Team:Alberta/Project

A microbial color wheel

This year, the University of Alberta team is developing a biological sensor circuit with three pigment colours to generate a multiple coloured output. We developed this idea because traditional biological reporters have been limited to a small set of fluorescent proteins and colour genes, which produce only an all-or-none output. Furthermore, having only a single channel of output limits the application of other sensors in a single biological device. Therefore, it is clear that the traditional sensor system requires easy-to-use bioreporters that are capable of responding to chemical gradients and mixing independent output channels. To construct the biosensor circuit, we used existing genetic parts pioneered by the 2009 and 2010 iGEM teams, which will result in the circuit being capable of responding to chemical gradients and producing a multi-coloured output in the form of a colour wheel. This biosensor will become the new age of easy–to-read reporters that are incorporated into new versions of the Genomikon kit, which directly impact both research and education usage.

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Stage one (June.20.2012)
We finished the first part of our biological circuit construction (promoter switching) and discovered that the red fluorescent protein (rfp) gene was only expressed from the two strongest promoters, 4 (second strongest) and 5 (strongest), out of nine different promoters tested. The blue pigment protein (bpp) gene was only expressed from promoter 2, which is the second weakest of the tested promoters..Expression of the green fluorescent protein (gfp) gene was not detected using any promoter, unexpectedly. These results indicate that the recruitment of the RNA polymerase to initiate transcription and expression of these pigment genes can be accomplished using promoter 2 or higher. However, the strength of the promoter does affect visible color development in E. coli colonies. A larger amount of RFP is required as expression was found to only be driven by the strongest promoter while BPP was produced at sufficient levels using a weaker promoter. This may also indicate that BPP is toxic at higher levels driven from stronger promoters. The lack of GFP production may be a result of cloning errors or secondary structure of mRNA blocking RBS from ribosome binding. Therefore, We have decided to construct plasmids to test the different strengths of RBS and investigate the toxicity of colour proteins. We have also begun cloning transcriptional repressors in order to modulate level of pigment gene expression.

Stage Two (June.20.2012)
Using theold Ribosome binding site (RBS) generated by a computer program and unique foreach colour gene, we achieved tiny, colorless or dull colonies. Therefore, weswitched the lacking RBS with a new known RBS, which was acquired from adifferent iGEM group.
The two differencesbetween the old and new plasmids are that all the colour genes now share anidentical RBS, and the novel RBS has been previously confirmed as working. Weattached the new RBS by performing PCR and transformed it in Top10 E.coli cells. As a result, the yellowcolonies expressed a vivid colour but still grew quickly, indicating that theoverexpression experienced with the blue gene’s original RBS was not a problemwith the yellow gene’s modified RBS. The blue gene with the new RBS, however,is not expressed. Overexpression, resulting in toxicity because of highconcentrations of pigments, and underexpression, resulting in death from lowconcentration of antibiotic resistance may be possible explanations. The redgene shows the same expression compared to the old RBS.

Step three

After completing the pieces that will produce the actual output we moved on to the more complicated process of constructing a regulatable promoter system that will produce the outputs that we are aiming for. After assembling the different pieces relevant, we started testing our regulation of the basic color gene RFP with the Lac operator. These experiments paved the way for the creation of a plasmid which had its copy number under control of an inducible repressor. We tested plasmids with the standard puc19 origin of replication promoter, and also a plasmid which was identical with the exception of this plasmid containing a much stronger promoter. This second plasmid was much more difficult to test, as a cell containing this plasmid is not viable without also having the LacI gene present; a feature not on our initial construct. The promoters were tested again once we had added the LacI gene to the plasmid, and the results were surprising. The cells grew in a large range of IPTG concentrations (1/20th to 1.6 times the recommended concentration), where we had been expecting a much more narrow window.

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