http://2013.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=20&target=Nousan&year=&month=2013.igem.org - User contributions [en]2024-03-29T05:51:02ZFrom 2013.igem.orgMediaWiki 1.16.5http://2013.igem.org/Team:HokkaidoU_Japan/RBS/ConclusionTeam:HokkaidoU Japan/RBS/Conclusion2013-10-29T03:57:37Z<p>Nousan: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header_Maestro}}<br />
<html><br />
<div id="common-header-bottom-background"><br />
<div class="wrapper"><br />
<h1 id="common-header-title">Maestro <span class="italic">E. coli</span></h1><br />
<h2 id="common-header-subtitle">RBS</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
</div><br />
</div><br />
<br />
<div class="wrapper"><br />
<div id="hokkaidou-contents"><br />
<!-- end header / begin contents --><br />
<br />
<h2>Conclusion</h2><br />
<p><br />
We were successful at making four RBS set with four strength levels.<br />
Construct with SD4 showed the strongest &beta;-Galactosidase activity.<br />
Second strongest was the SD8, followed by B0034 and SD6. SD2 had the weakest activity.<br />
</p><br />
<p><br />
Though we synthesized the RBSs based on previous reports, we got unexpected results.<br />
Preciously SD6 was reported as the strongest. However our results indicated SD4.<br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/1/1d/HokkaidoU2013_RBS_Conclusion_800.png"><br />
<div style="padding-bottom: 0;"><span class="bold">fig.1 The difference of expression level.&nbsp; </span>If there is A/U rich enhancer, SD sequence has mighty effect.</div><br />
</div><br />
<br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/2/2f/RBS_assay_HokkaidoU_2013.png"><br />
<div style="padding-bottom: 0;"><span class="bold">fig.2 LacZ&alpha; expression level.&nbsp; </span>Difference of SD sequence gives different amount of LacZ&alpha; expression.</div><br />
</div><br />
<br />
<br />
<p><br />
The sequence we synthesized was completely the same.<br />
Both plasmids had low copy number.<br />
One thing we changed was the reporter gene.<br />
In the previous experiments, GFP was used for the reporter.<br />
In contrast, we used LacZ&alpha;.<br />
The expected strength differed only by changing the coding sequence.<br />
</p><br />
<p><br />
It is well known fact that mRNA makes a secondary structure. The secondary structure of RNA takes an important role in the process of life.<br />
Identically, the secondary structure and the folding of mRNA is an important factor in translation efficiency.<br />
Kudla <span class="italic">et al.</span> (2009) showed that translation efficiency is determined by factors in Coding-Sequence.<br />
Overall, the translational efficiency is a correlation between RBS sequence and the Coding-Sequence.<br />
</p><br />
<p><br />
Our results show different levels of translation efficiency.<br />
We should try and repeat our assays with other reporter genes.<br />
The translation efficiency might change by choosing different coding sequences.<br />
The translation efficiency not depended on RBS but also influenced by coding sequence.<br />
This fact complicates designing biological devices.<br />
</p><br />
<p><br />
When regulating the expression it is important to have variability in RBSs strength.<br />
Our set RBSs proved to have different efficiencies in both GFP and LacZ&alpha; assays.<br />
From our results and the previous research we can expect that our RBSs will show variability in translational efficiency using any coding site.<br />
Therefore, we made a useful set of RBSs for regulating expression.<br />
</p><br />
<ol class="citation-list"><br />
<li id="cite-1">Grzegorz Kudla, <span class="italic">et al.</span> Coding-Sequence Determinants of Gene Expression in <span class="italic">Escherichia coli</span> (2009) Science</li><br />
</ol><br />
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{{Team:HokkaidoU_Japan/footer}}</div>Nousanhttp://2013.igem.org/Team:HokkaidoU_Japan/RBS/ConclusionTeam:HokkaidoU Japan/RBS/Conclusion2013-10-29T03:57:22Z<p>Nousan: </p>
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<div>{{Team:HokkaidoU_Japan/header_Maestro}}<br />
<html><br />
<div id="common-header-bottom-background"><br />
<div class="wrapper"><br />
<h1 id="common-header-title">Maestro <span class="italic">E. coli</span></h1><br />
<h2 id="common-header-subtitle">RBS</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
</div><br />
</div><br />
<br />
<div class="wrapper"><br />
<div id="hokkaidou-contents"><br />
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<br />
<h2>Conclusion</h2><br />
<p><br />
We were successful at making four RBS set with four strength levels.<br />
Construct with SD4 showed the strongest &beta;-Galactosidase activity.<br />
Second strongest was the SD8, followed by B0034 and SD6. SD2 had the weakest activity.<br />
</p><br />
<p><br />
Though we synthesized the RBSs based on previous reports, we got unexpected results.<br />
Preciously SD6 was reported as the strongest. However our results indicated SD4.<br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/1/1d/HokkaidoU2013_RBS_Conclusion_800.png"><br />
<div style="padding-bottom: 0;"><span class="bold">fig.1 The difference of expression level.&nbsp; </span>If there is A/U rich enhancer, SD sequence has mighty effect.</div><br />
</div><br />
<br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/2/2f/RBS_assay_HokkaidoU_2013.png"><br />
<div style="padding-bottom: 0;">><span class="bold">fig.2 LacZ&alpha; expression level.&nbsp; </span>Difference of SD sequence gives different amount of LacZ&alpha; expression.</div><br />
</div><br />
<br />
<br />
<p><br />
The sequence we synthesized was completely the same.<br />
Both plasmids had low copy number.<br />
One thing we changed was the reporter gene.<br />
In the previous experiments, GFP was used for the reporter.<br />
In contrast, we used LacZ&alpha;.<br />
The expected strength differed only by changing the coding sequence.<br />
</p><br />
<p><br />
It is well known fact that mRNA makes a secondary structure. The secondary structure of RNA takes an important role in the process of life.<br />
Identically, the secondary structure and the folding of mRNA is an important factor in translation efficiency.<br />
Kudla <span class="italic">et al.</span> (2009) showed that translation efficiency is determined by factors in Coding-Sequence.<br />
Overall, the translational efficiency is a correlation between RBS sequence and the Coding-Sequence.<br />
</p><br />
<p><br />
Our results show different levels of translation efficiency.<br />
We should try and repeat our assays with other reporter genes.<br />
The translation efficiency might change by choosing different coding sequences.<br />
The translation efficiency not depended on RBS but also influenced by coding sequence.<br />
This fact complicates designing biological devices.<br />
</p><br />
<p><br />
When regulating the expression it is important to have variability in RBSs strength.<br />
Our set RBSs proved to have different efficiencies in both GFP and LacZ&alpha; assays.<br />
From our results and the previous research we can expect that our RBSs will show variability in translational efficiency using any coding site.<br />
Therefore, we made a useful set of RBSs for regulating expression.<br />
</p><br />
<ol class="citation-list"><br />
<li id="cite-1">Grzegorz Kudla, <span class="italic">et al.</span> Coding-Sequence Determinants of Gene Expression in <span class="italic">Escherichia coli</span> (2009) Science</li><br />
</ol><br />
<br />
<br />
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<a href="https://2013.igem.org/Team:HokkaidoU_Japan/RBS/Results"><div class="arrow-div"></div><span>Results</span></a><br />
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{{Team:HokkaidoU_Japan/footer}}</div>Nousanhttp://2013.igem.org/Team:HokkaidoU_Japan/RBS/ConclusionTeam:HokkaidoU Japan/RBS/Conclusion2013-10-29T03:56:00Z<p>Nousan: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header_Maestro}}<br />
<html><br />
<div id="common-header-bottom-background"><br />
<div class="wrapper"><br />
<h1 id="common-header-title">Maestro <span class="italic">E. coli</span></h1><br />
<h2 id="common-header-subtitle">RBS</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
</div><br />
</div><br />
<br />
<div class="wrapper"><br />
<div id="hokkaidou-contents"><br />
<!-- end header / begin contents --><br />
<br />
<h2>Conclusion</h2><br />
<p><br />
We were successful at making four RBS set with four strength levels.<br />
Construct with SD4 showed the strongest &beta;-Galactosidase activity.<br />
Second strongest was the SD8, followed by B0034 and SD6. SD2 had the weakest activity.<br />
</p><br />
<p><br />
Though we synthesized the RBSs based on previous reports, we got unexpected results.<br />
Preciously SD6 was reported as the strongest. However our results indicated SD4.<br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/1/1d/HokkaidoU2013_RBS_Conclusion_800.png"><br />
<div style="padding-bottom: 0;"><span class="bold">fig.1 The difference of expression level.&nbsp; </span>If there is A/U rich enhancer, SD sequence has mighty effect.</div><br />
</div><br />
<br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/2/2f/RBS_assay_HokkaidoU_2013.png"><br />
</div><br />
<div><span class="bold">fig.2 LacZ&alpha; expression level.&nbsp; </span>Difference of SD sequence gives different amount of LacZ&alpha; expression.</div><br />
<p><br />
The sequence we synthesized was completely the same.<br />
Both plasmids had low copy number.<br />
One thing we changed was the reporter gene.<br />
In the previous experiments, GFP was used for the reporter.<br />
In contrast, we used LacZ&alpha;.<br />
The expected strength differed only by changing the coding sequence.<br />
</p><br />
<p><br />
It is well known fact that mRNA makes a secondary structure. The secondary structure of RNA takes an important role in the process of life.<br />
Identically, the secondary structure and the folding of mRNA is an important factor in translation efficiency.<br />
Kudla <span class="italic">et al.</span> (2009) showed that translation efficiency is determined by factors in Coding-Sequence.<br />
Overall, the translational efficiency is a correlation between RBS sequence and the Coding-Sequence.<br />
</p><br />
<p><br />
Our results show different levels of translation efficiency.<br />
We should try and repeat our assays with other reporter genes.<br />
The translation efficiency might change by choosing different coding sequences.<br />
The translation efficiency not depended on RBS but also influenced by coding sequence.<br />
This fact complicates designing biological devices.<br />
</p><br />
<p><br />
When regulating the expression it is important to have variability in RBSs strength.<br />
Our set RBSs proved to have different efficiencies in both GFP and LacZ&alpha; assays.<br />
From our results and the previous research we can expect that our RBSs will show variability in translational efficiency using any coding site.<br />
Therefore, we made a useful set of RBSs for regulating expression.<br />
</p><br />
<ol class="citation-list"><br />
<li id="cite-1">Grzegorz Kudla, <span class="italic">et al.</span> Coding-Sequence Determinants of Gene Expression in <span class="italic">Escherichia coli</span> (2009) Science</li><br />
</ol><br />
<br />
<br />
<br />
<div id="prev-page"><br />
<a href="https://2013.igem.org/Team:HokkaidoU_Japan/RBS/Results"><div class="arrow-div"></div><span>Results</span></a><br />
</div><br />
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{{Team:HokkaidoU_Japan/footer}}</div>Nousanhttp://2013.igem.org/Team:HokkaidoU_Japan/RBS/ConclusionTeam:HokkaidoU Japan/RBS/Conclusion2013-10-29T03:49:36Z<p>Nousan: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header_Maestro}}<br />
<html><br />
<div id="common-header-bottom-background"><br />
<div class="wrapper"><br />
<h1 id="common-header-title">Maestro <span class="italic">E. coli</span></h1><br />
<h2 id="common-header-subtitle">RBS</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
</div><br />
</div><br />
<br />
<div class="wrapper"><br />
<div id="hokkaidou-contents"><br />
<!-- end header / begin contents --><br />
<br />
<h2>Conclusion</h2><br />
<p><br />
We were successful at making four RBS set with four strength levels.<br />
Construct with SD4 showed the strongest &beta;-Galactosidase activity.<br />
Second strongest was the SD8, followed by B0034 and SD6. SD2 had the weakest activity.<br />
</p><br />
<p><br />
Though we synthesized the RBSs based on previous reports, we got unexpected results.<br />
Preciously SD6 was reported as the strongest. However our results indicated SD4.<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/1/1d/HokkaidoU2013_RBS_Conclusion_800.png"><br />
</div><br />
<div><span class="bold">fig.1 The difference of expression level.&nbsp; </span>If there is A/U rich enhancer, SD sequence has mighty effect.</div><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/2/2f/RBS_assay_HokkaidoU_2013.png"><br />
</div><br />
<div><span class="bold">fig.2 LacZ&alpha; expression level.&nbsp; </span>Difference of SD sequence gives different amount of LacZ&alpha; expression.</div><br />
<p><br />
The sequence we synthesized was completely the same.<br />
Both plasmids had low copy number.<br />
One thing we changed was the reporter gene.<br />
In the previous experiments, GFP was used for the reporter.<br />
In contrast, we used LacZ&alpha;.<br />
The expected strength differed only by changing the coding sequence.<br />
</p><br />
<p><br />
It is well known fact that mRNA makes a secondary structure. The secondary structure of RNA takes an important role in the process of life.<br />
Identically, the secondary structure and the folding of mRNA is an important factor in translation efficiency.<br />
Kudla <span class="italic">et al.</span> (2009) showed that translation efficiency is determined by factors in Coding-Sequence.<br />
Overall, the translational efficiency is a correlation between RBS sequence and the Coding-Sequence.<br />
</p><br />
<p><br />
Our results show different levels of translation efficiency.<br />
We should try and repeat our assays with other reporter genes.<br />
The translation efficiency might change by choosing different coding sequences.<br />
The translation efficiency not depended on RBS but also influenced by coding sequence.<br />
This fact complicates designing biological devices.<br />
</p><br />
<p><br />
When regulating the expression it is important to have variability in RBSs strength.<br />
Our set RBSs proved to have different efficiencies in both GFP and LacZ&alpha; assays.<br />
From our results and the previous research we can expect that our RBSs will show variability in translational efficiency using any coding site.<br />
Therefore, we made a useful set of RBSs for regulating expression.<br />
</p><br />
<ol class="citation-list"><br />
<li id="cite-1">Grzegorz Kudla, <span class="italic">et al.</span> Coding-Sequence Determinants of Gene Expression in <span class="italic">Escherichia coli</span> (2009) Science</li><br />
</ol><br />
<br />
<br />
<br />
<div id="prev-page"><br />
<a href="https://2013.igem.org/Team:HokkaidoU_Japan/RBS/Results"><div class="arrow-div"></div><span>Results</span></a><br />
</div><br />
<br />
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{{Team:HokkaidoU_Japan/footer}}</div>Nousanhttp://2013.igem.org/Team:HokkaidoU_Japan/Shuffling_Kit/ExamplesTeam:HokkaidoU Japan/Shuffling Kit/Examples2013-10-29T03:44:45Z<p>Nousan: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header_Maestro}}<br />
<html><br />
<div id="common-header-bottom-background"><br />
<div class="wrapper"><br />
<h1 id="common-header-title">Maestro <span class="italic">E. coli</span></h1><br />
<h2 id="common-header-subtitle">Shuffling Kit</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
</div><br />
</div><br />
<br />
<div class="wrapper"><br />
<div id="hokkaidou-contents"><br />
<!-- end header / begin contents --><br />
<h1>Demonstrations for Usecase Example</h1><br />
