Thymine Dependent Bacillus subtilis for Spore ContainmentJulissa van Renselaar

Figure 1. BGSC Library was utilized to order BKE and BKK strains. Using high fidelity PCR, the antibiotic cassette from BKK 21820 was amplified and used as a transformant. Figure shows PCR product [2].

Created with BioRender.comby AJ Walters.

Background§ Inactivation of thyA and thyB genes creates a complete dependence of

thymidine in Bacillus subtilis 168 [1]§ The double knockout strain has reduced fitness [1]§ Yeast extract in the growth media greater than 2 mg/mL has the potential

to affect transformation rate due to the interference of the folate pathway [1]

§ Through homologous recombination, the antibiotic cassette can be transformed in place of the gene of interest [2]

§ Bacillus subtilis has natural competence and can be efficiently transformed with linear DNA [3]

§ Bacillus subtilis 168 is tryptophan deficient [4]

AbstractThe GEAR UP lab at Utah State University (USU) was tasked with making a Bacillus subtilis classroom kit that would be safe and contained for student access. Bacillus subtilis is a sporulating bacteria that allow it to survive unfavorable conditions. A common method for disposal of endospores is autoclaving with conditions set at 121° C and 15 psi. These conditions are not favorable or accessible in a classroom setting. Proactive measures were taken by creating another condition of growth for Bacillus subtilis 168. In order to create this additional condition, a double knockout of the thymine coding genes was performed modeled after Hosseini et. al thymineless death paper [1]. With the adjustments of protocols for cell competence, transformation, and minimal media BKE 17680 ∆thyA:: erm ∆thyB:: kanwas successfully created. Further research utilizing the Cre plasmid, pDRR244 needs to be done to loop out the antibiotic cassettes as it will affect downstream processing [2].

Figure 2. PCR products: Q5 and KOD. Both polymerases are high fidelity but KOD produces a higher quantity of DNA. This was beneficial when preforming cell transformation. Cycling conditions were modeled after Sigma-Aldrich Protocol.

Methods1) Single knockouts of Bacillus subtilis 168 were ordered from Bacillus Genetic

Stock Center (BGSC)2) Primers for DNA construct of BKK 21820 ΔthyB::kan were designed using

Benchling and Primer3Plus3) Genomic DNA (gDNA) of BKK 21820 - ΔthyB::kan was extracted4) PCR with KOD polymerase5) PCR cleanup6) Cell competence of BKE 17680 ΔthyA::erm and transformation of BKK 21820

ΔthyB::kan DNA construct

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Figure 3. Cell Competence protocol was modeled after Yasbin et al. Bacillus subtilis competence protocol [5]. Adjustments were made with the utilization of the two growth medias. LB was shown to get to a desired competence faster and higher transformation efficiency.

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Results§ A double knockout strain of Bacillus subtilis 168 was successfully

created, BKE 17680 ∆thyA:: erm ∆thyB:: kan§ Growth on LB Media affected timeline of growth§ 100 ng of transformant was needed for a successful transformation hence

the need for KOD polymerase

Figure 4. Transformations plated on LB agar with both growth medias as LB media. Photo on the left was taken after 18 hours of incubation. Photo on the right was taken after 45 hours of incubation.

References[1] Hosseini, S., Curilovs, A., & Cutting, S. M. (2017). Biological Containment of Genetically Modified Bacillus subtilis. Applied and Environmental Microbiology, 84(3), 1–14. https://doi.org/10.1128/aem.02334-17[2] Koo, B.-M., Kritikos, G., Farelli, J. D., Todor, H., Tong, K., Kimsey, H., Wapinski, I., Galardini, M., Cabal, A., Peters, J. M., Hachmann, A.-B., Rudner, D. Z., Allen, K. N., Typas, A., & Gross, C. A. (2017). Construction and Analysis of Two Genome-Scale Deletion Libraries for Bacillus subtilis. Cell Systems, 4(3), 291-305.e7. https://doi.org/10.1016/j.cels.2016.12.013[3] Wu, G., Drufva, E., & Wu, K. (2019). Fast genome editing in Bacillus subtilis. Engineering in Life Sciences, 19(6), 471–477. https://doi.org/10.1002/elsc.201800164[4] Zeigler, D. R., Prágai, Z. ́., Rodriguez, S., Chevreux, B., Muffler, A., Albert, T., Bai, R., Wyss, M., & Perkins, J. B. (2008). The Origins of 168, W23, and Other Bacillus subtilis Legacy Strains. Journal of Bacteriology, 190(21), 6983–6995. https://doi.org/10.1128/jb.00722-08[5] Yasbin, R. E., Wilson, G. A., & Young, F. E. (1975). Transformation and transfection in lysogenic strains of Bacillus subtilis: evidence for selective induction of prophage in competent cells. Journal of Bacteriology, 121(1), 296–304. https://doi.org/10.1128/jb.121.1.296-304.1975

AcknowledgementsI would like to thank Dr. Ryan Jackson for his guidance and advice during the completion of this study. In addition, I would like to thank the USU GEAR UP undergraduates’ students including but not limiting to AJ Walters, Clark Riddle, Amanda Moravek, and David Hall for their recommendations and instruction that enabled this project to be successful. I would also like to thank Dr. Daniel Ziegler of the Bacillus Genetic Stock Center for his advice and direction on the Bacillus subtilis knockouts. USU STARS! GEAR UP played an essential role with their financial support and encouragement that made this project possible.

Ladder KOD Q5

Study conducted with funds from USU STARS! GEAR UP.

Department of Biological Engineering Utah State University Logan, UT

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