Goal: LB culture mediums were prepared to culture bacteria especially for Escherichia coli, which will be needed in latter experiment.

1. Materials

a. Reagent

Tryptone: 5g

Yeast extract: 2.5g

NaCl: 5g

Deionized Water: 500ml

b. Apparatus

balance

digital scale

autoclave sterilizer

beaker

test tubes

2.Procedure

1. A digital balance was used to get the exact amount of reagents that we need (2 g yeast extract, 4 g Tryptone, 4 g NaCl and 400 mL deionized water).

2. Placed all the material in a beaker.

3. Shook all reagents and let them mix completely.

4. Separated the solution into small test tubes.

5. Autoclaved EP tubes in a temperature of 121 ℃.

Goal: LB agarose plate was used to get monoclonal Escherichia coli.

1. Materials

A. Reagent

LB: 100 mL

agar: 1.5 g

antibiotic (Kana+): 100µg/mL

B. Apparatus

balance

beaker

2. Procedure

·A digital balance was used to get the exact amount of reagent that we need (1.5 g agar).

·Added 1.5 g agar into the LB

·Autoclaved LB (with agar) in a temperature of 121 ℃.

·Let the mixture cooled down until its temperature at roughly 50 ℃.

·Add antibiotic (100 µg Kana+) into the mixture and let them mixed completely.

·placed the solution into the plate and waited the agarose to solidify.

Goal: The aim of this experiment is to enlarge DNA fragments of I7L and ASFV-I7L to insert the plasmids.

1. Material

A. Reagent

Primer Front: 6.5 µL

Primer reverse: 6.5 µL

2x Heiff master mix: 130 µL

deionized water: 104 µL

plasmid/bacteria:13 µL

B. Apparatus

pipette

pipette tips

EP tubes

PCR thermo cycle:95 °C 5 min; 95 °C 30s, 55 °C 30s,72 °C 30, Cycles; 72 °C 10 min 4 °C

2. Procedure

• Choose a correct pipette and correct pipette.

• In the process of adding reagents (forward primer, reversed primer, 2x Heiff master mix, deionized water, plasmid/bacteria:), we followed an order that adding the one with the highest volume to lowest volume.L

• Remember to replace pipette tip after adding a reagent.

• Put test tube in PCR thermo cycle.

• Kept the DNA fragments enlarged production in -20 ℃ refrigerator.

1. Material

A. Reagent

Agarose: 1 g

deionized water: 100 mL

tae: 100 mL

nuclei acid dye: 5 mL

B. Apparatus

balance

pipette

mold

beaker

digital scale

microwave

1. Material

• A digital balance was used to get the exact amount of reagent that we need.

• Added agarose into a beaker with 100 mL tae buffer.

• Heated the beaker unit the reagents in the beaker turn clear.

• Placed the beaker under water and slowly shook it so that the mixture cooled down evenly.

• Add 5 mL nuclei acid dye into the reagents (be careful as it is poisonous).

• Put the solution into the mold and wait it to solidify.

1. Goal: recycle the target DNA sequence that we needed for latter experiment.

2. Material

a. Reagent

agarose gel

DNA Gel Extraction Kit

deionized water: 20 µL

wash solution:500 µL

b. Apparatus

pipette

pipette tips

metal bath instrument

3, Procedures

• Cut the gel block with the target segment from the agarose gel

• Add about three times buffer 2 respectively

• Placed it in a metal bath instrument for 10 min

• Add 20 µL of ddH2O between the adsorption membranes

• 2 rounds of wash solution and centrifugation.

1. Goal: Identify whether the target DNA fragments I7L/ASFV-I7L in E. coli are correct

2. Material

A. Reagent

seed liquid: 1µL

gel: 50 mL

B. Apparatus

pipette

pipette tips

EP tubes

3. Procedures

• Divided it into small tubes

• Added seed liquid using pipette and pipette tips.

• After inject the seed liquid, the pipette tips should be replaced.

• Remember the seed liquid has to be injected by insert the tips into the gel so that people could be sure that the seed liquid has been injected correctly.

1.Goal: Plasmids were extracted and transfected into BL21 and DH5α E. coli cells

2. Material

a. Reagent

Buffer SP1: 250 µL

bacteria liquid:

Buffer S: 500 µL

Buffer SP2: 250 µL

Buffer SP3: 350 µL

Buffer DW1: 500 µL

Wash Solution: 500 µL

Elution Buffer: 50 µL

b. Apparatus

centrifuge

EP tube

pipette

pipette tips

3. Procedure

1. Added Buffer S, centrifuge 12,000 xg for 1min, drain the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

2. Toke bacterial solution, centrifuge 8,000 xg for 2 min to collect bacteria, and discarded the medium.

3. Added Buffer SP1 to the precipitate and completely suspended the bacteria.

4. Added Buffer SP2 and immediately gently reversed the EP tube for 5-10 times for mixing well. Let stand at room temperature for 2-4 min.

5. Added Buffer SP3 and immediately gently reversed the EP tube for 5-10 times to mix.

6. Centrifugation (12,000 xg) for 5-10 min. Transfered the supernatant into the adsorption column, centrifuged at 8,000xg for 30 s, and drain the liquid in the collection tube.

7. Added Buffer DW1, centrifuged at 9,000 xg for 30 s, and drain the liquid in the collection tube.

8. Added Wash Solution, centrifuged at 9,000 xg for 30 s, and drain the liquid in the collection tube.

9. Repeat Step 8.

10. Centrifuged the empty adsorption column at 9,000 xg for 1 min.

11. Put the adsorption column into a clean 1.5 mL centrifuge tube, added Elution Buffer in the center of the adsorption film, stood at room temperature for 1 min, and centrifuged at 9,000 xg for 1 min. Kept the DNA solution in the tube.

Aim: to cultivate more bacteria for protein extraction

1. Aim: Transformation of plasmids containing I7L and ASFV-I7L fragments into DH5o and BL21 engineering bacteria, to preserve the plasmids and express the target proteins.

2. Material

a. Reagent

DH5o receptor cells: 200 µL

BL21 (DE3) receptor cells: 200µL

LB (no antibiotic): 1 mL

seed liquid: 100 µL, 5 µL

b. Apparatus

pipette

pipette tips

ice

water bath instrument

centrifuge

LB (with antibiotic)

3. Procedures

1. Warmed the water bath at 42 ℃ in advance

2. 200 μL DH5p cells was taken from the -80℃ refrigerator and thawed on ice. (100 µL per gene)

3. After thawing the receptive state, added the target DNA 1-10 ng, volume 《10 µL into the receptive cell suspension, gently rotated the centrifuge tube to mix the contents, and took an ice bath for 30 min.

4. Incubated in water bath at 42 ℃ for 90 s, and quickly put on ice to cool for 3min.

5. Added l mL LB liquid medium (no antibiotics!!) to the tube After mixing and oscillating culture 1h at 37℃, the bacteria were recovered.

6. After shaking the above bacterial solution, took 100 μL (can be concentrated if the receptive state efficiency is low) and smeared it on the screening plate containing K+, placed it face up for 30 min, and after the bacterial solution was completely absorbed by the medium, placed the petri dish at 37℃ for 16h - 24h.

* 2 controls simultaneously preparation

Negative control: DNA solution was replaced with equal volume of sterile double steaming water, and only 5 μL bacterial solution was taken and coated on LB plate containing antibiotics. There should be no colonies in this group under normal conditions. Positive control: A certain amount of known concentration of control DNA was added to the same amount of receptive cells, and other operations were the same as adding the connection system. Under normal circumstances, this control should have a relatively large number of colonies on the antibiotic containing LB plate.

1. Material

a. Reagent

IPTG:0.238 g

deionized water: 10 mL

b. Apparatus

pipette

pipette tips

balance

2. Procedures

• A digital balance was used to get the exact amount of reagent that we need

• Dissolved IPTG with deionized water

• Add IPTG solution to 100 mL I7L-BL21 and ASFV-I7L-BL21 bacterial fluid with the final concentration of 0.4 mM IPTG, and incubated for 10h at 14℃

1. Aim: The DNA double strand was broken and cut by “Nhel”, and “Xhol” enzyme to ensure that the I7L and ASFV-I7L sequence can be inserted into the plasmid in the correct orientation to prevent self-association.

2. Material

a. Reagent

b. Apparatus

test tubes

pipette

pipette tips

PCR thermal cycle

3. Procedures

• Operation on ice

• Digestion the I7L, ASFV-I7L and PET28a plasmid by NheI+XhoI, with in 2.1 cutsmart NEB buffer by the table.

• Double cleavage plasmid vector PET-28a and target gene PCR product by using NheI and XhoI for 30min at 37℃.

1. Aim: To ligate the I7L or ASFV-I7L cleavage fragments with plasmids by T4 ligase

2. Material

a. Reagent

plasmid: 20 ng

segment of the DNA (I7L and ASFV): 20 ng

10x T4 ligase buffer: 2 µL

T4 ligase Buffer: 0.2 µL

water: 20 µL

b. Apparatus

centrifuge tube

centrifuge

pipette

pipette tips

3. Procedures

1. The DNA was transferred to the sterile centrifuge tube, at least 1 molar amount of foreign DNA fragments was added.

2. Then 10 × T4 ligase buffer and T4 ligase buffer were added.

3. Sterile ultra-pure water was added to the volume of 10 μL,

4. Collected all the liquid at the bottom of the tube by microcentrifuge after full mixing. Connected at 16 °C for 1h

5. The DNA concentration of recombinant plasmid was measured after connection.

1. Aim: To obtain the monoclonal Escherichia coil which had been transformed into recombinant plasmid (PET-28a-I7L and ASFV-I7L-ASFV)

2. Material

a. Reagent

LB

b. Apparatus

shaker tubes

culture dishes

refrigerator

fire

EP tube

pipette

pipette tips

shaker

3. Procedures

• Took out some culture dishes and placed the shaker tubes near the fire for a few second in every open or close process in order to maintain the pure environment of the shaker tubes and to make sure that the tubes were not infected or polluted by anything

• Placed a pipette tip on fire for a few second to make sure a pure environment but also helped us to collect bacteria easier. Picked up bacteria with a with a pipette tip and transferred into shaker tubes with 5 mL LB

• Labeled the shaker tubes by the type of bacteria that we used

• Lastly, placed those shaker tubes into a Shaker and shake tubes for 5 hours.

