Difference between revisions of "Part:BBa K4166000"

Latest revision as of 02:19, 12 October 2022

Quorum sensing kill-switch

This is a kill switch that only permits cell growth at high concentrations of AHL (only high cell density)

Growth and MazF expression - Alma 2022

To test the efficacy and confirm the action of the kill switch, varying levels of HSL were used to prepare agar plates, which colonies were transplanted onto. The plates were either 10uL or a control plate. The top two colonies on the picture below were suspected to have successfully integrated the kill switch. This can be seen as there is slightly less growth in the first two columns of the first two rows on the control plate. Also, the colonies on the control plate took longer to grow. This difference is not great indicating that our kill switch works but isn’t very effective.

We wanted to quantitate our observations from the plates, and so we inoculated 3mL of LB with chloramphenicol, but without any AHL molecules, with different amounts of the starting cultures. Starting from an overnight culture, we pelleted the cells by centrifugation, washed them to remove any residual AHL molecules, and resuspended them at an OD600 of 0.1 (approximately 1x109 CFU/mL). Then, we used this to inoculate our fresh LB at two different levels – a relatively high density (OD600 of 1x10-5 or about 1x104 cells) or a lower density (OD600 of 1x10-8, or about 10 cells). We tracked their growth by optical density over the next day (If an OD of zero was read, we assumed it was 0.0005, which is below the limit of detection for our spectrophotometer). The results were as follows:

From the above we conclude that the kill switch impedes cell growth, but it is not sufficient to kill the cells or prevent growth outright. We wanted to examine this further and determine if there was a problem in expression of our MazF operon.

We prepared RNA from our competent cells using a RNA Mini Kit to become cDNA for qPCR to further confirm synthesis. We did this by running a qPCR with primers targeting specific genes in the sequence to determine how many PCR cycles it takes to reach a cDNA threshold. The lower the number of cycles means a greater amount of RNA/ cDNA expression in our plasmids. This is a way of quantitating the levels of expression for each specific part of the sequence. This further confirms proper synthesis, as each gene should have similar levels of expression to those on the same circuit. It also tells us how the levels of the toxin/ antitoxin/ control compare to each other, which is important data about the function of the kill switch. We ran 16 reactions and 5 controls, one for each of the 5 working parts, using RNA rather than cDNA to test for contamination. Representative results are shown below:

Our amplification plot and Ct values suggest that MazF (blue) expression lags behind that of the other genes on the same transcript (LuxR in gray, LuxI in green). It is not clear why this is the case! The values obtained in our qPCR experiment were validated by a disassociation curve, shown in the inset. Single peaks for this curve in each experiment indicate only one segement of the DNA was copied, as intended.

Sequence and Features