Designed by: Yuancheng Ding   Group: iGEM22_Imperial_College_London   (2022-09-30)



Introduction to this part

This part contains PsspB, GerAK_modified_chimera, and tL3S2P21

Our Experiment Design

Given the essential requirement of our sensing system to be orthogonal to chitin and chitin only, a strain featuring knockouts of all other germinant receptors had to be developed, to avoid spontaneous and off-target germination of the spore in the presence of other germinants readily available in nature.Initially, we set out to create both a GerA and GerK KO mutant harnessing vector pMAD2 from the SubtiToolKit. To that end, we designed primers for the amplification of homology regions (1kb in size) upstream and downstream of the target genomic loci where the germinant receptor operons are located. These were designed utilizing the SubtiWiki tool which allows users to browse the genome of B. subtilis 168. To screen for successful KO generation, our plan was to introduce a Km marker and a fluorescent readout, allowing us to pinpoint colonies without the receptors. We were able to successfully amplify homology arms for both GerA and GerK. However, when actually trying out the assembly of the KO inducing cassette, we failed to obtain a viable plasmid. Hence, we looked at other ways to source such a strain. The modified GerAK chimera has a modified overhang.

Our Experiment Result

We successfully amplified the homology arm1 and homology arm2 (HA1+HA2) of GerK. The knockout strain was acquired with the generous contribution of Dr. Graham Christie at the University of Cambridge, who agreed to share with the team a strain named Ger3 featuring gene knockouts of GerA, GerB and GerK.

Figure 1. The scheme of modified GerAK chimera
Figure 2. The PCR result of GerK KO



[2] Ramírez-Guadiana, F.H., Meeske, A.J., Wang, X., Rodrigues, C.D.A. & Rudner, D.Z. (2017) The Bacillus subtilis germinant receptor GerA triggers premature germination in response to morphological defects during sporulation. Molecular Microbiology. 105 (5), 689–704. doi:10.1111/mmi.13728.

[3] Setlow P. Spore germination. Curr OpinMicrobiol. 2003 Dec;6(6):550-6. doi: 10.1016/j.mib.2003.10.001. PMID: 14662349.

[4] Setlow P. Germination of spores of Bacillus species: whatwe know and do not know. J Bacteriol. 2014 Apr;196(7):1297-305. doi: 10.1128/JB.01455-13.Epub 2014 Jan 31. PMID: 24488313; PMCID: PMC3993344.

[5] Zhang JQ, Griffiths KK,Cowan A, Setlow P, Yu J. Expression level of Bacillus subtilis germinantreceptors determines the average rate but not the heterogeneity of sporegermination. J Bacteriol. 2013 Apr;195(8):1735-40. doi: 10.1128/JB.02212-12.Epub 2013 Feb 8. PMID: 23396907; PMCID: PMC3624553.

[6] Blinker S, Vreede J, Setlow P, Brul S. (2021) Predicting theStructure and Dynamics of Membrane Protein GerAB from Bacillus subtilis. Int JMol Sci. 2021 Apr 6;22(7):3793. doi: 10.3390/ijms22073793. PMID: 33917581;PMCID: PMC8038838.

[7] Artzi L, Alon A, Brock KP, Green AG, Tam A, Ramírez-GuadianaFH, Marks D, Kruse A, Rudner DZ. (2021) Dormant spores sense amino acidsthrough the B subunits of their germination receptors. Nat Commun. 2021 Nov25;12(1):6842. doi: 10.1038/s41467-021-27235-2. PMID: 34824238; PMCID:PMC8617281.

Sequence and Features

Assembly Compatibility:
  • 10
    Illegal EcoRI site found at 271
  • 12
    Illegal EcoRI site found at 271
  • 21
    Illegal EcoRI site found at 271
  • 23
    Illegal EcoRI site found at 271
  • 25
    Illegal EcoRI site found at 271
    Illegal NgoMIV site found at 66
  • 1000