Team:Shanghai high school/Experiments

Experiment 1: Plasmid transformation

1. Escherichia coli plasmid transformation

100μl of Escherichia coli DH5α competent cell which is kept in -80 ℃ was used. After the Escherichia coli melts on the ice, plasmid PHT43 was added and incubated on ice for 30 minutes.

And then, the Escherichia coli with the plasmid was heated in 42 ℃, for 90 seconds, so that the coli is able to absorb the plasmid.

And then, the cells were cooled on ice for another 3 to 4 minutes.

LB culture medium without antibiotic was added into the Escherichia coli DH5α cells, and then, incubated under 37 ℃ for 30 min on the shaker so that DH5α Escherichia coli will be able to express protein against ampicillin.

We then plated the DH5α Escherichia coli to the LA culture medium with antibiotic AMP to check if it is able to resist AMP.

If it grows, it proves that the plasmid was successfully transformed.

2. Plasmid electric transfer to Bacillus subtilis

1. Preparation of Bacillus subtilis competent cells

10 ml LB with sorbitol + 10 µ l B. subtilis

3. Electrotransformation

Cooled the Bacillus subtilis on ice for 10 min

The bacteria were then collected by centrifugation at 5000×g for 5 min at 4°C.

And then, the suspension was resuspended in 50 ml of precooled electrospun medium, centrifuged at 5000 × g for 5 min at 4°C to remove the supernatant.

Repeat the above process for three times.

The washed Bacillus were resuspended in 2 ml of the electrospun medium, and each 100 μl was transferred into a new EP tube.

8 μl of plasmid was added to 100 μl of competent cells, incubated on ice for 2 min, and then was added into a precooled electric cup.

Subtilis cell walls were electric shocked in a 1 mm cuvette (1 mm gap in the electrode)

Electric shock preparation, wipe clean the cuvettes without water

gap, 1 mm

200 ohm

25 μf

Electric shock once

Experimental group ①: 3.2 MS (eight microliters of plasmid added)

Experimental group ②: 3.6 MS (8 μl plasmid added)

Control ①: 3.9 MS (plus 8 μl of water)

After the electric shock, the cuvettes was removed and the bacteria were immediately added to 1 ml of RM (LB + 0.5mol sorbitol + 0.38mol of mannitol)

After resuscitation at 37°C for 3 h, the bacteria was incubated overnight.

The E. coli BL21 and the DH5α were bought from Beijing Zoman Biotechnology Company

Experiment 2: PCR

The first round PCR

20 μl of the PCR system

10 μl of enzyme (purple) Inc.: Vazyme

Two primers (white pet28a-vp7-ltb (both ends of complete gene sequence)) upstream and downstream, 1μl each.

Template (blue pET28a-vp7-Ltb intact plasmid) 4 μg dry powder (approx. 1 μl)

The remaining volume was filled up with water (7μl)

1. PCR procedure

① The DNA was denatured by 95 degrees for 3 min to break the duplex

② 66 degrees of "annealing" let DNA and "primer" complementary pairing for 1min and cycle 5 times

Cycles of 63 degrees for 1min and cycle 5 times

15 cycles of 60 degrees

③ Elongation at 72 degrees, replicating DNA

Put the target genes and the cut thread genes together and let them combine

④ Degree 4 preserved, program end

Second round PCR system 50 μl

The enzyme was given at 25 μl

2 μl of each primer (4 microliters total)

Template at 1 μl (the product of the first round of PCR)

The water added for total system of 20 μl

Vortex centrifugation

Put it into the PCR

2. PCR setup:

95 ° 3:00

{95 ° 30s

68 ° 30s

72 ° 1:00} repeat 5 times

{95 ° 30s

66 ° 30s

72 ° 1:00} repeat 5 times

{95 ° 30s

65 ° 30s

72 ° 1:00}repeat 15 times

72 Degrees 5:00

4 ° (preserved)

The enzyme was bought from Vazyme Biotech company

The primers were synthesized by Biosune company

Experiment 3: Enzymatic digestion

Plasmid pET28a used with the BamHI enzyme (a kind of restriction enzyme)

Two tubes of solution with a total of 40 μl : 30 μl plasmid + 6 μl BamH1+ 4 μl (10%) of Cutsmart (a solution to provide a good internal cutting environment).

