Experiments

LB （Lysogeny broth）is the most commonly used nutrient medium in microbiology experiments for culturing bacteria such as E. coli, and is divided into liquid medium and solid medium made by adding agar. LB medium with antibiotics added can be used to screen selected colonies with E. coli as the host.

The method for the preparation of 1 litre of LB:

• Measure out the following:
  • 10 g tryptone
  • 5 g yeast extract
  • 10 or 5 or 0.5 g NaCl as required (see Formulae above; some bacteria are sensitive to NaCl)
• Suspend the solids in ~800 mL of distilled or deionized water.
• Add further distilled water or deionized water, in a measuring cylinder to ensure accuracy, to make a total of 1 liter.
• Autoclave at 121°C for 20 mins.
• After cooling, swirl the flask to ensure mixing, and the LB is ready for use.

Because 1 L of LB is too much for our experiment, we’ve divided each dosage by 4

• 250 mL of distilled water
• 2.5 g of tryptone
• 1.25 g of yeast extract
• 2.5 g of NaCl

Yeast extract peptone dextrose (YEPD) is a complete medium for yeast growth. It contains yeast extract, peptone, double-distilled water, and glucose (dextrose). It can be used as solid medium by including agar. The yeast extract will typically contain all the amino acids necessary for growth.

Yeast extract 10 g

Tryptone 20 g

Dexyrose 20 g

1000 mL of distilled water

We’ve also divide each dosage by 4:

Yeast extract 2.5 g

Tryptone 5 g

Dexyrose 5 g

250 mL of distilled water

medium into petri dish in the clean bench.

PCR

Polymerase chain reaction (PCR) is a laboratory technique for rapidly producing (amplifying) millions to billions of copies of a specific segment of DNA, which can then be studied in greater detail. PCR involves using short synthetic DNA fragments called primers to select a segment of the genome to be amplified, and then multiple rounds of DNA synthesis to amplify that segment.

Principle to replicate a targeted sequence:

Components added:

Mix buffer-

dyed nucleotides(atcg)-5microlitres

Primers-

Primer 1 and primer 2-2microlitres*2

DNA-

targeted sequence(acc(1,2),fast 1,fast 2)-1 microlitre

Distilled water-

ddH₂O-10 microlitres

Method:

1. Primer is put into the centrifuge (get liquid off the sides);
2. Use specific micropipettes to add the components into PCR tube;
3. Complete solution is vibrated to mix evenly;
4. Complete solution is put into the centrifuge;
5. Complete solution is put into the PCR machine at a set temperature cycle (Repeated for the three different enzymes).

Analyze PCR

Make Electrophoresis：Nucleic acid dye, water, and TAE is microwaved to make an agarose gel electrophoresis.

Electrophoresis：Run for 30 mins

See the results：using the 8k DNA marker, read the results shown

Gel Purification

To use Gel DNA Extraction Kit to extract ACC1, FAS1, and FAS2.

1. Cut blocks containing the target fragment from the agarose gel into centrifuge tubes.
2. Weigh the amount needed.
3. Add 3-6 times the weight of Gel to Buffer B2, 50 degrees water bath for 5-10 minutes to dissolve the gel.
4. For fragments smaller than 500 bp, add one-third of the volume of buffer b2 with isopropanol.
5. Transfer the lysate into the adsorption column and centrifuge at 8000xg for 30 seconds. Pour off the liquid in the collection tube.
6. Add 500 µL wash solution, centrifuge at 9000xg for 30 seconds, pour off the liquid in the collection tube.
7. Empty the adsorption column centrifuged at 9000xg for 1 minute.
8. Place the adsorption column into a clean 1.5 mL centrifuge tube, add 15-40 µL of Elution buffer to the center of the adsorption membrane, let it stand at room temperature for 1 minute, then centrifuge for one minute to save the DNA solution in the tube.

DNA Recombination

1. Prepare: 2 uL ddH₂0, 5uL ClonExpress Mix, 2 uL linearized vector, 1uL insert.
2. Mix gently by pipetting (do not shake to mix) and collect the reaction solution to the bottom of the tube by brief centrifugation.
3. Single fragment reconstitution reaction, 50°C, 5 min: reduce to 4C or cool immediately on ice.

Transform the target genes into Escherichia coli:

1. Place chemo-sensitive cells for cloning on ice to thaw.
2. Add 5-10 µL of the recombinant product to 100 µL of the chemoreceptor cells, flick the wall of the tube to mix, and let stand on ice for 30 minutes.
3. Heat stimulate in a 42℃ water bath for 45 seconds, then immediately cool on ice for 2-3 minutes.
4. Add 900 µL of SOC or LB liquid medium and shake at 37℃ for 1 hour.
5. Pre-warm the corresponding resistant LB solid medium plates in a 37℃ incubator.
6. Centrifuge at 5000 rpm (2500xg) for five minutes and discard 900 µL of supernatant. Use the remaining medium to re-suspend the bacteria and apply gently with a sterile applicator stick on the plate containing the correct resistance
7. Incubate upside down in a 37℃ incubator for 12-16 hours

Detecting monoclonies

Pick monoclonal (receptor cells)

Use the micropipette tips to take out a colony and add it to the LB medium.

