Team:Wageningen UR/Description/Regulation
Detecting Mites
YOUR TEXT HERE
Quorum Sensing
Population dynamics
Overhauling population dynamics to improve toxin production
In our quest to save the honey bees from Varroa destructor we envisioned using Bacillus thuringiensis cry toxins. When activated and concentrated near the surface of cell membranes, Bt-toxins form pores inside the membrane, lysing the cell as a result1. High level constitutive Bt-endotoxin expression in Escherichia coli is known to inhibit growth and possibly kill the producing cells2. Our overall aim is to provide a better alternative for toxin production where production does not impair bacterial growth nor population survival.
Solutions to the problem of toxic expression are found in mechanisms that regulate protein production to minimize the negative effects on growth and survival3. Inducible expression for instance, is widely used to manually confine toxin expression in time to after the exponential growth phase of bacteria. We propose a regulation system that enables E. coli to separate the growth and the production phase by only expressing recombinant proteins when bacterial cell density is high. We cannot expect beekeepers to measure bacterial growth and induce expression themselves when the time is right.
Of course, synchronized toxin overexpression in the entire population still means death of all E. coli cells after the first wave of toxin expression. It would be more beneficial to increase the survival chances of some bacteria by keeping them as non-producers. If these cells can survive the production phase and last until the quorum sensing signal has died away, they would be able to initiate a new growth phase. It is important that these survivor cells are genetically identical to the toxin producers, otherwise a second growth phase is irrelevant.
Thomas designed a system that combines quorum sensing and non-producing subpopulations. He tested the design (figure 1) in discussions with other student- and supervisor team members and improved and simplified (the design once included both Cas9 and CPF1!) based on their feedback. The separate parts of the system were cloned in bacteria and tested with GFP and RFP reporters by Thomas.
