Team:SUSTech Shenzhen/Protocol
Part1. Lactobacillus Sensor System:
Plasmid Construction and Transformation1:
1. Materials & Apparatus
Liquid MRS media
MRS plate
MRS plate with 10mg/L erythromycin
Wash-solution (10mM MgCl2)
Electroporation Buffer (0.5mM Sucrose, 10% Glycerol)
Conservation buffer(12% Glycerol)
Gene transfection instrument(SCIENTZ-2C)
Bluepard biochemical incubator
Shock incubator(MQD-B2NR)
Centrifuge(Eppendorf 5424)
Refrigerated centrifuge (Thermo ST16R)
Protocol
A. Production of Competent Cells
1. Grow L.reuteri at 37 °C in MRS agar media overnight under stationary conditions
2. Cultures were inoculated (1:40) into MRS agar media until it OD600 becomes 0.8
3. Wash 2x with wash-solution
4. Wash 1X in Electroporation Buffer
5. Concentrate in conservation buffer and store in -80℃
B. Plasmids Transformation
1. Thaw bacteria in -80℃ fridge on ice for use. Precool electric shock cup
2. Add 5μL plasmid DNA and 50μL cells in an electric shock cup. Cool it for 5 minutes
3. Electroporate at 2.0kV/cm, 200Ω and 25μF
4. Recover cells by adding 950μL of MRS
5. Incubate for 2 hours at 37℃
6. Plate to media with 15mg/L erythromycin select
Part2. E.coli Effector System:
Plasmid Transformation and Extraction:
Plasmid Transformation:
1. Materials & Apparatus
LB media(10g tryptone, 10g NaCl, 5g yeast extract per liter)
0.1M CaCl2
50% Glycerol
0.1M CaCl2 + 15% glycerol
LB plate
LB plate with 50mg/L Kanamycin
Gene transfection instrument(SCIENTZ-2C)
Bluepard biochemical incubator
Shock incubator(MQD-B2NR)
Centrifuge(Eppendorf 5424)
Refrigerated centrifuge (Thermo ST16R)
2. Protocol
A. Production of Competent Cells
1. Grow E.coli(DH5α, Acella and Nissle 1917) on LB plates overnight at 37℃
2. Pick colony into 10mL LB media, grow overnight at 37°C. Take 2mL LB media saved for blank
3. Transfer 5mL overnight colony culture into 200mL LB media
4. Allow cell to grow at 37°C, 220 rpm until OD600 = 0.4
5. Transfer cells to several 50 mL centrifuge tube, and place cells on ice for 20 minutes
6. Centrifuge cells at 4°C for 10 minutes at 3000g
7. Pour off media and resuspend cells in 12 mL of cold 0.1M CaCl2, Transfer the suspended cells into a 50 mL centrifuge tube, and incubate on ice for 30 minutes
8. Centrifuge cells at 4°C for 10 mins at 3000g (2500 rpm)
9. Pour supernatant and resuspend cells (by pipetting) in 3.2 mL cold 0.1M CaCl2 contains 15% glycerol. Transfer 140 μL into 1.5 mL tubes
10. Stored in -80℃ fridge
B. Plasmids Transformation (heat shock for DH5α and Acella)
1. Add 2μL purchased plasmid into 50μL competent cell while adding 2μL ddH2O into another 50μL competent cell as the control group, then incubate on ice for 20~30 minutes
2. 42℃ water bath for 75 seconds
3. Incubate on ice for 2 minutes
4. Add 1ml LB, then incubate for 1 hour at 36°C, 220rpm
5. Centrifuge at lower than 6000rpm for 1 minute，then remove 900μL supernatant, resuspend cells and transfer it on LB plate with 50mg/L Kanamycin overnight
C. Plasmids Transformation (electrotransformation for Nissle 1917)
1. Thaw aliquots on ice for use
2. Add up to 5μL plasmid DNA
3. Electroporate at 9000kV/cm
4. Recover cells by adding 500μL of MRS with 80mM MgCl2
5. Incubate for 2 hours at 30°C
6. Plate to media with 10mg/L erythromycin select
Plasmid Extraction:
1. Materials & Apparatus
LB media(10g tryptone, 10g NaCl, 5g yeast extract per liter)
0.1M CaCl2
50% Glycerol
0.1M CaCl2 + 15% glycerol
LB plate
LB plate with 50mg/L Kanamycin
Gene transfection instrument(SCIENTZ-2C)
Thermostat water bath(JINGHONG DK-S28)
Bluepard biochemical incubator
Shock incubator(MQD-B2NR)
Centrifuge(Eppendorf 5424)
Refrigerated centrifuge (Thermo ST16R)
2. Protocol
1. Grow bacteria contains plasmids and stored in -80℃ fridge on LB plates overnight at 37°C
2. Pick colony into 5mL LB and incubate overnight in shake incubator at 37℃, 220rpm