Oscillate it for 12-15 hours to copy and duplicate itself.

1. Add mix, ddH₂O, two primers, and the colony of ACC1, FAS1, FAS2 into a PCR tube
2. Put into PCR machine to amplify and copy.

Making PCR gels：

Agarose gel electrophoresis

1. TAE (powder melted in 1L ddH₂O).
2. 0.24 g agarose (0.8% agarose gel) + 30 mL 1x TAE.
3. Microwave oven on high for one minute.
4. Add a small amount of nucleic acid fuel.
5. Pour into mold and solidify.
6. Weigh 135 volt electrophoresis for 30 minutes.

Run the Gels：

Add the marker, FAS1, FAS2 into the electrophoresis.

Run the electricity and wait for 30mins.

Check results

There were no results due to a error from one of the steps ahead.

Preparation of Saccharomyces cerevisiae receptor cells

1. Pick a fresh Saccharomyces cerevisiae EBY100 monoclonal from YPD plates into 10 mL YPD liquid medium and incubate at 30°C, 250 rpm overnight.
2. Determine the OD₆₀₀ value of the overnight culture to be between 3.0 and 5.0.
3. Dilute 10 mL of YPD overnight culture to an OD₆₀₀ value of 0.2~0.4.
4. Continue to incubate in a shaker at 28~30°C for 3~6 hr to reach an OD₆₀₀ value of 0.6~1.0.
5. Centrifuge the yeast cells for 5 min at 1500 g at room temperature and discard the supernatant.
6. Wash yeast cells with 10 mL of washing solution, followed by centrifugation at 1500 g for 5 min at room temperature to collect the cells, and discard the supernatant.
7. Resuspend yeast cells with 1 mL TE/LiAc in 50 μL portions per tube.

Make LB and YEDP media:

1. Take a small amount of yeast strain Y2H frozen stock and incubate it in line on YPDA plate medium, inverted at 30°C.
2. Pick a single colony of yeast in a 2.0 mL centrifuge tube, add 1 ml of YPD liquid medium and shake vigorously to disperse the clumps.
3. Transfer 1:100 ratio to the appropriate amount of YPD liquid medium, incubate at 30°C, 220 rpm for 18-24 h, until stable, OD₆₀₀>1.5.
4. Transfer the culture to YPD liquid medium at a ratio of 1:4 and incubate at 30°C, 220 rpm for 3 h until OD₆₀₀=0.2-0.3.
5. Transfer the yeast culture to a 1.5 mL centrifuge tube, centrifuge at 12000 rpm for 1 min at room temperature, and discard the supernatant.
6. Add 1 mL of TE buffer, re-suspend the precipitate, centrifuge at 12000 rpm for 30 s, and discard the supernatant.
7. Add 0.1 mL of TE-LiAc solution and re-suspend the precipitate.

Transformation of Saccharomyces cerevisiae cells

1. Take 50 µL of receptor cells, add 2 µL of each plasmid to be transformed, and mix well
2. Add 500 µL of transformation solution (PEG/LiAc, dimethyl sulfoxide), bounce on the wall of the tube and mix well.
3. Incubate in a shaker at 30°C for 1 hour, and mix by bouncing the wall of the tube at 15-minute intervals.
4. Add 1 mL of YPD culture solution and incubate for 1 hour at 30℃ in a shaker.
5. Centrifuge at 3500 g for 5 minutes, leave the sediment and discard the supernatant.
6. Re-suspend the precipitate with 150 μL TE, apply the corresponding SD plate and place in a 37℃ incubator for 30-60 minutes until the surface liquid has penetrated into the medium, then invert the plate and place in a 37℃ incubator overnight.

Verification of grown yeast cells using PCR and electrophoresis

1. Jump out the cultured yeast from the YPD plate and place in YPD solution.
2. Use a shaking incubator at 37℃ for 2-3 hours to grow more yeast cells.
3. PCR after taking out of the incubator.
4. 1 uL DNA, 5 uL Mix, 2 uL forward primer, 2 uL reverse primer, 10 uL ddH₂O.
5. Place on a shaker and beat well.
6. Centrifuge in a centrifuge for 1s.
7. put into thermal cycler for DNA replication.
8. Prepare solution: TE buffer, and agarose gel.
9. ddH₂O is added to the TE buffer solution, and the solution is then used as a solvent to melt the agar.
10. After adding agar, microwave for one minute.
11. Load the PCR mixture into the agarose gel for electrophoresis: total left to right Marker, FAS1, FAS2, ACC1.
12. Finally check the results to determine if the desired yeast cells are cultured.