Team:Alberta/Parts

The parts we have made are composed of protein generating open reading frames, ribosomal binding sites, promoters, and copy-number controlled vectors. We describe each of these classes of part in turn.

<groupparts>iGEM012 Alberta</groupparts>

We have made and tested parts for producing colors and repressors. The color proteins are red, based on E1010, yellow, based on amilGFP K592010, and blue, based on amilCP K592009. Repressor proteins are lambda CI, LacI, and TetR, based on C0051, C0012, and C0040 respectively. The sequences are unmodified except for removal of restriction sites, removal of degradation tags, and replacement of stop codons. The sequences were synthesized along with their RBSes.

Our original intent was to create a series of RBS sequences of similar strength that were each tailored to a particular ORF, and that showed significant sequence variation between each other to minimize the chances of intra-molecular recombination when used in combination. We used the online Salis RBS calculator (Salis et al, Nat Biotech 27: 946 2009) to calculate RBSes for each protein coding sequence with a target translation initiation strength set to a value of 50,000 for each of the ORFs shown above using a common length constraint that began with the common transcriptional start site (position -1 of the common promoter downstream region- Fig.1 below). When driven by Pr-2, the colour genes showed marked differences in expression and growth and were therefore not ideally suited to our purposes. We therefore created versions of the colour genes that each used the popular registry RBS, B0034. Although the LacI and TetR RBSes were never directly compared, they both functioned adequately in subsequent experiments.

                         Figure 1: Information of RBS protein parts

                         Figure 2: the list of promoters that were used in our project to regulate and tune colour expression.