Team:Macquarie Australia/Results

From 2013.igem.org

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<h7> <p> This page gives an overview of our results, which have provided large strides towards the production of Chlorophyll within <i>E. coli</i>. For more detail on our labwork and results, please see our  
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<center><h7> <p> This page gives an overview of our results, which have provided large strides towards the production of Chlorophyll within <i>E. coli</i>. For more detail on our labwork and results, please see our  
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<span style="color:#8B0000"><font size = 3><a class="three" href='https://2013.igem.org/Team:Macquarie_Australia/Notebook'><b>Notebook</b></a></font size></span>.</b></p></h7>
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<span style="color:#8B0000"><font size = 3><a class="three" href='https://2013.igem.org/Team:Macquarie_Australia/Notebook'><b>Notebook</b></a></font size></span>.</b></p></h7></center>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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<td></td><td><span style="color:#8B0000"><font size = 4><a class="three" a href="#2"><b>BioBrick Sequences</b></a></font size></span>  
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<td></td><td><span style="color:#8B0000"><font size = 4><a class="three" a href="#2"><b>BioBrick Information</b></a></font size></span>  
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<h1><center><a class="four" a name="2"><b>BioBrick Sequences</h1></b></h1></center>
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<h1><center><a class="four" a name="2"><b>BioBrick Information</h1></b></h1></center>
<p> All constructed BioBricks have been sent for sequencing to confirm fidelity with designs, and all sequences returned thus far have shown a 100% match with our gene design. Genes showing such a match have been submitted to iGEM via post, and we are currently awaiting sequences to be returned for our final three BioBricks. Click on gene names to be taken to the registry entry for that gene, with sequence information.</b></p></left>
<p> All constructed BioBricks have been sent for sequencing to confirm fidelity with designs, and all sequences returned thus far have shown a 100% match with our gene design. Genes showing such a match have been submitted to iGEM via post, and we are currently awaiting sequences to be returned for our final three BioBricks. Click on gene names to be taken to the registry entry for that gene, with sequence information.</b></p></left>
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<thead><tr><th>BioBricks Constructed</th><th>Gibson Composition</th><th>Sequence Confirmed</th><th>Submitted to iGEM</th></tr></thead>
<thead><tr><th>BioBricks Constructed</th><th>Gibson Composition</th><th>Sequence Confirmed</th><th>Submitted to iGEM</th></tr></thead>
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<tbody><tr><td><b><a href=http://parts.igem.org/wiki/index.php?title=Part:BBa_K1080007>POR</a></td><td><b>
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<tbody><tr><td><b><a class=three href=http://parts.igem.org/wiki/index.php?title=Part:BBa_K1080007>POR</a></td><td><b>
<center>2 fragments + vector</td><td><b>
<center>2 fragments + vector</td><td><b>
<center><img src = https://static.igem.org/mediawiki/2013/a/a2/Tick2.fw.png width = 30></td><td><b>
<center><img src = https://static.igem.org/mediawiki/2013/a/a2/Tick2.fw.png width = 30></td><td><b>
<center><img src = https://static.igem.org/mediawiki/2013/a/a2/Tick2.fw.png width = 30></td></tr>
<center><img src = https://static.igem.org/mediawiki/2013/a/a2/Tick2.fw.png width = 30></td></tr>
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<tr class="alt"><td><b>ChlG</td><td><b>
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<tr class="alt"><td><b><a href=http://parts.igem.org/wiki/index.php?title=Part:BBa_K1080009>ChlG</a></td><td><b>
<center>2 fragments + vector</td><td><b>
<center>2 fragments + vector</td><td><b>
<center><img src = https://static.igem.org/mediawiki/2013/a/a2/Tick2.fw.png width = 30></td><td><b>
<center><img src = https://static.igem.org/mediawiki/2013/a/a2/Tick2.fw.png width = 30></td><td><b>
<center><img src = https://static.igem.org/mediawiki/2013/a/a2/Tick2.fw.png width = 30></td></tr>
<center><img src = https://static.igem.org/mediawiki/2013/a/a2/Tick2.fw.png width = 30></td></tr>
