Difference between revisions of "Team:UMaryland/HokSok"

Revision as of 21:13, 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 140: / Line 140: @@
 <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>
-<li>3. We devised tests to measure maintenance over time.</li>
+<li>3. We devised tests to measure maintenance over time.</li></ul>
 </div>
 </div>
@@ Line 229: / Line 229: @@
 <div id='contentbox'>
 <a name="PCR"><p style="font-size:32px;text-align:center;font-family:Verdana, Geneva, sans-serif;"><b>Sequence Analysis</b></a>
-<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>
+<p style="font-size:24px">In order to find a definite answer as to the loss of fluorescence, we took minipreps of our non-fluorescent DH5α cultures, digested them to extract their inserts, and separated them using agarose gel electrophoresis in order to determine whether or not they were 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>
+<p style="font-size:24px;text-align:center;font-family:Verdana, Geneva, sans-serif;">Section Summary</p>
+<ul>
+<li>1. We wanted to determine a genetic reason for fluorescence loss.</li>
+<li>2. We analyzed insert sizes and sequenced plasmids in order to observe changes in plasmids over time</li></uL>
 </div>