Difference between revisions of "Team:UMaryland/HokSok"

Revision as of 05:47, 18 September 2015

Hok/Sok Construct

Section Summary

1. Full Hok-Sok sequence taken from Gerdes et al. Sequence is originally from the R1 plasmid of E. coli K-12.
2. g-Block of Hok-Sok sequence ordered from Integrated DNA Technologies, then assembled into pSB1C3 using the Gibson Assembly method.

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.

Fluorescence Studies

1. Hok-Sok was coupled to a constitutive generator of unstable RFP to form a larger composite part.
2. 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.
4. In order to test, five sets of cultures were grown in LB media for fluorescence studies.

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

5. Cultures were grown for 20 hours prior to fluorescence measurements.
6. Plate reader was used for fluorescence measurements.

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

Plating Studies

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:

1. Dilute each culture (1 : 10⁶) with LB media
2. Plate 10 µL of each diluted culture on a LB-agar + chloramphenicol plate
3. Incubate plates overnight
4. Count colonies the next day

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.

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.

@@ Line 135: / Line 135: @@
 </ul>
 <br>
-<p style="font-size:24px>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 <i>E. coli</i>, 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.</p>
+<p style="font-size:24px">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 <i>E. coli</i>, 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.</p>
 </div>