Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Rachelle Varga
Date created: 2016-10-08 11:00:00
Date modified: 2016-10-18 02:33:48
Xylose->comK with AmyE homology
| Types | DnaRegion |
| Roles | engineered_region Device |
| Sequences | BBa_K2008007_sequence (Version 1) |
Description
This part is an improvement on the comK gene with the intent to allow the the part to be chromosomally integrated in the B. subtilis genome for increased long-term stability.Internal restriction enzyme sites (one SpeI site located at base pairs 170-175 and two EcoRI sites located at base pairs 557-562 & 572-577 in the comK part submitted by UofC_Calgary 2014) have been removed. In our version of comK, the SpeI site (ACTAGT) was deleted entirely, as the site was contained within a spacer region between the xylose-inducible promoter and RBS. To remove EcoRI sites, the DNA bases of the new comK construct were changed, and codons were optimized for B. subtilis:
553 A>G
556 C>T
568 A>G
571 C>T
As the UofC_Calgary 2014 iGEM team discusses in their comK part (BBa_K1444018), comK is the master transcription factor involved in the competency of B. subtilis. ComK further affects the other competence factors (comC, comE, comF, comG and comK) to increase B. subtilis??? ability to be transformed (van Sinderen et al., 1995). As such, when additional comK is transcribed, it increases B. subtilis transformation efficiency.
Also like the UofC_Calgary 2014 iGEM team, we included a xylose-inducible promoter upstream of the comK coding sequence. This allows for induction of competency upon the addition of xylose, which is beneficial as the usual starvation method for transformation of B. subtilis is typically time-consuming and costly.
Note: This part is a composite part, not a basic part, but it could not be added as composite at the time of entry.
Notes
Two internal EcoRI restriction sites and one internal SpeI restriction site to allow for this part to be BioBrick compatible. Flanking AmyE regions of homology were added to allow for chromosomal integration and increased long-term stability of the comK construct.Source
This part was amplified from the pAX01-comK plasmid constructed by Zhang and Zhang, 2010, and was previously characterized by the UofC_Calgary 2014 iGEM team. Flanking AmyE regions of homology were derived from the B. subtilis 168 genome.| Sequence Annotation | Location | Component / Role(s) |
| 5' amyE PxylA RBS Start codon comK Stop codon BBa_B0010 BBa_B0012 3' amyE | 1,125 126,280 328,339 343,345 346,918 919,921 922,1001 1010,1036 1037,1161 | sequence_feature feature/dna feature/promoter promoter feature/rbs ribosome_entry_site feature/start start_codon CDS feature/protein feature/stop stop_codon stem_loop feature/stem_loop stem_loop feature/stem_loop sequence_feature feature/dna |