<p>We will show some interesting demonstrations of our kits, Promoter Selector and RBS Selector!</p><br />
<br />
<br />
<h2>Promoter Selector</h2><br />
<p>Let's select the best promoter for Kanamycin resistance by Promoter Selector.</p><br />
<p>For this demonstration we decided to use the expression of Kanamycin resistance. Changing the concentration of Kanamycin in agar plate, it is estimated that different promoter will be chosen by our Promoter Selector (fig.1).</p><br />
<br />
<p>If the concentration of Kanamycin was high, the colony with strong promoter would survive. Therefore, only one or two colors would appear and indicate the first and second biggest occupancy rate on the plate.<br />
If the concentration of Kanamycin was low, colonies with even weak promoters could be able to survive. So in this way many colors of colonies would appear (fig.2).<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/b/bb/Fig1_in_example_132029new_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.1 Different promoter express each colors.</span></div><br />
</div><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/2/2d/Fig2_in_example_HokkaidoU_2013.png"><br />
<div style="padding-bottom: 0;"><span class="bold">fig.2 Difference of Kanamycin concentration.</span></div><br />
</div><br />
<br />
<h3>Method</h3><br />
<br />
<p>Optimum concentration of Kanamycin: in LB is 50 mg/ml <br><br />
We prepared optimum and other 3 concentration plates. <br />
</p><br />
<br />
<ul><br />
<li>Plate A: Kanamycin 125 mg per plate</li><br />
<li>Plate B: Kanamycin 250 mg per plate</li><br />
<li>Plate C: Kanamycin 500 mg per plate (optimum concentration)</li><br />
<li>Plate D: Kanamycin 1000 mg per plate</li><br />
</ul><br />
<br />
<p>Gene<br />
Vector: pSB1C3<br />
</p><br />
<p>We cloned Kanamycin resistance gene from pSB3K3, by using BsaI adding primer. Used the Promoter Selector (K1084501, K1084502, K1084503, K1084504, K1084505 ).</p><br />
<br />
<p><br />
Culture: 37 &deg;C, for 48h<br><br />
</p><br />
<br />
<h3>Results</h3><br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/2/28/HokkaidoU_2013_Km_resistance_assay_summary_data.png"><br />
<div><span class="bold">fig.4 Graph of number and rate, and table of number of colonies size over 1mm diameter.</span></div><br />
</div><br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/c/ca/POK_DEMO_48h_newnew_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.3 Picture of plate B (Kanamycine 250 mg). The colonies showed several colors.</span></div><br />
</div><br />
<br />
<p>After 48h cultivation, around 300 colonies had appeared on each LBKC (Kanamycin and Chloramphenicol) plates. We prepared LacZ&alpha expression system in Promoter Selector as negative control to estimate the success of BsaI digestion. We got 0-7 blue colonies which was expressing LacZ&alpha.<br />
</p><br />
<br />
<p> Mixed colored colonies which would have been transformed by two or more Promoter Selector were also observed. The number and rate of colonies per each plate were graphed (fig.4), with rejecting these undesirable colonies.<br />
</p><br />
<br />
<br />
<p><br />
In fig.4, legend color corresponds to Promoter Selector’s part number. The sum of colony numbers is displayed above each bar, and rate is in these sections. Number in the table referrers the number of each Promoter Selector’s colony. These data are collected by n=1.<br />
</p><br />
<br />
<div class="clearfix"></div><br />
<br />
<h3>Conclusion</h3><br />
<p><br />
Big difference did not appear among each Kanamaicin concentration. <br />
<br />
In this experiment, number of colonies derived from K1084405 (containing K1084010 promoter) had the largest rate on each plate. This result suggests that the colonies which had K1084505 could cost little resource of transcription and translation for expression of Kanamycin resistance gene, and the rest of resource had been spared to cell growth. Thus, the number of colonies might be the largest. <br />
</p><br />
<br />
<p><br />
The other reason of why , colonies derived from K1084405 had the largest number may be, the DNA solution of K1084505 Promoter Selector used at ligation was simply larger than other DNA solution. Although the result is collected from assay of only once , higher concentration of Kanamycin and much number of trials than this time will be needed.<br />
</p><br />
<p><br />
From these result and the experimental fact, the existence of Km resistance gene in Promoter Selector’s BsaI cloning section is partially confirmed. Our Promoter Selector was successfully assembled. However it was not adopted to all colonies. As a result of assembling, we succeeded in making colorful colonies appear on one plate.<br />
</p><br />
<h2>RBS Selector</h2><br />
<h3>4 colors</h3><br />
<p>Let’s create all combinations by two reporter genes and make various colors on one plate!</p><br />
<br />
<p><br />
The RBS Selector we made, can randomize the strength of RBSs in the operon.<br />
For a demonstration, we decided to create all combinations by two genes; mRFP1 (BBa_E1010) and LacZ&alpha; (BBa_I732006) (fig.5). LacZ&alpha; makes the colony blue. mRFP1 makes the colony red.<br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/9/90/Fig4_in_example_new_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.5 Create all combinations by RBS of defferent stlength mRFP1 (BBa_E1010) and LacZα (BBa_I732006).</span></div><br />
</div><br />
<br />
<p>When the RBS of mRFP1 was stronger than that of LacZα, the colony would be red. When in the opposite case, the colony would be blue. And when the strengths of both RBSs were same, colony would be white or purple (fig.6).</p><br />
<br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/c/c8/Fig5_in_example_%2Boverhang_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.6 Each combinations of RBS make different colors.</span></div><br />
</div><br />
<br />
<h4>Method</h4><br />
<ul><br />
<li>Assembled promoter1 (BBa_K1084001), SD2 (BBa_K1084101), SD4 (BBa_K1084102).</li><br />
<li>Spread X-GAL(250 mg)on LBC plate.</li><br />
<li>Cultured at 37 &deg;C for 26h.</li><br />
</ul><br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/0/08/ROK_demo_new_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.7 The colonies showed red, blue, white, and purple.</span></div><br />
</div><br />
<h4>Results</h4><br />
<p><br />
We got colorful colonies; red, blue, white, and purple.<br />
<br />
<br />
</p><br />
<div class="clearfix"></div><br />
<br />
<h4>Conclusion</h4><br />
<br />
<br />
<p><br />
<br />
We can say that our RBS Selector worked exactly!!<br />
The RBSs upstream of 2 genes were randomly assembled and they had different expression level. <br />
<br />
</p><br />
<br />
<br />
<h3>64 colors</h3><br />
<br />
<p><br />
Let’s create all combinations by three reporter gene and using tandem RBS, make various colors on one plate!<br />
</p><br />
<br />
<p><br />
As the tandem RBS and the RBS selector demonstration, we randomized 64 patterns of RBS-CDS combination. By adding tandem RBS, three kinds of CDSs and GGA VACTOR, One-pot Golden Gate Assembly had done for making 64 kinds of different constructs.<br />
</p><br />
<br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/1/1d/64demo2_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.8 The 64 patterns of RBS-CDS combination constructed by RBS Selector.</span></div><br />
</div><br />
<br />
<p><br />
<h4>Method</h4><br />
<ul><br />
<li>Used tandem RBS (BBa_K1084302), GGA VECTOR that BsaI and overhang was added by PCR from K1084401, eforRed (BBa_K592012), aeBlue (BBa_K864401) and amilGFP (BBa_K592010) are assembled by Golden Gate Assembly.</li><br />
<li>Spread on LBC plate.</li><br />
<li>Cultured at 37 &deg;C, 48h.</li><br />
</ul><br />
</p><br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/5/55/HokkaidoU_2013_64ROK_result_1.JPG"><br />
<div><span class="bold">fig.9 The result plate of 64 pattern randomizing.</span></div><br />
</div><br />
<br />
<h4>Results</h4><br />
<br />
<p><br />
<br />
<br />
</p><br />
<br />
<p><br />
Many yellowish green (amilGFP) and red color (mRFP1) colonies are appeared, but these colonies are undesirable colonies. As a Golden Gate assembly result, green color (strong aeBlue and amilGFP expression) appeared, and the insert DNA length was confirmed that the insert DNA has same length of Golden Gate Assembly. other colonies without blue weren’t Golden Gate Assembly result. <br />
Other color colony, white, weak green, weak amilGFP expression colonies were observed. <br />
</p><br />
<br />
<h4>Conclusion</h4><br />
<br />
<br />
<p><br />
mRFP1 expression might cause by remaining of mRFP1 expression DNA used as template for GGA VECTOR producing by PCR (K1084401), thus mRFP1 expression colonies would appear.<br />
</p><br />
<br />
<p><br />
However, the blue and green colonies appeared on the plate has the insert DNA length same with ideal Golden Gate Assembling product. Our Shuffling kit partially worked.<br />
</p><br />
<br />
<br />
<br />
<div id="prev-page"><br />
<a href="https://2013.igem.org/Team:HokkaidoU_Japan/Shuffling_Kit/Primer_Designer"><div class="arrow-div"></div><span>Primer Designer</span></a><br />
</div><br />
<br />
<div id="next-page"><br />
<a href="https://2013.igem.org/Team:HokkaidoU_Japan/Shuffling_Kit/Future_Work"><div class="arrow-div"></div><span>Future Work</span></a><br />
</div><br />
<br />
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{{Team:HokkaidoU_Japan/footer}}</div>Nousanhttp://2013.igem.org/Team:HokkaidoU_Japan/Shuffling_Kit/ExamplesTeam:HokkaidoU Japan/Shuffling Kit/Examples2013-10-29T03:29:20Z<p>Nousan: </p>
<hr />
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<html><br />
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<h1 id="common-header-title">Maestro E.coli</h1><br />
<h2 id="common-header-subtitle">Shuffling Kit</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
</div><br />
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<br />
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<div id="hokkaidou-contents"><br />
<!-- end header / begin contents --><br />
<h1>Demonstrations for Usecase Example</h1><br />
<p>We will show some interesting demonstrations of our kits, Promoter Selector and RBS Selector!</p><br />
<br />
<br />
<h2>Promoter Selector</h2><br />
<p>Let's select the best promoter for Kanamycin resistance by Promoter Selector.</p><br />
<p>For this demonstration we decided to use the expression of Kanamycin resistance. Changing the concentration of Kanamycin in agar plate, it is estimated that different promoter will be chosen by our Promoter Selector (fig.1).</p><br />
<br />
<p>If the concentration of Kanamycin was high, the colony with strong promoter would survive. Therefore, only one or two colors would appear and indicate the first and second biggest occupancy rate on the plate.<br />
If the concentration of Kanamycin was low, colonies with even weak promoters could be able to survive. So in this way many colors of colonies would appear (fig.2).<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/b/bb/Fig1_in_example_132029new_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.1 Different promoter express each colors.</span></div><br />
</div><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/2/2d/Fig2_in_example_HokkaidoU_2013.png"><br />
<div style="padding-bottom: 0;"><span class="bold">fig.2 Difference of Kanamycin concentration.</span></div><br />
</div><br />
<br />
<h3>Method</h3><br />
<br />
<p>Optimum concentration of Kanamycin: in LB is 50 mg/ml <br><br />
We prepared optimum and other 3 concentration plates. <br />
</p><br />
<br />
<ul><br />
<li>Plate A: Kanamycin 125 mg per plate</li><br />
<li>Plate B: Kanamycin 250 mg per plate</li><br />
<li>Plate C: Kanamycin 500 mg per plate (optimum concentration)</li><br />
<li>Plate D: Kanamycin 1000 mg per plate</li><br />
</ul><br />
<br />
<p>Gene<br />
Vector: pSB1C3<br />
</p><br />
<p>We cloned Kanamycin resistance gene from pSB3K3, by using BsaI adding primer. Used the Promoter Selector (K1084501, K1084502, K1084503, K1084504, K1084505 ).</p><br />
<br />
<p><br />
Culture: 37 &deg;C, for 48h<br><br />
</p><br />
<br />
<h3>Results</h3><br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/2/28/HokkaidoU_2013_Km_resistance_assay_summary_data.png"><br />
<div><span class="bold">fig.4 Graph of number and rate, and table of number of colonies size over 1mm diameter.</span></div><br />
</div><br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/c/ca/POK_DEMO_48h_newnew_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.3 Picture of plate B (Kanamycine 250 mg). The colonies showed several colors.</span></div><br />
</div><br />
<br />
<p>After 48h cultivation, around 300 colonies had appeared on each LBKC (Kanamycin and Chloramphenicol) plates. We prepared LacZ&alpha expression system in Promoter Selector as negative control to estimate the success of BsaI digestion. We got 0-7 blue colonies which was expressing LacZ&alpha.<br />
</p><br />
<br />
<p> Mixed colored colonies which would have been transformed by two or more Promoter Selector were also observed. The number and rate of colonies per each plate were graphed (fig.4), with rejecting these undesirable colonies.<br />
</p><br />
<br />
<br />
<p><br />
In fig.4, legend color corresponds to Promoter Selector’s part number. The sum of colony numbers is displayed above each bar, and rate is in these sections. Number in the table referrers the number of each Promoter Selector’s colony. These data are collected by n=1.<br />
</p><br />
<br />
<div class="clearfix"></div><br />
<br />
<h3>Conclusion</h3><br />
<p><br />
Big difference did not appear among each Kanamaicin concentration. <br />
<br />
In this experiment, number of colonies derived from K1084405 (containing K1084010 promoter) had the largest rate on each plate. This result suggests that the colonies which had K1084505 could cost little resource of transcription and translation for expression of Kanamycin resistance gene, and the rest of resource had been spared to cell growth. Thus, the number of colonies might be the largest. Otherwise, the DNA solution of K1084505 Promoter Selector used at ligation was simply larger than other DNA solution. Although the result is collected from only one time assay, higher concentration of Kanamycin and much number of trials than this time will be needed.<br />
</p><br />
<br />
<p><br />
From these result and the experimental fact, the existence of Km resistance gene in Promoter Selector’s BsaI cloning section is partially confirmed. Our Promoter Selector was successfully assembled, but it does not adopted to all colonies. Then, as a result of assembling, we succeeded in making colorful colonies appear on one plate.<br />
</p><br />
<h2>RBS Selector</h2><br />
<h3>4 colors</h3><br />
<p>Let’s create all combinations by two reporter genes and make various colors on one plate!</p><br />
<br />
<p><br />
The RBS Selector we made, can randomize the strength of RBSs in the operon.<br />
For a demonstration, we decided to create all combinations by two genes; mRFP1 (BBa_E1010) and LacZ&alpha; (BBa_I732006) (fig.5). LacZ&alpha; makes the colony blue. mRFP1 makes the colony red.<br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/9/90/Fig4_in_example_new_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.5 Create all combinations by RBS of defferent stlength mRFP1 (BBa_E1010) and LacZα (BBa_I732006).</span></div><br />
</div><br />
<br />
<p>When the RBS of mRFP1 was stronger than that of LacZα, the colony would be red. When in the opposite case, the colony would be blue. And when the strengths of both RBSs were same, colony would be white or purple (fig.6).</p><br />
<br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/c/c8/Fig5_in_example_%2Boverhang_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.6 Each combinations of RBS make different colors.</span></div><br />
</div><br />
<br />
<h4>Method</h4><br />
<ul><br />
<li>Assembled promoter1 (BBa_K1084001), SD2 (BBa_K1084101), SD4 (BBa_K1084102).</li><br />
<li>Spread X-GAL(250 mg)on LBC plate.</li><br />
