Difference between revisions of "Part:BBa K1444018"

Revision as of 02:22, 16 October 2014

Xylose -> comK

Bacillus subtilis is a gram-positive bacterial species widely used in molecular biology labs around the world. It is capable of natural transformation under conditions of nutrient deprivation (energy starvation), and unlike many gram-negative species B. subtilis does not appear to require a specific uptake sequence. However, transformation through starvation may not be the most ideal process to implement in the lab as the protocols are very time-consuming, very sensitive to precise timings, and can be unreliable. Fortunately, all of the DNA uptake genes are under the control of a single transcription factor, so we can bypass the need for energy starvation by using cells derived from a specific strain of B. subtilis (SCK6) with the pAX01-comK plasmid constructed by Zhang (2010) Zhang.

This part contains a xylose-inducible promoter (pxylA), a ribosome binding site, and the comK coding sequence. This part is meant to be inserted into the genome of B. subtilis using an appropriate integration vector (unfortunately likely requiring the traditional transformation procedure). Once complete, you have a strain that can be transformed quickly and easily.

It must be noted that this sequence still contains 1 SpeI and 2 EcoRI sites. As such, cloning with this part would necessitate using either XbaI and PstI, or NotI (it is a self-contained generator, so the directionality in the genome is not important).

References

<biblio>

1. Zhang Zhang, X-Z., & Zhang Y-H. (2010). Simple, fast and high-efficiency transformation system for directed evolution of cellulase in Bacillus subtilis. Microbial Biotechnology, 4(1):98-105

</biblio>

Usage and Biology

Though the DNA was not submitted to the iGEM registry, we have characterized the strain that we amplified the part from.

Protocol

The protocol is described in detail [http://2014.igem.org/Team:Calgary/Notebook/ProtocolManual/Bsubtilis here], but in brief:

• Grow B. subtilis overnight at 37 C with shaking
• Subculture to an OD of 1.0
• Add xylose dissolved in LB to a final concentration (w/v) of 2%
• Grow for 2 hours with shaking
  • Freeze aliquots if desired at this point
• Add DNA (linearized plasmid or PCR product), grow for 2 hours with NO shaking
• Plate overnight with appropriate selection

Sequence and Features