Part:BBa_K4212009
GerAK_chimera
Synthetic operon designed with GerAA, GerKB and GerAC coding sequences from the B. subtilis genome. Designed to be transformed in a B. subtilis knockout strain so as to produce a germinant receptor that contains a binding site for glucose, and when activated triggers an orthogonal signalling cascade resulting in germination. The GerAA sequence features a C-terminus fusion to a GFPmut3b coding sequence. This is to enable visualisation of the chimeric receptor on the surface of the spore via fluorescence microscopy.
Usage and Biology
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. 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. 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. The checked no germination in response to NAGdry lab loop: megateriumwork towards formation of chimerastalked to experts about this, possible to engineer. Cristhie recommended adding a GFP fusion to allow for easy visualisaiton of receptor. Test with cocktail of germinants of KB, not AB.
References
[1] Wuytack, E.Y., Soons, J., Poschet, F. & Michiels, C.W. (2000) Comparative Study of Pressure- and Nutrient-Induced Germination of Bacillus subtilis Spores. Applied and Environmental Microbiology. 66 (1), 257–261. doi:10.1128/AEM.66.1.257-261.2000.
[2] Stewart, K.-A.V. & Setlow, P. (2013) Numbers of Individual Nutrient Germinant Receptors and Other Germination Proteins in Spores of Bacillus subtilis. Journal of Bacteriology. 195 (16), 3575–3582. doi:10.1128/JB.00377-13.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 2384
Illegal EcoRI site found at 4339
Illegal PstI site found at 533
Illegal PstI site found at 4290 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 2384
Illegal EcoRI site found at 4339
Illegal NheI site found at 4
Illegal PstI site found at 533
Illegal PstI site found at 4290 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 2384
Illegal EcoRI site found at 4339
Illegal BglII site found at 3871
Illegal XhoI site found at 640 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 2384
Illegal EcoRI site found at 4339
Illegal PstI site found at 533
Illegal PstI site found at 4290 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 2384
Illegal EcoRI site found at 4339
Illegal PstI site found at 533
Illegal PstI site found at 4290 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 3526
| None |