights; C. Positive control in bright field; D. K1051258 in exciting lights.</p><br />
<p>In picture, there are only obvious lights in the picture B, indicated the degradation rates are working.</p><br />
<p>预留酶标仪结果图和柱状图,降解效率换算</p><br />
<p>Enzyme - labelled meter detect the fluorescent protein intensity</p><br />
<p>First of all, take a certain amount of bacteria liquid, recovery to around OD0.6(600), ensuring the bacteria in the logarithmic growth phase. Diluted and then the bacteria transferred to 96 well plates, measured their fluorescence intensity. Red fluorescent protein using an excitation wavelength of 584nm, and its emission wavelength is 607nm.When measuring, we first detect the OD600 of each strain, removing the factor of bacteria number difference. Thus the fluorescence intensity cannot be altered by bacteria quantity. Then the measurement mode switching for measurement of fluorescence, fluorescence intensity.</p><br />
<!----------------------<br />
<img src="https://static.igem.org/mediawiki/2013/8/83/0917_t00_white_60mm.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/7/72/0917_t15_white_60mm.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/4/4e/0917_t30_white_60mm.jpg" /><br />
--------------------><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" /><br />
<p>The test results of BBa_K1051258 in chip. A,LB medium,O minuts; B, IPTG medium,9minutes; C,IPTG medium, 15 minutes</p><br />
<p>First of all, yeast will be measured after shaking to about OD2.0 (600)based on the chip which was washed by plasma water, vacuum pumping. The bacteria liquid was pushed into the chip, letting the cells enter the small triangle. Then use the constant flow pump culture medium into chip (the laboratory constant temperature, can not guarantee the training environment, 37 ° E. coli slower growth). The medium speed is about 200ul/h. Finally we test the data after yeast fulled in the triangles.</p><br />
<p>Using the IPTG medium, the new RFP expression was stopped and we can regard the lights as degradation tags'effecience. Obviously, the lights are decreasing along with the time. Thus, it indicates that the degradation tags work effectively</p><br />
<p>预留半衰期换算</p><br />
<br />
</div><br />
<br />
<div id="board5" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery5"><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/e4/09172_bright.jpg" title="E. coli captured" description="E. coli cells captured by microfluidics device."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/3/3f/Sc_bright_20.jpg" title="E. coli captured" description="Yeast cells captured by microfluidics device."/></li><br />
</ul></div><br />
<div id="box5" class="box"><br />
<h3>Cell Synchronization</h3><br />
<p>We made the chip as a platform for watching and synchronize the cells. First step is capture the cells by the chip. As showed in the two figures, both E.coli and budding yeast can be captured by the chip successfully.</p><br />
<img src="https://static.igem.org/mediawiki/2013/e/e4/09172_bright.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/3/3f/Sc_bright_20.jpg" /><br />
<p>Seven phosphorylation sites are mutated by PCR to make Sic1 mutant. However, it has been taking us too long time that we have been still working on right clones. Experiments results will be updated later.</p><br />
</div><br />
<br />
<div id="board6" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery6"><br />
<li><img src="https://static.igem.org/mediawiki/2013/b/b9/Blank-01.png" /><li><br />
</ul></div><br />
<div id="box6" class="box"><br />
<h3>Alternative Splicing</h3><br />
<p>In our project, we attempt to use intron as a switch. However, according to previous research, there are two splicing forms of SRC1 intron: one is complete splicing (5’S) while the other can leave 4bp at its 5’ end (5’L). The remaining 4-bp causes frame-shift and a stop codon in the region of adaptor, thus the following exon will not be translated. However, this kind of switch is not complete – the ratio of 5’L and 5’S varied between 40-60 and 85-15.<br />
</p><br />
<p></p><br />
<p>In order to make a complete switch, we abandoned the remaining 4bp of SRC1 intron to avoid producing 5’L mRNA. So the result comes out to be: when Hub1p are expressed, they bind to spliceosome to modify it, resulting to easy recognizing 5’S splice site and its splicing; when inhibit the expression of HUB1 by CRISPRi system, spliceosome can hardly recognize 5’S splice site, therefore no intron can be spliced. <br />
</p><br />
<p></p><br />
<p>However, things didn’t go as we expected. Our experiment showed that this 4bp plays important role in SRC1 intron splicing. So, to achieve our goal, we redesigned another three versions of SRC1 intron:<br />
