Team:BioBricks/Proof
To prove that our design worked, we built a daisy chain using two different fluorophores, testing to see whether both proteins were expressed and at what levels. Within our daisy chain, we built in a high BCD to ensure that the outcome was consistent across experiments. Within the Daisy Chain, we had mTurquoise2 (CFP) first with a Shine-Dalgarno site embedded within the end of the sequence to translationally couple the next fluorophore, mCherry (RFP) with a -1 frameshift. Using antibiotic selection, we selected for colonies that expressed both fluorescent proteins
To create the daisy chain, we gathered the genetic aspects to contorol transcriptional and translational coupling. We sourced the ptrc* promoter, double terminator, and BCD 5 from the BIOFAB plasmid set from Addgene. To ensure consistent expression, we used a high functioning promoter with a strong BCD. We then ended the sequence with a double terminator to insulate the expression of our system
The BCD we chose was proported to have a relatively high expression level with a narrow range of variability by unfolding the mRNA structure, which allows for more consistent ribosomal binding. We decided to use cyan fluorescent protein as the first gene of our daisy chain, and we embedded a ribosome binding site via codon degeneracy. Thus, our second gene coding for red fluorescent protein could be placed after the CFP with a -1 frameshift to allow for translational coupling. Such coupling allows for both genes' translation to be regulated as a single entity. While this experiment uses CFP and RFP, other combinations of genes can be used as well; however, they will be limited by the strength of the potential RBS optimized within the upstream gene.