3. Extract plasmid using E.Z.N.A. Plasmid Mini Kit I, The operating method is in the kit's instructions
Construction and Verification of Gene Expression Regulatory system:
EsaR and PhlF Expression Test(western blot):
1. Materials & Apparatus
TBS media
Tween-20
Loading dye
Purchased running buffer
CBB(Coomassie brilliant blue)
Antibody 1（1:2000 attenuation）
Antibody 2（1:2000 attenuation）
Purchased confining liquid
30% Acrylamide
1M Tris 8.8
dH2O
10% APS
TEMED 2.5μL
10% SDS 20μL
Shock incubator(MQD-B2NR)
Electrophoresis tank
Power supply(BIO-RAD PowerPac Basic)
Centrifuge(Eppendorf 5424)
Shaker
Multifunctional microporous plate detector(TECAN)
2. Protocol
A. protein Electrophoresis
1. Add IPTG into 5mL LB until its reaches 0.5 mM. Then add 10μL bacteria solution and shake with 220rpm, 37℃ in shake incubator overnight
2. Add Tween-20 into TBS media to make TBST media
3. Protein Electrophoresis（Make two copies once a time，One was used for coomassie bright blue staining and one was used for WB）:
① Making 1mm stacking gel(30% Acrylamide 0.335mL, 1M Tris 8.8 0.25mL, dH2O 1.38mL, 10% APS 15μL, TEMED 2.5μL, 10% SDS 20μL)
② Making separating gel(30% Acrylamide 4.0mL, 2M Tris 8.8 1.13mL, dH2O 0.82mL, 10% APS 30μL, TEMED 3μL, 10% SDS 60μL), insert the comb before the gel sets
③ Add 2μL loading dye in 10μL protein solution in the tube
④ Mark the tube and centrifuge in 6000rpm for 5min
⑤ 100℃ incubate protein solution for 5min
⑥ Respectively add 10μL protein and 3μL ladder in different holes
⑦ Power on, use 150V voltage for 60min
⑧ Wash one of the gels with CBB three times and soak the gel overnight at the last time
4. Cut the other gel with a membrane according to its sizes
5. Active membrane with methanal, then wash 15 seconds with ddH2O
6. Incubate membrane and filter paper with running buffer for 10 minutes
7. Transfer the protein to the membrane, place these materials into the machine from the bottom up: filter paper- The membrane that has been sheared off at a corner -gel-filter paper
8. Use a roller to remove bubbles
9. Close the lid, turn on the machine used to transfer the protein to the membrane, and adjust the list as required
10. Wash with TBS for 10 minutes after
11. Incubate with confining liquid in 4oC shaker
12. Wash with TBST 10 minutes three times
13. Combine with antibody(if test EsaR, antibody 1 should be used, otherwise antibody 2 should be used). Incubate on the shaker overnight
Fluorescence Test:
1. Materials & Apparatus
0.5M IPTG
AHL(first configurate 200mg/L AHL solution，for 10000 nM AHL，then ten times dilution four times for other concentration of AHL. Add 2.13μL solution with the corresponding concentration)
Liquid LB media
Shock incubator(MQD-B2NR)
Multifunctional microporous plate detector(TECAN)
2. Protocol
1. Culture the engineered bacteria E. coli Acella until OD equals 0.6
2. Transform media into 96-well plate (200μL/well), Add IPTG and AHL until the final concentration reach the following level
The 96-well plate is arranged as follows(the number in G and H line is the concentration of AHL-IPTG):
3. Measure the fluorescence level of both GFP and RFP using the multifunctional microporous plate detector for at least 24 hours
Construction and Verification of Colanic Acid(CA) Produce System:
Staining Test2
1. Materials & Apparatus
Nissle-1917 bacteria
Nissle-1917 bacteria with pBbE6k-RcsB plasmid
M9 agar medium (6.78 g/L Na2HPO4, 3 g/L KH2PO4, 4 g/L glucose, 1 g/L NH4Cl, 0.5 g/L NaCl, 0.24 g/L MgSO4, 0.011 g/L CaCl2, and 15g/liter Agar)
Fuchsin solution (mixture composed of 0.3 g basic fuchsin, 10 ml 95% ethanol, and 90 ml 5% phenol)
Mordant solution (a freshly prepared mixture containing 2 volumes of 0.3 g/liter FeCl3, 2 volumes of 1.5 g/liter tannins, and 5 volumes of a 2 g/liter saturated solution of potassium aluminum sulfate)
