Team:SJTU-BioX-Shanghai/Project/Subproject1/Results 3

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- Rare-Codon Switch
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Rare-Codon Switch

aaRS Modulator + Reporter for Qualitative Analysis

Results

To deprive AspRS of its anticodon specificity, we analyzed the structure of AspRS and expressed a truncated AspRS without anticodon recognition domain. Our design is shown below. This modified enzyme keeps its ability of aminoacylation while loses its activity of recognizing anticodon of tRNA.

Fig1. The design for modification is based on the crystal structure of E.coli AspRS coupled with tRNA (PDB ID: 1C0A). This figure shows E.coli aspartyl-tRNA synthetase with and without anticodon recognition domain. After this domain is depleted, the enzyme cannot bind tRNA^Asp anticodon. Yet the ability of aminoacylation is maintained.

We have used PT7-RFP-6AGG (BBa_K567017) as our Reporter. We have constructed tRNA^Asp-AGG and PT7-TDRS (AspRS without anticodon recognition domain) as the Modulator. tRNA^Asp-AGG, which can recognize rare codon AGG, is under constitutive promoter. Our device works as follows: Without Modulator, RFP with 6 consecutive AGG insertions can hardly be expressed. When Modulator works, tRNA^Asp-AGG can be charged with Asp by TDRS. tRNA^Asp-AGG recognizes rare codon AGG on the Reporter RFP-6AGG. The ribosome gets through this part of the mRNA more easily. RFP is expressed. Red fluorescence is observed. Our experiment results are shown below.

fig2. With our Modulator (BBa_K567012 and BBa_K567011), Reporter RFP-6AGG is expressed, demonstrating that our system works well.

fig3. aaRS Modulator + Reporter for Qualitative Analysis. aaRS Modulator + RFP-6AGG(the middle three) emit red fluorescence. Wild type RFP (the first one from the left) exhibits bright red fluorescenceis. Control (first one from the right) exhibits no red fluoresence.

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 [[image:11SJTU-ASPRS.jpg|600px|frame|center|Fig1. The design for modification is based on the crystal structure of ''E.coli'' AspRS coupled with tRNA (PDB ID: 1C0A). This figure shows ''E.coli'' aspartyl-tRNA synthetase with and without anticodon recognition domain. After this domain is depleted, the enzyme cannot bind tRNA<sup>Asp</sup> anticodon. Yet the ability of aminoacylation is maintained. ]]
-We have used BBa_K567017 PT7-RFP-6AGG as our Reporter. We have constructed tRNA<sup>Asp</sup>-AGG and PT7-TDRS (AspRS without anticodon recognition domain) as the Modulator. tRNA<sup>Asp</sup>-AGG, which can recognize rare codon AGG, is under constitutive promoter. Our device works as follows: Without Modulator, RFP with 6 consecutive AGG insertions can hardly be expressed. When Modulator works, tRNA<sup>Asp</sup>-AGG can be charged with Asp by TDRS. tRNA<sup>Asp</sup>-AGG recognizes rare codon AGG on the Reporter RFP-6AGG. The ribosome gets through this part of the mRNA more easily. RFP is expressed. Red fluorescence is observed. Our experiment results are shown below.
+We have used PT7-RFP-6AGG (BBa_K567017) as our Reporter. We have constructed tRNA<sup>Asp</sup>-AGG and PT7-TDRS (AspRS without anticodon recognition domain) as the Modulator. tRNA<sup>Asp</sup>-AGG, which can recognize rare codon AGG, is under constitutive promoter. Our device works as follows: Without Modulator, RFP with 6 consecutive AGG insertions can hardly be expressed. When Modulator works, tRNA<sup>Asp</sup>-AGG can be charged with Asp by TDRS. tRNA<sup>Asp</sup>-AGG recognizes rare codon AGG on the Reporter RFP-6AGG. The ribosome gets through this part of the mRNA more easily. RFP is expressed. Red fluorescence is observed. Our experiment results are shown below.
 [[image:11SJTU-ZBresult1.jpg|600px|frame|center|fig2. With our Modulator (BBa_K567012 and BBa_K567011), Reporter RFP-6AGG is expressed, demonstrating that our system works well. ]]