<li>Cultured at 37 &deg;C for 26h.</li><br />
</ul><br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/0/08/ROK_demo_new_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.7 The colonies showed red, blue, white, and purple.</span></div><br />
</div><br />
<h4>Results</h4><br />
<p><br />
We got colorful colonies; red, blue, white, and purple.<br />
<br />
<br />
</p><br />
<div class="clearfix"></div><br />
<br />
<h4>Conclusion</h4><br />
<br />
<br />
<p><br />
<br />
We can say that our RBS Selector worked exactly!!<br />
The RBSs upstream of 2 genes were randomly assembled and they had different expression level. <br />
<br />
</p><br />
<br />
<br />
<h3>64 colors</h3><br />
<br />
<p><br />
Let’s create all combinations by three reporter gene and using tandem RBS, make various colors on one plate!<br />
</p><br />
<br />
<p><br />
As the tandem RBS and the RBS selector demonstration, we randomized 64 patterns of RBS-CDS combination. By adding tandem RBS, three kinds of CDSs and GGA VACTOR, One-pot Golden Gate Assembly had done for making 64 kinds of different constructs.<br />
</p><br />
<br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/1/1d/64demo2_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.8 The 64 patterns of RBS-CDS combination constructed by RBS Selector.</span></div><br />
</div><br />
<br />
<p><br />
<h4>Method</h4><br />
<ul><br />
<li>Used tandem RBS (BBa_K1084302), GGA VECTOR that BsaI and overhang was added by PCR from K1084401, eforRed (BBa_K592012), aeBlue (BBa_K864401) and amilGFP (BBa_K592010) are assembled by Golden Gate Assembly.</li><br />
<li>Spread on LBC plate.</li><br />
<li>Cultured at 37 &deg;C, 48h.</li><br />
</ul><br />
</p><br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/5/55/HokkaidoU_2013_64ROK_result_1.JPG"><br />
<div><span class="bold">fig.9 The result plate of 64 pattern randomizing.</span></div><br />
</div><br />
<br />
<h4>Results</h4><br />
<br />
<p><br />
<br />
<br />
</p><br />
<br />
<p><br />
Many yellowish green (amilGFP) and red color (mRFP1) colonies are appeared, but these colonies are undesirable colonies. As a Golden Gate assembly result, green color (strong aeBlue and amilGFP expression) appeared, and the insert DNA length was confirmed that the insert DNA has same length of Golden Gate Assembly. other colonies without blue weren’t Golden Gate Assembly result. <br />
Other color colony, white, weak green, weak amilGFP expression colonies were observed. <br />
</p><br />
<br />
<h4>Conclusion</h4><br />
<br />
<br />
<p><br />
mRFP1 expression might cause by remaining of mRFP1 expression DNA used as template for GGA VECTOR producing by PCR (K1084401), thus mRFP1 expression colonies would appear.<br />
</p><br />
<br />
<p><br />
However, the blue and green colonies appeared on the plate has the insert DNA length same with ideal Golden Gate Assembling product. Our Shuffling kit partially worked.<br />
</p><br />
<br />
<br />
<br />
<div id="prev-page"><br />
<a href="https://2013.igem.org/Team:HokkaidoU_Japan/Shuffling_Kit/Primer_Designer"><div class="arrow-div"></div><span>Primer Designer</span></a><br />
</div><br />
<br />
<div id="next-page"><br />
<a href="https://2013.igem.org/Team:HokkaidoU_Japan/Shuffling_Kit/Future_Work"><div class="arrow-div"></div><span>Future Work</span></a><br />
</div><br />
<br />
<!-- end contents / begin footer --><br />
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</div><br />
</html><br />
{{Team:HokkaidoU_Japan/footer}}</div>Nousanhttp://2013.igem.org/Team:HokkaidoU_Japan/Shuffling_Kit/ExamplesTeam:HokkaidoU Japan/Shuffling Kit/Examples2013-10-29T03:27:41Z<p>Nousan: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header_Maestro}}<br />
<html><br />
<div id="common-header-bottom-background"><br />
<div class="wrapper"><br />
<h1 id="common-header-title">Maestro E.coli</h1><br />
<h2 id="common-header-subtitle">Shuffling Kit</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
</div><br />
</div><br />
<br />
<div class="wrapper"><br />
<div id="hokkaidou-contents"><br />
<!-- end header / begin contents --><br />
<h1>Demonstrations for Usecase Example</h1><br />
<p>We will show some interesting demonstrations of our kits, Promoter Selector and RBS Selector!</p><br />
<br />
<br />
<h2>Promoter Selector</h2><br />
<p>Let's select the best promoter for Kanamycin resistance by Promoter Selector.</p><br />
<p>For this demonstration we decided to use the expression of Kanamycin resistance. Changing the concentration of Kanamycin in agar plate, it is estimated that different promoter will be chosen by our Promoter Selector (fig.1).</p><br />
<br />
<p>If the concentration of Kanamycin was high, the colony with strong promoter would survive. Therefore, only one or two colors would appear and indicate the first and second biggest occupancy rate on the plate.<br />
If the concentration of Kanamycin was low, colonies with even weak promoters could be able to survive. So in this way many colors of colonies would appear (fig.2).<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/b/bb/Fig1_in_example_132029new_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.1 Different promoter express each colors.</span></div><br />
</div><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/2/2d/Fig2_in_example_HokkaidoU_2013.png"><br />
<div style="padding-bottom: 0;"><span class="bold">fig.2 Difference of Kanamycin concentration.</span></div><br />
</div><br />
<br />
<h3>Method</h3><br />
<br />
<p>Optimum concentration of Kanamycin: in LB is 50 mg/ml <br><br />
We prepared optimum and other 3 concentration plates. <br />
</p><br />
<br />
<ul><br />
<li>Plate A: Kanamycin 125 mg per plate</li><br />
<li>Plate B: Kanamycin 250 mg per plate</li><br />
<li>Plate C: Kanamycin 500 mg per plate (optimum concentration)</li><br />
<li>Plate D: Kanamycin 1000 mg per plate</li><br />
</ul><br />
<br />
<p>Gene<br />
Vector: pSB1C3<br />
</p><br />
<p>We cloned Kanamycin resistance gene from pSB3K3, by using BsaI adding primer. Used the Promoter Selector (K1084501, K1084502, K1084503, K1084504, K1084505 ).</p><br />
<br />
<p><br />
Culture: 37 &deg;C, for 48h<br><br />
</p><br />
<br />
<h3>Results</h3><br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/2/28/HokkaidoU_2013_Km_resistance_assay_summary_data.png"><br />
<div><span class="bold">fig.4 Graph of number and rate, and table of number of colonies size over 1mm diameter.</span></div><br />
</div><br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/c/ca/POK_DEMO_48h_newnew_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.3 Picture of plate B (Kanamycine 250 mg). The colonies showed several colors.</span></div><br />
</div><br />
<br />
<p>After 48h cultivation, around 300 colonies had appeared on each LBKC (Kanamycin and Chloramphenicol) plates. We prepared LacZ&alpha expression system in Promoter Selector as negative control to estimate the success of BsaI digestion. We got 0-7 blue colonies which was expressing LacZ&alpha.<br />
</p><br />
<br />
<p> Mixed colored colonies which would have been transformed by two or more Promoter Selector were also observed. The number and rate of colonies per each plate were graphed (fig.4), with rejecting these undesirable colonies.<br />
</p><br />
<br />
<br />
<p><br />
In fig.4, legend color corresponds to Promoter Selector’s part number. The sum of colony numbers is displayed above each bar, and rate is in these sections. Number in the table referrers the number of each Promoter Selector’s colony. These data are collected by n=1.<br />
</p><br />
<br />
<div class="clearfix"></div><br />
<br />
<h3>Conclusion</h3><br />
<p><br />
Big difference did not appear among each Kanamaicin concentration. <br />
<br />
In this experiment, number of colonies derived from K1084405 (containing K1084010 promoter) had the largest rate on each plate. This result suggests that the colonies which had K1084505 could cost little resource of transcription and translation for expression of Kanamycin resistance gene, and the rest of resource had been spared to cell growth. Thus, the number of colonies might be the largest. Otherwise, the DNA solution of K1084505 Promoter Selector used at ligation was simply larger than other DNA solution. Although the result is collected from only one time assay, higher concentration of Kanamycin and much number of trials than this time will be needed.<br />
</p><br />
<br />
<p><br />
From these result and the experimental fact, the existence of Km resistance gene in Promoter Selector’s BsaI cloning section is partially confirmed. Our Promoter Selector was successfully assembled, but it does not adopted to all colonies. Then, as a result of assembling, we succeeded in making colorful colonies appear on one plate.<br />
</p><br />
<h2>RBS Selector</h2><br />
<h3>4 colors</h3><br />
<p>Let’s create all combinations by two reporter genes and make various colors on one plate!</p><br />
<br />
<p><br />
The RBS Selector we made, can randomize the strength of RBSs in the operon.<br />
For a demonstration, we decided to create all combinations by two genes; mRFP1 (BBa_E1010) and LacZ&alpha; (BBa_I732006) (fig.5). LacZ&alpha; makes the colony blue. mRFP1 makes the colony red.<br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/9/90/Fig4_in_example_new_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.5 Create all combinations by RBS of defferent stlength mRFP1 (BBa_E1010) and LacZα (BBa_I732006).</span></div><br />
</div><br />
<br />
<p>When the RBS of mRFP1 was stronger than that of LacZα, the colony would be red. When in the opposite case, the colony would be blue. And when the strengths of both RBSs were same, colony would be white or purple (fig.6).</p><br />
<br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/c/c8/Fig5_in_example_%2Boverhang_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.6 Each combinations of RBS make different colors.</span></div><br />
</div><br />
<br />
<h4>Method</h4><br />
<ul><br />
<li>Assembled promoter1 (BBa_K1084001), SD2 (BBa_K1084101), SD4 (BBa_K1084102).</li><br />
<li>Spread X-GAL(250 mg)on LBC plate.</li><br />
<li>Cultured at 37 &deg;C for 26h.</li><br />
</ul><br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/0/08/ROK_demo_new_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.7 The colonies showed red, blue, white, and purple.</span></div><br />
</div><br />
<h4>Results</h4><br />
<p><br />
We got colorful colonies; red, blue, white, and purple.<br />
<br />
<br />
</p><br />
<div class="clearfix"></div><br />
<br />
<h4>Conclusion</h4><br />
<br />
<br />
<p><br />
<br />
We can say that our RBS Selector worked exactly!!<br />
The RBSs upstream of 2 genes were randomly assembled and they had different expression level. <br />
<br />
</p><br />
<br />
<br />
<h3>64 colors</h3><br />
<br />
<p><br />
Let’s create all combinations by three reporter gene and using tandem RBS, make various colors on one plate!<br />
</p><br />
<br />
<p><br />
As the tandem RBS and the RBS selector demonstration, we randomized 64 patterns of RBS-CDS combination. By adding tandem RBS, three kinds of CDSs and GGA VACTOR, One-pot Golden Gate Assembly had done for making 64 kinds of different constructs.<br />
</p><br />
<br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/1/1d/64demo2_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.8 The 64 patterns of RBS-CDS combination constructed by RBS Selector.</span></div><br />
</div><br />
<br />
<p><br />
<h4>Method</h4><br />
<ul><br />
<li>Used tandem RBS (BBa_K1084302), GGA VECTOR that BsaI and overhang was added by PCR from K1084401, eforRed (BBa_K592012), aeBlue (BBa_K864401) and amilGFP (BBa_K592010) are assembled by Golden Gate Assembly.</li><br />
<li>Spread on LBC plate.</li><br />
<li>Cultured at 37 &deg;C, 48h.</li><br />
</ul><br />
</p><br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/5/55/HokkaidoU_2013_64ROK_result_1.JPG"><br />
<div><span class="bold">fig.9 The result plate of 64 pattern randomizing.</span></div><br />
</div><br />
<br />
<h4>Results</h4><br />
<br />
<p><br />
<br />
<br />
</p><br />
<br />
<p><br />
Many yellowish green (amilGFP) and red color (mRFP1) colonies are appeared, but these colonies are undesirable colonies. As a Golden Gate assembly result, green color (strong aeBlue and amilGFP expression) appeared, and the insert DNA length was confirmed that the insert DNA has same length of Golden Gate Assembly. other colonies without blue weren’t Golden Gate Assembly result. <br />
Other color colony, white, weak green, weak amilGFP expression colonies were observed. <br />
</p><br />
<br />
<h4>Conclusion</h4><br />
<br />
<br />
<p><br />
mRFP1 expression might cause by remaining of mRFP1 expression DNA used as template for GGA VECTOR producing by PCR (K1084401), thus mRFP1 expression colonies would appear.<br />
<p><br />
<br />
<p><br />
However, the blue and green colonies appeared on the plate has the insert DNA length same with ideal Golden Gate Assembling product. Our Shuffling kit partially worked.<br />
</p><br />
<br />
<br />
<br />
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{{Team:HokkaidoU_Japan/footer}}</div>Nousanhttp://2013.igem.org/Team:HokkaidoU_Japan/Shuffling_Kit/ExamplesTeam:HokkaidoU Japan/Shuffling Kit/Examples2013-10-29T03:24:32Z<p>Nousan: </p>
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<h1 id="common-header-title">Maestro E.coli</h1><br />
<h2 id="common-header-subtitle">Shuffling Kit</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
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<h1>Demonstrations for Usecase Example</h1><br />
<p>We will show some interesting demonstrations of our kits, Promoter Selector and RBS Selector!</p><br />
<br />
<br />
<h2>Promoter Selector</h2><br />
<p>Let's select the best promoter for Kanamycin resistance by Promoter Selector.</p><br />
<p>For this demonstration we decided to use the expression of Kanamycin resistance. Changing the concentration of Kanamycin in agar plate, it is estimated that different promoter will be chosen by our Promoter Selector (fig.1).</p><br />
<br />
<p>If the concentration of Kanamycin was high, the colony with strong promoter would survive. Therefore, only one or two colors would appear and indicate the first and second biggest occupancy rate on the plate.<br />
If the concentration of Kanamycin was low, colonies with even weak promoters could be able to survive. So in this way many colors of colonies would appear (fig.2).<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/b/bb/Fig1_in_example_132029new_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.1 Different promoter express each colors.</span></div><br />
</div><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/2/2d/Fig2_in_example_HokkaidoU_2013.png"><br />
<div style="padding-bottom: 0;"><span class="bold">fig.2 Difference of Kanamycin concentration.</span></div><br />
</div><br />
<br />
<h3>Method</h3><br />
<br />
<p>Optimum concentration of Kanamycin: in LB is 50 mg/ml <br><br />
We prepared optimum and other 3 concentration plates. <br />
</p><br />
<br />
<ul><br />
<li>Plate A: Kanamycin 125 mg per plate</li><br />
<li>Plate B: Kanamycin 250 mg per plate</li><br />
<li>Plate C: Kanamycin 500 mg per plate (optimum concentration)</li><br />
<li>Plate D: Kanamycin 1000 mg per plate</li><br />
</ul><br />
<br />
<p>Gene<br />
Vector: pSB1C3<br />
</p><br />
<p>We cloned Kanamycin resistance gene from pSB3K3, by using BsaI adding primer. Used the Promoter Selector (K1084501, K1084502, K1084503, K1084504, K1084505 ).</p><br />
<br />
<p><br />
Culture: 37 &deg;C, for 48h<br><br />
</p><br />
<br />
<h3>Results</h3><br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/2/28/HokkaidoU_2013_Km_resistance_assay_summary_data.png"><br />
<div><span class="bold">fig.4 Graph of number and rate, and table of number of colonies size over 1mm diameter.</span></div><br />
</div><br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/c/ca/POK_DEMO_48h_newnew_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.3 Picture of plate B (Kanamycine 250 mg). The colonies showed several colors.</span></div><br />
