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Team:Cambridge/Project/VI02
From 2009.igem.org
Violacein Pigments
Design
The Vio operon had numerous forbidden restriction sites. We thus had to synthesize it, removing these restriction sites and optimizing codon usage for E. coli, to create the following biobrick:
As DNA2.0 very generously agreed to synthesise the entire operon for us, we designed it to include all the five genes, each preceded by a ribosome binding site, and flanked by the prefix and suffix. All forbidden restriction sites were removed from the operon. The final plan for the inserted operon is shown below:
This will be held under a repressible promoter on the pJexpress cloning cassette from DNA2.0. The restriction sites will allow us to easily remove the vioC and vioD which are the genes responsible for chaging pigment colour.
Creating colours
Once the violacein biobrick arrived we expressed in in TOP10 E. coli to create the purple pigment. We then carried out two more digests to see if we could create further colours:
- BamHI and BglII = removed vioC and produced a dark green pigment
- BglII and BclI = removed vioD and produced a light green pigment
Both of these new pigments were entered into the registry, along with the whole violacein operon for the purple pigment.
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