Plasmid PHT43 was with the XbaI enzyme (a kind of restriction enzyme),

Two tubes of solution with a total of 40 μl : 30 μl plasmid + 6 μlXbaI+ 4 μl (10%) of Cutsmart.

Place the four tubes at 37 degrees for 1h

And then prepare another 10 μl of uncut control group solution (9 μl of empty loading plasmid puls 1 μl of buffer without restriction enzyme )

Dye buffer [cutsmart], BamHI, and XbaI were all bought from company NEB (New England Biolabs)

Experiment 4: Plasmid extraction

(Note: The centrifuge speed has always been 12,000 rpm.

3ml Bacillus subtilis was pured by centrifugation for 1 min

1. plus 250 μl Solution I (degrading RNA for 2 min)

2. plus 250 μl Solution II. Lysis the cell wall for 4 min

3. 350 μl Solution III was added and mixed .The liquid (plasmid solution) was centrifuged after 10 min.

4. centrifugation and the remaining cell body were discarded

5. loaded the silicon basement membrane again centrifuged and absorbed DNA, then pour down the waste liquid. The unrelated compounds on the basement membrane were washed away with ethanol (pour 500μl of ethanol on the membrane and then put into a centrifuge for 1 min) leaving only plasmid DNA

6. Centrifuge for 2 min, and volatilize alcohol at room temperature for 10 min

7. Using an eluent of 60 degrees to shed the plasmid better from the basement membrane. (40 μl eluent)

8. Put into a centrifuge for 30s

Extracted plasmid concentration was measured

① 117.0 ng / μl L 1.82 2.09

② 130.9 ng / μl L 1.80 2.01

③ 169.1 ng / μl 1.77 1.92

④ 194.5 ng / μl 1.83 2.17

⑤ 170.1 ng / μl L 1.86 2.33

⑥ 170.9 ng / μl L 1.77 1.90

Experiment 5: DNA agarose gelelectrophoresis

Agarose Gels:

0.6g agarose + water = 60ml

Heat medium heat to boiling, take out and wait until it becomes clear and cool to 60 ° C.

Add the nucleic acid dye 6 μl

Voltage: 110 V

Time: 30 min

Current: 40 mA

Agarose Gels (Gene Company)

Nucleic acid dye (擎科生物公司TSINGKE)

Experiment 6: Expression of proteins

IPTG was added to 1 ml BL21 with a OD600 concentration of 1 mol/ml.

A total of 25 samples were obtained.

Proteins were extracted from 12 IPTG induced BL21 samples as well as three BL21 samples from control groups without IPTG addition.

IPTG dosage: 0.5 nmol growth time: 2 h 4 h 5 h 6 h and 0 h (control group) 1-1

IPTG dosage: 1.0 nmol growth time: 2 h 4 h 5 h 6 h and 0 h (control group) 2-1

IPTG dosage: 2.0 nmol growth time: 2 h 4 h 5 h 6 h and 0 h (control group) 3-1

IPTG dosage: 0.5 nmol growth time: 2 h 4 h 5 h 6 h and 0 h (no IPTG control group for E. coli vp7-LTB) 1-1

IPTG dosage: 2.0 nmol growth time: 2h 4h 5h 6h and 0h (no IPTG control group for E. coli vp7-LTB) 3-1


Experiment 7: Purify protein

Twenty samples with 150 μl IPTG induced bacteria solution were processed for protein purification.

There is a lysozyme that can help us get our proteins.

1. 5,000*g (7,500 rpm) centrifugation for 10 min to collect bacterial cell precipitation.

2. Add 0.5 ml of Extraction Reagent (0.1% DTT and 1% PMSF in ultrapure water) to every 100 mg of wet weight bacteria. Put them on the shaking table at 280 rpm room temperature for 20 min.

3. Centrifuge in a centrifuge of 15,000 g (13,000 rpm) at 4 ℃ for 5 min, collect the protein supernatant as the soluble part of bacteria, and keep the insoluble cell fragments and the precipitation of possible inclusion bodies for analysis or recovery. Repeat this step for 3 times.

The PMSF, DTT, DNase I, Lysozome lysis buffer are all bought from Sangon Biotech