The Rare Codon Switch can only regulate the amount of target protein production. To make our device a strict molecular switch that turns on/off protein biosynthesis, we need to eliminate the background.
In recent years, stop codons (codonST) and stop codon suppressor tRNAs (tRNASS) are used to incorporate unnatural amino acids in protein biosynthesis. Researchers like Peter Schultz and Kiga have made a lot of contributions in this field. The advantage of codonST and tRNASS is that they can incorporate target amino acids without background noise. We make use of codonST and tRNASS as the controlling element for protein biosynthesis. In E.coli, the rarest used stop codon is TAG.
We design a Stop-Codon Switch to turn on/off protein translation.
We can control the translation process by controlling whether the ribosome can get through the stop codon placed in the target protein's mRNA.
This process can be achieved by controlling the existence of charged tRNASS that recognizes the stop codon. This process is controlled by two elements:
The existence of tRNASS
aaRS that can charge tRNASS
Reporter: a stop codon is put immediately after the initial codon ATG in the target protein' s mRNA.
If the ribosome can get through the stop codon with the help of Stop-Codon Switch, luciferase can be expressed. Otherwise, luciferase cannot be expressed.
We have used Pbla-Luc-TAG (BBa_K567003) as our Reporter. The amount of luciferase produced is reflected using the bioluminescence emitted during the luciferin reaction. Our results demonstrate that TAG insertion into luciferase blocks luciferase production, which was shown in the control group. In the experimental group, with the help of PT7-TDRS(BBa_K567011) and tRNAAsp-TAG(BBa_K567013),luciferase was produced and bioluminescence was emitted during the luciferin reaction. These results proved that Stop-Codon Switch can turn on protein expression. Yet further work is needed to optimize the device.
We have successfully constructed the Stop-Codon Switch and have tested the device. Results proved Stop-Codon Switch as a strict molecular switch without background noise. tRNAAsp-TAG recognizes stop codon TAG and can be charged with Asp by modified AspRS.
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