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<tbody><tr><td><b>ChlP</td><td><b>
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<tbody><tr><td><b><a class=three href=http://parts.igem.org/wiki/index.php?title=Part:BBa_K1080008>ChlP</a></td><td><b>
<center>2 fragments + vector</td><td><b>
<center>2 fragments + vector</td><td><b>
<center><img src = https://static.igem.org/mediawiki/2013/a/a2/Tick2.fw.png width = 30></td><td><b>
<center><img src = https://static.igem.org/mediawiki/2013/a/a2/Tick2.fw.png width = 30></td><td><b>
<center><img src = https://static.igem.org/mediawiki/2013/a/a2/Tick2.fw.png width = 30></td></tr>
<center><img src = https://static.igem.org/mediawiki/2013/a/a2/Tick2.fw.png width = 30></td></tr>
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<tr class="alt"><td><b>ChlI2</td><td><b>
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<tr class="alt"><td><b><a href=http://parts.igem.org/wiki/index.php?title=Part:BBa_K1080011>ChlI2</a></td><td><b>
<center>2 fragments + vector</td><td><b>
<center>2 fragments + vector</td><td><b>
<center><img src = https://static.igem.org/mediawiki/2013/a/a2/Tick2.fw.png width = 30></td><td>
<center><img src = https://static.igem.org/mediawiki/2013/a/a2/Tick2.fw.png width = 30></td><td>
<center><img src = https://static.igem.org/mediawiki/2013/a/a2/Tick2.fw.png width = 30></td></tr>
<center><img src = https://static.igem.org/mediawiki/2013/a/a2/Tick2.fw.png width = 30></td></tr>
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<tbody><tr><td><b>YCF54</td><td><b>
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<tbody><tr><td><b><a class=three href=http://parts.igem.org/wiki/index.php?title=Part:BBa_K1080010>YCF54<a/></td><td><b>
<center>1 fragment + vector</td><td><b>
<center>1 fragment + vector</td><td><b>
<center><img src = https://static.igem.org/mediawiki/2013/a/a2/Tick2.fw.png width = 30></td><td><b>
<center><img src = https://static.igem.org/mediawiki/2013/a/a2/Tick2.fw.png width = 30></td><td><b>
<center><img src = https://static.igem.org/mediawiki/2013/a/a2/Tick2.fw.png width = 30></td></tr>
<center><img src = https://static.igem.org/mediawiki/2013/a/a2/Tick2.fw.png width = 30></td></tr>
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<tr class="alt"><td><b>CTH1</td><td><b>
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<tr class="alt"><td><b><a href=http://parts.igem.org/wiki/index.php?title=Part:BBa_K1080005>CTH1</a></td><td><b>
<center>3 fragments + vector</td><td><b>
<center>3 fragments + vector</td><td><b>
<center><img src = https://static.igem.org/mediawiki/2013/a/a2/Tick2.fw.png width = 30></td><td><b>
<center><img src = https://static.igem.org/mediawiki/2013/a/a2/Tick2.fw.png width = 30></td><td><b>
<center><img src = https://static.igem.org/mediawiki/2013/a/a2/Tick2.fw.png width = 30></td></tr>
<center><img src = https://static.igem.org/mediawiki/2013/a/a2/Tick2.fw.png width = 30></td></tr>
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<tbody><tr><td><b>ChlI1</td><td><b>
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<tbody><tr><td><b><a class=three href=http://parts.igem.org/wiki/index.php?title=Part:BBa_K1080000>ChlI1</a></td><td><b>
<center>2 fragments + vector</td><td><b>
<center>2 fragments + vector</td><td><b>
<center><img src = https://static.igem.org/mediawiki/2013/a/a2/Tick2.fw.png width = 30></td><td><b>
<center><img src = https://static.igem.org/mediawiki/2013/a/a2/Tick2.fw.png width = 30></td><td><b>
<center><img src = https://static.igem.org/mediawiki/2013/a/a2/Tick2.fw.png width = 30></td></tr>
<center><img src = https://static.igem.org/mediawiki/2013/a/a2/Tick2.fw.png width = 30></td></tr>
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<tr class="alt"><td><b>Gun4</td><td><b>
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<tr class="alt"><td><b><a href=http://parts.igem.org/wiki/index.php?title=Part:BBa_K1080003>Gun4</a></td><td><b>
<center>2 fragments + vector</td><td><b>
<center>2 fragments + vector</td><td><b>
<center><img src = https://static.igem.org/mediawiki/2013/a/a2/Tick2.fw.png width = 30></td><td><b>
<center><img src = https://static.igem.org/mediawiki/2013/a/a2/Tick2.fw.png width = 30></td><td><b>
<center><img src = https://static.igem.org/mediawiki/2013/a/a2/Tick2.fw.png width = 30></td></tr>
<center><img src = https://static.igem.org/mediawiki/2013/a/a2/Tick2.fw.png width = 30></td></tr>
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<tbody><tr><td><b>Plastocyanin</td><td><b>
