Difference between revisions of "Team:UMaryland/HokSok"

<p style="font-size:64px"><b>Project Design: Hok/Sok</b></p>

<p style="font-size:30px">"To maintain or not to maintain, that is the question." -- Every plasmid-carrying bacterium, ever</p>

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<p style="font-size:32px;text-align:center;font-family:Verdana, Geneva, sans-serif;"><b>Purpose</b></p>

<p style="font-size:24px">When setting up our experimental design, we focused on answering three main questions:</p>

<li>1. Can Hok-Sok maintain a plasmid over many generations? If so, how does its performance compare to classical antibiotic pressure systems?</li>

<li>2. Does the Hok-Sok cassette restrict the growth of the bacteria it resides in?</li>

<li>3. Do proximity effects occur between Hok-Sok and a neighboring expression construct? Alternatively, does the activity of Hok-Sok affect expression of a protein whose coding region is near the construct?</li>

<p align = "center"><img src = "https://static.igem.org/mediawiki/2015/4/4a/UMDHappyCell.jpeg"></p>

<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>1. We wanted to answer three main questions concerning the ability of Hok-Sok to maintain a plasmid.</li>

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

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<li>2. In order to test the ability of Hok-Sok to maintain protein expression, five sets of cultures were grown in LB media for fluorescence studies.</li>

<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:

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<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. We analyzed insert sizes and sequenced plasmids in order to observe changes in plasmids over time.</li></uL>

<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>