Team:Shanghai SFLS SPBS/Experiments
Culture medium preparation
The following is the recipe for 1 L culture medium. For solid plates, 10 g/L agar was added.
LB Medium
| Tryptone | 10.0 g/L |
| Yeast Extract | 5.0 g/L |
| NaCl | 10.0 g/L |
| ddH2O | To 1 L |
LBv2 Medium
| Tryptone | 10.0 g/L |
| Yeast Extract | 5.0 g/L |
| NaCl | 21.9 g/L |
| KCl | 0.31 g/L |
| MgCl2·6H2O | 4.70 g/L |
| ddH2O | To 1 L |
M9 Medium
| M9 minimal salts 5x | 11.28 g/L |
| glucose | 4 g/L |
| NaCl | 0.5 g/L |
| CaCl2 | 10 mg/L |
| MgSO4 | 0.5 g/L |
| CuSO4 | 40 mg/L |
| ddH2O | To 1 L |
Strain inoculation
For solid plates, a pipette tip or toothpick was used to pick a single colony from the plate and put into the culture medium. Bacterial solution was added 1:100 to the culture medium.
Strain preservation
After overnight culture, 500 μL (or 800 μL) bacterial solution was mixed with 500 μL (or 800 μL) 30% glycerol (sterilized) in a EP tube. The tube was preserved at the -80°C refrigerator.
Chemical transformation for E. coli
1. 50 μL of E. coli competent cells and 0.5-1 μL plasmid (2 μL for Gibson Assembly products) was mixed on ice. The tube was placed on ice bath for 20 min.
2. The tube was subjected to a heat shock at 42℃ for 60 s.
3. The tube was ice bathed for 2 min.
4. 300 μL LB culture medium without antibiotics was added, and the bacteria were cultured in a shaker at 37℃ for 60 min.
5. For plasmid transformation, 100 μL of bacterial solution was taken and spread on a solid plate containing antibiotics. The plates were cultured at 37℃ overnight. For the transformation of Gibson Assembly products, the bacterial solution was centrifuged at 4000 rpm for 1 min. 250 μL supernatant was removed and the bacterial pellet was resuspended with remaining LB medium. The resuspension was then inoculated on a solid plate containing antibiotics and incubated at 37℃ overnight.
V. natriegens competent cell preparation
1. V. Natriegens cells were grown in LBv2 medium overnight at 37℃, 220 rpm.
2. The cultures from Step 1 were used to inoculate (1:200 dilution) a second matched medium culture (100 mL), which was grown at 37℃, 220 rpm until an OD600 of 1.0.
3. The culture was split into centrifuge bottles and pelleted at 6465 g for 10 min at room temperature.
4. The supernatant was decanted, and the cell pellets were gently resuspended in 3.25 mL of prewarmed 1 M sorbitol (sterilized) and incubated at 37℃ for 1 h.
Chemical transformation for V. natriegens
1. Plasmid DNA (typically 1-2 μL of 100 ng/μL) and 200 μL chemically competent cells (in 1 M sorbitol) were combined and gently mixed on ice.
2. The mixture was transferred to a heat bath (42℃) and subjected to heat shock for 2.5 min.
3. The cells were immediately recovered in 500 μL of recovery medium (LBv2 substituted with 680 mM sucrose, sterilized) and transferred to a 15 mL conical tube.
4. Cells were incubated at 37℃, 220 rpm overnight.
5. 150 μL aliquots of the recovery medium were plated on warm agar plates containing the appropriate antibiotic.
6. The plates were incubated at 37℃ overnight (~10-12 h) for colony growth.
Plasmid extraction
TIANprep Mini Plasmid Kit (DP103 from Tiangen Biotech (Beijing) Co., Ltd.) was used for plasmid extraction.
1. A new Spin Column CP3 was placed in the collection tube. 500 μL Buffer BL was added to the column. The collection tube was centrifuged at 12,000 rpm for 1 min, and waste liquid was discarded.
2. 1-5 ml of overnight cultured bacteria solution was added to a centrifuge tube and centrifuge for 1 min at 12,000 rpm.
3. The supernatant was removed, and 250 μL Buffer P1 (with RNase A added) was added to the centrifuge tube with the bacterial pellet. The bacterial pellet was completely suspended.
