Knockout strain generation

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.

Figure 1.The gel image result of the amplified GerA and GerK HA1 and HA2. We used oSP029-32 for GerA HA1 and HA2, and oSP045-48 for GerK HA1 and HA2.

Figure 2. Result of restriction digestion using BsmbI of L1 plasmids containing GerA HA1 and HA2, and GerK HA1 and HA2

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.

Chimeric receptor assembly

Figure 3.The schematic view of the arrangement of the GerA operon, including the GerAA, GerAB, and GerAC subunit coding sequences, in the genome of B. subtilis 168.

Figure 4.The schematic view of our refactored chimeric operon, including coding sequences of the GerAA, GerKB and GerAC subunits.

Although the directed evolution approach towards engineering the GerA receptor resulted in being out of our reach within the scope of iGEM, we elected to explore a hypothesis of germinant receptors being orthogonal as a potential avenue towards achieving targeted germination. This chimeric receptor would be composed of: GerAA, GerAC and GerKB subunits, with the KB originating from a different operon, as outlined here. Our hypothesis is that if germinant receptors are modular in B. subtilis, the inclusion of KB will allow for full germination resulting from sensing of glucose, of which the binding receptor is found in this subunit. To test the possibility of modularity of these receptors, we would first need to assess whether these can form at all and localize in the spore coat, before looking into functionality. Thus, the chimeric operon was designed featuring a C’ terminal GFP fusion at the GerAA subunit. This would allow validation of the formation of the protein complex via fluorescence microscopy. Speaking to B. subtilis spore expert, Graham Christie, this was considered the most effective approach as extraction and purification of spore coat proteins is not feasible. This experiment would have to be done in the B. subtilis Ger3 strain which contains knockouts of all germinant receptor operons in the genome, so that we can be sure that the only subunits interacting are the ones we introduce.
The in silico designed chimeric operon, was ordered as 3 separate gblocks from IDT. Within the wet lab, we were able to successfully assemble the L0 plasmid where the three segments are joined scarlessly into one.

Figure 5.Result of restriction digestion of L0 Chimera plasmids with BsaI. Expected size of bands: 2080bp and 4458bp.

Figure 6.Result of restriction digestion of L1 Chimera plasmids with EcorI. Expected size of bands: 1955bp and 6749bp.

We were also able to successfully assemble a L1 EXP plasmid ready to be transformed in B. subtilis, featuring the PsspB sporulation-specific promoter and L3S2p21 (STK077) terminator. Native RBSs were included in the gblocks ordered, in order to ensure we maintained the correct ratio subunits. Unfortunately, transformation of this plasmid into the Ger3 strain proved to be a bottleneck, with two transformations attempted yet no colonies forming.
Within the B. subtilis family of germinant receptors, GerA is the only one capable of orthogonal germination and activated by a single germinant, meaning binding of the native ligand L-alanine to the receptor can result in full germination without other receptors having to be activated. This is not the same for other receptors such as GerK and GerB. However, these have a similar structure to GerA (3 sub-units), whilst having affinity to other ligands such as glucose and fructose, in their B sub-unit, the one proposed in literature as being home to the binding site [1]. The scientific question we are interested in exploring is whether the different Ger family subunits are modular. Would it be possible to build a chimeric receptor with GerA subunits involved in signal transduction and GerK subunit containing the binding site? Modularity in the Ger family would unlock several possibilities in targeted germination engineering, by enabling activation of the orthogonal signalling cascade belonging to GerA in response to alternative ligands. The B subunits of other germinant receptors would also be easier to engineer via directed evolution to respond to ligands structurally similar to their native ones, for example GerKB contains a binding site for glucose thus modifying its binding affinity towards another sugar would be easier than in the case of GerA which natively binds to an amino acid. Interestingly, modularity has already been observed in another Bacillus species, namely Bacillus megaterium.
We initially tested our hypothesis computationally, you can explore results of this work here.
Speaking to Bacillus subtilis experts Dr. Graham Christie and Prof. Rudner, we received some useful feedback that guided our design of a chimeric receptor to test in the wet lab, capable of triggering full germination in response to glucose.

References

[1]Ross C, Abel-Santos E. The Ger receptor family from sporulating bacteria. Curr Issues Mol Biol. 2010;12(3):147-58. PMID: 20472940; PMCID: PMC3081667.