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.1. Promoters Pr-1 to Pr-5 are variants of the Anderson promoters J23114, J23105, J23108 J23100 and J23119 (wt) that are described in the Registry. The five bp between the AvrII and NheI sites have been varied to minimize recombination events when used in combination. Strength refers to the original fluorescence measurements made using RFP as a reporter. Note that each promoter is roughly double the strength of the one that follows it in the list. Pr-0 is a negative control sequence that was designed from the complement of the sequence of Pr-1. The sequences for Tet-Pr, Lac-Pr and lCI-Pr are as originally reported in Cox et. Al (Molecular systems Biology 2007) where each operator sequence has been placed between the -35 and -10 regions. The downstream region indicated for each promoter is discussed below. Promoter DNA used in plasmid constructions was made by annealing plus and minus strand synthetic oligonucleotides (IDT). 2. Status indicates “made (+)/tested (+)/functional (+) as the best-case scenario. In our hands Pr-5 (wt) failed to produce viable colonies with RFP but worked when linked to LacI This may explain the N.D. result reported for its J23119 analog since strong colour development seems to be associated with slow growth.

LacO and TetO controlled Ori

These parts consist of a vector backbone with a pMB1 origin, modified to add a lactose or tetracycline operator sequence between the -10 and -35 boxes in the promoter for the RNA II gene. The RNA II gene is the primer which serves to begin replication of the plasmid, thus controlling its expression is an effective means of controlling plasmid reproduction and copy number.

The part is provided with an insert consisting of an RFP cassette and the appropriate repressor, so that the vector is functional out of the box. This repressor has the additional benefit of functioning as a positive selection marker when replacing the supplied insert: if the clones are plated on a plate lacking the appropriate inducer, the copy number of vectors not receiving the new insert will be repressed, leading to selection in favor of the user’s new insert. This necessitates plating in presence of the inducer during normal transformation and propagation, however.

Uses: