Goal: To create a functional and nutritional liquid or solid environment for bacteria to grow. Liquid media used for various purposes such as propagation of microorganism.

Materials: Sterilized Duran bottle\Autoclave\Tryptone\Yeast Extract\Sodium chloride\Double distilled H₂O\Lab Refrigerator.

Procedures for LB solution:

1. Prepare 20g of premix LB broth powder (Tryptone:Yeast extract:Sodium chloride-2:1:1).
2. Add into sterilized 1L bottle.
3. Add double distilled water to total volume of 1000ml.
4. Close the cap and autoclave it for 20 minutes at least.
5. Wait until it cools.

1. Obtaining target gene fragments by PCR

Goal: To amplify Papain\Abrus\Actinidia\Cajanu\Cicer fragments for later recombination.

Materials: 2xTaq PCR Mix*\Papain\Abrus\Actinidia\Cajanu\Cicer gene template\ddH₂O\Primer-R\Primer-F\centrifuge tube\ PCR thermal cycler.

* 2xTaq PCR Mix includes MgCl₂, dNTP, Taq DNA Polymerase, PCR buffer and loading buffer for PCR.

Procedure:

1. Add 2xTaq PCR Mix 25μl, papain template 2μl, primer-R 2μl, and primer-F 2μl to centrifuge tube. Add ddH₂O to the centrifuge tube until reaching 50μl.
2. Centrifuge the mixture for a few seconds to remove the bubbles.
3. Insert mixture into a PCR thermal cycler.
4. Perform the cycle,Step2-4 repeat 30 cycles.

94˚C	5 minutes	Step 1
94˚C	30 seconds	Step 2
55˚C	30 seconds	Step 3
72˚C	90 seconds	Step 4
72˚C	10 minutes	Step 5

5. Repeat step 1-5 for Abrus, Actinidia, Cajanus and Cicer.
2. Agarose gel construction, deployment and recycle

Goal: To produce a functional and well-shaped gel for electrophoresis.

Materials: Casting Tray\Well comb\Microwave oven\ 1x TAE\Agarose\10000x Nucleic acid gel stain\Erlenmeyer flask.

Procedure:

1. Dissolve 0.5g Agarose in Erlenmeyer flask.
2. Add 50μL of TAE (Running buffer).
3. Heat the material for 3 minutes in microwave oven.
4. Add 3μl nucleic acid gel stain (10000x).
5. Let it cool for 10 minutes.
6. Pour the agarose mixture into the casting tray.
7. Place the appropriate well comb to create the wells.
8. Wait to solidify 15 minutes.
9. Remove the comb and place the gel in the gel box.
3. Agarose gel electrophoresis

Goal: To verify the DNA bands' lengths and thus preparing for the next step of gel extraction to extract the bands that are of the correct length (corresponding with the length of Papain, Papain, Abrus, Actinidia, Cajanus and Cicer), eliminating the genetic fragments that didn't successfully undergo PCR.

Materials: Marker (DNA ladder)\Prepared gel\10x Loading buffer\Horizontal gel electrophoresis system.


Procedure:

1. Extract 10µl of every sample; add 5µl of loading buffer (10x) to each.
2. Place the gel in the horizontal electrophoresis system.
3. Extract 10µl of the mixed solution containing the sample, mix them till their colors are almost uniform.
4. Add the marker to the first well and load 10µl of each sample into the wells, the procedure runs at 180V for 20min.
5. Afterwards, take out the gel from the device and proceed to following steps.
   
4. DNA Gel Extraction

Goal: To recover DNA fragments in agarose gel.

Materials:clean scalpel\ 1.5 mL Eppendorf tube\pipettes and sterilized pipette heads\ hot water bath\buffer B2\purification column and collection tube\centrifuge\wash solution containing pure ethanol\ddH₂O.


