Translational_Unit

Part:BBa_K567014

Designed by: You Wang   Group: iGEM11_SJTU-BioX-Shanghai   (2011-09-29)
Revision as of 02:28, 6 October 2011 by ChobitParrot (Talk | contribs)

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. KanaR 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 Kana plate.

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.

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


Related Biobrick:

BBa_K567015

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]


[edit]
Categories
//cds/enzyme
Parameters
chassisER2566
functionanimo acylate tRNA(Met)
ligandsIsopropyl β-D-1-Thiogalactopyranoside (IPTG)