Experiments
A. Plasmid construction of pLY15-opt
1. Take the ATCC824 genome as the template, then thl, hbd, crt, and ter genes were amplified
by PCR using thl, hbd, crt and ter primers.
▶ Amplification
All reagents were melted at room temperature, mixed by shaking, centrifuged at 6000 rpm for 10 sec, and prepared according to the following system:
▶ The PCR program
All reagents were melted at room temperature, mixed by shaking, centrifuged at 6000 rpm for 10 sec, and prepared according to the following system:
| Name | Amount added | Final concentration |
|---|---|---|
| 2×TOROBlue Flash KOD DyeMix | 25 μL | 1× |
| 10 μM upstream primer | 1.5 μL | 0.3μM |
| 10 μM downstream primer | 1.5 μL | 0.3μM |
| DNA template | 5 μL | GenomeDNA10-200 ng; Plasmid1-50ng; cDNA ~750 ng; Living specimen, crude extract 5 μL |
| ddH2O | Add to 50 μL |
| Step | Temperature | Time | Numbers of circulation |
|---|---|---|---|
| Pre-denaturation | 98℃ | 2 min | |
| Denaturation | 98°C | 10 sec | 25-45 |
| Annealing | (Tm-5) °C | 5 sec | |
| Extension | 68°C | 1-10 sec/kb |
2. Agarose gel electrophoresis detection
▶ Prepare agarose gel: add 100 ml 1×TAE buffer into the conical flask, weigh 1.0 g agarose into a conical flask,
heat it in the microwave oven until it is completely melted, take it out and shake it well, then it is 1% agarose
gel.
▶ Add Dye: when agarose gel is cooled to 70°C, add 10 uL nucleic acid dye, and mix well.
▶ Preparation of gel board: waterproof tape is pasted on both ends of the glue bed to form an 8 mm high retaining wall, press the tape, and place it on the horizontal table. Insert the comb, and the lower end of the comb is about 1mm away from the bottom of the plate. Pour the 60℃ gel into the glue bed continuously, 3-4 mm high, avoid bubbles, and solidify at room temperature. After complete solidification, tear off the adhesive tape, place it in the electrophoresis tank, add the electrophoresis buffer, which is 1-2 mm higher than the glue surface, gently pull out the comb obliquely from one end to remove the bubbles generated.
▶ Sampling: add 5 μL DNA maker first, and then take 10 μL PCR reaction system for sample addition.
▶ Electrophoresis: cover the electrophoresis tank, the sampling hole is located at the negative pole of the electric field, and connects the power supply, 120V. When the front edge of the dye moves to the bottom edge for 1-2 mm, about 25-30 min, the electrophoresis ends.
▶ Observation results: turn off the power, take out the gel, put it in the UV detector (324 nm), and verify if there is a band of target DNA.
▶ Add Dye: when agarose gel is cooled to 70°C, add 10 uL nucleic acid dye, and mix well.
▶ Preparation of gel board: waterproof tape is pasted on both ends of the glue bed to form an 8 mm high retaining wall, press the tape, and place it on the horizontal table. Insert the comb, and the lower end of the comb is about 1mm away from the bottom of the plate. Pour the 60℃ gel into the glue bed continuously, 3-4 mm high, avoid bubbles, and solidify at room temperature. After complete solidification, tear off the adhesive tape, place it in the electrophoresis tank, add the electrophoresis buffer, which is 1-2 mm higher than the glue surface, gently pull out the comb obliquely from one end to remove the bubbles generated.
▶ Sampling: add 5 μL DNA maker first, and then take 10 μL PCR reaction system for sample addition.
▶ Electrophoresis: cover the electrophoresis tank, the sampling hole is located at the negative pole of the electric field, and connects the power supply, 120V. When the front edge of the dye moves to the bottom edge for 1-2 mm, about 25-30 min, the electrophoresis ends.
▶ Observation results: turn off the power, take out the gel, put it in the UV detector (324 nm), and verify if there is a band of target DNA.
3. Gel extraction.
▶ 1. After DNA electrophoresis, quickly cut the gel containing the target DNA fragment under UV light. It is recommended to blot the surface liquid of the gel with a paper towel and chop it, and try to remove the excess gel. The gel was weighed (removing the empty tube weight) and 100mg of gel was equivalent to 100 μL volume, which was used as a gel volume.