</div><br />
<br />
<p>After 48h cultivation, around 300 colonies had appeared on each LBKC (Kanamycin and Chloramphenicol) plates. We prepared LacZ&alpha expression system in Promoter Selector as negative control to estimate the success of BsaI digestion. We got 0-7 blue colonies which was expressing LacZ&alpha.<br />
</p><br />
<br />
<p> Mixed colored colonies which would have been transformed by two or more Promoter Selector were also observed. The number and rate of colonies per each plate were graphed (fig.4), with rejecting these undesirable colonies.<br />
</p><br />
<br />
<br />
<p><br />
In fig.4, legend color corresponds to Promoter Selector’s part number. The sum of colony numbers is displayed above each bar, and rate is in these sections. Number in the table referrers the number of each Promoter Selector’s colony. These data are collected by n=1.<br />
</p><br />
<br />
<div class="clearfix"></div><br />
<br />
<h3>Conclusion</h3><br />
<p><br />
Big difference did not appear among each Kanamaicin concentration. <br />
<br />
In this experiment, number of colonies derived from K1084405 (containing K1084010 promoter) had the largest rate on each plate. This result suggests that the colonies which had K1084505 could cost little resource of transcription and translation for expression of Kanamycin resistance gene, and the rest of resource had been spared to cell growth. Thus, the number of colonies might be the largest. Otherwise, the DNA solution of K1084505 Promoter Selector used at ligation was simply larger than other DNA solution. Although the result is collected from only one time assay, higher concentration of Kanamycin and much number of trials than this time will be needed.<br />
</p><br />
<br />
<p><br />
From these result and the experimental fact, the existence of Km resistance gene in Promoter Selector’s BsaI cloning section is partially confirmed. Our Promoter Selector was successfully assembled, but it does not adopted to all colonies. Then, as a result of assembling, we succeeded in making colorful colonies appear on one plate.<br />
</p><br />
<h2>RBS Selector</h2><br />
<h3>4 colors</h3><br />
<p>Let’s create all combinations by two reporter genes and make various colors on one plate!</p><br />
<br />
<p><br />
The RBS Selector we made, can randomize the strength of RBSs in the operon.<br />
For a demonstration, we decided to create all combinations by two genes; mRFP1 (BBa_E1010) and LacZ&alpha; (BBa_I732006) (fig.5). LacZ&alpha; makes the colony blue. mRFP1 makes the colony red.<br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/9/90/Fig4_in_example_new_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.5 Create all combinations by RBS of defferent stlength mRFP1 (BBa_E1010) and LacZα (BBa_I732006).</span></div><br />
</div><br />
<br />
<p>When the RBS of mRFP1 was stronger than that of LacZα, the colony would be red. When in the opposite case, the colony would be blue. And when the strengths of both RBSs were same, colony would be white or purple (fig.6).</p><br />
<br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/c/c8/Fig5_in_example_%2Boverhang_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.6 Each combinations of RBS make different colors.</span></div><br />
</div><br />
<br />
<h4>Method</h4><br />
<ul><br />
<li>Assembled promoter1 (BBa_K1084001), SD2 (BBa_K1084101), SD4 (BBa_K1084102).</li><br />
<li>Spread X-GAL(250 mg)on LBC plate.</li><br />
<li>Cultured at 37 &deg;C for 26h.</li><br />
</ul><br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/0/08/ROK_demo_new_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.7 The colonies showed red, blue, white, and purple.</span></div><br />
</div><br />
<h4>Results</h4><br />
<p><br />
We got colorful colonies; red, blue, white, and purple.<br />
<br />
<br />
</p><br />
<div class="clearfix"></div><br />
<br />
<h4>Conclusion</h4><br />
<br />
<br />
<p><br />
<br />
We can say that our RBS Selector worked exactly!!<br />
The RBSs upstream of 2 genes were randomly assembled and they had different expression level. <br />
<br />
</p><br />
<br />
<br />
<h3>64 colors</h3><br />
<br />
<p><br />
Let’s create all combinations by three reporter gene and using tandem RBS, make various colors on one plate!<br />
</p><br />
<br />
<p><br />
As the tandem RBS and the RBS selector demonstration, we randomized 64 patterns of RBS-CDS combination. By adding tandem RBS, three kinds of CDSs and GGA VACTOR, One-pot Golden Gate Assembly had done for making 64 kinds of different constructs.<br />
</p><br />
<br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/1/1d/64demo2_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.8 The 64 patterns of RBS-CDS combination constructed by RBS Selector.</span></div><br />
</div><br />
<br />
<p><br />
<h4>Method</h4><br />
<ul><br />
<li>Used tandem RBS (BBa_K1084302), GGA VECTOR that BsaI and overhang was added by PCR from K1084401, eforRed (BBa_K592012), aeBlue (BBa_K864401) and amilGFP (BBa_K592010) are assembled by Golden Gate Assembly.</li><br />
<li>Spread on LBC plate.</li><br />
<li>Cultured at 37 &deg;C, 48h.</li><br />
</ul><br />
</p><br />
<br />
<h4>Results</h4><br />
<br />
<p><br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/5/55/HokkaidoU_2013_64ROK_result_1.JPG"><br />
<div><span class="bold">fig.9 The result plate of 64 pattern randomizing.</span></div><br />
</div><br />
<br />
</p><br />
<br />
<p><br />
Many yellowish green (amilGFP) and red color (mRFP1) colonies are appeared, but these colonies are undesirable colonies. As a Golden Gate assembly result, green color (strong aeBlue and amilGFP expression) appeared, and the insert DNA length was confirmed that the insert DNA has same length of Golden Gate Assembly. other colonies without blue weren’t Golden Gate Assembly result. <br />
Other color colony, white, weak green, weak amilGFP expression colonies were observed. <br />
</p><br />
<br />
<h4>Conclusion</h4><br />
<br />
<br />
<p><br />
mRFP1 expression might cause by remaining of mRFP1 expression DNA used as template for GGA VECTOR producing by PCR (K1084401), thus mRFP1 expression colonies would appear.<br />
<p><br />
<br />
<p><br />
However, the blue and green colonies appeared on the plate has the insert DNA length same with ideal Golden Gate Assembling product. Our Shuffling kit partially worked.<br />
</p><br />
<br />
<br />
<br />
<div id="prev-page"><br />
<a href="https://2013.igem.org/Team:HokkaidoU_Japan/Shuffling_Kit/Primer_Designer"><div class="arrow-div"></div><span>Primer Designer</span></a><br />
</div><br />
<br />
<div id="next-page"><br />
<a href="https://2013.igem.org/Team:HokkaidoU_Japan/Shuffling_Kit/Future_Work"><div class="arrow-div"></div><span>Future Work</span></a><br />
</div><br />
<br />
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{{Team:HokkaidoU_Japan/footer}}</div>Nousanhttp://2013.igem.org/Team:HokkaidoU_Japan/Shuffling_Kit/ExamplesTeam:HokkaidoU Japan/Shuffling Kit/Examples2013-10-29T03:21:22Z<p>Nousan: </p>
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<h1 id="common-header-title">Maestro <span class="italic">E. coli</span></h1><br />
<h2 id="common-header-subtitle">Shuffling Kit</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
</div><br />
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<br />
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<div id="hokkaidou-contents"><br />
<!-- end header / begin contents --><br />
<h1>Demonstrations for Usecase Example</h1><br />
<p>We will show some interesting demonstrations of our kits, Promoter Selector and RBS Selector!</p><br />
<br />
<br />
<h2>Promoter Selector</h2><br />
<p>Let's select the best promoter for Kanamycin resistance by Promoter Selector.</p><br />
<p>For this demonstration we decided to use the expression of Kanamycin resistance. Changing the concentration of Kanamycin in agar plate, it is estimated that different promoter will be chosen by our Promoter Selector (fig.1).</p><br />
<br />
<p>If the concentration of Kanamycin was high, the colony with strong promoter would survive. Therefore, only one or two colors would appear and indicate the first and second biggest occupancy rate on the plate.<br />
If the concentration of Kanamycin was low, colonies with even weak promoters could be able to survive. So in this way many colors of colonies would appear (fig.2).<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/b/bb/Fig1_in_example_132029new_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.1 Different promoter express each colors.</span></div><br />
</div><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/2/2d/Fig2_in_example_HokkaidoU_2013.png"><br />
<div style="padding-bottom: 0;"><span class="bold">fig.2 Difference of Kanamycin concentration.</span></div><br />
</div><br />
<br />
<h3>Method</h3><br />
<br />
<p>Optimum concentration of Kanamycin: in LB is 50 mg/ml <br><br />
We prepared optimum and other 3 concentration plates. <br />
</p><br />
<br />
<ul><br />
<li>Plate A: Kanamycin 125 mg per plate</li><br />
<li>Plate B: Kanamycin 250 mg per plate</li><br />
<li>Plate C: Kanamycin 500 mg per plate (optimum concentration)</li><br />
<li>Plate D: Kanamycin 1000 mg per plate</li><br />
</ul><br />
<br />
<p>Gene<br />
Vector: pSB1C3<br />
</p><br />
<p>We cloned Kanamycin resistance gene from pSB3K3, by using BsaI adding primer. Used the Promoter Selector (K1084501, K1084502, K1084503, K1084504, K1084505 ).</p><br />
<br />
<p><br />
Culture: 37 &deg;C, for 48h<br><br />
</p><br />
<br />
<h3>Results</h3><br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/2/28/HokkaidoU_2013_Km_resistance_assay_summary_data.png"><br />
<div><span class="bold">fig.4 Graph of number and rate, and table of number of colonies size over 1mm diameter.</span></div><br />
</div><br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/c/ca/POK_DEMO_48h_newnew_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.3 Picture of plate B (Kanamycine 250 mg). The colonies showed several colors.</span></div><br />
</div><br />
<br />
<p>After 48h cultivation, around 300 colonies had appeared on each LBKC (Kanamycin and Chloramphenicol) plates. We prepared LacZa expression in Promoter Selector system as negative control to estimate the success of Golden Gate Assembly. We got 0-7 blue colonies which was expressing LacZ alpha.<br />
</p><br />
<br />
<p> Mixed colored colonies which would have been transformed by two or more Promoter Selector were also observed. The number and rate of colonies per each plate were graphed (fig.4), with rejecting these undesirable colonies.<br />
</p><br />
<br />
<br />
<p><br />
In fig.4, legend color corresponds to Promoter Selector’s part number. The sum of colony numbers is displayed above each bar, and rate is in these sections. Number in the table referrers the number of each Promoter Selector’s colony. These data are collected by n=!.<br />
</p><br />
<br />
<div class="clearfix"></div><br />
<br />
<h3>Conclusion</h3><br />
<p><br />
Big difference did not appear among each Kanamaicin concentration. <br />
<br />
In this experiment, number of colonies derived from K1084405 (containing K1084010 promoter) had the largest rate on each plate. This result suggests that the colonies which had K1084405 could cost little resource of transcription and translation for expression of Kanamycin resistance gene, and the rest of resource had been spared to cell growth. Thus, the number of colonies might be the largest. Otherwise, the DNA solution of K1084505 Promoter Selector used at ligation was simply larger than other DNA solution. Although the result is collected from only one time assay, higher concentration of Kanamycin and much number of trials than this time will be needed.<br />
</p><br />
<br />
<p><br />
From these result and the experimental fact, the existence of Km resistance gene in Promoter Selector’s BsaI cloning section is partially confirmed. Our Promoter Selector was successfully assembled, but it does not adopted to all colonies. Then, as a result of assembling, we succeeded in making colorful colonies appear on one plate.<br />
</p><br />
<h2>RBS Selector</h2><br />
<h3>4 colors</h3><br />
<p>Let’s create all combinations by two reporter genes and make various colors on one plate!</p><br />
<br />
<p><br />
The RBS Selector we made, can randomize the strength of RBSs in the operon.<br />
For a demonstration, we decided to create all combinations by two genes; mRFP1 (BBa_E1010) and LacZ&alpha; (BBa_I732006) (fig.5). LacZ&alpha; makes the colony blue. mRFP1 makes the colony red.<br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/9/90/Fig4_in_example_new_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.5 Create all combinations by RBS of defferent stlength mRFP1 (BBa_E1010) and LacZα (BBa_I732006).</span></div><br />
</div><br />
<br />
<p>When the RBS of mRFP1 was stronger than that of LacZα, the colony would be red. When in the opposite case, the colony would be blue. And when the strengths of both RBSs were same, colony would be white or purple (fig.6).</p><br />
<br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/c/c8/Fig5_in_example_%2Boverhang_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.6 Each combinations of RBS make different colors.</span></div><br />
</div><br />
<br />
<h4>Method</h4><br />
<ul><br />
<li>Assembled promoter1 (BBa_K1084001), SD2 (BBa_K1084101), SD4 (BBa_K1084102).</li><br />
<li>Spread X-GAL(250 mg)on LBC plate.</li><br />
<li>Cultured at 37 &deg;C for 26h.</li><br />
</ul><br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/0/08/ROK_demo_new_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.7 The colonies showed red, blue, white, and purple.</span></div><br />
</div><br />
<h4>Results</h4><br />
<p><br />
We got colorful colonies; red, blue, white, and purple.<br />
<br />
<br />
</p><br />
<div class="clearfix"></div><br />
<br />
<h4>Conclusion</h4><br />
<br />
<br />
<p><br />
<br />
We can say that our RBS Selector worked exactly!!<br />
The RBSs upstream of 2 genes were randomly assembled and they had different expression level. <br />
<br />
</p><br />
<br />
<br />
<h3>64 colors</h3><br />
<br />
<p><br />
Let’s create all combinations by three reporter gene and using tandem RBS, make various colors on one plate!<br />
</p><br />
<br />
<p><br />
As the tandem RBS and the RBS selector demonstration, we randomized 64 patterns of RBS-CDS combination. By adding tandem RBS, three kinds of CDSs and GGA VACTOR, One-pot Golden Gate Assembly had done for making 64 kinds of different constructs.<br />
</p><br />
<br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/1/1d/64demo2_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.8 The 64 patterns of RBS-CDS combination constructed by RBS Selector.</span></div><br />
</div><br />
<br />
<p><br />
<h4>Method</h4><br />
<ul><br />
<li>Used tandem RBS (BBa_K1084302), GGA VECTOR that BsaI and overhang was added by PCR from K1084401, eforRed (BBa_K592012), aeBlue (BBa_K864401) and amilGFP (BBa_K592010) are assembled by Golden Gate Assembly.</li><br />
<li>Spread on LBC plate.</li><br />
<li>Cultured at 37 &deg;C, 48h.</li><br />
</ul><br />
</p><br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/5/55/HokkaidoU_2013_64ROK_result_1.JPG"><br />
<div><span class="bold">fig.9 The result plate of 64 pattern randomizing.</span></div><br />
</div><br />
<br />
<h4>Results</h4><br />
<br />
<p><br />
<br />
</p><br />
<div class="clearfix"></div><br />
<br />
<br />
<br />
<div id="prev-page"><br />
<a href="https://2013.igem.org/Team:HokkaidoU_Japan/Shuffling_Kit/Primer_Designer"><div class="arrow-div"></div><span>Primer Designer</span></a><br />
</div><br />
<br />
<div id="next-page"><br />
<a href="https://2013.igem.org/Team:HokkaidoU_Japan/Shuffling_Kit/Future_Work"><div class="arrow-div"></div><span>Future Work</span></a><br />
</div><br />
<br />
<!-- end contents / begin footer --><br />
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</div><br />
</html><br />
{{Team:HokkaidoU_Japan/footer}}</div>Nousanhttp://2013.igem.org/Team:HokkaidoU_Japan/Shuffling_Kit/ExamplesTeam:HokkaidoU Japan/Shuffling Kit/Examples2013-10-29T03:19:36Z<p>Nousan: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header_Maestro}}<br />