4. 250 μL Buffer P2 was added to the centrifuge tube and the tube was gently inverted 6-8 times to fully lyse the cells.
5. 350 μL Buffer P3 was added to the centrifuge tube, which was immediately yet gently inverted 6-8 times. The system was centrifuged at 12,000 rpm for 10 min.
6. The supernatant collected in Step 5 was transferred to Spin Column CP3. The column was centrifuge at 12,000 rpm for 1 min and the waste was discarded.
7. 600 μL Buffer PW (with ethanol added) was added to the Spin Column CP3. The column was centrifuge at 12,000 rpm for 1 min and the waste was discarded.
8. Step 7 was repeated.
9. The column was placed in the collection tube and centrifuge at 12,000 rpm for 2 min to remove the residual Buffer PW.
10. The Spin Column CP3 was placed in a clean EP tube. 50-100 μL ddH2O was added. The column was placed at room temperature for 2 min, then centrifuged at 12,000 rpm for 2 min.
11. The purity and concentration of the extracted plasmids was measured.
Colony PCR
2x Taq PCR MasterMix (KT201 from Tiangen Biotech (Beijing) Co., Ltd.) was used for plasmid extraction.
1. The following reaction system was prepared. The system was gently mixed
| 2 x Taq PCR Mix | 10 μL |
| Forward Primer（10 μM） | 1 μL |
| Reverse Primer（10 μM） | 1 μL |
| Colony | 1 single colony (1 μL bacterial solution) |
| ddH2O | to 20 μL |
2. The PCR program was set as follows:
| Step | Temperature | Time | Cycle |
|---|---|---|---|
| Initial denaturation | 94℃ | 10 min | 1 cycle |
| Denaturation | 94℃ | 30 s | 30 cycles |
| Annealing | (Tm)-5~10℃ | 30 s | |
| Extension | 72℃ | 1 kb/min | |
| Final extension | 72℃ | 5 min | 1 cycle |
| Hold | ≤16℃ | ∞ | - |
Gibson Assembly
Golden Star T6 Super PCR Mix (from Beijing Tsingke Biotech Co., Ltd.) was used for the reaction.
1. The following reaction system was configured on ice.
| Golden Star T6 Super PCR Mix | 47 μL |
| Plasimd with interested gene | 1 μL |
| Forward primer | 1 μL |
| Reverse primer | 1 μL |
2. A pipette was used to gently mix the system.
3. A PCR was first carried out for the system. The program was set as follows:
| Step | Temperature | Time | Cycle |
|---|---|---|---|
| Initial denaturation | 98℃ | 2 min | 1 cycle |
| Denaturation | 98℃ | 10 s | 30 cycles |
| Annealing | (Tm)-5~10℃ | 10 s | |
| Extension | 72℃ | ~30 s/kb | |
| Final extension | 72℃ | 5 min | 1 cycle |
| Hold | 16℃ | ∞ | - |
4. After the PCR, the vector and the fragment were mixed. The reaction tubes was placed in the PCR machine and incubated at 50℃ for 15 min.
Agarose gel electrophoresis
1. 0.25 g/0.5g/1.0g agarose and 25 mL/50mL/100mL of 1×TAE buffer was mixed in a flask. The solution was heated in a microwave oven until agarose dissolves completely.
2. 2.5 μL/5 μL/10 μL SYBR safe DNA Gel Stain (10000×) was added. After mixing the solution, the agarose was poured into a gel tray with the well comb in place. The gel was left for 20-30 min to completely solidify.
3. After the liquid solidifies, the well comb was unplugged.
4. The gel was put into the electrophoresis unit. 1×TAE buffer was added to the unit until the gel was completely immersed in the buffer.
5. For PCR products with loading dye (e.g. those that used 2x Taq Mix), 3-6 μL DNA (PCR product) was loaded into each lane. For samples without loading dye, 4 μL DNA was mixed with 1 μL loading dye before being loaded into the lanes. One or multiple markers were loaded as needed.
6. The voltage was set to 120 V for 20-30 min.
DNA purification
TIANgel Midi Purification Kit (DP209 from Tiangen Biotech (Beijing) Co., Ltd.) was used for DNA purification.
