Part:BBa_K4166014
This is a control for our kill switch which is activated at low AHL concentrations
In this control, RFP takes the place of MazF, a toxin which kills the cells, and GFP takes the place of MazE, the anti-toxin. By measuring the relative amounts of each fluorescent protein, we can estimate how effective the kill switch should be.
Growth with or without HSL- Alma 2022
To test the efficacy and confirm the action of the kill switch (K4166000), as well as the control part (such as this one) 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.
One thing you can notice that is relevant here (and was replicated on other plates) is the slight coloration for the bottom row, which is this part. This shows that some RFP and GFP is being produced. This is especially pronouced in the plate with 10nM HSL.
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 918
Illegal NheI site found at 941 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1612
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 331
Illegal AgeI site found at 3035
Illegal AgeI site found at 3147 - 1000COMPATIBLE WITH RFC[1000]
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