Team:UMaryland/Results - 2015.igem.org

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.

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.

Red cells are red

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.

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 agent, such as a fluorescent protein, instead of an antibiotic resistance gene would permit selection of colonies containing the desired plasmid without ever using antibiotics. Further the Hok-Sok system could have a transformative role outside the lab. Increasingly synthetic biology projects are creating genetically modified bacteria which are designed for release into the soil, water sources, and ingestion by animals. 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 instead as a selecting agent and to maintain plasmids, it would reduce concern over increasing the number of antibiotic resistant bacterial strains.

Additionally 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 binding of Sok to Mok directly upsteam of Hok, 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 antibiotic, it would be wonderful and 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 or animal ingestion. Successful implementation of this would prove the efficacy of Hok-Sok as an alternative plasmid maintenance system and method for combating the widespread use of antibiotics and growing population of antibiotic resistant bacteria.