Conclusions and Future Plans
Conclusion
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.
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.
Moving Forward
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.
