Part:BBa_K567014

PT7-metGM

T7 promoter-metG(mutated). This biobrick is constructed by putting the mutated metG (Met-RS) under the control of T7 promoter and lac operator. We have cloned metG from E.coli and have used error-prone PCR to amplify the metG. KanR 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 LB Kanamycin plate.

Construction of BBa_K567014

In order to charge Met to tRNA^Met 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 KanR (BBa_K567020) are co-transformed into the cell, the cell is expected to survive Kan. We screened the MetRS obtained through error-prone PCR using Kanamycin and obtained one target mutant.

Characterization of BBa_K567015

When this part, metY-CGA (BBa_K567016) and KanR (BBa_K567020) are co-transformed into the cell, the cell is expected to survive Kanamycin. This enzyme lost specificity for tRNA^Met 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 Kanamycin resistance only when both modified MetRS (metGM) and modified tRNA^Met(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]

Related Biobrick:

PT7-metGN (BBa_K567015)

metY-CGA (BBa_K567016)

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 2257
  Illegal XbaI site found at 48
  Illegal PstI site found at 1508
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 2257
  Illegal PstI site found at 1508
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 2257
  Illegal BamHI site found at 1951
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 2257
  Illegal XbaI site found at 48
  Illegal PstI site found at 1508
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 2257
  Illegal XbaI site found at 48
  Illegal PstI site found at 1508
  
• 1000
  COMPATIBLE WITH RFC[1000]