Difference between revisions of "Team:UMaryland/Design"

Revision as of 17:03, 11 September 2015

Hok/Sok Construct

1. Full Hok-Sok sequence taken from Gerdes et al. Sequence is originally from the R-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 method.

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. We wanted to measure how fluorescence was retained over many generations in two E. coli strains: BL21 and DH5α.
4. 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.

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.

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Difference between revisions of "Team:UMaryland/Design"

Revision as of 17:03, 11 September 2015

Hok/Sok Construct

Fluorescence Studies

Plating Studies

Growth Curve

Parts Referenced