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<tbody><tr><td><b><a class=three href=http://parts.igem.org/wiki/index.php?title=Part:BBa_K1080006>Plastocyanin</a></td><td><b>
<center>1 fragment + vector</td><td><b>
<center>1 fragment + vector</td><td><b>
<center><img src = https://static.igem.org/mediawiki/2013/a/a2/Tick2.fw.png width = 30></td><td><b>
<center><img src = https://static.igem.org/mediawiki/2013/a/a2/Tick2.fw.png width = 30></td><td><b>
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<tr class="alt"><td><b>ChlM</td><td><b>
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<tr class="alt"><td><b><a href=http://parts.igem.org/wiki/index.php?title=Part:BBa_K1080004>ChlM</a></td><td><b>
<center>2 fragments + vector</td><td><b>
<center>2 fragments + vector</td><td><b>
<center>pending...</td><td></td></tr>
<center>pending...</td><td></td></tr>
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<tbody><tr><td><b>DVR1</td><td><b>
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<tbody><tr><td><b><a class=three href=http://parts.igem.org/wiki/index.php?title=Part:BBa_K1080012>DVR1</a></td><td><b>
<center>2 fragments + vector</td><td><b>
<center>2 fragments + vector</td><td><b>
<center>pending...</td><td></td></tr>
<center>pending...</td><td></td></tr>
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<tr class="alt"><td><b>ChlD</td><td><b>
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<tr class="alt"><td><b><a href=http://parts.igem.org/wiki/index.php?title=Part:BBa_K1080002>ChlD</a></td><td><b>
<center>3 fragments + vector</td><td><b>
<center>3 fragments + vector</td><td><b>
<center>pending...</td><td></td></tr>
<center>pending...</td><td></td></tr>
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<h1><center><a class="four" a name="3"><b>Characterisation</h1></b>
<h1><center><a class="four" a name="3"><b>Characterisation</h1></b>
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<br><br>
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<center><font size=4>Promoter Ligation</font size></center>
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<center><font size=4>Composite part creation</font size></center>
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<br><br>
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<img src="https://static.igem.org/mediawiki/2013/2/28/Complete_plates_plus_description.jpg" align="Left"  height="423" width="336">
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&nbsp;&nbsp; <img src="https://static.igem.org/mediawiki/2013/c/cd/Didn%27t_work_.jpg" align="right" height="250" width="526">
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<br><br>
<br><br>
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<b>Composite part creation: attaching promoters to genes</h1></b>
<br><br>
<br><br>
 +
We selected five of our genes for further characterisation: ChlD, ChlI1, ChlI2, Gun4, and Plastocyanin. A tac promoter BioBrick provided in the iGEM kit (part BBa_K864400) was digested with PstI and SpeI, gel cleaned, and used as a plasmid for ligation with XpeI and PstI digestions of these biobricks. These were ligated, transformed, and plated out, showing growth of hundreds of colonies per plate, with no growth on an unligated plasmid control (see plate image below).
 +
<br>
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<br>
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<p><center><img src="https://static.igem.org/mediawiki/2013/1/1d/New_plates_27_sept.jpg" height="350" width="483"><br><br></p></center>
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<br>
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Eight colonies were picked from each plate and grown up in LB+Chloramphenicol broth, and left to grow for 3 hours. 500ul of each broth was then pelleted, resuspended in 100ul water, and heated to 99 degrees for 5 minutes to lyse cells. PCR was then performed using lysed cell suspension as template, with BBVF and BBVR primers, with PCR products gelled (as shown below). Lanes with expected banding were used to identify successfully transformed colony cultures, which were then grown up in larger cultures for further investigation. Note that none of the eight ChlD colonies showed successful amplification - this may be due to the larger length of the ChlD gene.<br><br>
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 +
<center><p><img src="https://static.igem.org/mediawiki/2013/e/e2/Sucsess3.jpg"  height="348" width="336"><p></center>
<br><br>
<br><br>
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 +
