Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Alex Bina
Date created: 2011-10-22 11:00:00
Date modified: 2015-05-08 01:12:59
RecA without restriction sites or recombinant activity
| Types | DnaRegion |
| Roles | Other sequence_feature |
| Sequences | BBa_K648108_sequence (Version 1) |
Description
RecA is a protein used by E. coli to repair and maintain DNA. Many normal laboratory strains of E. coli, for example DH10B, are mutants capable of an inactivated form of RecA known as RecA1. RecA 1 is deficient in all known function of the RecA gene specifically in ATPase activity, binding with DNA in the presence of ATP, and changing conformation in the presence of ATP and repressor cleavage. In order to restore RecA1 to RecA and to reduce its recombination ability while maintaining its repressor cleavage activity a series of mutations was made.One such mutation occurs at amino acid 160, this adenine residue needed to be mutated to guanine to change RecA1 to RecA and restore its functions. In order to use the standard bio-bricking techniques we also had to remove naturally occurring enzymatic restriction sites. The Pst1 site CTGCAG needed to be converted to CTGCAA. Then the EcoR1 site GAATTC needed to be converted to GAATTT.
We also focused on how to mutate RecA so that it would detect damaged DNA without recombining the DNA. After literature research we identified two amino acids in the RecA sequence that were known to affect RecA???s ability to use recombination to repair the DNA. These sites were Arginine 243 and Lysine 286. Research suggested that changing Arginine 243 to Glutamine, and Lysine 286 to Asparagine would remove recombinant activity from RecA.
This strain has all five mutations.
Notes
Activate RecA activity from RecA1, remove restriction sites and recombinant activity.Source
The original strain of this part, RecA1, comes from the genomic sequence of the DH10B strain of Escherichia coli| Sequence Annotation | Location | Component / Role(s) |
| PstI mutation EcoRI mutation Lys286 mutation Arg243 mutation RecA activation mutation | 237,237 783,783 861,861 731,731 530,530 | feature/mutation sequence_alteration sequence_alteration feature/mutation sequence_alteration feature/mutation sequence_alteration feature/mutation feature/mutation sequence_alteration |