B.s. Chassis

Why Bacillus subtilis?

Bacillus subtilis is capable of natural transformation. Under conditions of nutrient deprivation (energy starvation), B. subtilis can be made to uptake foreign DNA. We can also bypass the need for energy starvation to make transformation even easier! By using cells derived from a specific strain of B. subtilis (SCK6) with the pAX01-comK plasmid (Zhang & Zhang, 2010), we can use xylose to induce competence and transform B. subtilis with our DNA constructs. Read more about this transformation process in our protocol manual for B. subtilis.

In addition, it is also easy to induce sporulation in B. subtilis. This leads to the generation of robust spores that can be activated when needed.

Transformation

Although B. subtilis is naturally transformable, the traditional transformation process via starvation is hard to control, time-consuming and labour-intensive. Fortunately, 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 & Zhang (2010).

The key feature conferred by this plasmid is the overexpression of comK, which is the master regulator of competence in B. subtilis. It is known as such because it encodes a transcription factor which upregulates the expression of these competence genes (comC, comE, comF, comG, nucA). All these genes contribute to the DNA uptake mechanism in B. subtilis.

In our “supercompetent” plasmid constructed by Zhang & Zhang (2010), the control of the master regulator comK is placed under the xylose-inducible promoter P_xylA. This means that if we add xylose to the cells, they will activate P_xylA and subsequently comK, resulting in the “turning on” of competence genes. The cells can then uptake foreign DNA without the need for energy starvation. The figure below depicts the pAX01-comK plasmid (Zhang & Zhang, 2010).

Figure 2. pAX01-comK vector map (Zhang & Zhang, 2010). Constructed plasmid for B. subtilis wherein the master regulator comK competence gene is placed under the control of P_xylA, a xylose-inducible promoter.

The transformation protocol utilized by Zhang & Zhang (2010) with the pAX01-comK plasmid in SCK6 included the addition of 1% xylose in LB to an overnight culture of SCK6 cells. The amount of xylose added was dictated by OD values. After taking an initial reading at OD₆₀₀¬, the 1% xylose in LB solution was added until the reading was 1.0; the culture was then grown for 2 hours to develop competence. This means that the final xylose concentration of cultures was not fixed, as it depended on the cell growth that occurred after the overnight incubation. Thus, we were curious as to the optimal incubation conditions (temperature, degree of shaking), and xylose concentration. To investigate the latter, we modified the protocol by diluting with only LB to obtain the desired OD reading. We then added the appropriate volume of a stock 20% xylose solution to obtain desired final xylose concentrations. The results of manipulating xylose concentration at different temperatures are shown below in Figure 2.

Figure 3. Colony growth of transformed B. subtilis at 30°C and 37°C for different xylose concentrations. Bacillus subtilis SCK6 pAX01-comK cells transformed with linearized plasmid DNA and incubated at 30°C and 37°C with different concentrations of xylose, followed by growth on selective media. Colony counts were higher for all concentrations of xylose when grown at 37°C. The conditions resulting in highest growth were an incubation temperature of 37°C and a xylose concentration of 2%.

In addition to manipulating incubation conditions for the optimization of the transformation protocol, we generated a “standard curve” of changes inn absorbance readings corresponding to different volumes of LB added to a given volume of B. subtilis overnight culture. The graph is shown below in Figure 5.

Figure 6. Decrease in absorbance reading at OD₆₀₀ for different volumes of LB added to overnight B. subtilis cultures. We added different volumes of LB to 500 μL of B. subtilis overnight culture and graphed the subsequent decrease in absorbance reading at OD₆₀₀.

Using Figure 6 means that there is no need to take an absorbance reading at every addition of a minute volume of LB. Instead, we can simply take an initial OD reading, note the difference to the desired reading of 1.0, and find the corresponding volume of LB that needs to be added to obtain this desired reading.

We thus optimized the transformation protocol developed by Zhang & Zhang (2010) by changing incubation conditions. We also developed tools for making transformation more convenient to carry out. Our modified protocol was used in order to transform B. subtilis more easily and obtain greater numbers of transformed colonies.

Sporulation