<html><br />
<div id="common-header-bottom-background"><br />
<div class="wrapper"><br />
<h1 id="common-header-title">Maestro <span class="italic">E. coli</span></h1><br />
<h2 id="common-header-subtitle">Shuffling Kit</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
</div><br />
</div><br />
<br />
<div class="wrapper"><br />
<div id="hokkaidou-contents"><br />
<!-- end header / begin contents --><br />
<h1>Demonstrations for Usecase Example</h1><br />
<p>We will show some interesting demonstrations of our kits, Promoter Selector and RBS Selector!</p><br />
<br />
<br />
<h2>Promoter Selector</h2><br />
<p>Let's select the best promoter for Kanamycin resistance by Promoter Selector.</p><br />
<p>For this demonstration we decided to use the expression of Kanamycin resistance. Changing the concentration of Kanamycin in agar plate, it is estimated that different promoter will be chosen by our Promoter Selector (fig.1).</p><br />
<br />
<p>If the concentration of Kanamycin was high, the colony with strong promoter would survive. Therefore, only one or two colors would appear and indicate the first and second biggest occupancy rate on the plate.<br />
If the concentration of Kanamycin was low, colonies with even weak promoters could be able to survive. So in this way many colors of colonies would appear (fig.2).<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/b/bb/Fig1_in_example_132029new_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.1 Different promoter express each colors.</span></div><br />
</div><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/2/2d/Fig2_in_example_HokkaidoU_2013.png"><br />
<div style="padding-bottom: 0;"><span class="bold">fig.2 Difference of Kanamycin concentration.</span></div><br />
</div><br />
<br />
<h3>Method</h3><br />
<br />
<p>Optimum concentration of Kanamycin: in LB is 50 mg/ml <br><br />
We prepared optimum and other 3 concentration plates. <br />
</p><br />
<br />
<ul><br />
<li>Plate A: Kanamycin 125 mg per plate</li><br />
<li>Plate B: Kanamycin 250 mg per plate</li><br />
<li>Plate C: Kanamycin 500 mg per plate (optimum concentration)</li><br />
<li>Plate D: Kanamycin 1000 mg per plate</li><br />
</ul><br />
<br />
<p>Gene<br />
Vector: pSB1C3<br />
</p><br />
<p>We cloned Kanamycin resistance gene from pSB3K3, by using BsaI adding primer. Used the Promoter Selector (K1084501, K1084502, K1084503, K1084504, K1084505 ).</p><br />
<br />
<p><br />
Culture: 37 &deg;C, for 48h<br><br />
</p><br />
<br />
<h3>Results</h3><br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/2/28/HokkaidoU_2013_Km_resistance_assay_summary_data.png"><br />
<div><span class="bold">fig.4 Graph of number and rate, and table of number of colonies size over 1mm diameter.</span></div><br />
</div><br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/c/ca/POK_DEMO_48h_newnew_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.3 Picture of plate B (Kanamycine 250 mg). The colonies showed several colors.</span></div><br />
</div><br />
<br />
<p>After 48h cultivation, around 300 colonies had appeared on each LBKC (Kanamycin and Chloramphenicol) plates. We prepared LacZa expression in Promoter Selector system as negative control to estimate the success of Golden Gate Assembly. We got 0-7 blue colonies which was expressing LacZ alpha.<br />
</p><br />
<br />
<p> Mixed colored colonies which would have been transformed by two or more Promoter Selector were also observed. The number and rate of colonies per each plate were graphed (fig.4), with rejecting these undesirable colonies.<br />
</p><br />
<br />
<br />
<p><br />
In fig.4, legend color corresponds to Promoter Selector’s part number. The sum of colony numbers is displayed above each bar, and rate is in these sections. Number in the table referrers the number of each Promoter Selector’s colony. These data are collected by n=!.<br />
</p><br />
<br />
<div class="clearfix"></div><br />
<br />
<h3>Conclusion</h3><br />
<p><br />
Big difference did not appear among each Kanamaicin concentration. <br />
<br />
In this experiment, number of colonies derived from K1084405 (containing K1084010 promoter) had the largest rate on each plate. This result suggests that the colonies which had K1084405 could cost little resource of transcription and translation for expression of Kanamycin resistance gene, and the rest of resource had been spared to cell growth. Thus, the number of colonies might be the largest. Otherwise, the DNA solution of K1084505 Promoter Selector used at ligation was simply larger than other DNA solution. Although the result is collected from only one time assay, higher concentration of Kanamycin and much number of trials than this time will be needed.<br />
</p><br />
<br />
<p><br />
From these result and the experimental fact, the existence of Km resistance gene in Promoter Selector’s BsaI cloning section is partially confirmed. Our Promoter Selector was successfully assembled, but it does not adopted to all colonies. Then, as a result of assembling, we succeeded in making colorful colonies appear on one plate.<br />
</p><br />
<h2>RBS Selector</h2><br />
<h3>4 colors</h3><br />
<p>Let’s create all combinations by two reporter genes and make various colors on one plate!</p><br />
<br />
<p><br />
The RBS Selector we made, can randomize the strength of RBSs in the operon.<br />
For a demonstration, we decided to create all combinations by two genes; mRFP1 (BBa_E1010) and LacZ&alpha; (BBa_I732006) (fig.5). LacZ&alpha; makes the colony blue. mRFP1 makes the colony red.<br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/9/90/Fig4_in_example_new_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.5 Create all combinations by RBS of defferent stlength mRFP1 (BBa_E1010) and LacZα (BBa_I732006).</span></div><br />
</div><br />
<br />
<p>When the RBS of mRFP1 was stronger than that of LacZα, the colony would be red. When in the opposite case, the colony would be blue. And when the strengths of both RBSs were same, colony would be white or purple (fig.6).</p><br />
<br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/c/c8/Fig5_in_example_%2Boverhang_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.6 Each combinations of RBS make different colors.</span></div><br />
</div><br />
<br />
<h3>Method</h3><br />
<ul><br />
<li>Assembled promoter1 (BBa_K1084001), SD2 (BBa_K1084101), SD4 (BBa_K1084102).</li><br />
<li>Spread X-GAL(250 mg)on LBC plate.</li><br />
<li>Cultured at 37 &deg;C for 26h.</li><br />
</ul><br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/0/08/ROK_demo_new_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.7 The colonies showed red, blue, white, and purple.</span></div><br />
</div><br />
<h3>Results</h3><br />
<p><br />
We got colorful colonies; red, blue, white, and purple.<br />
<br />
<br />
</p><br />
<div class="clearfix"></div><br />
<br />
<h3>Conclusion</h3><br />
<br />
<br />
<p><br />
<br />
We can say that our RBS Selector worked exactly!!<br />
The RBSs upstream of 2 genes were randomly assembled and they had different expression level. <br />
<br />
</p><br />
<br />
<br />
<h3>64 colors</h3><br />
<br />
<p><br />
Let’s create all combinations by three reporter gene and using tandem RBS, make various colors on one plate!<br />
</p><br />
<br />
<p><br />
As the tandem RBS and the RBS selector demonstration, we randomized 64 patterns of RBS-CDS combination. By adding tandem RBS, three kinds of CDSs and GGA VACTOR, One-pot Golden Gate Assembly had done for making 64 kinds of different constructs.<br />
</p><br />
<br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/1/1d/64demo2_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.8 The 64 patterns of RBS-CDS combination constructed by RBS Selector.</span></div><br />
</div><br />
<br />
<p><br />
<h4>Method</h4><br />
<ul><br />
<li>Used tandem RBS (BBa_K1084302), GGA VECTOR that BsaI and overhang was added by PCR from K1084401, eforRed (BBa_K592012), aeBlue (BBa_K864401) and amilGFP (BBa_K592010) are assembled by Golden Gate Assembly.</li><br />
<li>Spread on LBC plate.</li><br />
<li>Cultured at 37 &deg;C, 48h.</li><br />
</ul><br />
</p><br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/5/55/HokkaidoU_2013_64ROK_result_1.JPG"><br />
<div><span class="bold">fig.9 The result plate of 64 pattern randomizing.</span></div><br />
</div><br />
<br />
<h4>Results</h4><br />
<br />
<p><br />
<br />
</p><br />
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{{Team:HokkaidoU_Japan/footer}}</div>Nousanhttp://2013.igem.org/Team:HokkaidoU_Japan/Promoter/ResultsTeam:HokkaidoU Japan/Promoter/Results2013-10-28T23:48:42Z<p>Nousan: </p>
<hr />
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<html><br />
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<h1 id="common-header-title">Maestro E.coli</h1><br />
<h2 id="common-header-subtitle">Promoter</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
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<br />
<br />
<h1>Result</h1><br />
<h2>-35 region randomization</h2><br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/6/6b/HokkaidoU_2013_Parts-f1_2.png"><br />
<div><span class="bold">fig. 1 Randomized promoter sequences.</span></div><br />
</div><br />
<p>We randomized -35 region by PCR primers with random hexamer region. The template DNA was consensus_promtoer-B0034-mRFP1(E1010)-B0015 (about 1,000 bp). We assayed the constructed sequences and isolated 10 distinct promoters. We sequenced the randomized promoter sequences to confirm that only -35 regions was changed. Our consensus promoter is K1084001.</p><br />
</p><br />
<br />
<div class="clearfix"></div><br />
<h2>promoter assay; mRFP1, LacZ and Kanamycin resistance gene</h2><br />
<br />
<br />
<h3>mRFP1</h3><br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/7/73/HokkaidoU2013_promoter_Result-fig2.png"><br />
<div><span class="bold">fig. 2 mRFP1 assay result.</span></div><br />
</div><br />
<p>mRFP1(BBa_E1010) expressing JM109 colonies were resuspend to 2 ml LBC liquid culture.<br />
After cultivation (180 rpm shaking at 37C) for 12 hrs, we measured OD650 with micro titer plate reader. We avoided using 600 nm because mRFP1 absorbs 600 nm. mRFP1 expression was measured with fluorescence imaging machine.<br />
All 10 of the promoters were characterized. Five promoters were used as a reference.<br />
BBa_K1084010 and BBa_K1084009 couldn't be characterized by mRFP1 assay because of mutation at CDS.<br />
</p><br />
<br />
<p>Reference promoters are following<p><br />
<ul><br />
<li>BBa_R0010: pLac</li><br />
<li>BBa_R0040: pTetR</li><br />
<li>BBa_J23106: constitutive promoter family member (1185 arb. unit)</li><br />
<li>BBa_J23112: constitutive promoter family member ( 1 arb. unit)</li><br />
<li>Negative control: not protein expression construct</li><br />
</ul><br />
<br />
<br />
<br />
<br />
<h3>promoter selection by modeling</h3><br />
<br />
<p>We chose 5 of 10 promoters by the value of theoretical transcription efficiency (for theoretical explanation, see "Method page"). This efficiency is affected by binding energy in our assumption.</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/b/b2/HokkaidoU2013_promoter_Modeling_fig5.png"><br />
<div><span class="bold">fig. 3 Theoretical transcription efficiency distribution.</span></div><br />
</div><br />
<br />
<h3>LacZ&alpha;</h3><br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/1/1d/HokkaidoU2013_promoter_Result-fig4.png"><br />
<div><span class="bold">fig. 4 &beta;-Galactosidase assay result.</span></div><br />
</div><br />
<br />
<p><br />
We selected five promoters from our original family to model. LacZ&alpha;<br />
Only these promoters were characterized using LacZ assay.<br />
LacZ (&beta;-Galactosidase) activity was measured with &beta;-Galactosidase assay kit. (OZ Biogenesis<br />
http://www.funakoshi.co.jp/data/datasheet/OZB/GC-10002.pdf )<br />
DH5&alpha; strain was used.<br />
</p><br />
<br />
<p>Reference promoters are following<p><br />
<ul><br />
<li>BBa_R0010: pLac</li><br />
<li>BBa_R0040: pTetR</li><br />
<li>BBa_J23106: constitutive promoter family member (1185 arb. unit)</li><br />
<li>Negative control: not protein expression construct</li><br />
</ul><br />
<br />
<br />
<h3>Kanamycin resistance gene</h3><br />
<br />
<p>Kanamycin resistance gene is expressed by these promoters as Promoter Selector construct.<br />
</p><br />
<br />
<p><br />
As shown in the Shuffling_Kit/Examples page, Kanamycin resistance also differed by promoter. In the result, K1084010 has the best Kanamycin resistance activity. <br />
However, the result is containing some controversial problems, such like differences of concentration among used DNA solutions at ligation step, or the Kanamycin resistance measuring problem: the Kanamycin resistance gene activity wouldn't be measured by just counting colonies on a plate. There are needed more intermolecular assay for measuring the enzyme activity.<br />
</p><br />
<br />
<br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/e/eb/HokkaidoU2013_promoter_Result-fig5.png"><br />
<div><span class="bold">fig. 5 Comparison of assay results and modeling data.</span></div><br />
</div><br />
<h3>Comparison of assay results (Conclusion)</h3><br />
<p><br />
These data was compared with modeling data (logarithm of transcription efficiency, t. e.).<br />
BBa_K1084010 and BBa_K1084009 couldn't be characterized by mRFP1 assay because of mutation at CDS.<br />
</p><br />
<div class="clearfix"></div><br />
<br />
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{{Team:HokkaidoU_Japan/footer}}</div>Nousanhttp://2013.igem.org/Team:HokkaidoU_Japan/Promoter/ResultsTeam:HokkaidoU Japan/Promoter/Results2013-10-28T23:47:40Z<p>Nousan: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header_Maestro}}<br />
<html><br />
<div id="common-header-bottom-background"><br />
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<h1 id="common-header-title">Maestro E.coli</h1><br />
<h2 id="common-header-subtitle">Promoter</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
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<br />
<br />
<h1>Result</h1><br />
<h2>-35 region randomization</h2><br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/6/6b/HokkaidoU_2013_Parts-f1_2.png"><br />
<div><span class="bold">fig. 1 Randomized promoter sequences.</span></div><br />
</div><br />
<p>We randomized -35 region by PCR primers with random hexamer region. The template DNA was consensus_promtoer-B0034-mRFP1(E1010)-B0015 (about 1,000 bp). We assayed the constructed sequences and isolated 10 distinct promoters. We sequenced the randomized promoter sequences to confirm that only -35 regions was changed. Our consensus promoter is K1084001.</p><br />
</p><br />
<br />
<div class="clearfix"></div><br />
<h2>promoter assay; mRFP1, LacZ and Kanamycin resistance gene</h2><br />
<br />
<br />
<h3>mRFP1</h3><br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/7/73/HokkaidoU2013_promoter_Result-fig2.png"><br />
<div><span class="bold">fig. 2 mRFP1 assay result.</span></div><br />
</div><br />
<p>mRFP1(BBa_E1010) expressing JM109 colonies were resuspend to 2 ml LBC liquid culture.<br />
After cultivation (180 rpm shaking at 37C) for 12 hrs, we measured OD650 with micro titer plate reader. We avoided using 600 nm because mRFP1 absorbs 600 nm. mRFP1 expression was measured with fluorescence imaging machine.<br />
All 10 of the promoters were characterized. Five promoters were used as a reference.<br />
BBa_K1084010 and BBa_K1084009 couldn't be characterized by mRFP1 assay because of mutation at CDS.<br />
</p><br />
<br />
<p>Reference promoters are following<p><br />
<ul><br />
<li>BBa_R0010: pLac</li><br />
<li>BBa_R0040: pTetR</li><br />
<li>BBa_J23106: constitutive promoter family member (1185 arb. unit)</li><br />
<li>BBa_J23112: constitutive promoter family member ( 1 arb. unit)</li><br />
<li>Negative control: not protein expression construct</li><br />
</ul><br />
<br />
<br />
<br />
<br />
<h3>promoter selection by modeling</h3><br />
