Team:SJTU-BioX-Shanghai/Project/Subproject1-1

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Latest revision as of 00:38, 29 October 2011

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Project
- Rare-Codon Switch
- Application

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Parts

Rare tRNA Amount

Design

In this part we have overexpressed rare tRNA^Arg-AGG in the cell. The rare tRNA can recognize AGG codon on the mRNA.

tRNA^Arg-AGG([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567001 BBa_K567001]): tRNA^Arg-AGG is over expressed under the control of trc promoter (induced by IPTG).

This rare tRNA^Arg can be charged with Arg by native Arginyl-tRNA Synthetase(ArgRS) in E.coli.

Over expressed tRNA(Arg)-AGG is charged by native Arginyl-tRNA Synthetase(ArgRS)

RFP-6AGG([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567017 BBa_K567017]): we have inserted 6 AGG codons after the start codon ATG in the RFP gene.

6AGG codons are inserted after the start codon ATG in the reporter gene

Action

When rare tRNA^Arg-AGG is not over-expressed, RFP expression is hindered. When tRNA^Arg-AGG is over-expressed, this tRNA can recognize the AGG codon on the mRNA so a large amount of RFP is produced.

Result

Fig.1 Confocal Microscope examining RFP expression. RFP has been largely produced in cells overexpressing tRNA^Arg-AGG.

Fig.2 Cells over-expressing tRNA^Arg-AGG emit bright red fluorescence as wild type RFP (first one from the left). Control (first one from the right) exhibits no red fluorescence.

RFP has been largely produced in cells overexpressing tRNA^Arg. No RFP can be observed in cells without rare tRNA overexpression.

We have successfully controlled protein expression by controlling rare tRNA amount.

@@ Line 30: / Line 30: @@
 When rare tRNA<sup>Arg</sup>-AGG is not over-expressed, RFP expression is hindered. When tRNA<sup>Arg</sup>-AGG is over-expressed, this tRNA can recognize the AGG codon on the mRNA so a large amount of RFP is produced.
-[[image:11SJTU_rare_01.jpg|500px|center]]
+[[image:11SJTU_rare_01.jpg|center]]
 -----
 ===Result===
-[[File:11SJTU_rare_02.png|thumb|500px|center|''Fig 1''. Confocal Microscope examining RFP expression. '''RFP has been largely produced in cells overexpressing tRNA<sup>Arg</sup>-AGG.''']]
+[[File:11SJTU_rare_02.png|thumb|500px|center|''Fig.1'' Confocal Microscope examining RFP expression. '''RFP has been largely produced in cells overexpressing tRNA<sup>Arg</sup>-AGG.''']]
-[[File:11SJTU_rare_10.jpg|thumb|500px|center|''Fig 2'' Cells over-expressing tRNA<sup>Arg</sup>-AGG emit bright red fluorescence as wild type RFP (first one from the left). Control (first one from the right) exhibits no red fluorescence.]]
+[[File:11SJTU_rare_10.jpg|thumb|500px|center|''Fig.2'' Cells over-expressing tRNA<sup>Arg</sup>-AGG emit bright red fluorescence as wild type RFP (first one from the left). Control (first one from the right) exhibits no red fluorescence.]]
 RFP has been largely produced in cells overexpressing tRNA<sup>Arg</sup>. No RFP can be observed in cells without rare tRNA overexpression.