Translational_Unit

Part:BBa_K567014:Experience

Designed by: You Wang   Group: iGEM11_SJTU-BioX-Shanghai   (2011-09-29)
Revision as of 08:05, 5 October 2011 by Cathy zks (Talk | contribs) (→‎Applications of BBa_K567014)

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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.

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