Plating Studies
Along with measuring culture fluorescence, we also tested the ability of hok-sok to maintain a plasmid using daily chloramphenicol challenges. The protocol is as follows:
- 1. Dilute each culture (1 : 106) with LB media
- 2. Plate 10 µL of each diluted culture on a LB-agar + chloramphenicol plate
- 3. Incubate plates overnight
- 4. Count colonies the next day
The goal in doing this was to determine how many bacteria were surviving by retaining their plasmids. We took notice of the color of the colonies and the number of colonies on the plate.
For continuing generations of BL21 strain E. coli, we observed that on the plates for groups A and B, there was growth but no redness. If the bacteria were retaining the plasmids with the chloramphenicol resistance, the RFP gene should have been expressed and the colonies should fluoresce. We hypothesized that the chloramphenicol resistance gene was being recombined into the bacterial genome so the bacteria could therefore freely eject our inserted plasmids. As BL21 carries the gene for recombinase, it is possible. However, DH5α, as a common cloning strain, does not have recombinase. We created a new generation with every group (A, B, C, D, and E) to test whether the same plate would have similar results or once the bacteria stopped fluorescing, there would be no growth on the plates.