Procedure:

1. Cut the slice of gel containing Papain, Papain, Abrus, Actinidia, Cajanus and Cicer, cutting off as much unneeded gel as possible, and place it in an 1.5 mL Eppendorf tube.
2. Add 500uL of buffer B2 and put the tube into a 50°C hot water bath until gel has completely melted.
3. Transfer the solution containing melted gel into a purification column that's in a collection tube and perform centrifugation at 8000 g for 30 seconds.
4. Empty the collection tube, put the purification column back in, and add 500uL of wash solution containing pure ethanol. Perform centrifugation at 9000 g for 30 seconds, and empty collection tube again.
5. Repeat (4).
6. Perform centrifugation one more time at 9000 g for 2 minute, then open the cap of the tube and let it sit for one minute to allow the ethanol to evaporate.
7. Transfer purification column to a 1.5 mL EP tube. Add 20uL of ddH₂O at the center of the purification column.
8. Cap the lid and let the tube sit for 1 minute. Perform centrifugation at 9000 g for 1 minute.
9. Discard the purification column. Store DNA at 4°C.
5. Double digestion

Materials: 1.5mL Eppendorf tube\10×buffer\NdeI\HindⅢ\pET28a\ddH₂O.

Procedure:

1. Prepare the samples

Material	Amount
10×buffer	5µl
NdeI	1µl
HindⅢ	1µl
pET28a	10µl
ddH2O	33µl

React in 37℃ for 30 minutes, Store in 4℃.

6. Homologous recombination

Goal: add target gene into vector so that the gene could be transformed into competent cells for expression in the future.

Material: 2×CEⅡ buffer\Linear pET28a\pET28a-Papain/Abrus/Actinidia/Cajanus/Cicer

Procedure:

1. Prepare systems

Material	Amount
2×CEⅡ buffer	5µl
Linear pET28a	4µl
pET28a-papain\...	1µl

React in 50℃ for 30 minutes,Store in 4℃.

7. Extracted Plasmid

Materials: Centrifuge\1.5 ml EP tube\Collecting tube\Buffer S\Buffer sp1\Buffer sp2\Buffer sp3\Buffer DW1\Washing buffer 80% ethanol\ddH₂O

Procedure:

1. Take out the cultured bacteria (if LB medium is opaque, then the bacteria has grown),fill both with 1.5ml of the bacteria-containing culture,centrifuge at 8,000xg for 2mins.
2. Add 250µl of buffer sp1, gently blow the bacterium using the micropipette to resuspend the bacteria.
3. Add 250µl of buffer sp2,Immediately gently mix the mixture for 5~10 times,incubate for 2~4 mins (no more than 5min) at room temperature.
4. Add buffer sp3,Spin at a max of the centrifuge (greater or equal to 12,000xg) for 5~10 mins.
5. Carefully extract 700µl of the supernatant to the separating column (careful NOT to obtain any of the condensed DNA),Spin at 9,000xg for 30sec,Dispose of the liquid.
6. Add 500µl buffer DW1 to remove protein impurities,Spin at 9,000xg for 30sec,Dispose of the liquid.
7. Add 500µl wash solution and spin at 9,000xg for 30sec,Dispose of the liquid.
8. Repeat steps 7.
9. Spin at 9,000xg for 1min.
10. Use 20µl of 55℃ ddH₂O and drop it at the very center of the membrane to wash off the plasmid.
11. Preserve the plasmids obtained at -20℃ for future usage.

1. Culture

Goal: to acquire large amounts of target proteins.

Materials:liquid LB\pET28a-Abrus/Actinidia/Cajanus/Cicer E-coli fluid\Kanna antibiotic\isothermic shaker.

Procedure:

1. Add 100μl of pET28a-Papain E-coli fluid and 100μl of kana into 100mL of liquid LB.
2. Place the sample in the isothermic shaker for one night in 37°C, 220rpm.
3. Change to other E-coli-fluids. Repeat the steps.
2. Protein Extraction and SDS-PAGE

Goal: to verify the expression of desired proteins.