1. Aim: To get the protein of I7L and ASFV-I7L

2. Material

a. Reagent

ultrasonic cell disruptor

DNA fragments of I7L and ASFV-I7L

3. Procedures

1.The Escherichia coli was collected by centrifugation at 3,000 xg for 15 min

2. Added lysis solution into the tube with Escherichia coli and Placed the tube under the ultrasonic cell disruptor

3. The ultrasonic cell disruptor have to stop in every 10 s due to the temperature of the ultrasonic cell disruptor could get very high jurying the process. Without stop for a minute, it is relatively easy for cell to denature.

4. Ultrasonic cell disruptor have to attach to the lysate and the lysate has to be straight up.

Procedure:

1.Took the same volume of the lower rubber solution and the lower rubber buffer, 2.0/2.7/4.0 mL each, and mix well

2. Added 40/60/80 μL of modified coagulant to the mixture in Step 1 and mixed well

3. Injected the mixed solution of step 2 into the plastic glass plate so that the distance between the liquid level and the upper edge of the short glass plate is 0.5 cm longer than the comb length (note: This solution is excessive, do not inject all, can leave a little in the glue cup, to judge the glue setting condition), added appropriate amount of water or alcohol (such as isopropyl alcohol, positive

4. After the lower layer of glue solidifies (about 15 min), poured the upper layer of water or alcohol; Note: When there is a broken ray between water (alcohol) and glue, the gelatin has solidified.

5.Took the same volume of upper adhesive solution and color upper adhesive buffer solution, 0.5/0.75/1.0 mL each, and mixed well; Note: Due to the special physical and chemical properties of the dye, please shake well before use.

6.Add 10/15/201 L of modified coagulant to the mixture of step 5 and mixed well;

7.Injected the mixture of step 6 into the plastic glass plate and inserted the comb;

8.After the upper layer of glue solidifies (about 15 min), the comb teeth can be removed for electrophoresis.

1. Material

a. Reagent

b. Apparatus

2. Procedure

1. CL retention sample 20 μL

2. Took 1 mL of uniformly mixed 50% BeyoGold™ His-tag Purification Resin (reducation-resistant chelating type), centrifuged at 4℃ (1,000×g, 10 s) and discarded the storage solution. Added 0.5 mL of non-denaturing lytic solution to the gel and mix well to balance the gel. Centrifugation at 4℃ (1,000×g, 10 s) to discard the liquid, and then repeated the balance twice to discard the liquid. About 4 mL of the supernatant of the bacterial lysate was added into the mixture, and the supernatant was slowly shaken at 4℃ on a side shaking table or a horizontal shaking table for 60 min.

3. The mixture of the lysate and BeyoGold™ His-tag Purification Resin (reduction-resistant chelating type) was loaded into the empty affinity column tube provided with the kit. h. Opened the lid at the bottom of the purification column, let the liquid in the column flow out under the action of gravity, and collected about 20 μL of the draining liquid for subsequent analysis.

4. Washed the column three times, added 1 mL of non-denatured washing liquid each time, and collected about 20 μL of washing liquid through the column for subsequent analysis and detection.

5. Eluted the target protein 4 times with 0.5 mL of non-denaturing eluent. Each eluent was collected separately into a different centrifuge tube. The eluent obtained from the collection is the purified His label protein sample.

Material:

a. Reagent

b. Apparatus

Procedure:

1.100 nM I7L/ASFV-I7L/Control protein (BSA) protease, 0.4 mM EDTA, 4 mM DTT and 20% glycerol were added to a reaction buffer containing 20 mM HEPES, pH 6.5, 120 mM NaCl of the test compounds for 30 min at 30 °C.

2.Set up different concentrations of protein sample from 0.01,0.02,0.05,0.1mg/ml of protease and set up three parallel groups for each concentration group to reduce the error. At the time of statistical data, the average value of parallel groups was calculated as the reference fluorescence intensity.

3.Detection the baseline under a 320/380 nm filter.

4.Detection was carried out by adding 10 μM FRET- peptide substrate (MCA-T-N-A-G-T-C-TV-Lys(dnp)-K-K-NH2)) for a total of 1 h of incubation followed by detection under a 320/380 nm filter.

5.Analyzed the fluorescence intensity change and protein concentration with a linear regression analysis.