▶ 2. Add an equal volume of Buffer GDP. 50 ~ 55 '℃ water bath for 7-10min, adjust the time appropriately according to the gel size to ensure that the gel block is completely dissolved. Water bath period upside down mixing 2 times to accelerate sol. (Adding 1-3 times the volume of A Buffer GDP does not affect the DNA recovery rate. If the DNA fragment less than or equal to 100 bp is recovered, add 3 times the volume of Buffer GDP, add the same volume of isopropanol after water bath sol, mix and then proceed to the third step.)
▶ 3. Droplets on the wall of the tube were collected by brief centrifugation. The FastPure DNA Mini columns-G adsorption column was placed in the Collection Tubes2ml Collection tube, and ≤700 μL sol solution was transferred to the adsorption column, and centrifuged at 12,000 rpm (13,400×g) for 30-60 sec. If the sol volume is greater than 700 μL, put the adsorption column back in the collection tube, and the remaining sol solution is transferred to the column, 12,000 rpm (13,400×g) centrifugation 30-60 sec.
▶ 4. Discard the filtrate and place the column in the collection tube. Add 300 μL Buffer GDP to the column. Centrifugation at 12,000 rpm(13,400×g) for 30-60 sec.
▶ 5. Discard the filtrate and place the adsorption column in the collection tube. Add 700 μL buffer GW (absolute ethanol has been added) to the column. Centrifugation at 12,000rpm (13,400×g) for 30-60 sec. (Please add Buffer GW around the wall of the column, or mix the cover upside down for 2-3 times after adding Buffer GW to help completely wash the salt attached to the tube wall.)
▶ 6. Repeat Step 5. (Two flushes with Buffer GW can ensure that the salt coating is completely removed, eliminating the impact on subsequent experiments.)
▶ 7. Discard the filtrate and put the adsorption column back into the collection tube. Centrifuge at 12,000 rpm (13,400×g) for 2 min.
▶ 8. Place the column in a 1.5 ml sterilized centrifuge tube, add 20-30 μL Elution Buffer to the center of the adsorption column, and place for 2 min. Centrifuge at 12,000 rpm (13,400×g) for 1 min. The column was discarded and the DNA was stored at -20℃. (If maximum yield is required, it is recommended that the resulting solution be readded to the centrifugal adsorption column and step 8 is repeated for secondary elution. When recovering fragments larger than 3kb, it is recommended to preheat the Elution Buffer to 55 ° C to improve the recovery efficiency.)
4. One-step cloning.
▶ Recombinant plasmids were transformed into competent cells, and LB plates were screened by colony PCR.
▶ Preparation of selective medium plate: LB liquid medium is prepared with 15g agar powder /1000ml LB solid medium. After sterilization, it is cooled to below 55 ℃ and erythromycin is added. The final concentration is 300 μg/ mL, and immediately falls plate in disposable Petri dishes.
▶ Incubation on Ice: add recombinant plasmids into 50 μl prepared competent cells, mix well, and keep on ice for 30 minutes.
▶ Heat Shock: Put competent cells into 42℃ water bath for 42 seconds.
▶ Cooling: take it out and quickly keep it on ice for 2-3min.
▶ Resuscitation: 1 ml non-resistant LB liquid medium was added to each tube, 37℃, 150 rpm, 45 min.
▶ 5000rpm, 2min, suck and discard 700ul supernatant, resuspend the transformed competent cells or control samples in the medium and coat them to the Petri dishes containing ampicillin respectively.
▶ Preparation of selective medium plate: LB liquid medium is prepared with 15g agar powder /1000ml LB solid medium. After sterilization, it is cooled to below 55 ℃ and erythromycin is added. The final concentration is 300 μg/ mL, and immediately falls plate in disposable Petri dishes.
▶ Incubation on Ice: add recombinant plasmids into 50 μl prepared competent cells, mix well, and keep on ice for 30 minutes.
▶ Heat Shock: Put competent cells into 42℃ water bath for 42 seconds.
▶ Cooling: take it out and quickly keep it on ice for 2-3min.
▶ Resuscitation: 1 ml non-resistant LB liquid medium was added to each tube, 37℃, 150 rpm, 45 min.
▶ 5000rpm, 2min, suck and discard 700ul supernatant, resuspend the transformed competent cells or control samples in the medium and coat them to the Petri dishes containing ampicillin respectively.
5. The culture dish is turned upside down and incubated at 37℃ overnight.
6. Pick monoclonal culture seed solution.
7. Plasmid extraction
▶ Add absolute ethanol to rinsing solution PWT.
▶ Centrifugate the overnight culture of bacteria at 12,000 rpm (~ 13,400 × g) for 1 min and remove the supernatant.
