1.5μl of both primers pMTL-R and pMTL-F
1μl of template pMTL-Pthl-andE2
25μl (2X) of DNA polymerase
ddH2O
1. Add 1.5μl of both primers into a PCR tube.
2. Add 1μl of DNA template into the same tube.
3. Transfer 25μl of enzyme into the PCR tube.
4. Complete the system to 50μl by adding 21μl of ddH2O.
5. Put the prepared system into a PCR machine and set the protocol of PCR to be
The processfrom (2) to (4) will be repeated for 30 times before the product was stored at 4℃.
0.6g of agarose
60ml of TAE buffer
1. Add 0.6g agarose into 60ml TAE buffer and mix thoroughly.
2. Heat the container with the solution in a microwave oven.
3. Heat for 30sec and shake it to see if there are precipitates.
4. Repeat step 3 for several times until the solution is transparent.
5. Cool the agarose solution to room temperature and pour it into the mould.
6. Settle the baffle and comb in the mould and stand for 12min.
7. After the gel is solidified, remove the comb and the take the gel out of the mould.
Gel prepared previously
DNA loading buffer
DNA marker
Samples to be tested
1. Add 10μl of DNA loading buffer to each 50μl system in the PCR tube.
2. Mix them thoroughly by shaking the tube.
3. Add 5μl DNA marker into the very first well of the gel.
4. Add 5μl of sample into the consecutive well.
5. Repeat the step 4 until all the samples are loaded.
6. Move the gel into the electrophoresis machine and lose the lid.
7. Start the machine with a voltage of 120V and leave it for 20min.
DNA template pet25b
Digesting enzyme EcoRl
Digesting enzyme Ndel
1. Prepare the system of enzyme digestion using pet25b gene sequence as a model and EcoRl and Nedl enzymes to obtain a linear DNA plasmid. The whole reaction system is 25μl. Add 1.5μl of each enzyme (EcoRI and Ndel), 1μl of original DNA sequence and 21μl of ddH2O.
2. Use the PCR machine to give an optimum temperature of 37℃ for 30min to catalyze the reaction of digestion.
2.5μl of T4 DNA ligase
0.5μl of linear plasmid pet25b digested with Ndel and EcoRI
0.5μl of DNA fragment (CALB)
ddH2O
1. Add 2.5μl of T4 DNA ligase into the PCR tube.
2. Add 0.5μl of the linear plasmid pet25b and 0.5μl of the DNA fragment CALB.
3. Complete the system by adding 21μl of ddH2O.
4. Run the PCR machine at 50℃ for 15min.
10μl PCR Mix Master
0.5μl of each primer
ddH2O
1. Prepare a few 200μl PCR tubes with 20μl ddH2O in each.
2. Choose several single colonies from the agar gel medium to drop in each tube via pipette tips.
3. Stir the mixture thoroughly to suspend the cells.
4. Preparing a 20μl PCR system in PCR tubes: 10μl PCR Mix Master, 8.5μl ddH2O, 0. 5μl suspension, and 0.5μl of each primer.
5. Protocol of colony PCR: 95℃ for 5min; Replicate the following steps for 30 times: 95℃ for 30sec, 50℃ for 30sec,72℃ for 45sec;72 ℃ for 3min.
6. Store the products at 16℃.
10ml bacteria incubation solution (E coli)
Buffer P1, P2, P3, PW1, and PW2(all diluted with ethanol)
Elution buffer
ddH2O
(Plasmid extraction kit provided by Vazyme, China)
1. Transfer 5ml bacteria incubation solution into a centrifuge tube, centrifuge it at 10000rpm for 60sec, and discard the supernatant.
2. Add 250μl buffer P1 into the tube and use the pipette to blend the residue and the
buffer.3. Add 250μl buffer P2 and stir the tube to mix thoroughly.
4. Add 350μl buffer P3, shake the tube till white precipitates are observed, and then centrifuge the tube at 12000rpm for 600sec.
