Difference between revisions of "Team:Michigan/Results"

Experimental Results

Building pThermolyze*:

Four slightly different versions of pThermolyze were synthesized by Integrated DNA Technologies. Each version was synthesized from three linear parts. Part A had the TlpA promoter followed by T4 antiholin and the TlpA36 repressor protein, flanked by NheI and BglII restriction sites. Part B included ampicillin resistance gene and placed the plasmid’s origin of replication between BglII and HindIII restriction sites. There were four variations of Part C, each of which had T4 holin and T4 endolysin, but with a different constitutive promoter in front of them (or no promoter in one case). All four Part Cs were flanked by HindIII and NheI restriction sites.

Once the parts were synthesized, we performed the appropriate restriction digests on each part using NEB restriction enzymes. We then used a Qiagen PCR cleanup kit on each part before ligating them together using NEB T4 ligase. After ligating at room temperature for 48 hours, the reactions were transformed into NEB DH5a chemically competent E. coli cells. These transformations were plated on Amp-LB plates and grown overnight. 5mL liquid LB-Amp cultures were prepared from colonies picked from the plates the next day and themselves grown overnight. DNA was extracted from these liquid cultures using a Qiagen miniprep kit.

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*Initially our plan was to use gibson assembly, followed by several rounds of restriction digests and ligations to assemble our plasmid. Despite multiple attempts (see lab notebook), we decided to switch to the method described above.

Testing pThermolyze*:

Experiment #1: How do different promoters in pThermolyze affect cell growth at 37C and 25C?

For each of our four pThermolyze constructs (three promoters -T, L, and J- on part C and one with no promoter), two 50mL liquid LB-Amp cultures of E. coli transformed with our construct were grown in a shaking incubator at 37C until they had reached an OD of between 0.2 and 0.4 (read using a spectrophotometer at A540). The same was done for E. coli containing the pUC19 plasmid. When all ten of these cultures had OD between 0.2 and 0.4, initial readings at A540 were recorded. One of each of the five types of cultures (pUC19, no promoter, and promoters T, L, and J) was then shifted to a 25C shaking incubator. Every 20 minutes for 6 hours (or until a cultures OD went above the measurable limit of 2.5), A540 was measured and recorded.
This experiment was performed three times.

Experiment #2: Can pThermolyze transformed cells be stored as glycerol stocks and successfully recovered?

Glycerol stocks were produced from liquid cultures of E. coli transformed with each of our four pThermolyze constructs (three with different promoters -T, L, and J- on part C and one with no promoter on part C) and from a liquid culture of pUC19 transformed cells. After the glycerol stocks were stored for three days at -80C, prewarmed (37C) LB-Amp liquid cultures were inoculated with the glycerol stocks. These cultures were allowed to grow overnight in a 37C shaking incubator. 200uL of each culture was plated on LB-Amp plates and grown overnight in a 37C incubator. Plates were counted the next day.

Experiment #3: Can pThermolyze transformed cells be recovered from liquid cultures after storing at room temperature for 4 hours?

Liquid cultures of E. coli transformed with each of our four pThermolyze constructs (three with different promoters -T, L, and J- on part C and one with no promoter on part C) and a liquid culture of pUC19 transformed cells were each separated into five 1mL aliquots. 200uL of 1 of one aliquot of each type was plated on LB-Amp plates and incubated at 37C. Every hour, another 200uL of a new aliquot of each type was plated and incubated at 37C. After 24 hours since it was plated, each plate was examined and colonies were counted.

Experiment #4: Can pThermolyze transformed cells be recovered from plates after storing at 4C for 16 hours?

200uL of liquid cultures of E. coli transformed with each of our four pThermolyze constructs (three with different promoters -T, L, and J- on part C and one with no promoter on part C) and a liquid culture of pUC19 transformed cells were each plated on LB-Amp plates. These plates were stored at 4C. After 16 hours, 3 colonies were picked from each plate and used to start 5mL LB-Amp liquid cultures. These cultures were grown overnight at 37C and inspected for growth.

Protocols

Agarose Gel Electrophoresis:

1. Fill an erlenmeyer flask with 50ml of 1X TAE buffer.
2. Weigh out and add 0.75g of agarose to make 1.5% gel. Mix by swirling the flask
3. Microwave for ~2.5 minutes without boiling. Then run the beaker under cold water until the glass has cooled.
4. Add 5uL of GelRed and mix gently by swirling.
5. Pour the beaker’s contents into the gel mold.
6. Add plastic “combs” to the mold to make wells.
7. Push any large bubbles to the side walls if possible using a spare comb or pipet tip.
8. Let the gel cool and solidify (~10-20 min).
9. Once the gel is solid, move it into the electrophoresis device. Make sure the ‘top’ end of the gel (with the wells) is on the same side as the negative terminal.
10. Pour 1X TAE buffer to the fill line.
11. Slowly and gently remove the plastic combs from the wells.
12. Select a ladder to use based on what kind of samples you’re running.
13. Load 10uL of the ladder into the leftmost well.
14. For each sample, in a small PCR tube, add 10uL of the DNA sample and 2uL of 6X purple loading dye and mix by pipetting up and down.
15. Load each sample into a well in the same way you loaded the ladder.
16. Run the gel.