Team:SJTU-BioX-Shanghai/Project/Subproject1/Results 2

Rare-Codon Switch

tRNA Modulator + Reporter for Quantitative Analysis

tRNA Modulator and Reporter for Quantitative Analysis work together to regulate protein biosynthesis.

tRNA Modulator controls rare tRNA amount. In our project, we use sulA promoter (BBa_K567002 sulA promoter-tRNA^Asp-AGG) or trc promoter(BBa_K567001 lacI-Ptrc-tRNAArg) to control rare tRNA amount. tRNA Modulator: BBa_K567002 sulA promoter-tRNA^Asp-AGG BBa_K567001 lacI-Ptrc-tRNA^Asp-AGG

Reporter can control two elements: the number of rare codons inserted into luciferase after start codon ATG and the strength of target protein promoter. In our project, 2, 4, 6, 8 rare codon AGGs are inserted into the Reporter gene. T7 promoter or bla promoter are used to control target protein mRNA amount.

Reporter: BBa_K567004 Pbla-Luc-2AGG BBa_K567005 Pbla-Luc-4AGG BBa_K567006 Pbla-Luc-6AGG BBa_K567007 Pbla-Luc-8AGG BBa_K567008 PT7-Luc-2AGG BBa_K567009 PT7-Luc-4AGG BBa_K567019 PT7-Luc-6AGG BBa_K567010 PT7-Luc-8AGG

Luc for luciferase

We have tested different combination of tRNA Modulators and Reporters and analyzed the influence of promoter strength for rare tRNA, number of rare codons in target protein mRNA and promoter strength for target protein gene respectively.

1.The Working Curve of tRNA Modulator We have tested luciferin reaction in cells with different combinations of tRNA Modulators and Reporters. We examined the changes in luciferase enzyme activity over time after rare tRNA expression is induced. The amount of luciferase is reflected indirectly by the bioluminescence emitted from the luciferin reaction. Results are shown below:

Here we use the following two curves as examples to characterize the working curve of tRNA Modulator. Both curves fits typical titration curve, indicating that tRNA Modulator can function as a regulating tool. The rest of the working curves are shown here: Pbla

PT7

From this experiment, we noticed that the typical working curve of tRNA Modulator can be better observed with IPTG induced tRNA Modulator (lacI-Ptrc-tRNA^Asp) compared with UV excitation induced tRNA Modulator (BBa_K567002 sulA promoter-RNA^Asp), though sulA promoter-tRNA^Asp responded quicker to signals. We step further to test the influence of different Reporters with tRNA Modulator lacI-Ptrc-tRNA^Asp.