▶ Add 150 μL solution P1 to the centrifuge tube to resuspend bacterial precipitation.
▶ Add 150 μl solution P2 into the centrifuge tube, and the tubes were gently turned up and down for 6-8 times.
▶ Add 350 μL solution P3 into the centrifuge tube, immediately and quickly mix up and down for 12-20 times, 12,000 rpm (~13,400 × g) centrifuge for 2 min.
▶ The supernatant collected in the previous step is transferred to the adsorption column CP3 with a pipette. 12,000 rpm (~ 13,400 × g) was centrifuged for 30 sec, the waste liquid in the collecting pipe was poured out, and the adsorption column CP3 was put into the collecting pipe.
▶ 300 mL of rinsing solution PWT was added into the column CP3, and 12,000 rpm (~ 13,400 × g) was centrifuged for 30 sec. Discard the filtrate and reuse the collection tube.
▶ The adsorption column CP3 was put into the collection tube, and centrifuged at 12,000 rpm (~ 13,400 × g) for 1 min. Discard the filtrate and reuse the collection tube.
▶ Place the adsorption column CP3 in a clean centrifuge tube and add 50-100μL elution buffer TB drops to the middle of the adsorption membrane, The plasmid solution was collected into a centrifuge tube after centrifugation at 12, 000 rpm (~13400 × g) for 30 seconds.
▶ Centrifugate the overnight culture of bacteria at 12,000 rpm (~ 13,400 × g) for 1 min and remove the supernatant.
▶ Add 150 μL solution P1 to the centrifuge tube to resuspend bacterial precipitation.
▶ Add 150 μl solution P2 into the centrifuge tube, and the tubes were gently turned up and down for 6-8 times.
▶ Add 350 μL solution P3 into the centrifuge tube, immediately and quickly mix up and down for 12-20 times, 12,000 rpm (~13,400 × g) centrifuge for 2 min.
▶ The supernatant collected in the previous step is transferred to the adsorption column CP3 with a pipette. 12,000 rpm (~ 13,400 × g) was centrifuged for 30 sec, the waste liquid in the collecting pipe was poured out, and the adsorption column CP3 was put into the collecting pipe.
▶ 300 mL of rinsing solution PWT was added into the column CP3, and 12,000 rpm (~ 13,400 × g) was centrifuged for 30 sec. Discard the filtrate and reuse the collection tube.
▶ The adsorption column CP3 was put into the collection tube, and centrifuged at 12,000 rpm (~ 13,400 × g) for 1 min. Discard the filtrate and reuse the collection tube.
▶ Place the adsorption column CP3 in a clean centrifuge tube and add 50-100μL elution buffer TB drops to the middle of the adsorption membrane, The plasmid solution was collected into a centrifuge tube after centrifugation at 12, 000 rpm (~13400 × g) for 30 seconds.
B. Preparation of natural competence
1. Cultivate pIB184: LB medium (resistance: Erythromycin), 37° C, 220 rpm overnight. The plasmid was extracted the
next day.
2. Culture of Streptococcus Brevis ATCC367: Glycerol-preserving bacteria were isolated at 37° C for 24 ~ 48 h, and single colonies were cultured in the MRS medium overnight. Overnight culture of 2 ml was seeded into 50 ml GMRS (Mixed with 1% glycine) and incubated at 37 ° C until OD600 reached ~ 0.6. The cells were collected by centrifugation at 4 ° C, 4000 rpm, 10 min. The supernatant was removed and the cell particles were washed twice with 1 mM MgCl2. Cells were then suspended in 0.5 ml SM buffer (952 mM sucrose and 3.5 mM MgCl2), and 100μL of aliquots were stored at -80 ° C.
2. Culture of Streptococcus Brevis ATCC367: Glycerol-preserving bacteria were isolated at 37° C for 24 ~ 48 h, and single colonies were cultured in the MRS medium overnight. Overnight culture of 2 ml was seeded into 50 ml GMRS (Mixed with 1% glycine) and incubated at 37 ° C until OD600 reached ~ 0.6. The cells were collected by centrifugation at 4 ° C, 4000 rpm, 10 min. The supernatant was removed and the cell particles were washed twice with 1 mM MgCl2. Cells were then suspended in 0.5 ml SM buffer (952 mM sucrose and 3.5 mM MgCl2), and 100μL of aliquots were stored at -80 ° C.
