Difference between revisions of "Part:BBa K1444018"

Revision as of 21:51, 16 October 2014

Xylose -> comK

Xylose-induced activation of the B. subtilis natural transformation system.

Sequence and Features

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).

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).

Usage and Biology

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

Transformation 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
• Dilute the culture to an OD₆₀₀ 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

Diluting a B. subtilis culture

Figure 1: The decrease in optical density from addition of fresh LB media to an overnight culture of B. subtilis.

The second step in the protocol above calls for the overnight culture of B. subtilis. This can be rather tedious, so we generated a standard curve of how the OD₆₀₀ decreased with the addition of LB media. 500 µL of an overnight culture was diluted, and the decrease in OD₆₀₀ was noted. This allows a user to measure the cell density of their overnight culture, and then consult the graph to determine how much fresh LB must be added to drop the absorbance to 1.0.

Effects of temperature and xylose

Figure 2: The effects of temperature and xylose concentration on the transformation efficiency of B. subtilis.

We have tested the original transformation protocol, and characterized several aspects of it. Here, we modified the concentration of xylose that the B. subtilis cells were exposed to, as well as the temperature that they were cultured in after addition of linearized plasmid DNA containing an antibiotic resistance cassette. After a 2-hour period, the cultures were spread onto selective media, and the colonies counted.

References

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