Difference between revisions of "Team:UMaryland/HokSok"

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

<p style="font-size:24px">We hypothesized that Hok-Sok 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>

<p style="font-size:24px">Unstable LVA-tagged RFP has a half-life of 1 hour and allows for more current measurements of protein production.</li>

<li>3. For our first testing method to see if Hok-Sok was capable of maintaining a plasmid, we wanted to measure how RFP fluorescence was retained over many generations in two <i>E. coli</i> strains: BL21 and DH5α.</li>

<li>A. Constitutive unstable RFP grown <b>with</b> chloramphenicol (33 µg/mL) in media <b>(+ Control)</b></li>

<li>B. Constitutive unstable RFP grown <b>without</b> chloramphenicol</li>

<li>C. Unstable RFP without promoter <b>with</b> chloramphenicol <b>(- Control)</b></li>

<li>D. Hok-Sok + Constitutive unstable RFP grown <b>with</b> chloramphenicol</li>

<li>E. Hok-Sok + Constitutive unstable RFP grown <b>without</b> chloramphenicol</li>

<ol>

<li>A. 200 µL of undiluted culture was pipetted into each well.</li>

<li>B. Excitation wavelength was 555 nm. Emission wavelength was 584 nm.</li>

</ol>

<li>3. Cultures were grown for 20 hours overnight prior to fluorescence measurements using a plate reader.</li>

 

<p style = "font-size:24px">Along with measuring culture fluorescence, we also tested the ability of hok-sok to maintain a plasmid using daily chloramphenicol challenges. The protocol is as follows:</p>

<p style = "font-size:24px">The goal in doing this was to determine how many bacteria were surviving by retaining their plasmids. We took notice of the color of the colonies and the number of colonies on the plate.</p>

<p style = "font-size:24px">For continuing generations of BL21 strain E. coli, 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>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>We started growing 250 mL cultures and monitored the OD at 600nm using a spectrophotometer over the span of 7.5 hours. </p>

<p style="font-size:24px">Interested as to why our cells were losing fluorescence in the span of a week, we increased the level of protein expression in order to observe this effect on a larger scale. This was done by switching cell lines from DH5α to BL21, which is optimized for protein expression due to the removal of several proteases. We repeated plating experiments in triplicate in order to determine if fluorescence loss would be as dramatic in such a small span of time.</p>  

<p style="font-size:24px">In order to find a definite answer as to the loss of fluorescence, we took samples of our non-fluorescent DH5α cultures, miniprepped them, and cut out the BioBrick insert in order to ascertain whether or not it was the proper size. For samples that contained an insert of the proper size, the miniprepped plasmid was then sequenced in order to determine the genetic reason as to the fluorescence loss.</p>

 

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