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Before we get started:
"Control" is so-called because it is used to control the number of S. mutans, instead of killing it completely off, which wouldn't help the matter as indicated in [Modeling: Competition]. When the number of S. mutans exceeds the threshold that causes cavities, the circuit will be activated, thus killing the excess S. mutans.
In order to find and confirm the threshold that causes cavities, we designed different kinds and numbers of terminators (each with different leakage rates) to create the threshold. Moreover, we combined both wet lab results and modelling to decide which design is more suitable.
Over the years, it has been found that the competence stimulating peptide, CSP, a quorum sensing chemical, is released in every competence S. mutans. Thus, we can detect the number of S. mutans by marking the amount of CSP.
In S. mutans the CSP bind to the membrane receptor, "comD", thereby phosphorylating the response regulator, "comE". The phosphorylated comE will activate numerous vital biological mechanisms in Streptococcus mutans such as biofilm formation and the release of mutacin, an antibiotic peptide. Compared with all the other mechanisms involved with CSP, it is found that the promoter of gene "nlmC"( non-lantibiotic mutacin C) in S. mutans has the best performance under the stimulation of CSP.
At this point, the two main organisms we adapted for our project are our probiotics and M102 bacteriophage. We used one of the most commonly seen quorum sensing chemicals, AHL, N-Acyl-homoserine-lactone, as our indicator between phage and E.coli. Due to the idea of phage threshold control of the number of S. mutans in prevention of the second-dominant strain in the oral cavity, we envision a possibility to trigger another killing system to reach synergistic effect on diminishing S. mutans. If the threshold terminators were passed, the downstream signaling pathway would be switched on and contact with the engineered probiotics.
When the amount of the S. mutans is so large that the phages couldn't afford the loading, CSP would pass the threshold value and turn on the luxR-luxI system. LuxI acts as a gene which would generate AHL-synthase that yielding the quorum-sensing signal AHL right after the threshold level of S. mutans is exceeded. LuxR is a protein which forms a complex with AHL, and then turns into an activated form which will afterwards bind with the promoter pLuxR and trigger it. The signal sequence and endolysin downstream would be secreted and eliminate the excess S. mutans.
Figure 1. Gram positive bacteria have only one layer of cell wall outside of cell membrane, composing of peptidoglycan, while the cell wall of gram negative bacteria has additional protection outside peptidoglycan cell wall.[1]
Endolysin is an enzyme expressed by phage-infected bacteria. The C-terminus bind to the host cell wall, while the N-terminus is enzymatic domain. Working with holin, which lyses cell membrane, endolysin could break the cell wall of bacteria. Usually serve as peptidase, endolysin tend to work on gram positive species, which have layers of peptidoglycan cell wall. Though originally intended to be utilized inside the cell wall, endolysin can also be applied outside cell according to previous research. Furthermore, it is even less harmful to gram negative bacteria this way because of the lipid layer of those bacteria composes cell wall.
So how did we do it?
In our circuit, we incorporate nlmC promoter and different combination of terminators to create the threshold.
When the amount of S. mutans is so large that the phages couldn't afford the loading, CSP would pass the threshold value and turn on the luxR-luxI system. LuxI in S. mutans will be triggered by nlmC and activate AHL which will combine with LuxR produced by probiotics continuously. When LuxR combine with AHL to form AHL-LuxR complex, it will induce pLuxR promoter and trigger the Killer Module.
Killer Module
YebF is a protein that is naturally secreted by E.coli. It would transport proteins attached to it to the periplasm (space between cell membrane and cell wall), and later secrete it through porin on the cell wall. We chose YebF for its two advantages: one, it can be secreted in experimental strains which usually do not secrete proteins, along with other diverse passenger characters. Two, YebF can prevent endolysin from toxicating E. coli.
Putting it to the test!
Our result
Reference
- http://www.expertsmind.com/topic/microbiology/bacterial-cell-wall-92313.aspx
