Part:BBa_K4166001

mazE/RFP control "kill switch"

This kill switch was created in order to gauge the expression of RFP as a readout of mazF expression in part BBa_K4166000.

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.

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 444
  Illegal NheI site found at 467
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1138
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 2561
  Illegal AgeI site found at 2673
  
• 1000
  COMPATIBLE WITH RFC[1000]