<center><font size=4><u>Further investigation: ChlD</u></font size></center>
<br><br>
<br><br>
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A simple assay is available to experimentally validate the function of our ChlD Biobrick. The basis of this test is to provide all proteins required to form the Magnesium Chelatase complex, except for ChlD. When the ChlD protein is present, Magnesium-protoporphyrin is formed from protoporphyrin already present in the E. coli. This formation can be observed via fluorescence, and we have performed this assay with the same setup as used in Zhou et al, 2010:<br><br>
 +
 +
The final concentrations of assay components were 50 mM Tricine–NaOH (pH 8.0), 15 mM MgCl2, 2 mM dithiothreitol (DTT), 1 mM ATP (assay buffer), 2000 nM Proto, 200 nM CrChlI1 and 200nM CrChlI2, 500 nM CrChlH, 500 nM CrGUN4. ChlD Extract was added to CrI1 and CrI2 at 2x assay concentration in reaction buffer. Assay was started by adding 25 uL of Cr-ChlH and Cr-GUN4 at 2x assay concentration to 25 uL of the Cr-I1 Cr-I2 ChlD extract in assay buffer.<br><br>
 +
 +
ChlD expressing E. coli cells were lysed with B-PER bacterial cell lysis reagent (Pierce) as per instructions. 2 ul of this extract gave the equivalent of 2.1ng of purified ChlD activity when added to the assay. The B-PER protein concentration was approximately 10mg/ml, indicating a low expression of the gene. Improvements of expression level may be obtained by using alternate promoters. The control used was a ChlI1 expressing cell extract at the same concentration (ie with ChlD activity).
 +
 +
Assay was monitored every 2 seconds for 100 seconds using fluorescence detection in a Pherastar plate reader with a 420nm excitation filter and a 590nm emission filter.<br><br>
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Graph 1 shows the fluorescence tracking for our control E. coli (without ChlD activity):<br><br>
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<center><img src=https://static.igem.org/mediawiki/2013/d/d8/MQ_control_ChlD.jpg width=900></center>
<br><br>
<br><br>
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<br><br>
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<br><br>
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Graph 2 shows the fluorescence of two of our ChlD subcultures, with increasing fluorescence detected, indicating Magnesium-protoporphyrin-IX formation. Red line shows ChlD2 activity, green line shows ChlD4 activity:<br><br>
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<br><br>
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<br><br>
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<center><img src=https://static.igem.org/mediawiki/2013/5/58/MQ_ChlD_2_4.jpg width=900><br><br></center>
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<b>References:</b>
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<b>1st Attempt at ligation:</h1></b>
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<br>
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<br>
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Promoter was ligated onto the genes and plated up on LB + CAM plates, we observed a lot of colony growth.
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Zhou, S., Sawicki, A., Willows, R. D., & Luo, M. (2012). C-terminal residues of<i> Oryza sativa</i> GUN4 are required for the activation of the ChlH subunit of magnesium chelatase in chlorophyll synthesis. FEBS letters, 586(3), 205-210.
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We inoculated LB + CAM broth with a colony from the corresponding plate. A variety of growth was
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<br>
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observed, a few plates corresponding to a certain genes had a lot more colonies than others, this
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can be observed from the image with the description order of genes.
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More colonies corresponding to a gene displayed more colony growth than others which can be observed from
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the image description showing the order of genes on the agar.  
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<br>
<br>
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The PCR showed that the ligation was not successful, we derived the reason of this problem to either an improper clean up and therefore self ligation or improper restriction enzyme digest.
 
