Team:UMaryland/HokSok
Project Design: Hok/Sok
"To maintain or not to maintain, that is the question." -- Every plasmid-carrying bacterium, ever
Purpose
When setting up our experimental design, we focused on answering three main questions:
Can this cell living with Hok-Sok remain happy?
We hypothesized that Hok-Sok could maintain recombinant plasmids, as it does natural ones. We also hypothesized that Hok-Sok would have a slight negative effect on bacterial growth rate, in line with other alternative maintenance systems such as sRNBC (K817015), as well as the amount of protein expression due to competing parallel promoters. In order to answer these questions, we set up a variety of testing procedures, as shown below.
Section Summary
Hok/Sok Construct
Prior to experimentation, we had to insert the Hok-Sok construct into pSB1C3 in order to make it a BioBrick. We originally planned to PCR amplify the cassette out of the R1 plasmid of E. coli, but we were unable to find an suitable wild-type strain that was easily available. Instead, we turned to synthesizing the construct as a gBlock from IDT. As a 580 bp dsDNA fragment, it was suitable for addition via Gibson Assembly into pSB1C3. After subsequent transformation, miniprep, and confirmation sequencing, we had the first piece of our testing puzzle.
Section Summary
Fluorescence Studies
Unstable LVA-tagged RFP has a half-life of 1 hour and allows for more current measurements of protein production.
Section Summary
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:
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.
Growth Curve
We created a growth curve of Hok/Sok in comparison to controls to test the effectiveness of the Hok/Sok system in keeping the bacteria alive. We had four groups:
We started growing 250 mL cultures and monitored the OD at 600nm using a spectrophotometer over the span of 7.5 hours.
Parts Referenced
References