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| − | ===Construction of BBa_K567015===
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| − | Rational design of MetRS: we have obtained a truncated MetRS without anticodon recognition domain. The structure of the truncated protein is shown below. This modified MetRS can charge tRNA<sup>Met</sup> without recognizing its anticodon.
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| − | [[image:11SJTU-MetRS.jpg|frame|center|fig. 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 (BBa_K567015), based on this design. ''']]
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| − | ===Characterization of BBa_K567015===
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| − | When this part, ''metY''-CGA (BBa_K567016) and KanaR (BBa_K567020) are co-transformed into the cell, the cell is expected to survive Kana. We tested the activity of the truncated MetRS PT7-''metG''N (BBa_K567015)and found that the truncated MetRS acted as expected, losing specificity for tRNA<sup>Met</sup> anticodon while maintaining aminoacylation ability.
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| − | [[image:11SJTU-initial_codon_result.jpg|frame|center|fig. 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 Kana.]]
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| − | Cell growth shows that the cells show Kana resistance only when both modified MetRS (''metG''N) 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
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| − | For more information concerning this part, please see [http://2011.igem.org/Team:SJTU-BioX-Shanghai 2011 SJTU-BioX-iGEM]
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| | ===User Reviews=== | | ===User Reviews=== |
Latest revision as of 10:35, 5 October 2011
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