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. 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.
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:
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 2257
Illegal XbaI site found at 48
Illegal PstI site found at 1508 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 2257
Illegal PstI site found at 1508 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 2257
Illegal BamHI site found at 1951 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 2257
Illegal XbaI site found at 48
Illegal PstI site found at 1508 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 2257
Illegal XbaI site found at 48
Illegal PstI site found at 1508 - 1000COMPATIBLE WITH RFC[1000]
chassis | ER2566 |
function | animo acylate tRNA(Met) |
ligands | Isopropyl β-D-1-Thiogalactopyranoside (IPTG) |