Team:SJTU-BioX-Shanghai/Project/Subproject3
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We can control the translation process by controlling whether the ribosome can get through the changed initial codon placed in the target protein's mRNA. In ''E.coli'', the original initial codon is AUG. If the initial codon is changed to any other codon, translation cannot be initiated. Based on this, a strict switch can be achieved by controlling the existence of charged tRNA<sup>fMet</sup>-TCG base pairing codon CGA. This process is controlled by two elements: | We can control the translation process by controlling whether the ribosome can get through the changed initial codon placed in the target protein's mRNA. In ''E.coli'', the original initial codon is AUG. If the initial codon is changed to any other codon, translation cannot be initiated. Based on this, a strict switch can be achieved by controlling the existence of charged tRNA<sup>fMet</sup>-TCG base pairing codon CGA. This process is controlled by two elements: | ||
- | *The existence of tRNA<sup>fMet</sup>-TCG | + | *The existence of '''tRNA<sup>fMet</sup>-TCG''' |
- | *MetRS that can charge tRNA<sup>fMet</sup>-TCG | + | *'''MetRS''' that can charge tRNA<sup>fMet</sup>-TCG |
- | *KanR-CGA: the initial codon of KanR gene is changed from AUG to CGA. | + | *'''KanR-CGA''': the initial codon of KanR gene is changed from AUG to CGA. |
[[image:11SJTU_Initial_01.jpg|center|Design of initial-codon switch]] | [[image:11SJTU_Initial_01.jpg|center|Design of initial-codon switch]] | ||
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- | [[image:11SJTU-MetRS.jpg|frame|center|''Fig | + | [[image:11SJTU-MetRS.jpg|frame|center|''Fig.1'' This design is based on the crystal structure of methionyl-tRNA synthetase complex with tRNA (PDB ID:2CSX). We have superimposed the crystal structure of methionyl-tRNA synthetase from ''E.coli'' and obtained the overlay structure after kinetics optimization. Above is the picture showing ''E.coli'' methionyl-tRNA synthetase with (left) and without (right) anticodon recognition domain. The picture proposed that ''E.coli'' methionyl-tRNA synthetase will lose the ability to bind tRNA<sup>Met</sup> anticodon if anticodon recognition domain is deleted, thus losing anticodon specificity while maintaining aminoacylation ability. '''We have built the truncated MetRS, PT7-''metG''N ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567015 BBa_K567015]), based on this design. ''']] |
''metY''-CGA: We have cloned operon ''metY'' containing tRNA<sup>Met</sup> from ''E.coli'' to pACYC184. The anticodon of tRNA<sup>Met</sup> was mutated to TCG (base pairing codon CGA). | ''metY''-CGA: We have cloned operon ''metY'' containing tRNA<sup>Met</sup> from ''E.coli'' to pACYC184. The anticodon of tRNA<sup>Met</sup> was mutated to TCG (base pairing codon CGA). | ||
- | We inserted ''bla'' operon from pUC18 into pET28a, endowing pET28a with | + | We inserted ''bla'' operon from pUC18 into pET28a, endowing pET28a with Kan and Amp resistance. Then the start codon of KanR gene is mutated from ATG to CGA. We then inserted MetRS obtained from error-prone PCR into the MCS of this pET28a. When this plasmid is co-transformed with ''metY''-CGA, we can screen the mutants with Kanamycin resistance. The same method is also used to test the function of the truncated MetRS PT7-''metG''N ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567015 BBa_K567015]). |
We screened the MetRS obtained through error-prone PCR and obtained one target mutant. We have also tested the activity of the truncated MetRS PT7-''metG''N ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567015 BBa_K567015])and found that the truncated MetRS acted as expected, losing specificity for tRNA<sup>Met</sup> anticodon while maintaining aminoacylation ability. | We screened the MetRS obtained through error-prone PCR and obtained one target mutant. We have also tested the activity of the truncated MetRS PT7-''metG''N ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567015 BBa_K567015])and found that the truncated MetRS acted as expected, losing specificity for tRNA<sup>Met</sup> anticodon while maintaining aminoacylation ability. | ||
- | [[image:11SJTU-initial_codon_result.jpg|frame|center|''Fig | + | [[image:11SJTU-initial_codon_result.jpg|frame|center|''Fig.2'' Growth of ER2566 with a. ''metG''N + ''metY''-CGA, b. ''metG''M + ''metY''-CGA, c. + ''metG''N, d. + ''metG''M. Growth medium (left): LB Kana+Tet. Growth medium (right): LB Kanamycin.'''Cells survived Kanamycin with our device. Without our device, cells cannot survive.''' ]] |
- | Cell growth shows that the cells have | + | Cell growth shows that the cells have Kanamycin resistance only when both modified MetRS (''metG''N or ''metG''M) and modified tRNA<sup>Met</sup>(''metY''-CGA) are transformed into the cell, '''proving that tRNA ''metY''-CGA can transfer fMet to CGA when it is used as the start codon and that ''metG''N and ''metG''M work well. ''' |
'''Initial-Codon Switch proved successful. ''' | '''Initial-Codon Switch proved successful. ''' |
Latest revision as of 00:42, 29 October 2011