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Transcription/Translation Circuit

The translation/transcription circuit consists of an arabinose inducible promoter followed by a ribosomal binding site and the protein of interest (these two parts will be cloned into the circuit by the user). The protein of interest will be followed by a wild type Red Fluorescent Protein (RFP) which contains a signal peptide that allows it to fold in the periplasmic region of the cell. Dr. Shawn Lewenza's lab specializes in research pertaining to fluorescent lipoproteins and provided us with this “ppRFP”. The advantage of using this variant RFP is that it has the ability to fold correctly in both the cytoplasm and the periplasm. If regular cytoplasmic folding RFP was used, false negatives could be obtained even if the amino acid chain folded properly but traveled to the periplasm post-transationally. The ppRFP is followed by the composite part I13504 which consists of a ribosomal binding site, GFP and double terminators. We debated which promoter would be ideal to incorporate in this circuit and eventually agreed on I0500 (an Arabinose Inducible promoter). The main advantage of an inducible promoter over a constitutive promoter is that protein expression can be controlled to a higher degree with an inducible promoter. Protein expression needs to be strictly controlled in this circuit especially as over-expression can also lead to misfolded proteins, which would result in a false positive in either the Cpx or the DegP pathways.

This is a schematic of our system. RFP output indicates that translation is successful and GFP output indicates that transcription is successful.

The next cause of concern arose in regards to the ribosomal binding site before the GOI and the GOI itself. Initially, we had planned on cloning together the arabinose inducible with B0034 and the user would simply be required to clone in their GOI. However, due to spacing issues that could cause a possible frameshift resulting in incorrect protein production we decided it would be best for the user to clone in a DNA fragment containing their own ribosomal binding site with the GOI. Another advantage of this particular set up is that the ribosomal binding site would be native to the GOI and therefore would be optimal for the GOI. Many researchers have attempted to and have successfully used similar circuits in order to test for transcription and translation (Kim et al. 2009).

1) Kim, HR, Shay, T, O'Shea, EK, & Regev, A. (2009). Transcriptional regulatory circuits: predicting numbers from alphabets. Science Online, 325(5939), Retrieved from doi: 10.1126/science.1171347