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Bacillus subtilis

Limited Extracellular Ligand Accessibility - Possible Solutions

One of the main advantages of using B. subtilis as a living biosensor lies in the presence of a single outer membrane. Theoretically, this would allow direct access of a membrane protein to extracellular ligands. However, B. subtilis has a thick peptidoglycan layer surrounding it, providing additional support and protection. Since this layer is fairly permeable and porous, it is estimated that globular proteins of up to 50kD are able to pass through without problem. However, if the target ligand is a larger protein, the thickness of the cell wall can pose an issue (1).

The ideal solution would involve using a cell wall-deficient B. subtilis, such as strain PDC204 and derivatives. These strains can easily be converted from the wildtype phenotype to the L-form state, which does not possess a cell wall. The operon MurE (involved in the creation of the cell wall) was modified to be under the control of a xylose-inducible promoter. Thus, bacteria grown in an osmotically stabilized medium without xylose will be devoid of a cell wall (2, 3). We have added background information about both the cell wall problematic and the strain PDC204 to the iGEM registry.

By using this strain as the chassis for a biosensor, B. subtilis can still be easily deliverable in either lyophilized or sporulated state. When the user has a sample to analyse, these bacteria would be reactivated in the media provided with the kit, and only then would they be converted to L-forms.

However, L-forms are fairly fragile, and this reduction in the viability of the cells could result in complications when employed as a biosensor. An alternative solution would be to supplement the media present in the detection kit with antibiotics such as trichokonin VI, which has a concentration-dependent effect on the cell wall of B. subtilis. This would reduce the density of the cell wall while preventing a significant reduction in the viability of the cells (4).

Protein Secretion Chassis - B. subtilis KO7 Strain

In recent years, B. subtilis rose in popularity within the industrial setting as a protein secretion chassis. This bacterial species is known to secrete a wide variety of proteins, making it a more desirable model organism for this purpose when compared to its gram-negative counterpart – E. coli. This characteristic is also desirable in biosensors or other catalytic systems where one or more steps involves the secretion of a given protein.

However, B. subtilis also secretes a considerable number of proteases to the extracellular medium. These enzymes will end up reducing both the half-life and the total amount of secreted protein – an undesirable outcome of any biotechnological application. In an industrial process, this issue leads to significant economic losses; in the case of a biosensor, it reduces the sensitivity of the system.

In earlier versions of NANOFLEX, the output of our biosensor involved the secretion of a signal protein to the extracellular medium. When looking for ways to further optimize our engineered signaling pathway, we came across the work of Dr. Daniel R. Zeigler – the director of Bacillus Genetic Stock Center, BGSC. He realized that a biotechnology project seeking a higher efficiency would naturally gravitate towards a B. subtilis strain where extracellular proteolytic activity is less prominent. Thus, he developed the B. subtilis KO7 strain, in which 7 genes for secreted proteases have been knocked out.

After being introduced to our project, BGSC kindly provided us with the KO7 strain, free of charge! Since we ended up designing a system where protein secretion was not necessary, we did not work with this strain. However, we decided to upload this background information about KO7 to the iGEM registry all the same, because we really believe this strain will be a major asset for future teams working with B. subtilis! You can also learn more about KO7 and other Bacillus-related products at BGSC.

Optimization of a B. subtilis Transformation Protocol

As soon as we decided to work with B. subtilis, we started contacting researchers in the field to collect more information on how to work with this bacterial species. One of them was a researcher at the New University of Lisbon (Isabel Sá-Nogueira), who kindly provided us with a transformation protocol.

After some trial and error, we managed to successfully transform B. subtilis! However, we came to realize that we were not experienced enough to conduct this protocol with an acceptable efficiency. With that in mind, we created an optimized version of the original protocol, more suitable for iGEM teams with little to no hands-on experience with B. subtilis. If you would like to have access to both the original and the new protocols, as well as learn more about the optimization, check out our handbook to the right, done in a collaboration with Generation Mendel, an iGEM team from Czech Republic!

Bookshelf

In addition to the B.subtilis Handbook, iGEM Uppsala 2020 presents:

References

1. Demchick, P., and Koch, A. L. (1996) The permeability of the wall fabric of Escherichia coli and Bacillus subtilis. Journal of bacteriology. 178, 768–773
2. Wolf, D., Domínguez-Cuevas, P., Daniel, R. A., and Mascher, T. (2012) Cell Envelope Stress Response in Cell Wall-Deficient L-Forms of Bacillus subtilis. Antimicrob. Agents Chemother. 56, 5907–5915
3. Domínguez-Cuevas, P., Mercier, R., Leaver, M., Kawai, Y., and Errington, J. (2012) The rod to L-form transition of Bacillus subtilis is limited by a requirement for the protoplast to escape from the cell wall sacculus: L-form escape. Molecular Microbiology. 83, 52–66
4. Su, H.-N., Chen, Z.-H., Song, X.-Y., Chen, X.-L., Shi, M., Zhou, B.-C., Zhao, X., and Zhang, Y.-Z. (2012) Antimicrobial Peptide Trichokonin VI-Induced Alterations in the Morphological and Nanomechanical Properties of Bacillus subtilis. PLoS ONE. 7, e45818