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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. 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.
BioBrick Construction
BioBrick Sequences
Characterisation
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.
BioBricks Constructed Sequence Confirmed Submitted to iGEM POR
ChlG
ChlP ChlI2 YCF54 CTH1 ChlI1 Gun4 Plastocyanin ChlM DVR1 ChlD
1st Attempt at ligation:
Promoter was ligated onto the genes and plated up on LB + CAM plates, we observed a lot of colony growth.
We inoculated LB + CAM broth with a colony from the corresponding plate. A variety of growth was
observed, a few plates corresponding to a certain genes had a lot more colonies than others, this
can be observed from the image with the description order of genes.
More colonies corresponding to a gene displayed more colony growth than others which can be observed from
the image description showing the order of genes on the agar.
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.
Attempt 2:
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, the results are to the left.
PCR of 2nd attempt:
The PCR showed that the ligation attempts were successful this time round, we exclaimed for joy. We used the BioBrick to inoculate E.coli .
