Abstract
Site-directed mutagenesis is a key tool in the analysis of biological mechanisms. We have established an efficient and systematic gene targeting strategy for Bacillus subtilis based on the Golden Gate cloning methodology. Our approach permits the introduction of single or multiple point mutations or of heavily engineered alleles into the endogenous gene locus in a single step using a 96-well microtiter plate format. We have successfully applied this system for high-throughput functional screening of resized variants of the Structural Maintenance of Chromosome (Smc) protein and for exhaustive cysteine cross-linking mutagenesis. Here we describe, in detail, the experimental setup for high-throughput introduction of modifications into the B. subtilis chromosome. With minor modifications, the approach should be applicable to other bacteria and yeast.
Key words
- Golden Gate assembly
- Bacillus subtilis
- Gene targeting
- High-throughput screening
- Cysteine scanning
This is a preview of subscription content, access via your institution.
Buying options
Springer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Spizizen J (1958) Transformation of biochemically deficient strains of Bacillus Subtilis by deoxyribonucleate. Proc Natl Acad Sci U S A 44(10):1072–1078
Hamoen LW, Smits WK, Ad J, Holsappel S, Kuipers OP (2002) Improving the predictive value of the competence transcription factor (ComK) binding site in Bacillus subtilis using a genomic approach. Nucleic Acids Res 30(24):5517–5528
Tomasz A, Hotchkiss RD (1964) Regulation of the transformability of pneumococcal cultures by macromolecular cell products. Proc Natl Acad Sci U S A 51(3):480–487
Alexander HE, Leidy G (1950) Transformation of type specificity of Haemophilus influenzae. Proc Soc Exp Biol Med 73:485–487
Meibom KL, Blokesch M, Dolganov NA, Wu C-Y, Schoolnik GK (2005) Chitin induces natural competence in Vibrio cholerae. Science 310(5755):1824
Mell JC, Redfield RJ (2014) Natural competence and the evolution of DNA uptake specificity. J Bacteriol 196(8):1471
Blokesch M (2016) Natural competence for transformation. Curr Biol 26(21):R1126–R1130. https://doi.org/10.1016/j.cub.2016.08.058
Engler C, Kandzia R, Marillonnet S (2008) A one pot, one step, precision cloning method with high throughput capability. PLoS One 3(11):e3647. https://doi.org/10.1371/journal.pone.0003647
Engler C, Gruetzner R, Kandzia R, Marillonnet S (2009) Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS One 4(5):e5553. https://doi.org/10.1371/journal.pone.0005553
Diebold-Durand ML, Lee H, Ruiz Avila LB, Noh H, Shin HC, Im H, Bock FP, Burmann F, Durand A, Basfeld A, Ham S, Basquin J, Oh BH, Gruber S (2017) Structure of full-length SMC and rearrangements required for chromosome organization. Mol Cell 67(2):334–347.e335. https://doi.org/10.1016/j.molcel.2017.06.010
Burmann F, Basfeld A, Vazquez Nunez R, Diebold-Durand ML, Wilhelm L, Gruber S (2017) Tuned SMC arms drive chromosomal loading of prokaryotic condensin. Mol Cell 65(5):861–872.e869. https://doi.org/10.1016/j.molcel.2017.01.026
Aizenman E, Engelberg-Kulka H, Glaser G (1996) An Escherichia coli chromosomal “addiction module” regulated by 3′,5′-bispyrophosphate: a model for programmed bacterial cell death. Proc Natl Acad Sci 93(12):6059
Gerdes K, Christensen SK, Løbner-Olesen A (2005) Prokaryotic toxin–antitoxin stress response loci. Nat Rev Microbiol 3:371. https://doi.org/10.1038/nrmicro1147
Cegłowski P, Boitsov A, Chai S, Alonso JC (1993) Analysis of the stabilization system of pSM19035-derived plasmid pBT233 in Bacillus subtilis. Gene 136(1):1–12. https://doi.org/10.1016/0378-1119(93)90441-5
Lioy VS, Machon C, Tabone M, Gonzalez-Pastor JE, Daugelavicius R, Ayora S, Alonso JC (2012) The ζ toxin induces a set of protective responses and dormancy. PLoS One 7(1):e30282. https://doi.org/10.1371/journal.pone.0030282
Bieniossek C, Imasaki T, Takagi Y, Berger I (2012) MultiBac: expanding the research toolbox for multiprotein complexes. Trends Biochem Sci 37(2):49–57. https://doi.org/10.1016/j.tibs.2011.10.005
Benoit RM, Ostermeier C, Geiser M, Li JSZ, Widmer H, Auer M (2016) Seamless insert-plasmid assembly at high efficiency and low cost. PLoS One 11(4):e0153158. https://doi.org/10.1371/journal.pone.0153158
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Additional information
Author Contributions
F.B. and S.G. designed, established, and optimized the Golden Gate cloning and allelic replacement strategy. F.B. wrote code for automated primer design. M.-L.D.-D. wrote the draft manuscript, while F.B. and S.G. commented on the manuscript.
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Diebold-Durand, ML., Bürmann, F., Gruber, S. (2019). High-Throughput Allelic Replacement Screening in Bacillus subtilis . In: Badrinarayanan, A. (eds) SMC Complexes. Methods in Molecular Biology, vol 2004. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9520-2_5
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9520-2_5
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9519-6
Online ISBN: 978-1-4939-9520-2
eBook Packages: Springer Protocols