Team:Calgary/Project/BsDetector/BsChassis

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B.s. Chassis

Why Bacillus subtilis?

Bacillus subtilis is capable of natural transformation. Under conditions of nutrient deprivation (energy starvation), B. subtilis efficiently uptakes 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 easy to induce sporulation in B. subtilis. This leads to the generation of robust spores that can be activated back to their normal state when needed.

Transformation

Although B. subtilis is naturally transformable, the traditional transformation process via starvation is difficult 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.

Figure 1: Diagram of DNA uptake within B. subtilis (Maier et al., 2004). Five genes are involved in the mechanism of competency seen in B. subtilis; comC, comE, comF, comG and nucA (Hamoen, Venema, Kuipers & 2003). comG encodes several proteins which assemble to form a pilus structure. comC facilitates the correct assembly of this structure. comE encodes for a polytopic transmembrane protein (ComEC), which forms a pore for DNA entry. dsDNA is unwound and converted into ssDNA upon entry by a protein (resembling DNA-helicase) encoded by comF. ssDNA is eventually integrated into the genome through the function of the general DNA-recombination factor RecA and the DNA-helicase AddAB.

In our “supercompetent” plasmid constructed by Zhang & Zhang (2010), the control of the master regulator comK is placed under the xylose-inducible promoter PxylA. This means that if we add xylose to the cells, they will activate PxylA 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 PxylA, 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%.

We further optimized incubation conditions by investigating whether or not shaking had an adverse effect on colony growth. As previously described above in Figure 1, DNA uptake in B. subtilis involves a set of machinery working together with the pilus to allow foreign DNA to cross the membrane. We postulated that excessive shaking can lead to disturbance of this uptake process. Our findings are presented below.

Figure 4: The effect of shaking on colony growth. Following addition of DNA to competent B. subtilis cells, the tubes were incubated at 37°C either without shaking (left) or shaken at 200 rpm (right). The lack of shaking resulted in greater colony growth on selective plates. Given that no shaking results in more transformed colonies, we implemented this change in protocol.

Besides the optimization of incubation conditions outlined above, we also spent time investigating how the entire process carries over in small liquid volume form. This is because the “small, liquid volume” model will most closely approximate the transformation process occurring within our physical B. s. detection device. We quantified results of optimization via OD readings on an ELISA plate reader. These results are quantified below in Figure 7.

Figure 5: Absorbance readings of small volume B. subtilus in liquid volume for different xylose concentrations. B. subtilis SCK6 pAX01-comK cells in small liquid volume transformed with linearized plasmid DNA and incubated at 37°C with different amounts of xylose, followed by growth and addition of selection marker (antibiotic). All transformation occurred in liquid. The negative control comprised of untransformed cells (killed by antibiotic), while for positive control, antibiotic was not added to untransformed cells. There was a slight increase as xylose concentration increased.

In addition to manipulating incubation conditions for the optimization of the transformation protocol, we generated a “standard curve” of changes in OD 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 OD reading at 600 nm 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 OD600.

Using Figure 6 means that there is no need to take an OD 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.

In addition to optimization of the B. subtilis transformation process, we also investigated the length of DNA required. We began by studying existing literature, and found that 70 bp is the minimum size for successful uptake. There is a linear relationship between substrate length and transformation efficiency (Khasanov et al, 1992). We were interested in finding out the required DNA length because it needed to be compatible with our isothermal PCR process.

We designed specific primers spanning the thrC region. These primers amplified the spec and RFP casettes with varying lengths of homologous sequence. The smallest length resulting in successful transformation with the desired product was 125 bp.

Figure 7: B. subtilis was transformed with DNA containing various lengths of homologous sequence and was plated on selective spectinomycin plates. Plate A shows B. subtilis colonies transformed with digested thrC vector (700, 1300 bp of homology). Plate B, C and D show B. subtilis transformed with PCR product 2,3 and 4 respectively (215 bp, 190 bp and 125 bp of homology).

These experiments were necessary to carry out, because the results enabled us to optimize isoPCR as well. That is, determining the proper length of uptake DNA informs the timing and duration of the amplification step.

Sporulation

One compelling reason for choosing this organism was its tendency to sporulate, a process by which bacteria enter a dormant state in order to survive adverse conditions such as starvation, irradiation, and heat (Eichenberger, 2012). B. subtilis has two types of states: vegetative and spore. In the vegetative state, bacteria are able to use nutrients and can actively divide. For the spore state, bacteria are metabolically inactive with the genetic material stored and protected in the spore coat (Eichenberger, 2012). When bacteria sense insufficient nutrients, they convert from the metabolically active vegetative state to the metabolically inactive spore state (Eichenberger, 2012). Once sufficient nutrients are available, spores switch back to the vegetative state.

The ability of B. subtilis to form spores under extreme conditions is beneficial for our purposes because the durability of the bacteria facilitates transportation of our diagnostic tool around the world.

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