</p><br />
<p>1. Intron with GG at its 5’ end</p><br />
<p>2. Intron with CGG at its 5’ end</p><br />
<p>3. Intron with its original 6bp at both end</p><br />
<p></p><br />
<p>However, due to the lack of time, we could not connect all these parts to our test parts.<br />
</p><br />
<br />
</div><br />
<br />
</div></div>BGI K2http://2013.igem.org/Team:Shenzhen_BGIC_ATCG/resultsTeam:Shenzhen BGIC ATCG/results2013-09-27T21:18:18Z<p>BGI K2: </p>
<hr />
<div><!----------------------<br />
<br />
<br />
Title: Results<br />
Team: Shenzhen_BGIC_ATCG<br />
Author: BGI_K2<br />
Twitter: @BGI_K2<br />
Email: im.ss.kk@gmail.com<br />
<br />
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<div class="navleftholder"><br />
<ul class="navleft"><br />
<li><a id="navleftsub1" class="navleftsub" href="#board1">The Magic</a></li><br />
<li><a id="navleftsub2" class="navleftsub" href="#board2">Promoter Verification</a></li><br />
<li><a id="navleftsub3" class="navleftsub" href="#board3">Reporter Locating</a></li><br />
<li><a id="navleftsub4" class="navleftsub" href="#board4">Degradation Rate</a></li><br />
<li><a id="navleftsub5" class="navleftsub" href="#board5">Cell Synchronization</a></li><br />
<li><a id="navleftsub6" class="navleftsub" href="#board6">Alternative Splicing</a></li><br />
</ul></div><br />
<br />
<div id="board1" class="board"></div><br />
<div class="media"><br />
<br />
<ul id="myGallery1"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/></li><br />
<li><img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" title="Degradation Tags" description="Degradation tags testing device for E. coli."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" title="Microfluidics" description="Microfluidics device for degradation test."/></li><br />
<br />
</ul></div><br />
<div id="box1" class="box"><br />
<h3>The Magic</h3><br />
<p>Our project “Cell Magic", a complex work has been completed partly,due to the time limitation. The results we have gotten are listed below:</p><br />
<p>We verified some of the cyclin promoters. The clb6 promoter, which is supposed to be expressed in G1 phase, has been verified perfectly.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." width=50%/><br />
<p>Targeting peptides to three locations have been verified. Others are still being constructed and tested.</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. " width=50%/><br />
<p>We got good data for degradation tags for <i>E. coli</i>. Construction of degradation devices for yeast have been finished and being tested.</p><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" width=50%/><br />
<p>Microfluidics device has been successfully developed for measurement and cell synchronization.</p><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" width=50%/><br />
<p>Cell synchronization device and alternative splicing device are still in construction, related results will be updated later.</p><br />
<video src="https://static.igem.org/mediawiki/2013/c/cc/Microfluidic.mp4" controls="controls" /><br />
<br />
<br />
</div><br />
<br />
<div id="board2" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery2"><br />
<li><img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" title="G1 Phase Promoter" description="clb6 promoter for G1 phase has been verified." /></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/1/19/Flu.jpg" title="S Phase Promoter" description="cln3 promoter for S phase has been verified using flow cytometry." /></li><br />
</ul></div><br />
<div id="box2" class="box"><br />
<h3>Promoter Verification</h3><br />
<p> Using the GFP as reporter and morphological alteration as cell cycle representation, we verified the Clb6 can be activated in G1 phase in the yeast.</P><br />
<p> As shown in the picture in normal lights, there are some yeast were budding. These budded yeast cells and small size budding yeasts (within the red circles) are assumed in the G1 phase of cell cycle. And the next picture was taken under activation light, thus the green lights can representing the Clb6 promoter expressing. The two pictures indicated the Clb6 were expressed in the G1 phase as we expected when designing the experiments </p><br />
<p> PS: we modified the contrast ratio to lower the lights of neighbor cells, thus our results looking better.</p><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Clb6.jpg" /><br />
<p>We also captured fluorescence on cln3 promoter (S phase) test device, for the fluorescence is quite weak, we utilize micro plate reader and flow cytometry to verify the promoter.</p><br />
