Difference between revisions of "Part:BBa K567014:Experience"

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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.
 
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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For more information concerning this part, please see [http://2011.igem.org/Team:SJTU-BioX-Shanghai 2011 SJTU-BioX-iGEM]
  
 
===User Reviews===
 
===User Reviews===

Revision as of 08:12, 5 October 2011

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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.

fig. Growth of ER2566 with a. metGN + metY-CGA, b. metGM + metY-CGA, c. + metGN, d. + metGM. Growth medium (left): LB Kana+Tet. Growth medium (right): LB Kana.

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.

For more information concerning this part, please see [http://2011.igem.org/Team:SJTU-BioX-Shanghai 2011 SJTU-BioX-iGEM]

User Reviews

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