Part:BBa_K567015

PT7-metGN

T7 promoter-metG(truncated). This biobrick is constructed by putting the truncated metG (Met-RS) under the control of T7 promoter and lac operator. We have cloned metG from E.coli and the tRNA recognition domain of metG is truncated. Kana gene with start codon substituted for CGA is used to testify the function of mutated metG. When this biobrick and metY-CGA (BBa_K567016) are co-transformed into the cell, the cells can survive on the LBKana plate.

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) and modified tRNA^Met(metY-CGA) are transformed into the cell, proving that tRNA metY-CGA can transfer fMet to CGA when it is used as the start codon

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

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1265
  Illegal EcoRI site found at 1437
  Illegal XbaI site found at 48
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1265
  Illegal EcoRI site found at 1437
  Illegal NheI site found at 1226
  Illegal NotI site found at 1290
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1265
  Illegal EcoRI site found at 1437
  Illegal BamHI site found at 1259
  Illegal XhoI site found at 1211
  Illegal XhoI site found at 1299
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1265
  Illegal EcoRI site found at 1437
  Illegal XbaI site found at 48
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1265
  Illegal EcoRI site found at 1437
  Illegal XbaI site found at 48
  
• 1000
  COMPATIBLE WITH RFC[1000]