|
|
| (One intermediate revision by the same user not shown) |
| Line 3: |
Line 3: |
| | how you used this part and how it worked out. | | how you used this part and how it worked out. |
| | | | |
| − | ===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<sup>Met</sup> without recognizing its anticodon.
| |
| − |
| |
| − | [[image:11SJTU-MetRS.jpg|frame|center|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<sup>Met</sup> anticodon if anticodon recognition domain is deleted, thus losing anticodon specificity while maintaining aminoacylation ability. '''We have built the truncated MetRS, PT7-''metG''N (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-''metG''N (BBa_K567015)and found that the truncated MetRS acted as expected, losing specificity for tRNA<sup>Met</sup> anticodon while maintaining aminoacylation ability.
| |
| − |
| |
| − | [[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.]]
| |
| − |
| |
| − | Cell growth shows that the cells show Kana resistance only when both modified MetRS (''metG''N) and modified tRNA<sup>Met</sup>(''metY''-CGA) are transformed into the cell, proving that ''metG''N works well.
| |
| | | | |
| | ===User Reviews=== | | ===User Reviews=== |
Latest revision as of 10:35, 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.
User Reviews
UNIQ081cd9cbfa10089e-partinfo-00000000-QINU
UNIQ081cd9cbfa10089e-partinfo-00000001-QINU