<br />
<p>We chose 5 of 10 promoters by the value of theoretical transcription efficiency (for theoretical explanation, see "Method page"). This efficiency is affected by binding energy in our assumption.</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/b/b2/HokkaidoU2013_promoter_Modeling_fig5.png"><br />
<div><span class="bold">fig. 3 Theoretical transcription efficiency distribution.</span></div><br />
</div><br />
<br />
<h3>LacZ&alpha;</h3><br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/1/1d/HokkaidoU2013_promoter_Result-fig4.png"><br />
<div><span class="bold">fig. 4 &beta;-Galactosidase assay result.</span></div><br />
</div><br />
<br />
<p><br />
We selected five promoters from our original family to model. LacZ&alpha;<br />
Only these promoters were characterized using LacZ assay.<br />
LacZ (&beta;-Galactosidase) activity was measured with &beta;-Galactosidase assay kit. (OZ Biogenesis<br />
http://www.funakoshi.co.jp/data/datasheet/OZB/GC-10002.pdf )<br />
DH5&alpha; strain was used.<br />
</p><br />
<br />
<p>Reference promoters are following<p><br />
<ul><br />
<li>BBa_R0010: pLac</li><br />
<li>BBa_R0040: pTetR</li><br />
<li>BBa_J23106: constitutive promoter family member (1185 arb. unit)</li><br />
<li>Negative control: not protein expression construct</li><br />
</ul><br />
<br />
<br />
<h3>Kanamycin resistance gene</h3><br />
<br />
<p>Kanamycin resistance gene is expressed by these promoters as Promoter Selector construct.<br />
</p><br />
<br />
<p><br />
As shown in the Shuffling_Kit/Examples page, Kanamycin resistance also differed by promoter. In the result, K1084010 has the best Kanamycin resistance activity. <br />
However, the result is containing some controversial problems, such like differences of concentration among used DNA solutions at ligation step, or the Kanamycin resistance measuring problem: the Kanamycin resistance gene activity wouldn't be measured by just counting colonies on a plate. There are needed more intermolecular assay for measuring the enzyme activity.<br />
</p><br />
<br />
<h3>Comparison of assay results (Conclusion)</h3><br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/e/eb/HokkaidoU2013_promoter_Result-fig5.png"><br />
<div><span class="bold">fig. 5 Comparison of assay results and modeling data.</span></div><br />
</div><br />
<br />
<p><br />
These data was compared with modeling data (logarithm of transcription efficiency, t. e.).<br />
BBa_K1084010 and BBa_K1084009 couldn't be characterized by mRFP1 assay because of mutation at CDS.<br />
</p><br />
<div class="clearfix"></div><br />
<br />
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{{Team:HokkaidoU_Japan/footer}}</div>Nousanhttp://2013.igem.org/Team:HokkaidoU_Japan/Shuffling_Kit/Future_WorkTeam:HokkaidoU Japan/Shuffling Kit/Future Work2013-10-28T18:25:26Z<p>Nousan: </p>
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<h1 id="common-header-title">Maestro E.coli</h1><br />
<h2 id="common-header-subtitle">Optimization Kit</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
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<h1>Maestro E.coli Future Work</h1><br />
<br />
<h2>To make Promoter Selector better kit</h2><br />
<p>To make Promoter Selector better, we are going to put pigment producing constructs between two PstI sites. After you confirm promoter, you can remove pigment producing constructs by PstI.</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/1/12/Fig11_new2_800_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.1 How to make our kit better.</span></div><br />
</div><br />
<br />
<h2>Application of RBS Selector</h2><br />
<p>We can get a lot of operons by RBS Selector. RBS Selector provides 16 operons in case of 2 CDS, 64 operons in case of 3 CDS. What can we do using these number of operons? We expect industrial applications of RBS Selector as future works.</p><br />
<br />
<p>For example PHB <br />
(polyhydroxybutyrate) ,3 enzymes synthesize PHB from substrate.PHB is one of biodegradable plastic. You can learn more about PHB at <a href="https://2012.igem.org/Team:HokkaidoU_Japan/Project/PHB_Synthesis">iGEM HokkaidoU_Japan 2012 wiki</a>. However, you know by our wiki, CDS has each optimum RBS. It is possibility to find more efficient construct from 64 constructs by assay results. It is possible to use RBS Selector to medicine or vaccine etc.<br />
We hope applications of our project like making world happy.<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2012/3/38/HokkaidoU_PHB_Fig3.jpg"><br />
<div><span class="bold">fig.2 P(3HB) synthesis pathway in R. eutropha.</span></div><br />
</div><br />
<br />
<br />
<h2>Select Promoter & RBS</h2><br />
<p>To select proteins expressions in wider range, you can use Promoter Selector and RBS Selector at the same time! The combination number is 320 patterns!</p><br />
<br />
<br />
<br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/3/3b/Maestro_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.3 Making various combination patarns.</span></div><br />
</div><br />
<br />
<p>Besides, you can create promoter and terminator by yourself and assemble them with our RBS Selector and GGA VECTOR (K1084301) by Golden Gate Assembly! </p><br />
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{{Team:HokkaidoU_Japan/footer}}</div>Nousanhttp://2013.igem.org/Team:HokkaidoU_Japan/Shuffling_Kit/ExamplesTeam:HokkaidoU Japan/Shuffling Kit/Examples2013-10-28T18:04:46Z<p>Nousan: </p>
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<h1 id="common-header-title">Maestro E.coli</h1><br />
<h2 id="common-header-subtitle">Optimization Kit</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
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<h1>Demonstrations for Usecase Example</h1><br />
<p>We will show some interesting demonstrations of our kits, Promoter Selector and RBS Selector!</p><br />
<br />
<br />
<h2>Promoter Selector</h2><br />
<p>Let's select the best promoter for Kanamycin resistance by Promoter Selector.</p><br />
<p>For a demonstration we decided to optimize the expression of Kanamycin resistance. Changing the concentration of Kanamycin in agar plate, it is estimated that different promoter will be chosen by our Promoter Selector (fig.1).</p><br />
<br />
<p>If the concentration of Kanamycin was high, the colony with strong promoter will survive. Therefore, only one or two colors indicate the first and second biggest occupancy rate on the plate.<br />
If the concentration of Kanamycin was low, colonies with weak promoters will be able to survive. This way many colors of colonies would appear (fig.2).<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/b/bb/Fig1_in_example_132029new_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.1 Different promoter express each colors.</span></div><br />
</div><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/2/2d/Fig2_in_example_HokkaidoU_2013.png"><br />
<div style="padding-bottom: 0;"><span class="bold">fig.2 Difference of Kanamycin concentration.</span></div><br />
</div><br />
<br />
<h4>Method</h4><br />
<br />
<p>Optimum concentration of Kanamycin: in LB is 50 mg/ml<br />
We prepared 3 different concentration plates. <br />
</p><br />
<br />
<ul><br />
<li>Plate A: Kanamycin 125 mg per plate</li><br />
<li>Plate B: Kanamycin 250 mg per plate</li><br />
<li>Plate C: Kanamycin 500 mg per plate (optimum concentration)</li><br />
<li>Plate D: Kanamycin 1000 mg per plate</li><br />
</ul><br />
<br />
<p>Gene<br />
Vector: pSB1C3<br />
</p><br />
<p>We cloned Kanamycin resistant gene from pSB3K3, by using BsaI adding primer. Used the Promoter Selector (K1084501, K1084502, K1084503, K1084504, K1084505 ).</p><br />
<br />
<p><br />
Culture: 37 &deg;C, for 48h<br />
</p><br />
<br />
<h4>Results</h4><br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/2/28/HokkaidoU_2013_Km_resistance_assay_summary_data.png"><br />
<div><span class="bold">fig.4 Graph of number and rate, and table of number of colonies size over 1mm diameter.</span></div><br />
</div><br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/c/ca/POK_DEMO_48h_newnew_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.3 Picture of plate B (Kanamycine 250 mg). The colonies showed several colors.</span></div><br />
</div><br />
<br />
<p>After 48h cultivation, around 300 colonies had appeared on each LBKC (Kanamycin and Chloramphenicol) plates. We prepared LacZa expression in Promoter Selector system as negative control to estimate the success of Golden Gate Assembly, and only 7 to 0 colonies are expressed LacZa. Mixed colored colonies which would have been transformed by two or more Promoter Selector were also observed. The number and rate of colonies per each plate were graphed (fig.4), with rejecting these undesirable colonies.<br />
</p><br />
<br />
<p><br />
In (fig.4), legend color corresponds to Promoter Selector’s part number. The sum of colony numbers is displayed above each bar, and rate is in these sections. Number in the table is the number of each Promoter Selector’s colonies. These data are collected from only one time Kanamycin resistance assay result.<br />
</p><br />
<br />
<div class="clearfix"></div><br />
<br />
<h4>Conclusion</h4><br />
<p><br />
There is no difference from lowest and highest Kanamycin concentration. In these colonies, number of colonies derived from K1084405 (containing K1084010 promoter ) has the most largest rate on each plate. This result suggests that the colonies expressed the lowest amount of Kanamycin resistance gene, and the resorce of transcription and translation could be spared to cell growth,thus the number of colonies may have been largest. Otherwise, the DNA solution of K1084505 Promoter Selector used at ligation was simply larger than other DNA solution. Although the result is collected from only one time assay, higher conscentration of Kanamycin and much number of trials than this time will be needed.<br />
</p><br />
<br />
<p><br />
From these result and the experimental fact, the existence of Km resistance gene in Promoter Selector’s BsaI cloning section is partially confirmed. Our Promoter Selector was successfully assembled, but it does not adopted to all colonies. Then, as a result of assembling, we succeeded in making colorful colonies appear on one plate.<br />
</p><br />
<h2>RBS Selector</h2><br />
<h3>4 colors</h3><br />
<p>Let’s create all combinations by two reporter genes and make various colors on one plate!</p><br />
<br />
<br />
<p><br />
The RBS Selector we made, can randomize the strength of RBSs in the operon.<br />
For a demonstration, we decided to create all combinations by two genes; mRFP1 (BBa_E1010) and LacZ&alpha; (BBa_I732006) (fig.5). LacZ&alpha; makes the colony blue. mRFP1 makes the colony red.<br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/9/90/Fig4_in_example_new_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.5 Create all combinations by RBS of defferent stlength mRFP1 (BBa_E1010) and LacZα (BBa_I732006).</span></div><br />
</div><br />
<br />
<p>When the RBS upstream of mRFP1 was strong and the RBS upstream was weak, the colony should be red. When the RBS upstream of mRFP1 was weak, and the RBS upstream was strong, the colony should be blue. So when if the strength of RBS upstream both genes were the same, colony will be white, purple (fig.6).</p><br />
<br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/c/c8/Fig5_in_example_%2Boverhang_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.6 Each combinations of RBS make different colors.</span></div><br />
</div><br />
<br />
<h4>Method</h4><br />
<ul><br />
<li>Used promoter1 (BBa_K1084001), SD2 (BBa_K1084101), SD4 (BBa_K1084102) and assembled with.</li><br />
<li>Spread X-GAL(250 mg)on LBC plate.</li><br />
<li>Cultured for 37 &deg;C, 26h.</li><br />
</ul><br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/0/08/ROK_demo_new_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.7 The colonies showed red, blue, white, and purple.</span></div><br />
</div><br />
<h4>Results</h4><br />
<p><br />
We got many colored colonies,red, blue, white, and purple.<br />
<br />
<br />
</p><br />
<div class="clearfix"></div><br />
<br />
<h4>Conclusion</h4><br />
<br />
<br />
<p><br />
<br />
We can say that our RBS Selector worked!!<br />
The RBSs uptsream 2 genes were randomized and they had many levels of expressions. <br />
<br />
</p><br />
<br />
<h3>64 colors</h3><br />
<br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/1/1d/64demo2_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.8.</span></div><br />
</div><br />
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{{Team:HokkaidoU_Japan/footer}}</div>Nousanhttp://2013.igem.org/File:Fig1_in_example_132029new_HokkaidoU_2013.pngFile:Fig1 in example 132029new HokkaidoU 2013.png2013-10-28T18:04:12Z<p>Nousan: </p>
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<div></div>Nousanhttp://2013.igem.org/File:Fig1_in_example_132029_HokkaidoU_2013.pngFile:Fig1 in example 132029 HokkaidoU 2013.png2013-10-28T18:02:26Z<p>Nousan: </p>
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<div></div>Nousanhttp://2013.igem.org/Team:HokkaidoU_Japan/Overview/BackgroundTeam:HokkaidoU Japan/Overview/Background2013-10-28T15:17:33Z<p>Nousan: </p>
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<h1 id="common-header-title">Maestro E.coli</h1><br />
<h2 id="common-header-subtitle">Overview</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
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<h1>Background</h1><br />
<br />
<p><br />
Synthetic biology is one of the most interesting fields in 21st century.<br />
Its goal is to comprehend and reproduce the marvels of living things.<br />
As members of syn-bio community, a lot of iGEMers have found interesting proteins.<br />
However, the focuses of these projects are “qualities” of proteins.<br />
As well as the uniqueness of proteins, the expression levels of proteins contribute to the marvelous functions of living things.<br />
The next goal of syn-bio is to control “quantities” of proteins.<br />
</p><br />
<br />
<p><br />
To control protein quantities in the organism, we must control transcription, a step from DNA to mRNA and translation, a step from mRNA to protein.<br />
Transcription is regulated by promoter region.<br />
Translation is regulated by ribosome binding site: RBS.<br />
Depending on promoters, the transcription rate varies about 1000 fold.<br />
Similarly, depending on RBSs, the translation rate about 100 fold.<br />
So, to control quantity, it is essential to figure out the characteristics of these expression-regulatory regions and adjust them.<br />
</p><br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2013/8/8a/HokkaidoU2013_Motivation_figure4_new.png"><br />
</div><br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2013/2/28/HokkaidoU2013_Motivation_figure5.png"><br />
</div><br />
<p><br />
Recently, many studies have been done to theoretically predict the amount of gene expression by the sequences of expression-regulatory regions.<br />
There are some accurate in vivo expression efficiency predictions.<br />
Besides, even if you were able to predict it exactly, you could not adjust it to an optimal level because you don't know the best rate for the bacteria.<br />
</p><br />
<br />
<p><br />
Suppose you are planning to express beneficial product, like antibodies.<br />
You may think that the strongest promoter and RBS lead the maximal yield of the proteins.<br />
But actually, selecting the strongest one isn't always the best strategy.<br />
Overexpressed proteins can be perceived as detrimental by bacterial immune system and packed into inclusion bodies.<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/2/2d/HokkaidoU2013_Motivation_figure1.png"><br />
</div><br />
<p><br />
Another example is a multiple expression, such as mixing color of chromoprotein.<br />
Theoretically, three different chromoproteins can express any kind of colors by fixing each expression level.<br />
But this adjustment is too sensitive to be made instinctively.<br />
</p><br />
<div class="fig fig300"><br />
