Week 1 experimental preparation
Experiment Plan: prepare culture medium and experiment consumables, activate plasmid-carrying strain
Experiment:
1. The use of ultra-clean Table: open the ultra-clean table, place the centrifuge tube plate, tube rack, Marker pen, alcohol disinfection sterilization, ultraviolet irradiation, irradiation for 30 minutes.
2. Sterilization: Prepare Eppendorf tubes, PCR tubes, centrifuge tubes, and wrap with gauze. Pack 2 boxes of large, medium, small and gun heads, using Kraft paper to wrap the case, using rope/rubber band to bind the gun head boxes. Pre-prepared LB medium was used, 20 tubes were packed and 50 mL x 2 vials of LB liquid medium were arranged. The above articles were sterilized at 121 ° C for 15 min (about 1.5 h) . An experiment accident happened during the course of packing liquid culture medium: the culture medium was poured on the super-clean table and was cleaned up afterwards.
3. The activated strain included one tube of E. coli DH5Α/empty vector 1 and one tube of DH5Α/carrying CEP94 gene. 1 E. coli DH5 α/empty vector 2,1 E. coli DH5 α/carrying cex, CENA gene, 1 E. coli DH5 α/empty vector 3,1 E. coli DH5 α/carrying CEP94A, 1 E. coli DH5 α/carrying ACSAB. The sterilized LB tube was placed in the center of the ultra-clean table and ventilated. The alcohol lamp was lit, and the tube mouth and plug were burned. 100 ΜL of preserved bacteria solution was inserted into each tube, and 2.5 ΜL Amp mother liquor was added, cauterize the tube opening and plug again. After the tube was closed, the tube was bundled and cultured overnight in a shaker (150 rpm, 37 ° C) .
At the second week, the template was extracted and the plasmid was constructed
Experiment Plan: to extract plasmid and amplify the target fragment by PCR
Experiment:
1. Plasmid extraction: 6 plasmids were extracted according to the instructions of plasmid extraction kit. The bacterial solution was centrifuged for 1 min and then precipitated. P 1 suspension, p 2 upside down and p 3 upside down were added respectively, and centrifuged for 10 min. BL reagent 500μl was added to the column, the waste liquor was poured, and the supernatant was added to the column after centrifugation. 50 μl EB solution was added to the adsorption column membrane and centrifuged for 2 min. DNA was stored at -20 ° C using Nanodrop to determine the concentration of recovered DNA.
2. Diluting primers
The dry powder primers were centrifuged at 12000 rpm for 1 min, and the corresponding amount of distilled water was added to dilute the primers to the appropriate concentration
3.PCR reaction:
(1) the target gene was amplified by high-fidelity PFU. The reaction system is as follows:
Pfu 0.5 μL
10xPfu buffer 5 μL
Primer 11 ΜL
The primers were 21 ΜL
Template (plasmid extracted above) : 1 ΜL
dNTP Mixture 25μL
Sterilized water 20 ΜL
(2) PCR amplification was performed
98℃，5 min
The following 3-step reaction takes place in 30 cycles
98℃ 10s
55℃ 5s
72℃ 1min/kb
72℃ 8 min
The plasmids, primers and products required for PCR are shown in the following table:
In the 3rd week, the gel was recovered by agarose gel electrophoresis, double enzyme digestion and recovery
Experiment Plan: Agarose gel electrophoresis, gel recovery, double enzyme digestion, recovery
Experiment:
1. Agarose gel electrophoresis: the above PCR products were added to a previously prepared agarose gel, 5 ΜL DL 5000 DNA marker was added, and electrophoresis was performed (120 V, 20 min) . After electrophoresis, the target DNA gel was cut and placed in a 2ml centrifuge tube for labeling.
