• Combine all components:

• Place the solution into PCR machine
• Start machine with following settings:

• For pMD19-xynA (xynA) plasmid:

• Add 0.4g Agarose Regular to 40ml TAE Buffer
• Mix together and bring to a boil in a microwave (stop immediately once boiling)
• When the solution is cool enough to touch, remove from microwave and add 5μl GelRed
• Cool the solution away from light.
• Pour into gel electrophoresis mold with the comb inserted.
• Remove when solidified and place in gel electrophoresis machine. The end with indents left by the comb should be facing the cathode.

• Add 20μl PCR products, enzymatic plasmid cutting products, and marker (control group) into individual indents in the agarose gel.
• Conduct electrophoresis at 140V for 30 minutes.
• Observe the gel under blue light. Bands of DNA show up as brightly lit.
• Slice the desired DNA fragment from the gel.
• Place the gel in a labeled microfuge tube.
• Add 650μl gel dissolving buffer (adjust amounts according to instructions on the kit) to the tube and immerse in 56˚C water bath.
• Transfer solution to adsorption columns and centrifuge at 5100g for 5 minutes, 4 times (add 650μl Wash Buffer in 2-3 times)

• Mix the solution and let it sit for 1 hour in 16°C.

• For Xyloglucosidase (Xyl3A) and Acetylxylan esterase gene (CcXynA)
• Mix the solution and wait 30min in 37°C

• Prepare liquid and solid culture media in separate flasks

• Add 100μl pET28A-xynA, pET28-PKC, pET28a-ccXynA, and pET28a-xyI3A into separate test tubes each containing 100μl TOP10 competent cells and place on ice for 30 minutes.
• Immerse in 42°C water bath for 60 seconds.
• Place on ice for 2 minutes.
• Add 1ml LB culture media (liquid) to each tube and let sit for 1 hour.
• Inoculate the TOP10 competent cells onto solid LB culture media for 12 hours.
• Pick single colonies and inoculate into 5ml liquid LB culture media that contain 250μg Kanamycin and allow bacteria to grow for 12 hours.

• Add 250μl Buffer S into an absorption column, then centrifuge at 12000g for 1 minute. • Centrifuge the 5ml liquid culture medium at 8000g for 2 minutes and discard the resulting liquid (keep the sediment).
• Add 250μl of SP1 Buffer into the tube and mix well.
• Add 250μl of SP2 Buffer into the liquid and mix gently until it becomes clear.
• Add 350μl SP3 Buffer and mix gently until precipitates form, then centrifuge the tube at 12000g for 10 minutes
• Add resulting liquid to the absorption column, then centrifuge at 8000g for 1 minute and discard the liquid.
• Add 500μl of DW1 Buffer and centrifuge at 9000g for 1 minute and discard the liquid.
• Add 500μl Wash solution and centrifuge at 9000g for 1 minute and discard the liquid. Repeat this step again (total 2 times)
• Centrifuge the absorption column at 9000g for 2 minutes and discard any liquid.
• Add 50μl Elution Buffer and centrifuge at 9000g for 1 minute. Keep the resulting liquid.
• Use the resulting liquid to check for the density of plasmids
• Serialization and Identification of DNA sequences.

• Create the following solutions:

• Mix 100 ng of pET28A-xynA, pET28-PKC, pET28a-ccXynA, and pET28a-xyI3A with 100μl of E.coli BL21(AI) Strain competent cells (BL21 competent cells) in separate tubes. Place on ice for 30 minutes.
• Immerse in 42°C water bath for 60 seconds.
• Place on ice for 2 minutes.
• Add 1ml liquid LB culture media and let the culture grow for 12 hours.
• Inoculate the BL21 competent cells onto solid LB culture medium for 12 hours.
• Pick single colonies and inoculate into 5ml liquid LB culture media containing 250μg Kanamycin for 12 hours
• Inoculate into 100ml liquid LB culture media at room temperature for 2-3 hours.
• When the detected OD is at 0.4-0.6, add 0.24mM IPTG and culture overnight.

• Centrifuge 100ml cultures at 1530g for 15min. Discard the liquid (keep sediment).
• Add 20ml of His Buffer A and mix well so that there is no more sediment at the bottom of the tubes.
• Pack ice in a container and place the tube containing the solution in it, then sonicate the solution for 5 minutes with settings for 3-second work and 2-second pause; collect the liquid.
• Centrifuge at 9000g or 20 minutes. Keep the liquid but discard of the sediment (keep 50μl samples of both)
• Add 500μl of Ni-NTA beads and let sit for 2 hours at 4˚C.
• Centrifuge at 500g for 5 minutes and collect the sediment (discard the liquid)
• Add 5ml of Buffer A and mix so that the sediment is suspended, then transfer to beads column.
• Add 1ml of His Buffer A 5 times, waiting for all the liquid to filter before each addition.
• Add 500μl of His Buffer B 6 times, waiting for all the liquid to filter before each addition. Collect the final liquid.
• Use His buffer B to elute protein 500μl (by 5 times) and collect the liquid.
• Measure the concentration of protein in the liquid.

• Mix protein gel solution (components shown below) and solidify to a thickness of 1.5mm

• Mix 500ml of protein gel buffer

• Mix 100ml of coomassie brilliant blue decolorizing solution

• Inject samples and a marker into the wells.
• Add 12.5μl of G250 Loading into target protein and inject into a well
• Electrophoresis at 180V for 1 hour.
• Use coomassie brilliant blue to dye the gel for 1 hour and decolorize for 1 hour.
• Imaging of gel

• Prepare 50ml of MnCl2 solution at a concentration of 5mM
• Add 0.15g Xylan into 30ml of the MnCl2 solution.
• Prepare 1ml each of xynA, PKC, xyl3A, and cCxynA
• Divide the experiment into four groups