Plating Tests - Counting Colonies to Confirm Chloramphenicol Resistance
Over time, all cultures appeared to maintain antibiotic resistance, except for the negative control, which experienced no pressure during its growth. The following figures demonstrate the presence of resistant colonies over many hundreds of bacterial generations. Most importantly, the Hok-Sok culture grown without antibiotic was shown to maintain chloramphenicol resistance. This sets up one of our major conclusions: the Hok-Sok cassette is capable of maintaining a recombinant plasmid for an extended period of time.
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.
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.
Our fluorescence studies supported the findings of our plating tests. We were able to observe that fluorescence was rapidly lost in the negative control. This was expected due to plating tests demonstrating the loss of plasmid from that group. We were pleasantly surprised to observe that Hok-Sok was able to maintain fluorescence for a longer period of time than typical chloramphenicol pressure.
DH5α Cell Line
Figure 6. Fluorescence measurements of K1783002 (Constitutive unstable RFP) in DH5α cells grown in media containing antibiotic. All measurements are blank-subtracted. Fluorescence is initially high, but rapidly diminishes over time.
Figure 7. K1783002 (Constitutive unstable RFP) in DH5α cells grown in media without antibiotic. Fluorescence is initially low and rapidly diminishes over time. This result supports the notion that cells grown without any pressure quickly lose their plasmids.
Figure 8. K1783003 (Hok-Sok+Constitutive unstable RFP) in DH5α cells grown in media with antibiotic. Fluorescence is initially moderate and diminishes over time. The use of two maintenance systems does not appear to better maintain fluorescence levels than chloramphenicol alone. We suggest that the proximity of the Hok-Sok cassette to the RFP construct somewhat represses expression of RFP due to promoter interference, leading to a lower initial reading.
Figure 9. K1783003 (Hok-Sok+Constitutive unstable RFP) in DH5α cells grown in media without antibiotic. Fluorescence is initially moderate, but remains relatively constant over time, a one-time spike notwithstanding.
Figure 10. Fluorescence measurements of K1783002 (Constitutive unstable RFP) in BL21 cells grown in media containing antibiotic. All measurements are blank-subtracted. Fluorescence is variable throughout testing suggesting an uncorrected factor influencing results. Note: Our initial tests using the BL21 cell line were inconclusive due the need to calibrate our testing protocol.
Figure 11. Fluorescence measurements of K1783002 (Constitutive unstable RFP) in BL21 cells grown in media without antibiotic.
Figure 12. Fluorescence measurements of K1783003 (Hok-Sok+Constitutive unstable RFP) in BL21 cells grown in media with antibiotic.
Figure 13. Fluorescence measurements of K1783003 (Hok-Sok+Constitutive unstable RFP) in BL21 cells grown in media without antibiotic.
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.
Figure . Representative gram stain of culture. Gram staining of our samples confirms that our samples are gram-negative bacilli, making it highly unlikely that our cultures have been contaminated.
Top right bands of above gel are of negative control in DH5-alpha. They demonstrate that the cells no longer have the plasmid of interest. Gels generally show that plasmids are kept whenever a form of pressure is placed on the cell. Why then, is RFP not being expressed? Our sequencing results showed random mutations in the promoter and coding region of the RFP construct. This is a valuable lesson that, with any BioBrick construct, mutations and evolution is inevitable. However, plasmids that were maintained with Hok-Sok alone (no chloramphenicol) did not display mutations in the RFP construct. The large difference in protein expression over multiple days, as shown by our fluorescence and plating tests, suggests to us that the presence of Hok-Sok, combined with the absence of chloramphenicol pressure, is putting a smaller evolutionary pressure on the bacterium.
Conclusions and Future Plans
Through our plating tests and fluorescence measurements, we were able to successfully observe the plasmid maintenance ability of the Hok-Sok system. In comparison to a negative control grown without chloramphenicol pressure, cells containing Hok-Sok were able to maintain their antibiotic resistance for a significant period of time, as shown from our plating tests. In addition, we noted the ability of Hok-Sok had a negative effect on protein production, as shown from our fluorescence study. However, this level of production was consistent over a long period of time, contrasting with traditional pressure systems where mutations were able to build up quickly, shutting down production of RFP, which has no biological usefulness for the cell. We thus suggest that the downregulation of RFP production via Hok-Sok is capable of decreasing the evolutionary pressure against the removal on non-essential genes. This effect will be further studied in later experiments, where the distance between the hok-sok cassette and a gene of interest is lengthened. Overall, we suggest use of the Hok-Sok system as an effective method for internal plasmid maintenance without the use of antibiotics.
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.
In the future there is great potential to use the Hok-Sok system both in vitro and in vivo. In the laboratory, using plasmids which contain the Hok-Sok cassette along with some sort of positive selection marker, such as a fluorescent protein, instead of an antibiotic resistance gene would permit for positive selection of desired colonies without the use of antibiotics.
Furthermore, the Hok-Sok system could have a transformative role outside the lab. Synthetic biology projects often rely upon genetically modified bacteria which are designed for release into the soil, water sources, and ingestion by animals. Our 2014 project, for example, involved E. coli that could one day detect an oyster pathogen, and the University of Toulouse 2014 iGEM team was interested in designing bacteria capable of defending trees against fungal attack. If these bacteria carried antibiotic resistance genes, there would be ample concern about other bacteria acquiring this resistance through horizontal gene transfer. However, if the Hok-Sok system was used to maintain plasmids, it would reduce concern over increasing the number of antibiotic resistant bacterial strains.
In addition, there is potential to take advantage of the Hok-Sok cassette as a method of post-transcriptional control over a variety of genes. As hok is indirectly regulated by the upstream binding of sok to mok, there is potential to control protein expression through using the interplay of sok and mok to prevent the translation of various mRNA transcripts. Although we did not focus on this, there is great potential for experimentation.
As mentioned earlier, many synthetic biology projects, including those within iGEM, intend to release their final product into nature. As our testing supports the conclusion that Hok-Sok can successfully be implemented to maintain plasmids without antibiotics, it would be intriguing to test this further ourselves or see other iGEM teams test this by implementing the Hok-Sok cassette as the maintenance system on a project where the final product is intended for release into nature. Successful implementation of this would further prove the efficacy of Hok-Sok as an alternative plasmid maintenance system and as a method for combating the widespread use of antibiotics and growing population of antibiotic resistant bacteria.