Distilled water
1% Methylene blue alcohol saturated solution
2.5% glutaraldehyde
1% osmium tetroxide vapor
10 mM phosphate-buffered saline (PBS, adjust pH to 7.2)
Immersion lens
2. Protocol
1. Grows normal Nissle-1917 bacteria and Nissle-1917 bacteria with pBbE6k-RcsB plasmid on M9 agar medium at 30°C for 2 days
2. Fixed cells on slides by smearing and drying and then treated with fuchsin solution for 3 min
3. Add mordant solution for 3 min and then washed with distilled water
4. Use 1% g/dl Methylene blue alcohol saturated solution(Put the 37 ℃ water bath 30 minutes in advance to preheat) for 30s prior to examination of the samples under an oil immersion lens. The cells were stained red and the exopolysaccharides blue
CA Expression Test
1. Materials & Apparatus
M9 media (6.78 g/L Na2HPO4, 3 g/L KH2PO4, 4 g/L glucose, 1 g/L NH4Cl, 0.5 g/L NaCl, 0.24 g/L MgSO4, and 0.011 g/L CaCl2)
IPTG
0.75% (wt/vol) streptomycin sulfate
1% glacial acetic acid (v/v)
Dichloromethane-methanol (2:1 [vol/vol])
Distilled water
trifluoroacetic acid solution
0.3 M NaOH
Monosaccharide (50 μL, 0.05 M) or 100 μL polysaccharide hydrolysate
0.5 M 1-phenyl-3-methyl-5-pyrazolone (PMP) methanol solution
0.3 M HCl
Dichloromethane
0.05 M phosphate buffer (pH 6.9)
Acetonitrile (0.05 M phosphate buffer + 10% (v/v) acetonitrile)
High-performance liquid chromatography(SHIMADZU)
AKTA purifier
Centrifuge(Eppendorf 5424)
Shock incubator(MQD-B2NR)
2. Protocol
1. Incubate bacteria in M9 media at 37℃ for 2 days with 220 rpm shaking while using 0.5M IPTG induced
2. Centrifugate in 18000 g for 45 minutes. The supernatant was collected and mixed with 3 volumes of ice-cold anhydrous ethanol
3. Precipitate at 4℃ overnight, the solid product was resuspended in water to a concentration of 100 mg/L
4. UPrecipitate nucleic acids by adding 0.75% (wt/vol) streptomycin sulfate and were removed by centrifugation at 18000 g for 30 min
5. To eliminate LPS, treat supernatant with 1% glacial acetic acid (v/v) at 100°C for 2 h and then centrifuge at 18000 g for 30 min at 4°C
6. Extract the supernatant with 1 volume of dichloromethane-methanol (2:1 [vol/vol]), and collect the aqueous phase(top phase)
7. Dialyze against distilled water (molecular weight cutoff, 10000) for 48 h
8. Using vacuum centrifugation evaporator(1.5 hours, 60℃, 1mL sample)
1. Mix 15 mg CA with 2 ml of 2M trifluoroacetic acid solution in a tube and heated at 110°C for 4 h
2. Cool down The mixture to room temperature and centrifuged at 12,000 × g for 5 minutes. Adjust The supernatant to pH 7.0 with 0.3M NaOH
3. Mix monosaccharide (50μL, 0.05M) or 100 μL polysaccharide hydrolysate with 50 μL 0.5M 1-phenyl-3-methyl-5-pyrazolone (PMP) methanol solution and 0.3M NaOH, incubate in water at 70 °C for 30 min. Then hold at room temperature for 20 min, and neutralize to pH 7.0 with 50 μL 0.3 M HCl
4. Extract the reaction solution with 1 mL dichloromethane, shook for 2 min, and centrifuged at 12,000g for 5 minutes. Do this operation three times
5. Dilute them into 0.125, 0.25, 0.5 mM (water) solution, and 0.4 μL supernatant was subjected for UHPLC analysis. The mobile phase was 0.05 M phosphate buffer (pH 6.9) and acetonitrile (solvent A: 0.05 M phosphate buffer + 10% (v/v) acetonitrile) at a flow rate of 0.2 mL/min, 10 min
6. Absorbance at 245nm is measured and derive standard curves
Antibacterial System:
Gibson Assembly:
1. Materials & Apparatus
Q5 2x PCR enzyme Mix
Arabinose promoter and designed plasmid
Primer for Arabinose promoter and designed plasmid
ddH2O
Gibson assembly Master mix
50% glycerin
1*TAE
Agarose
EB
1kb DNA ladder
LB medium
Glycerin
Centrifuge(Eppendorf 5424)
Thermostat water bath(JINGHONG DK-S28)
Microwave
PCR amplifier(Bio-Rad C100)
Shock incubator(MQD-B2NR)
Power supply(BIO-RAD PowerPac Basic)
Thermostat water bath(JINGHONG DK-S28)
Vortexer