<img src="https://static.igem.org/mediawiki/2013/1/19/Flu.jpg" /><br />
<p>While the promoters are natural ones without optimization, and our shuttle plasmid is pSR416 which is a single copied one, fluorescence is too weak to be captured by microfluidics device. That is a main reason for why we currently cannot show you a perfect cell magic movie. We are now constructing all devices with high copy number vectors, and try to optimize the junction near kozak sequence to make a better magic.</p><br />
<br />
</div><br />
<br />
<div id="board3" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery3"><br />
<li><img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/></li><br />
</ul></div><br />
<div id="box3" class="box"><br />
<h3>Reporter Locating</h3><br />
<p>Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose.</p><br />
<img src="https://static.igem.org/mediawiki/2013/0/0d/Reporter.jpg" title="Modified Reporters" description="Fluorescence proteins have been modified to RFC[23], and stop codons have been removed for fusion protein purpose."/><br />
<p> The four pictures shows the reporters can rightly locate to Mitochondria, Nucleus and Vacuole,respectively. Picture A is the negative control, all yeast cells are lighted with GFP. And figure B is the reporter to the mitochondria, we can saw there are several light spots in one cells. Figure C is the reporter located in nucleus, the green spots are small and there is only one in a yeast cell.The last picture shows the reporter of Vacuolar membrane, the green lights were discrete in cells which was as expected.</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2013/6/6e/Target2.jpg" title="Reporter Targeting" description="Test for targeting peptides to mitochondria, nucleus and vacuolar. "/><br />
</div><br />
<br />
<div id="board4" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery4"><br />
<li><img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" title="E. coli Version" description="Degradation tags testing device for E. coli"/></li><br />
</ul></div><br />
<div id="box4" class="box"><br />
<h3>Degradation Rate</h3><br/><br />
<p>In E.coli, the adaptor SspB tethers ssrAtagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. Which means, a variation of the WT SsrA tag sequence (K1051206, K1051207 and K1051208) will accelerate the degradation of proteins when fused to their C-terminal. Thus the degradation rates are dependent on concentration of proteases and binding mediators.</p><br />
<p>We constructed the measurement pathway of each tag (K1051257, K1051258 and K1051259) to test the rates of degradation of tagged proteins respectively. J04450 was used as positive control because of the same promoter and fluorescent protein. </p><br />
<img src="https://static.igem.org/mediawiki/2013/6/68/Growth-Curve.jpg" /><br />
<p>In every curcuits measurement, we firstly test the growth curve through detecting the absorbance. Taken small amount of bateria, inoculate into 400ul LB medium, ensuring the initial concentration between 0.02-0.05 (OD600). Cultured in the room temperature, detect the OD600 value every 20 minutes. Y axis represents the logarithm of bacteria number, X axis represents the growth time. </p><br />
<br />
<img src="https://static.igem.org/mediawiki/2013/e/e9/Degladder.jpg" /><br />
<p>From right to left, the negative control,BBa_K1051257, BBa_K1051258, BBa_K1051259, J04450 as Positive Control. As the pictures showed, the lights of RFP within three degradation tags are decreasing. </p><br />
<!----------------------<br />
<img src="https://static.igem.org/mediawiki/2013/a/a6/IMG_0804.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/c/ca/IMG_0817.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/M0050bright.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/0/06/M0050strong.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/8/89/M0050weak.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/6/63/Flo.jpg" /><br />
--------------------><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg" /><br />
<p>The test results for K1051258 and positive control. A. K1051258 in bright field; B. K1051258 in exciting lights; C. Positive control in bright field; D. K1051258 in exciting lights.</p><br />
<p>In picture, there are only obvious lights in the picture B, indicated the degradation rates are working.</p><br />
<p>预留酶标仪结果图和柱状图,降解效率换算</p><br />
<p>Enzyme - labelled meter detect the fluorescent protein intensity</p><br />