2. Glue recovery: the DNA gel of PCR product is recovered according to the instructions of the glue recovery kit
Add 500 microliters of equilibrium solution BL into the adsorption column Ca2, centrifuge for 1 min, dump the waste liquid in the collection tube, and put the adsorption column back into the collection tube. A single target DNA strip was cut from the agarose gel and placed in a net centrifuge tube. Add equal volume solution PN to the rubber block, place in 50 °C water bath, until the rubber block completely dissolved. The solution obtained from the previous step was added to another adsorption column CA2 and placed at room temperature for 2 min and centrifuged for 1 min. Add PW (600 microlitre) to adsorption column Ca2(check whether anhydrous alcohol is added before use) , centrifuge for 1 min, rinse twice, and air dry. A proper amount of elution buffer EB was added to the middle of the membrane and left at room temperature for 2 min. After centrifugation for 2 min, DNA solution was collected. The concentration of recovered DNA was determined using Nanodrop.
3. Double digestion: the plasmid pET23b and the target DNA fragment were digested by EcoRI and XHOI. 5x20 ΜL reaction. The 20 ΜL digestion system is as follows:
DNA 1 μg
EcoRI 1 μL
XhoI 1 μL
10xY buffer 4 μL
H2O up to 20μL
37 ° C for 4 H
Four. Agarose gel electrophoresis: enzyme-digested products were added to the agarose gel, 5 ΜL DL 5000 DNA marker was added, and electrophoresis was performed (120 V, 20 min) . After electrophoresis, the target DNA gel was cut and placed in a 2ml centrifuge tube for labeling. The product was extracted successfully, but the amount was less. After the second glue recovery.
Week 4 preparation of Natural competence
Experiment: activation of E. Coli DH5 alpha and Rosetta, prepare the Natural competence
Experiment:
1. Activated strains: including E. coli dh5α and Rosetta. The sterilized LB tube was placed in the center of the ultra-clean table and ventilated. The alcohol lamp was lit, and the tube mouth and tube plug were burned. 100 ΜL of preserved bacteria liquid was inserted into each tube, and the tube mouth and tube plug were burned again. After the tube was closed, the tube was tied and cultured overnight in a shaker (220 rpm, 37 ° C) .
Experiment:
Preparation of Natural competence:
(1) the ultra-clean table was opened, and the 1ml gun head, 200μl gun head, 5ml gun head, 2ML and 30ml centrifuge tube, 2x 50ml LB medium were put in for UV irradiation. 0.1 m CaCl2 and 15% 0.1 m Cacl2 solution were precooled in 4 ° C refrigerator.
(2) preparation of DH5Α and Rosetta Natural competence by adding prepackaged dh5α and Rosetta bacteria into a centrifuge at 4 °C, centrifugation at 5000 g for 10 min, discarding the supernatant and adding 800 μl 0.1 m CaCl2 solution, ice Bath for 40 min. After centrifugation (4000 g, 10 min) , the supernatant was discarded, 100 μl 15% glycerol 0.1 m Cacl2 was added into each tube, and the bacteria were resuspended. Divide the Natural competence on ice into 2 mL centrifuges, 100 μl each. Label and store the Natural competence in a -80 â ° C refrigerator.
Week 5 connection, transformation
The experimental plan: to prepare solid agar plate containing antibiotics, carry out the linking reaction and transform the linking product
Experiment:
1. Preparation of antibiotic plates: LB solid medium (4.5 g agar in 300 mL LB liquid medium) , 121 ° C, autoclaved for 15 min. When cooled to about 60 ° C, add 150 ΜL 100 mg/mL Amp antibiotic, shake well and pour into disposable culture dish (about 10-15 plates)
2. Ligating reaction: the vector is ligated with the target fragment by double enzyme digestion. The link system is as follows (Note: This system is an experience system, and the best link carrier/target segment volume ratio can also be calculated according to the instructions) :
T4 DNA ligase 1 μL
10 x ligation buffer 2 μL
Linear plasmid 1 μL
Insert DNA 6 μL
16 ° C of PCR apparatus was used to connect for 1H. The PET23B-INSERT is obtained and the join product is stored at ~ 20 ° C.
3. Transformation of ligated products: 10 ΜL of the ligated products were added to 100 μl of Natural competence solution, and then ice bath for 30 min, 42 ° C water bath for 60 s, Ice Bath for 5 min, and 1 mL LB liquid medium was added, after incubation for 1 h, resuscitation (37 ° C, 150 RPM) was performed. 100μl resuscitative solution was used to coat Amp antibiotic plate with aseptic coating rod.
