<p>The fourth pillar of our project is the biocontainment of our therapeutic organism. Since we intend to apply our bacterium in-situ in the lungs of chickens where the expressed and secreted nanobodies can be diffused into the lung mucus. Such an application, though theoretically safe, will require a robust biocontainment strategy to prevent the bacterium from escaping the chicken lungs. For this purpose we designed a series of TlpA-based temperature kill-switches and a CarH-based light kill-switch. These cause the expression of lethal CRISPR-Cas9 complexes targeting 16s rRNA when the temperature drops below 36℃, 39℃ and 41℃, or when exposed to light respectively.</p>
<p>Though the assembly of these final constructs was not managed in time, we did construct a series of temperature sensor test-constructs, where the 6 temperature sensor variants control mRFP1 expression. These were all transformed into DH5α, and some in BL21 E. coli, and the 36℃ constructs were tested at 37℃ for activity. This showed that the cells became fluorescent at this temperature. We also showed that one of the 4 constitutive promoter components was functional, and is affected by temperature changes as well.</p>
<p>Though the assembly of these final constructs was not managed in time, we did construct a series of temperature sensor test-constructs, where the 6 temperature sensor variants control mRFP1 expression. These were all transformed into DH5α, and some in <i>E. coli</i> BL21, and the 36℃ constructs were tested at 37℃ for activity. This showed that the cells became fluorescent at this temperature. We also showed that one of the 4 constitutive promoter components was functional, and is affected by temperature changes as well.</p>
<p>The next steps would have been further transformation into the <i>E. coli</i> BL21 strain, which had shown better expression, and then electroporation transformation into <istyle="color: white;">L. reuteri</i> DSM 20016. The microtiter plate fluorescence experiment has been mostly optimized, so we could then compare fluorescence at different temperatures in the 3 bacterial strains.</p>
<p>We had some trouble synthesizing the light sensor, and would need to get into contact with the original creators to acquire the template. We also need the CRISPR-Cas9 expression cassettes from the original creators, as we could not get them in time.</p>
<p>Finally we could then assemble all these components into the final kill-switches and test their effects on growth-rate at different temperatures and light-exposure. Additionally, the kill-switches could be incorporated into <istyle="color: white;">L. reuteri</i> DSM 20016 genome to create the final OR-gated containment strain.</p>