<p>First of all, take a certain amount of bacteria liquid, recovery to around OD0.6(600), ensuring the bacteria in the logarithmic growth phase. Diluted and then the bacteria transferred to 96 well plates, measured their fluorescence intensity. Red fluorescent protein using an excitation wavelength of 584nm, and its emission wavelength is 607nm.When measuring, we first detect the OD600 of each strain, removing the factor of bacteria number difference. Thus the fluorescence intensity cannot be altered by bacteria quantity. Then the measurement mode switching for measurement of fluorescence, fluorescence intensity.</p><br />
<!----------------------<br />
<img src="https://static.igem.org/mediawiki/2013/8/83/0917_t00_white_60mm.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/7/72/0917_t15_white_60mm.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/4/4e/0917_t30_white_60mm.jpg" /><br />
--------------------><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg" /><br />
<p>The test results of BBa_K1051258 in chip. A,LB medium,O minuts; B, IPTG medium,9minutes; C,IPTG medium, 15 minutes</p><br />
<p>First of all, yeast will be measured after shaking to about OD2.0 (600)based on the chip which was washed by plasma water, vacuum pumping. The bacteria liquid was pushed into the chip, letting the cells enter the small triangle. Then use the constant flow pump culture medium into chip (the laboratory constant temperature, can not guarantee the training environment, 37 ° E. coli slower growth). The medium speed is about 200ul/h. Finally we test the data after yeast fulled in the triangles.</p><br />
<p>Using the IPTG medium, the new RFP expression was stopped and we can regard the lights as degradation tags'effecience. Obviously, the lights are decreasing along with the time. Thus, it indicates that the degradation tags work effectively</p><br />
<p>预留半衰期换算</p><br />
<br />
</div><br />
<br />
<div id="board5" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery5"><br />
<li><img src="https://static.igem.org/mediawiki/2013/e/e4/09172_bright.jpg" title="E. coli captured" description="E. coli cells captured by microfluidics device."/></li><br />
<li><img src="https://static.igem.org/mediawiki/2013/3/3f/Sc_bright_20.jpg" title="E. coli captured" description="Yeast cells captured by microfluidics device."/></li><br />
</ul></div><br />
<div id="box5" class="box"><br />
<h3>Cell Synchronization</h3><br />
<p>We made the chip as a platform for watching and synchronize the cells. First step is capture the cells by the chip. As showed in the two figures, both E.coli and budding yeast can be captured by the chip successfully.</p><br />
<img src="https://static.igem.org/mediawiki/2013/e/e4/09172_bright.jpg" /><br />
<img src="https://static.igem.org/mediawiki/2013/3/3f/Sc_bright_20.jpg" /><br />
<p>Seven phosphorylation sites are mutated by PCR to make Sic1 mutant. However, it has been taking us too long time that we have been still working on right clones. Experiments results will be updated later.</p><br />
</div><br />
<br />
<div id="board6" class="board"></div><br />
<div class="media"><br />
<ul id="myGallery6"><br />
<li><img src="https://static.igem.org/mediawiki/2013/b/b9/Blank-01.png" /><li><br />
</ul></div><br />
<div id="box6" class="box"><br />
<h3>Alternative Splicing</h3><br />
<p>In our project, we attempt to use intron as a switch. However, according to previous research, there are two splicing forms of SRC1 intron: one is complete splicing (5’S) while the other can leave 4bp at its 5’ end (5’L). The remaining 4-bp causes frame-shift and a stop codon in the region of adaptor, thus the following exon will not be translated. However, this kind of switch is not complete – the ratio of 5’L and 5’S varied between 40-60 and 85-15.<br />
</p><br />
<p></p><br />
<p>In order to make a complete switch, we abandoned the remaining 4bp of SRC1 intron to avoid producing 5’L mRNA. So the result comes out to be: when Hub1p are expressed, they bind to spliceosome to modify it, resulting to easy recognizing 5’S splice site and its splicing; when inhibit the expression of HUB1 by CRISPRi system, spliceosome can hardly recognize 5’S splice site, therefore no intron can be spliced. <br />
</p><br />
<p></p><br />
<p>However, things didn’t go as we expected. Our experiment showed that this 4bp plays important role in SRC1 intron splicing. So, to achieve our goal, we redesigned another three versions of SRC1 intron:<br />
</p><br />
<p>1. Intron with GG at its 5’ end</p><br />
<p>2. Intron with CGG at its 5’ end</p><br />
<p>3. Intron with its original 6bp at both end</p><br />
<p></p><br />
<p>However, due to the lack of time, we could not connect all these parts to our test parts.<br />
</p><br />
<br />
</div><br />
<br />
</div></div>BGI K2http://2013.igem.org/File:Flu.jpgFile:Flu.jpg2013-09-27T21:07:23Z<p>BGI K2: </p>
<hr />
<div></div>BGI K2