Difference between revisions of "Team:UMaryland/HokSok"

<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>  

<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. We transformed this Biobrick onto both Dh5 alpha and BL21 strains for testing. We tested fluorescence of 3 biological and 3 technical replicates of the 5 groups listed below using a microplate reader. The 5 groups and their replicates were picked off of plates and incubated in 5 mLs of LB in culture tubes. A 1000x chloramphenicol concentration was added to groups A, C, and D. There was no chloramphenicol added to groups B and E. After 20 hours, 200 uL of each overnight culture was transferred onto a 96 well plate and the fluorescence data was recorded. 4 hours later, 50 uL of this overnight was inoculated in a new culture tube containing 5 mL of LB. These new cultures were the new generation, and they were incubated for 20 more hours for more testing. This process was repeated for several generations. </li>

<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.  

<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>

<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>