At the 6th week, the two constructed plasmids were verified and extracted
Experiment Plan: Colony PCR verification, expand culture positive clone, extract the correct plasmid and transformation
Experiment:
1. PCR validation: 6 colonies per plate were labeled. Half of the colonies were selected as template, and Colony PCR was performed with Taq enzyme. The primers were TF, TR. The reaction system and procedure are carried out according to the instructions. A 50 mL agarose gel was configured, and electrophoresis was performed using DL 2000 DNA marker. The correct size of the band indicates the successful construction of the plasmid
2. Expansion Culture: the correct colonies were picked out and added to 5ml LB liquid medium, and 2.5 ΜL Amp antibiotic was added to expand culture.
3. Plasmid extraction: 2 plasmids were extracted according to the instructions of plasmid extraction kit. The bacterial solution was centrifuged for 1 min and then precipitated. P 1 suspension, p 2 upside down and p 3 upside down were added respectively, and centrifuged for 10 min. BL reagent 500μl was added to the column, the waste liquor was poured, and the supernatant was added to the column after centrifugation. 50 μl EB solution was added to the adsorption column membrane and centrifuged for 2 min. DNA was stored at -20 ° C using Nanodrop to determine the concentration of recovered DNA. This step most personnel successfully extracts the plasmid.
Four. Transformation: verify that the correct plasmid 1 is transformed into expression type E. coli.
The exo-glucanase CEX was tested at week 7
The exo-glucanase cex gene was synthesized and cloned into PET23B vector. The linker was first transformed into E. coli DH5 α and screened by antibiotic selection on LB agar plate. Positive clones were verified by PCR and DNA sequencing. The validated recombinant plasmid was transformed into E. coli Rosetta for subsequent protein expression and enzyme activity assays. The transformants were cultured overnight in LB medium containing ampicillin. The cells were collected by centrifugation, resuspended in 10 mL PBS buffer, and then sonicated to obtain 10 mL supernatant. The exo-glucanase activity was measured using p-nitrophenol-β-d-cellulose diglycoside (pNPC) as substrate. The 100 μl reaction mixture contained 50 mM sodium acetate buffer (pH 5.0) , 1 mM PNPC and 10 μl crude enzyme solution. The reaction was incubated at 50 ° C for 30 min. The release of P-nitrophenol at 405 nm was measured. A unit of enzyme activity was defined as the amount of 1 μmol p-nitrophenol released per minute. The specific activity was expressed as U/mg protein. Control experiments were performed using E. coli Rosetta cells transformed with empty pET23b vector. All experiments were repeated three times. Data are presented as mean ± standard deviation. T-test was used to analyze the difference, p < 0.05 that the difference was statistically significant.
Cena of endoglucanase was tested in the 8th week
Endo-glucanase cenA gene was synthesized and cloned into PET23B vector. The linker was first transformed into E. coli DH5 α and screened by antibiotic selection on LB agar plate. Positive clones were verified by PCR and DNA sequencing. The validated recombinant plasmid was transformed into E. coli Rosetta for subsequent protein expression and enzyme activity assays. The transformants were cultured overnight in LB medium containing ampicillin. The cells were collected by centrifugation, resuspended in 10 mL PBS buffer, and then sonicated to obtain 10 mL supernatant. Endo-glucanase activity was performed by measuring the resulting glucose in a 100 ΜL reaction system consisting of 1% carboxymethyl cellulose (CMC) , 10 ΜL of enzyme solution, and 50 mM acetate buffer (pH = 6.0) . At 50 ° C for 30 minutes. The release of reducing sugar was measured by DNS method. A unit of enzyme activity was defined as the amount of enzyme releasing 1 μmol of reducing sugar per minute. The specific activity was expressed as U/mg protein. Control experiments were performed using E. coli Rosetta cells transformed with empty pET23b vector. All experiments were repeated three times. Data are presented as mean ± standard deviation. T-test was used to analyze the difference, p < 0.05 that the difference was statistically significant.
