Background

The xylose inducible promoter BBa_K733018 was of interest to our group as a means to control expression of a toxin that would function as a counter-selective marker for marker free integration of large pieces of DNA into Bacillus subtilis. Our idea was to use it to select for transfer of the 40.8kb tyrocidine operon from Brevibacillus parabrevis to Bacillus subtilis. The limitation of this repressor and promoter control system is that, as in E. coli, the repressor is leaky [1]. When used as a control for expression of toxins, it is necessary that the repressor is tight. Bhavsar et al. identified an alternative xylose inducible promoter from Bacillus subtilis W23 that showed tighter repression in Bacillus subtilis than the promoter from B. megaterium (BBa_K733018). Lin et al. used this improved repressor to control expression of the native E. coli toxin in Bacillus subtilis. Using this system, they were able to do marker-less gene deletions. We investigated the repressor activity in E. coli with the intention of using it for selecting for transfer of large gene clusters.

Experimental Design and Methods

Generation of promoter constructs

The gblock and BBa_K733018 were amplified with primers containing 20-25 overhangs with plasmid or synthesized repressor, respectively. The two pieces of DNA were assembled by Gibson assembly according to the manufacture’s protocol. Transformants were selected and an overnight culture started. Plasmids were purified from overnight cultures and digested with restriction enzymes to confirm the correct insert.

 

Results

All transformants were tested in triplicate and the standard deviation is included in the error bars on the graph. The absorbance readings were averaged and corrected for background by subtracting the values of the media only samples. It is interesting to note that all samples grown in 1% xylose had a higher amount of biomass and indicates that E. coli can use it as a nutrient source. This appears to verify the hypothesis of {link to previous group’s wiki} regarding utilization of xylose as a nutrient source. GFP expression appears to begin after 0.5 hours of growth and reaches a maximum rate of synthesis by 2.5-3 hours of growth.

Figure 1 Biomass Absorbance readings measured at 620 nm were unstable for the initial two hours of the experiment so the Biomass measurements start at 2.2 hours in this graph. All samples had an initial OD600 of 0.1 at the start of the experiment.

Figure 2 Average GFP measurements corrected for the background of the media at an excitation of 488 nm and absorbance of 520 nm

Discussion and Conclusion

The optimized promoter for expression control in Bacillus subtilis did not have the proposed function in E. coli. Whether this is the same in Bacillus subtilis was not tested in this experiment, but according to previous findings, the promoter is optimized for control in B. subtilis [?,?]. While we set out to improve a BioBrick function, we were only able to improve the description of the existing BioBrick by providing more detailed data for induction GFP synthesis from BBa_K7330018 in a 1% xylose solution.

References

1. Bhavsar, A, Zhao, X, Brown, E. Development and Characterization of a Xylose-Dependent System for Expression of Cloned Genes inBacillus subtilis: Conditional Complementation of a Teichoic Acid Mutant. Appl. Environ. Microbiol. January 2001 vol. 67 no. 1 403-410.