5. Put the adsorption column into another centrifuge tube and transfer the pellucid liquid in the upper part of the tube into the adsorption column. Centrifuge it at 12000rpm for 30sec and discard the liquid in the bottom of the tube.
6. Add 500μl buffer PW1 into the adsorbing column, centrifuge it at 12000rpm for 30sec, and discard the liquid collected at the bottom the tube.
7. Add 600μl buffer PW2 into the column, centrifuge it at 12000rpm for 30sec, and discard the liquid collected.
8. Repeat step 7 to ensure that all other distractions are eliminated.
9. Put the empty tube into the centrifuge and centrifuge it at 12000rpm for 120sec to make sure that no liquid remains on the adsorbing membrane.
10. Add 30μl elution buffer into the column and centrifuge it at 12000rpm for 120sec to wash the DNA plasmid down into the collecting tube.
11. Test the concentration and purity of the extracted DNA plasmids by the spectrum analyzer.
Competent cells (E.Coli)
DNA sample
Sterile medium
LB agar
1. Remove competent E. coli from -80°C refrigerator (original 10-15μl), and quickly insert it into the ice box to dissolve it.
2. Add DNA sample and mix gently. Leave on ice for 30min.
3. Heat shock the tube by 42°C water bath for 45 sec, and quickly put the tube back on ice for 2min (be careful not to shake).
4. Add 700μl sterile medium without antibiotics and mix well. The culture was shaken at 160-225 rpm, 37°C for 1 hour.
5. Coat come of the cultured bacteria onto an LB agar medium plate containing the corresponding antibiotics. Stand at 37°C until the cell culture is absorbed.
6. Incubate the plate in inverted position at 37℃ overnight.
The culture medium used for cultivating E. coli was Luria Bertani (LB) medium, which consisted of 1% peptone, 1% sodium chloride, 0.5% yeast powder, and the fermentation medium for E. coli consisted of 1.75% mannitol and 2.25% peptone.
The enriched medium for cultivating C. tyrobutyricum consisted of 1% peptone, 1% beef extract, 0.5% sodium chloride, 0.5% glucose, 0.3% anhydrous sodium acetate, 0.3% yeast powder, 0.1% soluble starch, and 0.05% cysteine hydrochloride (pH 6.5).
The fermentation medium TGY of C. tyrobutyricum was shrimp shell powder medium, which consisted of 2.5%-3% shrimp shell powder, 0.5% sodium chloride peptone, 0.15% potassium hydrogen phosphate, 0.06% magnesium sulfate heptahydrate, 0.003% ferrous sulfate heptahydrate, 2% glucose, 1% yeast powder, and 0.13% cysteine hydrochloride, pH 6.0.
E. coli was cultured at 37℃ 200 rpm in a triangular flask, induced fermentation at 20℃ 200 rpm in a triangular flask, and C. tyrobutyricum was cultured in an anaerobic flask at 37℃.
1.Take the bacterial solution freshly cultured to OD600~2.0, centrifugate it at 10000×g for 1 min to remove the supernatant, and then resuspend it with 1mL PBS buffer, pH 7.0.
2.Centrifugate it at 10000×g for 1 min to remove the supernatant, and then add 0.5mL PBS buffer for resuspension.
3.Break the cells by ultrasound for 10 min, and centrifuge the broken transparent protein solution for 5 min at 10000×g to remove the residual cell fragments and insoluble substances.
4.Take the supernatant 160μl，add 4×SDS protein electrophoresis buffer solution and heat it at 100℃ for 10 min. After it is cooled to room temperature, sample it for protein electrophoresis.
1.Use butyric acid and butanol (5 g/L each) as substrates, and use hexadecane as extractant through two-phase catalysis.
2.Measure butyl butyrate generated in unit time by gas phase to determine the activity of lipase. 1 U (μmol/min) is defined as the amount of the enzyme that catalyzes the conversion of one μmol of substrate per minute.
Mix 2 g of chitin pellets with 20 mL of free lipase with chitin binding domain (CALB-ChBD fusion protein), and adsorb fully for 2 h at 20℃ 100 rpm in a shaker to obtain chitin lipase pellets.