Part:BBa_K567015:Experience

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Construction of BBa_K567015

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^Met without recognizing its anticodon.

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^Met anticodon if anticodon recognition domain is deleted, thus losing anticodon specificity while maintaining aminoacylation ability. We have built the truncated MetRS, PT7-metGN (BBa_K567015), based on this design.

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. We tested the activity of the truncated MetRS PT7-metGN (BBa_K567015)and found that the truncated MetRS acted as expected, losing specificity for tRNA^Met anticodon while maintaining 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 (metGN or metGM) and modified tRNA^Met(metY-CGA) are transformed into the cell, proving that metGN works well.

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