Results:Hok/Sok
Plating Tests
Negative Control (No Pressure)
Figure 1. Negative control was K1783002 (constitutive unstable RFP) grown in media without chloramphenicol. After 2-3 days, plates demonstrate fewer than 10 colonies per plate, suggesting that antibiotic resistance is now only minimally present in the culture.
Chloramphenicol Pressure Only
Figure 2. K1783002 (constitutive unstable RFP) grown in media with chloramphenicol. Colonies persist through several days of plating, suggesting that antibiotic resistance was maintained throughout the test.
Chlor + Hok-Sok Pressure
Figure 3. K1783003 (constitutive unstable RFP under Hok-Sok regulation) grown in media with chloramphenicol. Colonies persist through several days of plating, suggesting that antibiotic resistance was maintained throughout the test.
Hok-Sok Pressure Only
Figure 4. K1783003 (constitutive unstable RFP under Hok-Sok regulation) grown in media without chloramphenicol. Colonies persist through several days of plating, suggesting that antibiotic resistance was maintained throughout the test.
Growth Curve
Figure 5. Growth curves of parallel cultures grown with and without chloramphenicol. The presence of Hok-Sok on the inserted plasmid does not appear to have a major effect on the bacterial growth rate. This is in line with our qualitative observations, where we do not observe any major difference in cell density during plating or fluorescence studies.
Fluorescence Studies
Analysis of Fluorescence Loss
Over time, colonies appear to stop producing RFP.
This effect occurs even when the culture is under chloramphenicol pressure.
This effect occurs much more slowly when the Hok-Sok cassette is adjacent to the RFP construct.
With or without added chloramphenicol, Hok-Sok appears to be able to maintain a relatively constant level of fluorescence.
Sequence Analysis
Future Plans
Considerations
PCR of Hok-Sok Construct
Amplification of the Hok-Sok cassette is difficult due to the high inherent secondary structure in the construct. Hok ssDNA is capable of naturally folding into a stable secondary structure, resulting in early terminations and other side products. We recommend that a reaction buffer suitable for difficult templates be used, such as Phusion GC buffer + added DMSO.
Measuring OD of RFP Cultures
Due to the close proximity of the emission wavelength of RFP (584 nm) and the classical absorbance wavelength for measuring cell density (600 nm), it is difficult to accurately determine the cell density of cultures that are expressing RFP. Given more time to calibrate our testing measurements, we would either have used an alternative wavelength for measuring OD (>600 nm), used a hemocytometer as an alternate counting method, or switched to GFP as an alternative fluorescent marker whose emission wavelength differs from 600 nm by a greater amount.
