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Team:Macquarie Australia/Results
From 2013.igem.org
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
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 Constructed | Gibson Composition | Sequence Confirmed | Submitted to iGEM |
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| POR |
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| ChlG |
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| ChlP |
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| ChlI2 |
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| YCF54 |
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| CTH1 |
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| ChlI1 |
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| Gun4 |
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| Plastocyanin |
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| ChlM |
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| DVR1 |
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| ChlD |
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Characterisation
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.
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, and white/yellow on regular LB plates:
