Results of Components

pUS201 - Low Copy Backbone

Characterization of the low-copy backbone was unfortunately unsuccessful. Despite careful planning and verification of correct insertion and sequence the low-copy backbone displayed high copy replication characteristics. See the pUS201 Parts page for more info.

pUS203 - Controllable Integrase

Without pUS201 the experimental design had to adapt. The controllable Integrase system from pUS203 should ideally be in a low copy backbone but the the iGEM supplied low-copy plasmid was not an option as the anti-biotic resistance interfered with the experimental set up. At this point in time we did not have time to alter the experiment to such a degree as to accommodate the change. Instead we set about validating the controllable integrase system in the submission plasmid to try and determine whether it was producing integrase but the initial experiment failed and follow up experiments to troubleshoot problems were not possible due to time constraints. See the pUS203 Parts page for more info.

pUS204 - Cassette in pSB1C3

The cassette was functionally proven although it did not express the aeBlue gene but did confer gentamycin resistance. After sequencing we have found that a deletion near the end of the part seems to have been the cause for the absence of blue from the colonies expressing the cassette. Despite the lack of blue reporter phenotype being observed, the Gm Resistance was real. This part can be considered as ready to use for further experiments involving integrons.

Conclusion of Project

At the time of the Wiki Freeze we have been unable to functionally validate our controllable integrase BioBrick and so cannot determine if it expressing Integrase under the action of arabinose or not. Troubleshooting of the original experiment and designing a new experiment to allow us to use the low-copy plasmid would give us evidence for which part of the araC-pBAD system isn't working but due to time constraints we could not perform these tests before the end of the competition.

The low-copy plasmid has been shown to be high-copy and so we believed it was not worth submitting as a part as it was functionally incorrect.

The cassette worked but with only the Gentamicin resistance as a selectable marker. We view this as a great success as it's a definite step toward creation of our Integrase cloning system if another iGEM team or next years USyd team could validate a controllable Integrase BioBrick!