C. Electroporation and Inoculation of pIB184 and streptococcus Brevis ATCC367
1. Plasmids were transformed into Streptococcus Brevis ATCC367 by electroporation.
2. During electroporation, competent cells were mixed with DNA (about 1 µ g, 5 µ L). Incubated on ice for 5min, and then transferred to a pre-cooled 2mm electric rotating cup for electroporation under the conditions of 2kV, 25UF,200 Ω.
3. After electric shock, add 1ml fresh broth (add 500 mM sucrose, 20 mM MgCl2 and 2 mM CaCl2 to MRS), and resuscitate at 37 ° C and 220rpm for 2~3h.
4. After that, the cells were inoculated in medium containing antibiotics and incubated at 37 ° C for 24~48h.
2. During electroporation, competent cells were mixed with DNA (about 1 µ g, 5 µ L). Incubated on ice for 5min, and then transferred to a pre-cooled 2mm electric rotating cup for electroporation under the conditions of 2kV, 25UF,200 Ω.
3. After electric shock, add 1ml fresh broth (add 500 mM sucrose, 20 mM MgCl2 and 2 mM CaCl2 to MRS), and resuscitate at 37 ° C and 220rpm for 2~3h.
4. After that, the cells were inoculated in medium containing antibiotics and incubated at 37 ° C for 24~48h.
D. Butanol fermentation and product detection
1. After electroporation, the isolated colonies were cultured in MRS overnight and added with antibiotics.
2. Transfer 0.7 ml of overnight cultured medium to 70 ml of deoxy-MRS and seeded into an anaerobic chamber for overnight incubation.
3. After culture, the cells were removed from the anaerobic chamber and incubated at 25 ° C for about 24 hours.
4. The cell density was measured with a DU730 photometer. Butanol and ethanol were detected by gas chromatography.
2. Transfer 0.7 ml of overnight cultured medium to 70 ml of deoxy-MRS and seeded into an anaerobic chamber for overnight incubation.
3. After culture, the cells were removed from the anaerobic chamber and incubated at 25 ° C for about 24 hours.
4. The cell density was measured with a DU730 photometer. Butanol and ethanol were detected by gas chromatography.
E. Extraction and detection of intracellular metabolites
1. The cells were cultured at 25 ° C, and 70 ml of fermentation broth was filtered by 0.45 μm filter. The
metabolic reaction of the sample on a filter using methanol: Acetonitrile: WATER = 40:40:20 was cooled at -4℃ for
20 min.
2. The samples were then transferred to a tube containing a quarter volume of 0.1 mm SiO2 dioxide beads and ground three times for 3 minutes at 2,000 rpm.
3. The supernatant was collected by centrifugation at 12,000 rpm for 30 min at 4℃. The above extraction steps are repeated, and the supernatant is merged and filtered prior to metabolite detection.
4. Metabolites were analyzed using High-performance liquid chromatography, UPLC and Q Exactive Orbitrap mass spectrometer. The chromatographic column is T3 column (2.1×150 mM, 3 μm, Waters) , mobile phase A was 10 mM ammonium acetate, mobile phase B was 100% acetonitrile, flow rate was 0.2 ml/min, elution gradient of Phase B was 0 min, 0% ; 1 min, 0% ; 1.1 min, 5% ; 7 min, 25% ; 8 min, 40% ; 11 min, 40% ; 11. 1min,0%；15 min,0%.
5. Full scans were performed at M/z 700-1300 using electrospray ionization (ESI) in a positive ion mode, and all acquired data were analyzed with XCalibur 4.0
2. The samples were then transferred to a tube containing a quarter volume of 0.1 mm SiO2 dioxide beads and ground three times for 3 minutes at 2,000 rpm.
3. The supernatant was collected by centrifugation at 12,000 rpm for 30 min at 4℃. The above extraction steps are repeated, and the supernatant is merged and filtered prior to metabolite detection.
4. Metabolites were analyzed using High-performance liquid chromatography, UPLC and Q Exactive Orbitrap mass spectrometer. The chromatographic column is T3 column (2.1×150 mM, 3 μm, Waters) , mobile phase A was 10 mM ammonium acetate, mobile phase B was 100% acetonitrile, flow rate was 0.2 ml/min, elution gradient of Phase B was 0 min, 0% ; 1 min, 0% ; 1.1 min, 5% ; 7 min, 25% ; 8 min, 40% ; 11 min, 40% ; 11. 1min,0%；15 min,0%.
5. Full scans were performed at M/z 700-1300 using electrospray ionization (ESI) in a positive ion mode, and all acquired data were analyzed with XCalibur 4.0