Team:UMaryland/HokSok - 2015.igem.org

Hok/Sok Construct

1. Full Hok-Sok sequence taken from Gerdes et al. Sequence is originally from the R1 plasmid of E. coli K-12.
2. g-Block of Hok-Sok sequence ordered from Integrated DNA Technologies, then assembled into pSB1C3 using the Gibson method.

1. Hok-Sok was coupled to a constitutive generator of unstable RFP to form a larger composite part.
2. Unstable LVA-tagged RFP has a half-life of 1 hour and allows for more current measurements of protein production.
3. For our first testing method to see if Hok-Sok was capable of maintaining a plasmid, we wanted to measure how RFP fluorescence was retained over many generations in two E. coli strains: BL21 and DH5α.
4. In order to test, five sets of cultures were grown in LB media for fluorescence studies.

A. Constitutive unstable RFP grown with chloramphenicol (33 µg/mL) in media (+ Control)
B. Constitutive unstable RFP grown without chloramphenicol
C. Unstable RFP without promoter with chloramphenicol (- Control)
D. Hok-Sok + Constitutive unstable RFP grown with chloramphenicol
E. Hok-Sok + Constitutive unstable RFP grown without chloramphenicol

5. Cultures were grown for 20 hours prior to fluorescence measurements.
6. Plate reader was used for fluorescence measurements.

A. 200 µL of undiluted culture was pipetted into each well.
B. Excitation wavelength was 555 nm. Emission wavelength was 584 nm.

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.

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:

• Hok/Sok without chloramphenicol
• Hok/Sok with chloramphenicol
• RFP without chloramphenicol
• RFP with chloramphenicol

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

1. Gerdes, K., Thisted, T., & Martinussen, J. (1992). Mechanism of post-segregational killing by the hok/sok system of plasmid R1: Sok antisense RNA regulates formation of a hok mRNA species correlated with killing of plasmid-free cells. Molecular Microbiology, 223(1), 1807-1818. doi:10.1016/0022-2836(92)90714-U
2. Mitsuoki Kawano (2012) Divergently overlapping cis-encoded antisense RNA regulating toxin-antitoxin systems from E. coli, RNA Biology, 9:12, 1520-1527, DOI: 10.4161/rna.22757