Team:Alberta/Project/promotor

description

Description

Stage One

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

we replaced constitutive promotor into inducible lac promotor to control the color gene expression and introduced (consetitutive) promotor 1, 2, 3, or 4 to control Lac I repressor expression. According to one of our experiment results, we have transformed

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.