<img src="https://static.igem.org/mediawiki/2013/7/76/HokkaidoU2013_Motivation_figure3_big.png"><br />
</div><br />
<p><br />
Over these challenging problems in syn-bio, we put forward one solution like a Columbus's egg.<br />
"No more prediction, let's experiment!" ---- that is, we should try several promoters or RBSs and select the best one or the best combination.<br />
We don't know the best answer, but bacteria do.<br />
</p><br />
<br />
<br />
<p><br />
This is surely the best way, but trying every pattern costs a lot of time and labor.<br />
We overcame this obstacle by using a one-pot DNA shuffling method, namely, "Golden Gate Assembly".<br />
And then, we made two kits to optimize transcription and translation.<br />
For this kit, we made artificial promoter and RBS families.<br />
They are created based on concrete philosophies and characterized well.<br />
Please refer <a href="https://2013.igem.org/Team:HokkaidoU_Japan/Promoter">Promoter</a> and <a href="https://2013.igem.org/Team:HokkaidoU_Japan/RBS">RBS</a> for details.<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/a/ac/HokkaidoU2013_Motivation_figure2.png"><br />
</div><br />
<p><br />
We named these kits "Promoter Selector" and "RBS Selector".<br />
<dl><br />
<dt>Promoter Selector</dt><dd>Promoter Selector is for adjustment of relatively simple expression, like a production of antibodies reffered above.</dd><br />
<dt>RBS Selector</dt><dd>RBS Selector is for adjustment of multiple expressions, like complex metabolisms.</dd><br />
</dl><br />
We named these two devices ''Maestro <span class="italic">E.coli</span>'' Random Operon Shuffling Kit! As a maestro harmonizes music, let's harmonize proteins in the bacteria! <br />
<br />
</p><br />
<br />
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<a href="https://2013.igem.org/Team:HokkaidoU_Japan/Overview/Achievements"><div class="arrow-div"></div><span>Achievements</span></a><br />
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{{Team:HokkaidoU_Japan/footer}}</div>Nousanhttp://2013.igem.org/Team:HokkaidoU_Japan/Overview/BackgroundTeam:HokkaidoU Japan/Overview/Background2013-10-28T15:16:55Z<p>Nousan: </p>
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<h1 id="common-header-title">Maestro E.coli</h1><br />
<h2 id="common-header-subtitle">Overview</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
</div><br />
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<div id="hokkaidou-contents"><br />
<!-- end header / begin contents --><br />
<h1>Background</h1><br />
<br />
<p><br />
Synthetic biology is one of the most interesting fields in 21st century.<br />
Its goal is to comprehend and reproduce the marvels of living things.<br />
As members of syn-bio community, a lot of iGEMers have found interesting proteins.<br />
However, the focuses of these projects are “qualities” of proteins.<br />
As well as the uniqueness of proteins, the expression levels of proteins contribute to the marvelous functions of living things.<br />
The next goal of syn-bio is to control “quantities” of proteins.<br />
</p><br />
<br />
<p><br />
To control protein quantities in the organism, we must control transcription, a step from DNA to mRNA and translation, a step from mRNA to protein.<br />
Transcription is regulated by promoter region.<br />
Translation is regulated by ribosome binding site: RBS.<br />
Depending on promoters, the transcription rate varies about 1000 fold.<br />
Similarly, depending on RBSs, the translation rate about 100 fold.<br />
So, to control quantity, it is essential to figure out the characteristics of these expression-regulatory regions and adjust them.<br />
</p><br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2013/8/8a/HokkaidoU2013_Motivation_figure4_new.png"><br />
</div><br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2013/2/28/HokkaidoU2013_Motivation_figure5.png"><br />
</div><br />
<p><br />
Recently, many studies have been done to theoretically predict the amount of gene expression by the sequences of expression-regulatory regions.<br />
There are some accurate in vivo expression efficiency predictions.<br />
Besides, even if you were able to predict it exactly, you could not adjust it to an optimal level because you don't know the best rate for the bacteria.<br />
</p><br />
<br />
<p><br />
Suppose you are planning to express beneficial product, like antibodies.<br />
You may think that the strongest promoter and RBS lead the maximal yield of the proteins.<br />
But actually, selecting the strongest one isn't always the best strategy.<br />
Overexpressed proteins can be perceived as detrimental by bacterial immune system and packed into inclusion bodies.<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/2/2d/HokkaidoU2013_Motivation_figure1.png"><br />
</div><br />
<p><br />
Another example is a multiple expression, such as mixing color of chromoprotein.<br />
Theoretically, three different chromoproteins can express any kind of colors by fixing each expression level.<br />
But this adjustment is too sensitive to be made instinctively.<br />
</p><br />
<div class="fig fig300"><br />
<img src="https://static.igem.org/mediawiki/2013/7/76/HokkaidoU2013_Motivation_figure3_big.png"><br />
</div><br />
<p><br />
Over these challenging problems in syn-bio, we put forward one solution like a Columbus's egg.<br />
"No more prediction, let's experiment!" ---- that is, we should try several promoters or RBSs and select the best one or the best combination.<br />
We don't know the best answer, but bacteria do.<br />
</p><br />
<br />
<br />
<p><br />
This is surely the best way, but trying every pattern costs a lot of time and labor.<br />
We overcame this obstacle by using a one-pot DNA shuffling method, namely, "Golden Gate Assembly".<br />
And then, we made two kits to optimize transcription and translation.<br />
For this kit, we made artificial promoter and RBS families.<br />
They are created based on concrete philosophies and characterized well.<br />
Please refer <a href="https://2013.igem.org/Team:HokkaidoU_Japan/Promoter">Promoter</a> and <a href="https://2013.igem.org/Team:HokkaidoU_Japan/RBS">RBS</a> for details.<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/a/ac/HokkaidoU2013_Motivation_figure2.png"><br />
</div><br />
<p><br />
We named these kits 'Promoter Selector' and 'RBS Selector'.<br />
<dl><br />
<dt>Promoter Selector</dt><dd>Promoter Selector is for adjustment of relatively simple expression, like a production of antibodies reffered above.</dd><br />
<dt>RBS Selector</dt><dd>RBS Selector is for adjustment of multiple expressions, like complex metabolisms.</dd><br />
</dl><br />
We named these two devices ''Maestro <span class="italic">E.coli</span>'' Random Operon Shuffling Kit! As a maestro harmonizes music, let's harmonize proteins in the bacteria! <br />
<br />
</p><br />
<br />
<div id="prev-page"><br />
<a href="https://2013.igem.org/Team:HokkaidoU_Japan/Overview"><div class="arrow-div"></div><span>Project Description</span></a><br />
</div><br />
<br />
<div id="next-page"><br />
<a href="https://2013.igem.org/Team:HokkaidoU_Japan/Overview/Achievements"><div class="arrow-div"></div><span>Achievements</span></a><br />
</div><br />
<br />
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{{Team:HokkaidoU_Japan/footer}}</div>Nousanhttp://2013.igem.org/Team:HokkaidoU_Japan/Shuffling_Kit/How_To_UseTeam:HokkaidoU Japan/Shuffling Kit/How To Use2013-10-28T15:13:46Z<p>Nousan: </p>
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<html><br />
<div id="common-header-bottom-background"><br />
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<h1 id="common-header-title">Maestro E.coli</h1><br />
<h2 id="common-header-subtitle">Optimization Kit</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
</div><br />
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<br />
<br />
<h1>How to use</h1><br />
<h2>What users should prepare</h2><br />
<p><br />
To use the kit, the protein sequence you chose must have specific prefix/suffix which contains a BsaI site and produce overhang. Therefore, users must design a primer to add it. To reduce time and trouble, we automated the design by creating program <a href="https://2013.igem.org/Team:HokkaidoU_Japan/Optimization/Primer_Designer">"Primer Designer for Maestro"!</a><br />
</p><br />
<br />
<h2>Promoter Selector</h2><br />
<h3>What our kit contains</h3><br />
<p><br />
Our Promoter Selector consists of 5 different plasmids (table.1). Each has a promoter with a different strength. Downstream of the RBS there is a BsaI site to insert the protein sequence. There is a color expression construct downstream of protein insertion site (fig.1). Each color is paired with different strength promoter. The pairings are shown on the table below.<br />
</p><br />
<div class="fig fig800"><br />
<style type="text/css"><br />
table color expression construct downstream of protein insertion site { border: 1px solid #a4a4a4; margin: 0 auto; }<br />
td, th { text-align: center; }<br />
</style><br />
<table><br />
<tr><br />
<th>Part number</th><th>Promoter</th><th>Promoter strength</th><th>Paired protein</th><th>Protein color</th><br />
</tr><br />
<tr><br />
<td><a href="http://parts.igem.org/Part:BBa_K1084501">BBa_K1084501</a></td><td><a href="http://parts.igem.org/Part:BBa_K1084001">BBa_K1084001</a></td><td>Strongest</td><td>amilGFP</td><td>yellowish green</td><br />
</tr><br />
<tr><br />
<td><a href="http://parts.igem.org/Part:BBa_K1084502">BBa_K1084502</a></td><td><a href="http://parts.igem.org/Part:BBa_K1084002">BBa_K1084002</a></td><td>Stronger</td><td>aeBlue</td><td>strong blue</td><br />
</tr><br />
<tr><br />
<td><a href="http://parts.igem.org/Part:BBa_K1084503">BBa_K1084503</a></td><td><a href="http://parts.igem.org/Part:BBa_K1084005">BBa_K1084005</a></td><td>Medium</td><td>amilCP</td><td>Purple</td><br />
</tr><br />
<tr><br />
<td><a href="http://parts.igem.org/Part:BBa_K1084504">BBa_K1084504</a></td><td><a href="http://parts.igem.org/Part:BBa_K1084009">BBa_K1084009</a></td><td>Weaker</td><td>mRFP</td><td>Pink</td><br />
</tr><br />
<tr><br />
<td><a href="http://parts.igem.org/Part:BBa_K1084505">BBa_K1084505</a></td><td><a href="http://parts.igem.org/Part:BBa_K1084010">BBa_K1084010</a></td><td>Weakest</td><td>eforRED</td><td>red</td><br />
</tr><br />
</table><br />
<br />
<div><span class="bold">table.1 Matching list of promoters and their colors.</span></div><br />
</div><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/6/60/Fig1_131027_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.1 The matching color for respective promoters.</span></div><br />
</div><br />
<p><br />
The color always expresses. LacZ&alpha; reporter is placed between two BsaI sites (fig.2). The LacZ&alpha; expressing construct will be replaced by chosen sequence using BsaI.<br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/c/cc/Fig.2_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.2 Insertion of LacZα reporter between two BsaI sites.</span></div><br />
</div><br />
<br />
<h2>How it works</h2><br />
<div class="fig fig300"><br />
<img src="https://static.igem.org/mediawiki/2013/2/27/HokkaidoU2013_optimization_Fig7_ver2_400.png"><br />
<div><span class="bold">fig.3 Digestion by BsaI.</span></div><br />
</div><br />
<br />
<p><br />
1. Have BsaI site and specific overhang added to your protein sequence (fig.3). PCR with primers designed with <a href="https://2013.igem.org/Team:HokkaidoU_Japan/Optimization/Primer_Designer">Primer Designer for Maestro</a> should do the trick.<br />
</p><br />
<br />
<p><br />
2.<br />
Digest and ligate your protein coding sequence and all our Promoter Selector together (fig.4). This is accomplished by "Golden Gate Assembly" reaction. The detailed recipe is shown in Engler (2009)<sup><a href="#cite-1">[1]</a></sup>. All the protein coding sequence will be inserted in the plasmid.<br />
<!-- add more detail --><br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/a/ac/Fig4_131027_2_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.4 How to insert CDS into protein coding sequence.</span></div><br />
</div><br />
<br />
<p><br />
3. You should get the construct shown below after Golden Gate Assembly (fig.5).<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/a/a1/Fig5_131027_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.5 Constructs you can get.</span></div><br />
</div><br />
<p><br />
4. Transform the ligated DNA to <span class="italic">E. coli</span>, and spread it on plate. Then, you will get colonies with five colors easily because you can see 5 colors by naked eyes (fig.6). The colors are paired with the promoters, so you will know what promoter you are using without sequencing all the colonies. You can pick up colonies and have an assay.</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/0/07/Assay_new_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.6 Method after transformation.</span></div><br />
</div><br />
<p><br />
<br />
<br />
<h2>RBS Selector</h2><br />
<h3>What our kit contains</h3><br />
<p><br />
Our kit contains tandem RBS (fig.7) and acceptor plasmid (fig.8).<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/2/22/RBS_strength_2_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.7 The sequence of tandem RBS.</span></div><br />
</div><br />
<p><br />
In this part, 4 strength levels of RBSs[BBa_K1084101, BBa_ K1084102, BBa_ K1084103, BBa_ K1084104] are connected in tandem. To optimize up to 3 coding sequence expressions in the operon this part has three sets of RBSs with different overhangs are connected together.<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/d/d5/HokkaidoU2013_optimization_Fig13_800.png"><br />
<div><span class="bold">fig.8 The acceptor part of the RBS and protein coding region.</span> It has a BsaI site for the parts to be assembled.</div><br />
</div><br />
</p><br />
<br />
<h3>How to use</h3><br />
<br />
<p><br />
1. Have BsaI site and specific overhang added to your protein sequence. Again, <a href="https://2013.igem.org/Team:HokkaidoU_Japan/Optimization/Primer_Designer">Primer Designer for Maestro</a> should help your primers design (fig.9). Also when you want to optimize more than one protein coding sites, add BsaI sites and overhang to them too. Be careful not to choose the same overhangs.<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/e/ef/HokkaidoU2013_optimization_Fig14_800.png"><br />
<div><span class="bold">fig.9 Insertion CDS anf digestion by BsaI.</span></div><br />
</div><br />
<p><br />
2.<br />
Digest and ligate your protein coding sequence and all RBS Selector together. This reaction is also accomplished by Golden Gate Assembly. DNA fragments will be assembled in the desired order (fig.10).<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/7/7d/HokkaidoU2013_optimization_Fig15_ver.2_800.png"><br />
<div><span class="bold">fig.10 How to use Golden Gate Assembly.</span></div><br />
</div><br />
<br />
<p><br />
3. Transform the ligated DNA to <span class="italic">E. coli</span>. If you are optimizing three different proteins, you will get 64 different kinds of constructs.<br />
</p><br />
<br />
<p><br />
We will submit these standard methods as RFC to BioBrick Foundation.<br />
</p><br />
<br />
<br />
<ol class="citation-list"><br />
<li id="cite-1">C. Engler <span class="italic">et al.</span> Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes (2009) PLoS ONE</li><br />
</ol><br />
<br />
<div id="prev-page"><br />
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</div><br />
<br />
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{{Team:HokkaidoU_Japan/footer}}</div>Nousanhttp://2013.igem.org/Team:HokkaidoU_Japan/Shuffling_Kit/How_To_UseTeam:HokkaidoU Japan/Shuffling Kit/How To Use2013-10-28T15:12:59Z<p>Nousan: </p>
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<h1 id="common-header-title">Maestro E.coli</h1><br />
<h2 id="common-header-subtitle">Optimization Kit</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
</div><br />
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<br />
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<div id="hokkaidou-contents"><br />
<!-- end header / begin contents --><br />
