Team:Macquarie Australia/Results

From 2013.igem.org

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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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<tr class="alt"><td><b><a href=http://parts.igem.org/wiki/index.php?title=Part:BBa_K1080011ChlI2</a></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>
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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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<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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&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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<b>Composite part creation: attaching promoters to genes</h1></b>
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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).
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<b>1st Attempt at ligation:</h1></b>
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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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We inoculated LB + CAM broth with a colony from the corresponding plate. A variety of growth was
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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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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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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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<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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Revision as of 00:02, 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.




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