At that time, the class said“Anthrone sulfuric acid method”, this is wrong, it should be“DNS Law”. The anthrone-sulfuric acid method is for measuring polysaccharides, and the DNS method is for measuring reducing sugars
Week 9 test of cellobiose phosphorylase CEP94A
CEP94A gene was synthesized and cloned into PET23B vector. The linker was first transformed into E. coli DH5 α and screened by antibiotic selection on LB agar plate. Positive clones were verified by PCR and DNA sequencing. The validated recombinant plasmid was transformed into E. coli Rosetta for subsequent protein expression and enzyme activity assays. The transformants were cultured overnight in LB medium containing ampicillin. The cells were collected by centrifugation, resuspended in 10 mL PBS buffer, and then sonicated to obtain 10 mL supernatant. Cellobiose phosphorylase activity was performed by measuring the glucose produced: a 100 ΜL reaction system consisted of 1 mM cellobiose, 10 ΜL enzyme solution, and 20 mM citrate buffer (pH = 4.8) . At 50 ° C for 30 minutes. Glucose content was measured using the GOD-POD analysis kit (Yuanye, China)(instructions given) . Using the standard curve of glucose, the amount of glucose produced in the reaction was calculated according to the absorbance value. Enzyme activity can be defined as the number of micromoles of glucose produced per minute. E. coli Rosetta strain carrying the empty plasmid was used as a control. All experiments were repeated at least three times. Data are presented as mean ± standard deviation. T-test was used to compare the two groups. P value < 0.05 that the difference was statistically significant.
The synergistic effect of cellulolytic enzymes was observed at week 10
In order to investigate whether the crude enzyme solution of cex, Cena and CEP94A can increase glucose yield, we carried out enzymatic hydrolysis experiment. The crude enzyme solutions of cex, Cena and CEP94A were prepared by ultrasonic treatment of corresponding E. coli Rosetta transformants and centrifugation of lysates to remove cell residues. Enzymatic hydrolysis was carried out in a 100 μl reaction mixture consisting of 1% (wav) carboxymethyl cellulose (CMC) , 50 mM citrate buffer (pH 4.8) and 10 μl crude enzyme solution. Reaction mixtures were prepared: cex-only, cenA-only, cep94A-only, cex, Cena, and CEP94A combinations, and control without enzymes. The mixture was incubated at 50 ° C for 30 minutes. Glucose production was quantified using the DNS method. In short, mix 20 ΜL of the reaction mixture with 30 ΜL of DNS reagent and bring to a boil for 10 minutes. The absorbance at 540 nm was measured with a microplate reader. The concentration of reducing sugar in the reaction mixture was calculated using the standard curve of glucose. Enzyme activity is expressed as the amount of glucose produced per minute. All experiments were repeated three times. Data are presented as mean ± standard deviation. T-test was used to analyze the difference, p < 0.05 that the difference was statistically significant.
The cellulose synthesis ability of ACSAB was tested at week 11
To assess the cellulose synthesis capacity of E. coli Rosetta transformants carrying the ACSAB gene, we synthesized the ACSAB gene encoding cellulose synthase and cloned it into the PET23B vector. The recombinant plasmid was then transformed into E. coli Rosetta cells. The transformants were selected on LB agar plates containing ampicillin. Positive clones were confirmed by PCR and DNA sequencing. In order to determine cellulose synthesis, engineering E. coli was cultured at 37 °C. After 48 hours, 100 mL bacterial solution was collected. LB medium samples were collected by centrifugation. The bacterial precipitates were resuspended with equal amount of PBS and broken up by ultrasonic wave. The bacterial precipitation samples and LB medium samples were added with 100 mL 4m NaOH and incubated at 80 ° C for 2 hours. This step dissolves the non-cellulose component and precipitates the cellulose. Subsequently, cellulose pellets were collected after centrifugation at 4 ° C, 15,000 g conditions for 30 min. The precipitated cellulose is washed repeatedly with distilled water until pH reaches 7.0 to remove residual NaOH. The washed cellulose was dried at 60 °C to a constant weight. Weigh the dried cellulose to determine the cellulose content. In bacterial precipitation samples, engineering E. Coli produced about 1.37 g/L of bacterial cellulose in LB medium, but almost no cellulose was detected in the culture medium samples.
