Project Design
Our project design for measuring crosstalk between pathways needed something that was easy to build, affordable in in terms of new reagents, and modular enough to be used with other constructs we may work with. With that in mind, we went on to learn about the two main kinds of crosstalk in quorum sensing pathways.
One is what is called "chemical crosstalk", the interaction between an Homoserine Lactone(HSL) and a receptor protein. As the HSLs used in quorum sensing are quite similar, sometimes a small change in the number of carbons in a hydrocarbon tail can cause the same protein to end up recognizing and becoming active through non-cognate binding, creating crosstalk.
Another way of losing orthogonality is genetic crosstalk. This happens when proteins from a quorum sensing pathway activate and lead to the transcription of genes of promoters belonging natively to other pathways. As chemical crosstalk is more thorughly characterized in the literature, we focused on genetic crosstalk and had to design experiments combining different receptor proteins and promoters. Considering this, our design consisted of an auto-inducible, two plasmid system. In the first plasmid, we have our signal synthase and receptor protein genes. We called this composite construct our Sender system.
The promoter for the receptor gene is constitutive (BBa_J23104), so the protein would always be present throughout our experiments, being capable of readily forming a complex with the HSL molecules and triggering transcription of our reporter genes. This sequence would be downstream of a HSL synthase gene, which is what would start the quorum sensing signal. The choice of using an HSL synthase instead of synthetic purified HSLs was to represent a closer approximation of what an experiment in a bioreactor or real applications would look like. Our point of view is that in real-world applications, many of the HSL concentrations tested in the literature are not realistic coming from the premise of using synthases. And besides, the high price of these molecules that didn't quite fit our project's budget. To be able to control when and where our quorum sensing signal would be initiated, the promoter used for this gene was pBad, coupled with an araC gene for an initial negative inducible state (BBa_I0500).
The second plasmid was called our Receiver system, composed of two genes. One was a reporter gene with a quorum-sensing-activated promoter encoding for EYFP (BBa_E0030). The other gene (BBa_I13602) expressed ECFP - with an LVA tail for degradation - constitutively and served as a control for extrinsic variations in the fluorescence measurement. With this double-reporter system, we would be able to have a ratiometric measurement of YFP/CFP fluorescence that would more finely represent the effect of our other system's activation, instead of presenting variation due to plasmid copy number, cell number, medium differences, or other external factors.
With this two plasmid layout, we would put our six quorum sensing systems along twelve plasmids, and co-transform them into E. coli to get a complete and functional system, with 36 possibilities of Sender-Receiver combinations, testing for the system's cognate and non-cognate response. The choice to separate the system in two plasmids was made to give more modularity to our project, making it capable of being used along with other Sender or Receiver modules from other projects, and to lower the number of constructs we would have to do. We used pSB1A3 as the vector backbone for Sender plasmids and pRSF for Receivers. The choice was made to have compatibility between replication origins, along with having one plasmid with a smaller copy number and lower the metabolic load.
For empty plasmid controls, we had one for both occasions. When using an empty Sender plasmid, we co-transformed our systems with pSB1A3 with only pBad/araC, making our experiments with this control report only promoter leaking. When using an empty Receiver, we co-transformed with a pRSF backbone only with our part BBa_K2771020 and no promoter, so this control showed only cell autofluorescence, while still growing at a rate similar to our test cells.
Some of our promoters to be tested (pLux, pLas, pTra, pRpa) were ordered from IDT because of modifications we wanted to test, coming from two different previous works (Grant et al. for pLux and pLas, and Scott, Hasty for pTra and pRpa). The thought process behind this choice was to intertwine this two project's works, and check if two parallel optimizations could still retain orthogonality.
References
- Grant, Paul K et al. “Orthogonal Intercellular Signaling for Programmed Spatial Behavior.” Molecular Systems Biology 12.1 (2016): 849. PMC. Web. 16 Oct. 2018.
- Spencer R. Scott and Jeff Hasty. “Quorum Sensing Communication Modules for Microbial Consortia” ACS Synthetic Biology 5.9 (2016), 969-977 doi: 10.1021/acssynbio.5b00286
- N.Kylilis, Z.A. Tuza, G. Stan, K.M. Polizzi. “Tools for engineering coordinated system behaviour in synthetic microbial consortia” Nature Communications, volume 9 (2018), Article number: 2677.
- R.J.Case, M.Labbate, S.Kjelleberg. “AHL-driven quorum-sensing circuits: their frequency and function among the Proteobacteria” The ISME Journal, volume 2, pages 345–349 (2008)
- T.J.Rudge, J.R.Brown, F.Federici, N.Dalchau, A.Phillips, J.W.Ajioka, J.Haseloff. “Characterization of Intrinsic Properties of Promoters” ACS Synthetic Biology 5 (1), 89-98, (2016) doi:10.1021/acssynbio.5b00116