Difference between revisions of "Part:BBa K567014:Experience"
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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter | This experience page is provided so that any user may enter their experience using this part.<BR>Please enter | ||
how you used this part and how it worked out. | how you used this part and how it worked out. | ||
| − | === | + | ===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. | ||
===User Reviews=== | ===User Reviews=== | ||
Revision as of 08:05, 5 October 2011
This experience page is provided so that any user may enter their experience using this part.
Please enter
how you used this part and how it worked out.
Construction of BBa_K567014
In order to charge Met to tRNAMet 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.
Characterization of BBa_K567015
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 tRNAMet anticodon while maintained aminoacylation ability.
Cell growth shows that the cells show Kana resistance only when both modified MetRS (metGM) and modified tRNAMet(metY-CGA) are transformed into the cell, proving that metGM works well.
User Reviews
UNIQdc3e4c3cdaba993b-partinfo-00000000-QINU UNIQdc3e4c3cdaba993b-partinfo-00000001-QINU