Regulation of toxin expression

A problem that is encountered when expressing Bt toxins is that overexpression can be lethal to the bacterial chassis, in our case Escherichia coli. We aimed to prevent premature lysis of BeeT and achieve higher toxin yield by separating the growth phase from the toxin-producing phase. Toxin expression in coupled to quorum-sensing: only when a sufficient number of bacteria are present, the toxin is produced. Furthermore, we aimed to create a subpopulation of bacteria that do not produce the toxin, even when the bacterial density is high. When the toxin-producing bacteria perish, the subpopulation survives. As the survivors are genetically identical to the rest of the population, they are able to initiate a new growth phase and subsequently new toxin production.

The high toxin expression is needed to induce significant damage to the Varroa population. It is known that longer-term, low-dose exposure of a pesticide to the target organism can cause resistance (Tabashnik, Brévault & Carrière, 2013). Ideally, the toxin is only expressed when the target organism is present. To further optimize toxin expression, two measures to regulate expression were explored. BeeT was designed to sense the presence of Varroa destructor through the use of two riboswitches: one senses guanine, the other senses vitamin B12. Both are normally not present in beehives, but indicate the presence of the mite as guanine is a major component of mite faeces and B12 is present in the haemolymph of the bees. When a mite attaches itself to a bee to feed on the haemolymph, BeeT will be able to sense it.

Lastly, a toggle switch for toxin expression was designed, by combining the riboswitch and part of the light killswitch employed in our safety system (hyperlink). A hybrid promoter was designed that both facilitates continuous toxin production after transient sensing of the mite, and shuts off production when BeeT escapes from the hive and is exposed to light.

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.

The luxI protein is a synthase that produces acyl-homoserine lactones (AHLs). AHLs are small compound that diffuse across cell-membranes and function as autoinducers, the molecules that bacteria secrete to signal population density. A single cell produces insufficient autoinducer molecules to start quorum sensing. When there is a high density of bacteria, all producing AHL, the AHL concentration in the growth medium increases. AHL can then reach high enough cytoplasmic concentrations to effectively bind the luxR protein. The luxR protein is a cytoplasmic receptor for AHLs, regulating gene expression depending on whether AHL molecules are bound. In our design, the luxR-AHL complex represses expression of two cI genes that work antagonistically on the same promoter. As 434 cI in this system has a higher protein turnover than lambda cI, transcriptional repression of both cI genes causes a shift in the balance between the two proteins. The idea behind the subpopulation system is that - with the right tuning - the protein balance shift causes inhomogeneity in the protein ratios throughout the bacterial population. Because the cI proteins work antagonistically on the same promoter, this causes diversity between cells in the transcription of mRFP (‘toxin 1’ in figure 1). Positive feedback on the promoter regulating mRFP, makes substantial mRFP expression last. This we hypothesized can be used to create bacteria that behave differently from the rest of the population: a subpopulation. Because the forming of the subpopulation is designed to be dependent on quorum sensing, the bacteria are expected to, upon high bacterial density, switch to a state were part of the cells produce toxin, while others do not. This should then improve the expression of the toxin because the toxicity has less influence on the growth and longevity of the bacterial population. Though both systems were designed to be used together, we decided to first assemble and test both systems separately: quorum sensing and subpopulation formation

[collapsable details of the quorum sensing and subpopulation circuit] The luxR-AHL complex also regulates expression of the two lux quorum sensing genes. LuxI transcription is induced by the AHL bound luxR protein. LuxR transcription on the other hand was found to be induced by free luxR proteins, but not by luxR bound to AHL⁴. Effectively the luxR-AHL complex forming thus promotes LuxI but represses LuxR transcription. As mentioned before, positive feedback is included for the mRFP operon, this was done by including a gene encoding rapidly degradable lambda cI protein, the activator of the promoter regulating the mRFP operon.

Quorum Sensing

To test density dependent expression, a two plasmid quorum sensing system was assembled in and transformed to E. coli DH5alpha cells. One plasmid contains the actual quorum sensing system BBa_K546000 in the pSB4K5 backbone, while the other plasmid contains the newly made GFP quorum sensing reporter BBa_K1913014 in the pSB1C3 backbone. Bacteria that contain both plasmids should be able to communicate their cell density and produce GFP once cell density gets high enough. These quorum sensing bacteria were found to color green, preliminary indicating functional quorum sensing and reporting hereof (figure 3).

Figure 3. Cell pellets of 10ml LB cultures of (left) DH5alpha cells containing both the quorum sensing system and the reporter plasmid. (right) DH5alpha cells containing only the reporter plasmid, unable to substantially activate expression of GFP.

To follow the dynamics of the quorum sensing, plate reader measurements were done for the previously mentioned 2 plasmid quorum sensing E. coli. Fluorescence intensity was assessed by exciting at 480 nm and measuring light intensity of 510 nm. In addition, the optical density (absorbance at 600nm) was measured to relate fluorescence intensity to cell density (figure 4). Fluorescence over OD₆₀₀ is plotted to correct the fluorescence intensity for the amount of cells that produce the fluorescence. Starting from an OD₆₀₀ of 0.8 there is a sharp increase in fluorescence intensity per cell for the quorum sensing system. There is a very small increase in fluorescence/OD₆₀₀ for the reporter only control as well.

figure 4. Fluorescence and absorbance data for E. coli DH5alpha growing overnight. Continuous line: cells containing both the quorum sensing system and a GFP quorum sensing reporter. Dashed line: cells containing only the reporter. For both strains every value displayed is the average of at least three technical replicates and for each, three biological repeats were measured and plotted (the negative control lines show great overlap).

Subpopulation formation

Bravo, A., Gómez, I., Porta, H., García-Gómez, B. I., Rodriguez-Almazan, C., Pardo, L., & Soberón, M. (2013). Evolution of Bacillus thuringiensis Cry toxins insecticidal activity. Microbial Biotechnology, 6(1), 17–26. https://doi.org/10.1111/j.1751-7915.2012.00342.x Douek, J., Einav, M., & Zaritsky, A. (1992). Sensitivity to plating of Escherichia coli cells expressing the cryA gene from Bacillus thuringiensis var. israelensis. MGG Molecular & General Genetics, 232(1), 162–165. https://doi.org/10.1007/BF00299149 Saida, F., Uzan, M., Odaert, B., & Bontems, F. (2006). Expression of Highly Toxic Genes in E. coli: Special Strategies and Genetic Tools. Current Protein and Peptide Science, 7(1), 47–56. https://doi.org/10.2174/138920306775474095 Pearson, B., Lau, K. H., DeLoache, W., Penumetcha, P., Rinker, V. G., Allen, A., … Campbell, A. M. (2011). Bacterial Hash Function Using DNA-Based XOR Logic Reveals Unexpected Behavior of the LuxR Promoter. Interdisciplinary Bio Central, 3(3), 1–8. https://doi.org/10.4051/ibc.2011.3.3.0010

Toggle Switch

YOUR TEXT HERE This is bold text
This is italic text

1. EXAMPLE AUTHOR, EXAMPLE TITLE, ETC….
2. EXAMPLE AUTHOR, EXAMPLE TITLE, ETC….