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| − | ===Construction of BBa_K567014===
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| − | In order to charge Met to tRNA<sup>Met</sup> with mutated anticodon, we need to deprive MetRS of its anticodon specificity. Directed Evolution Strategy is used. Error-prone PCR is used to introduce random mutations into MetRS.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 screened the MetRS obtained through error-prone PCR using Kana and obtained one target mutant.
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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. This enzyme lost specificity for tRNA<sup>Met</sup> anticodon while maintained 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''M) and modified tRNA<sup>Met</sup>(''metY''-CGA) are transformed into the cell, proving that ''metG''M works well.
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| | ===User Reviews=== | | ===User Reviews=== |
Latest revision as of 10:37, 5 October 2011
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