E.Z.N.A. Gel extraction kit
2. Protocol
1. Add 2.5μL two types of primer, 1μL of vector, 12.5μL Q5 *2 enzyme in a PCR tube, then add ddH2>O until its volume becomes 25μL
2. Put the tube in PCR amplifier and set the parameters:
Fragment: 98℃*30s→30 cycles*[98℃*10s→72℃*30s→72℃*40s]→72℃,2min
Vector: 98℃*30s→30 cycles*[98℃*10s→72℃*30s→72℃*320s]→72℃,2min
3. Stored at 4℃
1. Add 40mL 1*TAE and 0.4g agarose into a 100mL conical flask
2. Cover the neck of the conical bottle with tissue. Microwave it for 30 seconds, then continue to heat it and oscillate it every 10 seconds until it meltdown completely
3. Rinse outside of the conical bottle, then add 4mL EB and shakeup
4. Pour the liquid into the rubber stand, place it in the comb, and wait to cool
5. Put the gel into the electrophoresis tank with 1*TAE. Respectively add all product and 1μL 1kb DNA ladder into the gel
6. Start electrophoresis in 120V voltage for 45min
7. Observe under ultraviolet light to see if necessary bands are present(1300kb for fragment and about 8000kb for vector)
8. Cut the gel contains DNA product, put it in a 1.5mL centrifuge tube
9. LB plate with 50mg/L Kanamycin
1. Minimize the gel contains DNA
2. Use E.Z.N.A Gel Extraction Kit to extract DNA. The operating method is in the kit's instructions
1. Add 1μL PCR Fragment(≈55ng/μL), 2μL PCR vector(≈65ng/μL), 5μL Gibson assembly Master mix. Then add ddH2O until its volume becomes 10μL
2. Incubate samples in a thermocycler at 50°C for 15 minutes. Following incubation, store samples on ice or at -20°C for subsequent transformation
1. Add 2μL product plasmid into 50μL competent cell while adding 2μL ddH2O into another 50μL competent cell as the control group, then incubate on ice for 20~30 minutes
2. 42℃ water bath for 75 seconds
3. Incubate on ice for 2 minutes
4. Add 1ml LB, then incubate for 1 hour at 36°C, 220rpm
5. Centrifuge at lower than 6000rpm for 1 minute，then Remove 900μL supernatant, resuspend cells and transfer it on LB plate with 50mg/L Kanamycin overnight. If there are colonies, used pipette tips to pick out them into 5mL LB medium, growth overnight, mixed with Isovolume glycerin, and stored in -80℃
Lactoferrin Verification and Purification:
1. Materials & Apparatus
1L LB media(10g tryptone, 10g NaCl, 5g yeast extract)
0.5M IPTG
50mM Kanamycin
Protein buffer(basic buffer: 50mM NaHPO4 7.1g, 300mM NaCl 17.53g, 10% glycero 100mL. Adjust pH to 7.4, filtration with 0.22mm filter. Add 1mM DTT)
1mM PMSF(Dissolve with isopropyl alcohol or methanol)
1M Imidazole
ddH2O
Refrigerated centrifuge (Thermo ST16R)
Ni column(Shirin-pack Scepter C18-120/1.9um, Dimension:2.1*50mm)
AKTA purifier
Refiner
Shock incubator(MQD-B2NR)
2. Protocol
1. Active the bacteria with 10μL bacterial suspension and 10ml LB in 220rpm shake incubator
2. Inoculate bacteria in residual LB, incubate it until its OD reach 0.8~0.9
3. Add IPDG until 0.5mM concentration to induce its expression 160rpm, 20℃
4. Cold centrifuge and refiner
5. Centrifuge in 8000rmp for 10min
6. Resuspension with protein buffer. Add PMSF
7. Disrupt cell with refiner to 20000bar for 6~10 cycle
8. Wash refiner with ddH2O two times and ethanol one times
9. LB plate with 50mg/L Kanamycin
10. Filtration with 0.22μm filter paper
11. Wash the tube with water
12. Wash the Ni column with water
13. Start Ni-sepharose purification
14. Gel electrophoresis according to peak value
15. Centrifuge 4000rmp for 15~30min
16. Wash the molecular sieve and wash the loop. Push the protein into the loop
17. Filtered through molecular sieves
Reference
[1]方明建; 王磊; 陈玉林, 6株羊源乳酸杆菌电转化条件的优化. 中国畜牧兽医 2013, 40 (09), 80-85.
[2]Ren, G.; Wang, Z.; Li, Y.; Hu, X.; Wang, X.; Mullineaux, C. W., Effects of Lipopolysaccharide Core Sugar Deficiency on Colanic Acid Biosynthesis in Escherichia coli. Journal of Bacteriology 2016, 198 (11), 1576-1584.