Part:BBa_K2008007

Xylose->comK with AmyE homology

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. The regions of homology are 125 bp in length and are derived from the amyE gene in B. subtilis, a nonessential gene which encodes the alpha-amylase subunit required for starch degradation.

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 version submitted by UofC_Calgary 2014 (https://parts.igem.org/Part:BBa_K1444018) 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, (https://parts.igem.org/Part:BBa_K1444018), comK is the master transcription factor involved in the competency of B. subtilis. The transcription factor it codes for, ComK, upregulates other competence factor genes (comC, comE, comF, and comG) to increase B. subtilis’ ability to be transformed (van Sinderen et al., 1995). As such, when additional comK is transcribed and translated, 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, costly, and inefficient.


References

van Sinderen, D., Luttinger, A., Kong, L., Venema, G., and Hamoen, L. (1995). comK encodes the competence transcription factor, the key regulatory protein for competence development in Bacillus subtilis. Molecular Microbiology, 15(3), 455-462.

Note: This part is a composite part, not a basic part, but it could not be added as composite at the time of entry.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 322
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 862