Difference between revisions of "Team:UMaryland/HokSok"

Revision as of 21:29, 18 September 2015

Purpose

When setting up our experimental design, we focused on answering three main questions:

1. Can Hok-Sok maintain a plasmid over many generations? If so, how does its performance compare to classical antibiotic pressure systems?
2. Does the Hok-Sok cassette restrict the growth of the bacteria it resides in?
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?

4. Can this cell living with Hok-Sok stay happy?

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 (K817015), 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.

Section Summary

1. We wanted to answer three main questions concerning the ability of Hok-Sok to maintain a plasmid.
2. We hypothesized that Hok-Sok would be able to replicate its natural maintenance ability for a recombinant plasmid.
3. We devised tests to measure maintenance over time.

Hok/Sok Construct

Prior to experimentation, we had to insert the Hok-Sok construct into pSB1C3 in order to make it a BioBrick. We originally planned to PCR amplify the cassette out of the R1 plasmid of E. coli, but we were unable to find an suitable wild-type strain that was easily available. Instead, we turned to synthesizing the construct as a gBlock from IDT. As a 580 bp dsDNA fragment, it was suitable for addition via Gibson Assembly into pSB1C3. After subsequent transformation, miniprep, and confirmation sequencing, we had the first piece of our testing puzzle.

Proof that at least some of our cloning worked

Section Summary

1. Full Hok-Sok sequence taken from Gerdes et al. Sequence is originally from the R1 plasmid of E. coli.
2. g-Block of Hok-Sok sequence ordered from Integrated DNA Technologies, then assembled into pSB1C3 using the Gibson Assembly method.

Fluorescence Studies

Unstable LVA-tagged RFP has a half-life of 1 hour and allows for more current measurements of protein production.

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 E. coli strains: BL21 and DH5α.

A. Constitutive unstable RFP grown with chloramphenicol (33 µg/mL) in media (+ Control)
B. Constitutive unstable RFP grown without chloramphenicol
C. Unstable RFP without promoter with chloramphenicol (- Control)
D. Hok-Sok + Constitutive unstable RFP grown with chloramphenicol
E. Hok-Sok + Constitutive unstable RFP grown without chloramphenicol

A. 200 µL of undiluted culture was pipetted into each well.
B. Excitation wavelength was 555 nm. Emission wavelength was 584 nm.

Section Summary

1. Hok-Sok was coupled to a constitutive generator of unstable RFP to form a larger composite part.
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.
3. Cultures were grown for 20 hours overnight prior to fluorescence measurements using a plate reader.

@@ Line 219: / Line 219: @@
 <div id='contentbox'>
 <a name="PCR"><p style="font-size:32px;text-align:center;font-family:Verdana, Geneva, sans-serif;"><b>Fluorescence Loss Analysis</b></a>
-<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. Plates were exposed to UV light using a transilluminator in order to visually observe fluorescence loss over many generations.</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. Plates were exposed to UV light using a transilluminator in order to visually observe fluorescence loss over many generations.<br>In addition to our observations, we also wanted to make sure that our cultures had not been contaminated with a non-fluorescent, chloramphenicol resistant bacteria that had out-competed our intended culture. We thus performed a gram stain in order to verify that our bacteria were gram negative bacilli.</p>
 <p style="font-size:24px;text-align:center;font-family:Verdana, Geneva, sans-serif;">Section Summary</p>
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 <li>1. We wanted to see whether fluorescence loss was independent of cell strain.</li>
 <li>2. We switched to a better cell line for protein production, BL21, in order to magnify the effects of fluorescence loss.</li>
+<li>3. We performed a gram stain to confirm our colonies were descendants of our initial <i>E. coli</i> colony and not contamination.</li>
 </ul>