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<br><br>
 
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<center><font size=4><u>Further investigation: Plastocyanin</u></font size></center>
<br><br>
<br><br>
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<br><br>
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<img src="https://static.igem.org/mediawiki/2013/1/1d/New_plates_27_sept.jpg" align="Left"  height="350" width="483">
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Our tac+plastocyanin composite biobrick theoretically expresses a copper containing protein. Colonies containing this biobrick should turn blue in the presence of inducer (IPTG) and copper. To test this, we plated this culture out on a regular LB plate, and a copper + IPTG LB plate. We also plated out a control plate with both Plasto and CHLI1 expressing colonies. <br>
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&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2013/e/e2/Sucsess3.jpg" align="right" height="348" width="336">
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The first image below show plastocyanin expressing colonies growing blue on LB+copper+IPTG plates seen on the right, and white/yellow on regular LB plates shown on the left: <br><br>
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<center><img src=https://static.igem.org/mediawiki/2013/4/49/MQ_copper_no_copper_comparison_cropped.JPG><br><br></center>
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The second image shows a LB+copper+IPTG plate with plastocyanin colonies growing on one half, and non-expressing colonies (ChlI1) on the other half:<br><br>
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<center><img src=https://static.igem.org/mediawiki/2013/5/50/MQ_copper_control2_cropped.JPG></center><br><br>
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<b>Attempt 2:</h1></b>
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We re-attempted the ligation after a light change in the process, the vector was properly cleaned and restricted. The result was plated on LB + CAM plates as seen top left. The plate cultures from the second attempt show growth on all plates and slightly blobby looking colonies for the +Cu plate designated to plastocyanin. The control plate did not have any growth and this was expected as it was only the cut circular plasmid and therefore would not have incorporated the antibiotic resistance required for growth.
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The PCR showed that the ligation attempts were successful this time round, we exclaimed for joy. We used the BioBrick to inoculate <i/> E.coli </i>
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Latest revision as of 04:14, 28 September 2013



Results and Characterisation

This page gives an overview of our results, which have provided large strides towards the production of Chlorophyll within E. coli. For more detail on our labwork and results, please see our Notebook.


BioBrick Construction             BioBrick Information             Characterisation


BioBrick Construction



Twelve BioBricks were successfully constructed, complying with all requirements set out by iGEM. An electrophoresis gel was run on EcoRI + PstI digests of all constructions, with bands confirming expected part sizes. Note that the part size in ChlD is roughly the same as the plasmid fragment, so only one band is visible in this lane.



BioBrick Information

All constructed BioBricks have been sent for sequencing to confirm fidelity with designs, and all sequences returned thus far have shown a 100% match with our gene design. Genes showing such a match have been submitted to iGEM via post, and we are currently awaiting sequences to be returned for our final three BioBricks. Click on gene names to be taken to the registry entry for that gene, with sequence information.


BioBricks ConstructedGibson CompositionSequence ConfirmedSubmitted to iGEM
POR
2 fragments + vector
ChlG
2 fragments + vector
ChlP
2 fragments + vector
ChlI2
2 fragments + vector
YCF54
1 fragment + vector
CTH1
3 fragments + vector
ChlI1
2 fragments + vector
Gun4
2 fragments + vector
Plastocyanin
1 fragment + vector
ChlM
2 fragments + vector
pending...
DVR1
2 fragments + vector
pending...
ChlD
3 fragments + vector
pending...