Materials:bacteria culture containing desired protein\50 mL centrifuge tubes\beaker containing ice\Centrifuge\sonicator

Procedure:

1. add samples into 50 mL centrifuge tubes, perform centrifugation at 50% power for 10 minutes.
2. partially bury a centrifuge tube into a beaker filled with ice, then put the beaker and tube into the sonicator.
3. sonicate for 10 minutes in 50% power, turning on 3 seconds and stopping for 3 seconds for one circle.
4. repeat steps 2 and 3 for all samples.
5. the remaining liquid will contain desired proteins.
3. SDS-PAGE:

Materials:The SDS-PAGE color preparation kit\pipette\distilled water\electrophoresis buffer\protein ladder\vertical electrophoresis system

Procedure：

1. using a pipette, add and mix 4 mL of 2X separating gel solution, 4 mL of 2X separating gel buffer, and 80 μL of the catalyst in a plastic cup.
2. slowly inject mixture into casting stand and frame to avoid bubbles.
3. add 1 mL water to flatten out the top.wait 8 minutes for the gel to set.
4. discard the water that was added previously.
5. using a pipette, add and mix 1000 μL of 2X stacking gel solution, 1000 μL of 2X stacking gel buffer, and 20μL of catalyst in a plastic cup.
6. add stacking gel mixture until the cast is completely filled, then slowly insert comb without producing any air bubbles.
7. wait 12 minutes for the gel to set, carefully remove the comb, and wash the wells with electrophoresis buffer.
8. add the protein ladder into the first well, then load samples in each successive well.transfer gel into vertical electrophoresis system.
9. set the voltage to 180V and run it for 40 minutes.
10. stain the gel with Coomassie blue by submerging it for 10 minutes.

1. Hydrolysis of casein

Goal: Ensure the enzyme have the ability to hydrolysis the casein

Material: milk\The enzymes

Procedure:

1. Add acetic acid into the milk. Make the milk precipitate.
2. Add the enzyme
3. Measure the rate of casein hydrolysis
2. Hydrolysis of FN1 protein

Goal: to ensure the enzyme have the ability to hydrolysis the FN1 protein

Materials: Papain samples\Human fibronectin (FN1)\Activation buffer

Procedures:

1. Dissolve papain in the activation buffer. Let it stand at room temperature for 10 minutes to activate the enzyme activity.
2. Treatment of FN1 substrate dilute FN1 to 1 mg/mL with the activation buffer.
3. Add 10 μL of the activated enzyme solution to the FN1 substrate tube.
4. Immediately place it in a 37°C water bath for incubation for 2 hours after mixing.
5. Add 20 μL of SDS - PAGE loading buffer and heat at 95°C for 5 minutes. After precipitation on ice for 10 minutes, centrifuge at 12,000 rpm for 10 minutes. Discard the supernatant and resuspend the precipitate in the buffer.
6. Load 20 μL of the terminated sample for SDS - PAGE detection.
3. ELISA

Goal:To test the efficiency when using different combinations of enzymes

Material:Enzyme detector (450nm)\37°C incubator\double distilled water\20×buffer\substrate A\substrate B\stop buffer

Procedure:

1. Take the required strips from the aluminum foil bag that has been balanced at room temperature for 20 minutes. The remaining strips should be sealed in self-sealing bags and returned to 49°C.
2. Set up the standard sample wells and sample wells. Add 50 mL of samples of different concentrations to each standard sample well.
3. Add 50 mL of the sample to the sample wells; the blank wells do not receive any addition.
4. Except for the blank wells, add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each well of the standard sample wells and the sample wells. Seal the reaction wells with a sealing film and incubate at 37°C in a water bath or an incubator for 60 minutes.
5. Discard the liquid, blot dry with absorbent paper, and fill each well with washing solution (350 μL). Let it stand for 1 minute, then remove the washing solution and blot dry with absorbent paper. Repeat this process of washing the plates 5 times (or use a plate washer).
6. Add 50 μL of substrate A and B to each well. Incubate at 37°C in the dark for 15 minutes.
7. Add 50 μL of stop solution to each well. Measure the OD value of each well at 450 nm within 15 minutes.