<br />
<br />
<h1>How to use</h1><br />
<h2>What users should prepare</h2><br />
<p><br />
To use the kit, the protein sequence you chose must have specific prefix/suffix which contains a BsaI site and produce overhang. Therefore, users must design a primer to add it. To reduce time and trouble, we automated the design by creating program <a href="https://2013.igem.org/Team:HokkaidoU_Japan/Optimization/Primer_Designer">"Primer Designer for Maestro"!</a><br />
</p><br />
<br />
<h2>Promoter Selector</h2><br />
<h3>What our kit contains</h3><br />
<p><br />
Our Promoter Selector consists of 5 different plasmids (table.1). Each has a promoter with a different strength. Downstream of the RBS there is a BsaI site to insert the protein sequence. There is a color expression construct downstream of protein insertion site (fig.1). Each color is paired with different strength promoter. The pairings are shown on the table below.<br />
</p><br />
<div class="fig fig800"><br />
<style type="text/css"><br />
table color expression construct downstream of protein insertion site { border: 1px solid #a4a4a4; margin: 0 auto; }<br />
td, th { text-align: center; }<br />
</style><br />
<table><br />
<tr><br />
<th>Part number</th><th>Promoter</th><th>Promoter strength</th><th>Paired protein</th><th>Protein color</th><br />
</tr><br />
<tr><br />
<td><a href="http://parts.igem.org/Part:BBa_K1084501">BBa_K1084501</a></td><td><a href="http://parts.igem.org/Part:BBa_K1084001">BBa_K1084001</a></td><td>Strongest</td><td>amilGFP</td><td>yellowish green</td><br />
</tr><br />
<tr><br />
<td><a href="http://parts.igem.org/Part:BBa_K1084502">BBa_K1084502</a></td><td><a href="http://parts.igem.org/Part:BBa_K1084002">BBa_K1084002</a></td><td>Stronger</td><td>aeBlue</td><td>strong blue</td><br />
</tr><br />
<tr><br />
<td><a href="http://parts.igem.org/Part:BBa_K1084503">BBa_K1084503</a></td><td><a href="http://parts.igem.org/Part:BBa_K1084005">BBa_K1084005</a></td><td>Medium</td><td>amilCP</td><td>Purple</td><br />
</tr><br />
<tr><br />
<td><a href="http://parts.igem.org/Part:BBa_K1084504">BBa_K1084504</a></td><td><a href="http://parts.igem.org/Part:BBa_K1084009">BBa_K1084009</a></td><td>Weaker</td><td>mRFP</td><td>Pink</td><br />
</tr><br />
<tr><br />
<td><a href="http://parts.igem.org/Part:BBa_K1084505">BBa_K1084505</a></td><td><a href="http://parts.igem.org/Part:BBa_K1084010">BBa_K1084010</a></td><td>Weakest</td><td>eforRED</td><td>red</td><br />
</tr><br />
</table><br />
<br />
<div><span class="bold">table.1 Matching list of promoters and their colors.</span></div><br />
</div><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/6/60/Fig1_131027_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.1 The matching color for respective promoters.</span></div><br />
</div><br />
<p><br />
The color always expresses. LacZ&alpha; reporter is placed between two BsaI sites (fig.2). The LacZ&alpha; expressing construct will be replaced by chosen sequence using BsaI.<br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/c/cc/Fig.2_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.2 Insertion of LacZα reporter between two BsaI sites.</span></div><br />
</div><br />
<br />
<h2>How it works</h2><br />
<div class="fig fig300"><br />
<img src="https://static.igem.org/mediawiki/2013/2/27/HokkaidoU2013_optimization_Fig7_ver2_400.png"><br />
<div><span class="bold">fig.3 Digestion by BsaI.</span></div><br />
</div><br />
<br />
<p><br />
1. Have BsaI site and specific overhang added to your protein sequence (fig.3). PCR with primers designed with <a href="https://2013.igem.org/Team:HokkaidoU_Japan/Optimization/Primer_Designer">Primer Designer for Maestro</a> should do the trick.<br />
</p><br />
<br />
<p><br />
2.<br />
Digest and ligate your protein coding sequence and all our Promoter Selector together (fig.4). This is accomplished by "Golden Gate Assembly" reaction. The detailed recipe is shown in Engler (2009)<sup><a href="#cite-1">[1]</a></sup>. All the protein coding sequence will be inserted in the plasmid.<br />
<!-- add more detail --><br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/a/ac/Fig4_131027_2_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.4 How to insert CDS into protein coding sequence.</span></div><br />
</div><br />
<br />
<p><br />
3. You should get the construct shown below after Golden Gate Assembly (fig.5).<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/a/a1/Fig5_131027_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.5 Constructs you can get.</span></div><br />
</div><br />
<p><br />
4. Transform the ligated DNA to <span class="italic">E. coli</span>, and spread it on plate. Then, you will get colonies with five colors easily because you can see 5 colors by naked eyes (fig.6). The colors are paired with the promoters, so you will know what promoter you are using without sequencing all the colonies. You can pick up colonies and have an assay.</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/0/07/Assay_new_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.6 Method after transformation.</span></div><br />
</div><br />
<p><br />
<br />
<br />
<h2>RBS Selector</h2><br />
<h3>What our kit contains</h3><br />
<p><br />
Our kit contains tandem RBS (fig.7) and acceptor plasmid (fig.8).<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/2/22/RBS_strength_2_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.7 The sequence of tandem RBS.</span></div><br />
</div><br />
<p><br />
In this part, 4 strength levels of RBSs[BBa_K1084101, BBa_ K1084102, BBa_ K1084103, BBa_ K1084104] are connected in tandem. To optimize up to 3 coding sequence expressions in the operon this part has three sets of RBSs with different overhangs are connected together.<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/d/d5/HokkaidoU2013_optimization_Fig13_800.png"><br />
<div><span class="bold">fig.8 The acceptor part of the RBS and protein coding region.</span> It has a BsaI site for the parts to be assembled.</div><br />
</div><br />
</p><br />
<br />
<h3>How to use</h3><br />
<br />
<p><br />
1. Have BsaI site and specific overhang added to your protein sequence. Again, <a href="https://2013.igem.org/Team:HokkaidoU_Japan/Optimization/Primer_Designer">Primer Designer for Maestro</a>should help your primers design (fig.9). Also when you want to optimize more than one protein coding sites, add BsaI sites and overhang to them too. Be careful not to choose the same overhangs.<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/e/ef/HokkaidoU2013_optimization_Fig14_800.png"><br />
<div><span class="bold">fig.9 Insertion CDS anf digestion by BsaI.</span></div><br />
</div><br />
<p><br />
2.<br />
Digest and ligate your protein coding sequence and all RBS Selector together. This reaction is also accomplished by Golden Gate Assembly. DNA fragments will be assembled in the desired order (fig.10).<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/7/7d/HokkaidoU2013_optimization_Fig15_ver.2_800.png"><br />
<div><span class="bold">fig.10 How to use Golden Gate Assembly.</span></div><br />
</div><br />
<br />
<p><br />
3. Transform the ligated DNA to <span class="italic">E. coli</span>. If you are optimizing three different proteins, you will get 64 different kinds of constructs.<br />
</p><br />
<br />
<p><br />
We will submit these standard methods as RFC to BioBrick Foundation.<br />
</p><br />
<br />
<br />
<ol class="citation-list"><br />
<li id="cite-1">C. Engler <span class="italic">et al.</span> Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes (2009) PLoS ONE</li><br />
</ol><br />
<br />
<div id="prev-page"><br />
<a href="https://2013.igem.org/Team:HokkaidoU_Japan/Optimization"><div class="arrow-div"></div><span>Optimization Kit Top</span></a><br />
</div><br />
<br />
<div id="next-page"><br />
<a href="https://2013.igem.org/Team:HokkaidoU_Japan/Optimization/Primer_Designer"><div class="arrow-div"></div><span>Primer Designer</span></a><br />
</div><br />
<br />
<br />
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{{Team:HokkaidoU_Japan/footer}}</div>Nousanhttp://2013.igem.org/Team:HokkaidoU_Japan/Shuffling_Kit/How_To_UseTeam:HokkaidoU Japan/Shuffling Kit/How To Use2013-10-28T15:10:08Z<p>Nousan: </p>
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<h1 id="common-header-title">Maestro E.coli</h1><br />
<h2 id="common-header-subtitle">Optimization Kit</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
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<br />
<div class="wrapper"><br />
<div id="hokkaidou-contents"><br />
<!-- end header / begin contents --><br />
<br />
<br />
<h1>How to use</h1><br />
<h2>What users should prepare</h2><br />
<p><br />
To use the kit, the protein sequence you chose must have specific prefix/suffix which contains a BsaI site and produce overhang. Therefore, users must design a primer to add it. To reduce time and trouble, we automated the design by creating program <a href="https://2013.igem.org/Team:HokkaidoU_Japan/Optimization/Primer_Designer">"Primer Designer for Maestro"!</a><br />
</p><br />
<br />
<h2>Promoter Selector</h2><br />
<h3>What our kit contains</h3><br />
<p><br />
Our Promoter Selector consists of 5 different plasmids (table.1). Each has a promoter with a different strength. Downstream of the RBS there is a BsaI site to insert the protein sequence. There is a color expression construct downstream of protein insertion site (fig.1). Each color is paired with different strength promoter. The pairings are shown on the table below.<br />
</p><br />
<div class="fig fig800"><br />
<style type="text/css"><br />
table color expression construct downstream of protein insertion site { border: 1px solid #a4a4a4; margin: 0 auto; }<br />
td, th { text-align: center; }<br />
</style><br />
<table><br />
<tr><br />
<th>Part number</th><th>Promoter</th><th>Promoter strength</th><th>Paired protein</th><th>Protein color</th><br />
</tr><br />
<tr><br />
<td><a href="http://parts.igem.org/Part:BBa_K1084501">BBa_K1084501</a></td><td><a href="http://parts.igem.org/Part:BBa_K1084001">BBa_K1084001</a></td><td>Strongest</td><td>amilGFP</td><td>yellowish green</td><br />
</tr><br />
<tr><br />
<td><a href="http://parts.igem.org/Part:BBa_K1084502">BBa_K1084502</a></td><td><a href="http://parts.igem.org/Part:BBa_K1084002">BBa_K1084002</a></td><td>Stronger</td><td>aeBlue</td><td>strong blue</td><br />
</tr><br />
<tr><br />
<td><a href="http://parts.igem.org/Part:BBa_K1084503">BBa_K1084503</a></td><td><a href="http://parts.igem.org/Part:BBa_K1084005">BBa_K1084005</a></td><td>Medium</td><td>amilCP</td><td>Purple</td><br />
</tr><br />
<tr><br />
<td><a href="http://parts.igem.org/Part:BBa_K1084504">BBa_K1084504</a></td><td><a href="http://parts.igem.org/Part:BBa_K1084009">BBa_K1084009</a></td><td>Weaker</td><td>mRFP</td><td>Pink</td><br />
</tr><br />
<tr><br />
<td><a href="http://parts.igem.org/Part:BBa_K1084505">BBa_K1084505</a></td><td><a href="http://parts.igem.org/Part:BBa_K1084010">BBa_K1084010</a></td><td>Weakest</td><td>eforRED</td><td>red</td><br />
</tr><br />
</table><br />
<br />
<div><span class="bold">table.1 Matching list of promoters and their colors.</span></div><br />
</div><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/6/60/Fig1_131027_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.1 The matching color for respective promoters.</span></div><br />
</div><br />
<p><br />
The color always expresses. LacZ&alpha; reporter is placed between two BsaI sites (fig.2). The LacZ&alpha; expressing construct will be replaced by chosen sequence using BsaI.<br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/c/cc/Fig.2_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.2 Insertion of LacZα reporter between two BsaI sites.</span></div><br />
</div><br />
<br />
<h2>How it works</h2><br />
<div class="fig fig300"><br />
<img src="https://static.igem.org/mediawiki/2013/2/27/HokkaidoU2013_optimization_Fig7_ver2_400.png"><br />
<div><span class="bold">fig.3 Digestion by BsaI.</span></div><br />
</div><br />
<br />
<p><br />
1. Have BsaI site and specific overhang added to your protein sequence (fig.3). PCR with primers designed with <a href="https://2013.igem.org/Team:HokkaidoU_Japan/Optimization/Primer_Designer">Primer Designer for Maestro</a> should do the trick.<br />
</p><br />
<br />
<p><br />
2.<br />
Digest and ligate your protein coding sequence and all our Promoter Selector together (fig.4). This is accomplished by "Golden Gate Assembly" reaction. The detailed recipe is shown in Engler (2009)<sup><a href="#cite-1">[1]</a></sup>. All the protein coding sequence will be inserted in the plasmid.<br />
<!-- add more detail --><br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/a/ac/Fig4_131027_2_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.4 How to insert CDS into protein coding sequence.</span></div><br />
</div><br />
<br />
<p><br />
3. You should get the construct shown below after Golden Gate Assembly (fig.5).<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/a/a1/Fig5_131027_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.5 Constructs you can get.</span></div><br />
</div><br />
<p><br />
4. Transform the ligated DNA to <span class="italic">E. coli</span>, and spread it on plate. Then, you will get colonies with five colors easily because you can see 5 colors by naked eyes (fig.6). The colors are paired with the promoters, so you will know what promoter you are using without sequencing all the colonies. You can pick up colonies and have an assay.</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/0/07/Assay_new_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.6 Method after transformation.</span></div><br />
</div><br />
<p><br />
<br />
<br />
<h2>RBS Selector</h2><br />
<h3>What our kit contains</h3><br />
<p><br />
Our kit contains tandem RBS (fig.7) and acceptor plasmid (fig.8).<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/2/22/RBS_strength_2_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.7 The sequence of tandem RBS.</span></div><br />
</div><br />
<p><br />
In this part, 4 strength levels of RBSs[BBa_K1084101, BBa_ K1084102, BBa_ K1084103, BBa_ K1084104] are connected in tandem. To optimize up to 3 coding sequence expressions in the operon this part has three sets of RBSs with different overhangs are connected together.<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/d/d5/HokkaidoU2013_optimization_Fig13_800.png"><br />
<div><span class="bold">fig.8 The acceptor part of the RBS and protein coding region.</span> It has a BsaI site for the parts to be assembled.</div><br />
</div><br />
</p><br />
<br />
<h3>How to use</h3><br />
<br />
<p><br />
1. Have BsaI site and specific overhang added to your protein sequence. Again, our program should help your primers design (fig.9). Also when you want to optimize more than one protein coding sites, add BsaI sites and overhang to them too. Be careful not to choose the same overhangs.<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/e/ef/HokkaidoU2013_optimization_Fig14_800.png"><br />
<div><span class="bold">fig.9 Insertion CDS anf digestion by BsaI.</span></div><br />
</div><br />
<p><br />
2.<br />
Digest and ligate your protein coding sequence and all RBS Selector together. This reaction is also accomplished by Golden Gate Assembly. DNA fragments will be assembled in the desired order (fig.10).<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/7/7d/HokkaidoU2013_optimization_Fig15_ver.2_800.png"><br />
<div><span class="bold">fig.10 How to use Golden Gate Assembly.</span></div><br />
</div><br />
<br />
<p><br />
3. Transform the ligated DNA to <span class="italic">E. coli</span>. If you are optimizing three different proteins, you will get 64 different kinds of constructs.<br />
</p><br />
<br />
<p><br />
We will submit these standard methods as RFC to BioBrick Foundation.<br />
</p><br />
<br />
<br />
<ol class="citation-list"><br />
<li id="cite-1">C. Engler <span class="italic">et al.</span> Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes (2009) PLoS ONE</li><br />
</ol><br />
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