Difference between revisions of "Team:UMaryland/HokSok"

<p style = "font-size:18px" align = "center"><b>4. Can this cell living with Hok-Sok stay happy?</b></p>

<p style="font-size:24px">The ability to use plasmids as vectors to introduce genes of interest in <i>E. coli</i> is one of the most essential bioengineering tools. However, one of the limitations of transforming a bacterium with a plasmid, is that the organism will eventually eject the plasmid over time. To counter this, scientists add a positive selective pressure on <i>E. coli</i> to retain plasmids carrying resistance genes through the use of antibiotics. While this technique has proven to be reliable and effective, there are many limitations. The prevalent use of antibiotics both for medical and agricultural purposes has rapidly increased the number of pathogens that harbor antibiotic resistant genes. As a result, there is a pressing need to find an alternative to antibiotic use for plasmid maintenance to prevent the spread of antibiotic resistant genes. Many synthetic biology projects which focus of solving health or environmental issues are confined to the lab because of these limitations. The University of Maryland iGEM team seeks to solve this problem by developing an alternative plasmid maintenance system that should liberate other iGEM teams from the dependance on antibiotic usage. We hypothesized that Hok-Sok, our plasmid maintenance system, could maintain recombinant plasmids, as it does natural ones. We also hypothesized that Hok-Sok would have a slight negative effect on bacterial growth rate, in line with other alternative maintenance systems such as sRNBC (<a href = "http://parts.igem.org/Part:BBa_K817015">K817015</a>), as well as the amount of protein expression due to competing parallel promoters. In order to answer these questions, we set up a variety of testing procedures, as shown below.</p>  

<li>2. We hypothesized that Hok-Sok would be able to replicate its natural maintenance ability for a recombinant plasmid.</li>

<li>3. We devised tests to measure maintenance over time.</li></ul>

<p style="font-size:24px">In order to determine if Hok/Sok was capable of maintaining a plasmid without antibiotic pressure, we decided to use a visual reporter gene to quantify the ability of Hok/Sok to maintain plasmids over many generations. We decided to use a RFP along with a degradation tag as the reporter gene. The most suitable candidate was an unstable LVA-tagged RFP that has a half-life of 1 hour. The shorter half life allows for more frequent measurements of protein production that would not aggregate over time. Therefore we combined a constitutive promoter and RBS to the LVA-tagged RFP through 3A assembly. We transformed this construct to <i>E. coli</i> DH5a to confirm the effectiveness of this reporter gene and its expression through increased fluorescence. Afterwords we ordered a g-block of our Hok/Sok+reporter construct. The expression of this reporter gene is proportional to plasmid number. Therefore, we concluded that if the cells containing a plasmid with both Hok/Sok and reporter gene could maintain fluorescence over many generations without the positive pressure of antibiotics compared to our controls, Hok/Sok can be used as a viable plasmid maintenance system.  

 

<p style="font-size:24px">We chose unstable Red Fluorescent Protein (RFP) as a marker for all our test groups to represent whether or not the inserted plasmid is still present in the bacteria. If the plasmid is maintained, the RFP is expressed and the overall fluorescence of the culture is greater. In contrast, if the bacteria does not feel enough pressure to keep the plasmid and ejects it, the measured fluorescence is on the lower end. From this data, we can gather whether or not the maintenance system is effective in preserving a plasmid in bacteria that is not beneficial to its survival, such as the aforementioned RFP.

<p style="font-size:24px">The reason for using unstable RFP is that the half-life of the proteins is shorter than a stable protein, therefore we can tell in real-time, or at least more so, whether or not the plasmids are present. The RFP degrades and unless the plasmid is maintained, the fluorescence in the cells actively declines.

<p style="font-size:24px">We used two <i>E. coli</i> strains for our testing. Originally, we used BL21 strain of <i>E. coli</i> because it is known to be the best for testing because the cell lacks proteases; the protein expression is optimal because the proteins are not digested by the enzymes. After testing BL21, we transitioned to the DH5a strain of <i>E. coli</i> because the cells lack recombinase.

<li>3. Cultures were grown for 20 hours overnight prior to fluorescence measurements using a plate reader. Excitation at 555 nm, emission at 584 nm. </li>

<p style = "font-size:24px">While our fluorescence studies were effective at measuring protein expression over time, we wanted a second test that would more directly measure whether or not plasmids were being maintained throughout generations. Using the identical cultures as the fluorescence tests, we devised a plating protocol involving a challenge of chloramphenicol every 24 hours.</p>

<p style = "font-size:24px">The goal in doing this was to determine how many bacteria were surviving by retaining their plasmids. We did not discriminate between the color of colonies.</p>

<p style = "font-size:24px">For continuing generations of BL21 strain <i>E. coli</i>, 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.</p>

<p style = "font-size:24px">While the Hok-Sok cassette may help to maintain a plasmid, it may also impact the rate of cell growth. We were unsure of the level of stress that came from additional hok and sok translation, and we decided to measure whether or not cultures containing the Hok-Sok construct would grow at a similar rate to control. 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:

</ul></p>

<p style = "font-size:24px">We started growing 250 mL cultures and monitored the OD at 600nm using a spectrophotometer over the span of 7.5 hours. </p>

<li>2. Mitsuoki Kawano (2012) Divergently overlapping cis-encoded antisense RNA regulating toxin-antitoxin systems from <i>E. coli</i>, <i>RNA Biology</i>, 9:12, 1520-1527, DOI: 10.4161/rna.22757</li>