Characterisation


Composite part creation


Composite part creation: attaching promoters to genes

We selected five of our genes for further characterisation: ChlD, ChlI1, ChlI2, Gun4, and Plastocyanin. A tac promoter BioBrick provided in the iGEM kit (part BBa_K864400) was digested with PstI and SpeI, gel cleaned, and used as a plasmid for ligation with XpeI and PstI digestions of these biobricks. These were ligated, transformed, and plated out, showing growth of hundreds of colonies per plate, with no growth on an unligated plasmid control (see plate image below).




Eight colonies were picked from each plate and grown up in LB+Chloramphenicol broth, and left to grow for 3 hours. 500ul of each broth was then pelleted, resuspended in 100ul water, and heated to 99 degrees for 5 minutes to lyse cells. PCR was then performed using lysed cell suspension as template, with BBVF and BBVR primers, with PCR products gelled (as shown below). Lanes with expected banding were used to identify successfully transformed colony cultures, which were then grown up in larger cultures for further investigation. Note that none of the eight ChlD colonies showed successful amplification - this may be due to the larger length of the ChlD gene.



Further investigation: ChlD


A simple assay is available to experimentally validate the function of our ChlD Biobrick. The basis of this test is to provide all proteins required to form the Magnesium Chelatase complex, except for ChlD. When the ChlD protein is present, Magnesium-protoporphyrin is formed from protoporphyrin already present in the E. coli. This formation can be observed via fluorescence, and we have performed this assay with the same setup as used in Zhou et al, 2010:

The final concentrations of assay components were 50 mM Tricine–NaOH (pH 8.0), 15 mM MgCl2, 2 mM dithiothreitol (DTT), 1 mM ATP (assay buffer), 2000 nM Proto, 200 nM CrChlI1 and 200nM CrChlI2, 500 nM CrChlH, 500 nM CrGUN4. ChlD Extract was added to CrI1 and CrI2 at 2x assay concentration in reaction buffer. Assay was started by adding 25 uL of Cr-ChlH and Cr-GUN4 at 2x assay concentration to 25 uL of the Cr-I1 Cr-I2 ChlD extract in assay buffer.

ChlD expressing E. coli cells were lysed with B-PER bacterial cell lysis reagent (Pierce) as per instructions. 2 ul of this extract gave the equivalent of 2.1ng of purified ChlD activity when added to the assay. The B-PER protein concentration was approximately 10mg/ml, indicating a low expression of the gene. Improvements of expression level may be obtained by using alternate promoters. The control used was a ChlI1 expressing cell extract at the same concentration (ie with ChlD activity). Assay was monitored every 2 seconds for 100 seconds using fluorescence detection in a Pherastar plate reader with a 420nm excitation filter and a 590nm emission filter.

Graph 1 shows the fluorescence tracking for our control E. coli (without ChlD activity):



Graph 2 shows the fluorescence of two of our ChlD subcultures, with increasing fluorescence detected, indicating Magnesium-protoporphyrin-IX formation. Red line shows ChlD2 activity, green line shows ChlD4 activity:



References:

Zhou, S., Sawicki, A., Willows, R. D., & Luo, M. (2012). C-terminal residues of Oryza sativa GUN4 are required for the activation of the ChlH subunit of magnesium chelatase in chlorophyll synthesis. FEBS letters, 586(3), 205-210.

Further investigation: Plastocyanin


Our tac+plastocyanin composite biobrick theoretically expresses a copper containing protein. Colonies containing this biobrick should turn blue in the presence of inducer (IPTG) and copper. To test this, we plated this culture out on a regular LB plate, and a copper + IPTG LB plate. We also plated out a control plate with both Plasto and CHLI1 expressing colonies.

The first image below show plastocyanin expressing colonies growing blue on LB+copper+IPTG plates seen on the right, and white/yellow on regular LB plates shown on the left:



The second image shows a LB+copper+IPTG plate with plastocyanin colonies growing on one half, and non-expressing colonies (ChlI1) on the other half:




Retrieved from "http://2013.igem.org/Team:Macquarie_Australia/Results"