1. 500 μL Buffer BL was added into a Spin Column CA2, which was centrifuged at 12,000 rpm for 1 minute. Waste liquid was discarded from the collection tube.
2. As much agarose gel as possible was carefully removed from the target DNA strip. The DNA strip was placed in a clean centrifuge tube for weighing.
3. The same amount of Buffer PN was added. The mixture was placed in 50°C water bath for 10 min. The tube was gently turned to ensure that the gel was fully dissolved in the buffer.
4. The solution obtained from Step 3 was added into a Spin Column CA2 and left at room temperature for 2 min. The solution was centrifuged at 12,000 rpm for 1 min, and the waste liquid was discarded.
5. 600 μL Buffer PW (with ethanol added before use) was added to the column. The column was centrifuged at 12,000 rpm for 1 min. The waste liquid was discarded.
6. Step 5 was repeated.
7. The Spin Column CA2 was centrifuged at 12,000 rpm for another 2 min to remove any remaining buffer. It was then placed at room temperature for a few minutes to dry completely.
8. The column was moved onto a clean EP tube. 50-100 μL ddH2O was added. The column was place at room temperature for a few minutes and then centrifuge it at 12,000 rpm for 2 min.
9. The purity and concentration of the recovered DNA was measured and recorded.
Protein electrophoresis
BeyoGel™ Plus Precast PAGE Gel (4-20%, 15 wells) from Beyotime Biotechnology was used as the SDS-PAGE gel for protein electrophoresis.
1. The precast gel was fixed in the electrophoresis tank. The comb was gently pulled out.
2. The electrophoresis buffer was prepared using SDS-PAGE Electrophoresis Buffer with Biofuraw™ Pre-Cassette Gel-Running Buffer.
3. The electrophoresis tank was filled with electrophoresis buffer. The buffer was poured into the inner tank until it overflowed to the outer tank and submerged the anode at the bottom of the tank.
4. 4-10 μL of each sample was gently loaded into one lane. One or multiple markers were loaded as needed.
5. The voltage was set to 120 V for 20-30 min.
6. The precast gel was taken out of the tank. A blade was used to separate the gel from the cast.
7. An appropriate amount of protein dye (Biofuraw™ LDS Sample Buffer, With Reducing, diluted from 4X) was added so that the staining solution could cover the gel and the liquid level is at least three times higher than the thickness of the gel. Dyeing was performed on a shaker at room temperature (20-25°C) for 20-30 min.
Hair bleach
1. Bleach powder and hydrogen peroxide milk were mixed 1:1.
2. Black hair was added and let stand for 50 min.
Hair dye
1. pH=9 Ca(OH)2 (for melanin, polydopamine, indigo, and dopaxanthin) or pH=5 sodium acetate buffer (for indoline-betacyanin and laccase+precursors) was prepared.
2. An appropriate amount of hair was added to the solution and let stand at 50℃ for 40 min to open the cortex of the hair.
3. The solution was discarded and 10 mg/mL pigment solution was added.
4. The system was let stand at 50℃ for another 40 min.
5. The hair was washed with excessive water.
Production & measurement of pigments
Single colonies or preserved bacteria (1:100) was added to 5 mL culture medium (LB for E. coli, LBv2 for Vibrio natriegens) and cultured for approximately 12 h. Next, 500 μL bacterial solution was added to 50 mL matching culture medium, the appropriate substrates are added, and this time is recorded as 0 h.
Melanin
1. 50 mg/L L-DOPA and 8 mg/L L-Tyrosine was added at 0 h. The bacteria were cultured at 37℃, 220 rpm.
2. Every 12 h, 2 mL bacterial solution was moved to a EP tube, the tube was centrifuged, and absorbance at 400 nm was measured and recorded.
3. The production curve was measured over 72 h.
Indigo
1. 100 mg/L L-Tryptophan was added at 0 h. The bacteria were cultured at 37℃, 220 rpm.
2. Every 12 h, 2 mL bacterial solution was moved to a EP tube
3. The tube was centrifuged for 1 min, the supernatant was discarded, and 2 mL DMSO was added to resuspend the indigo.
4. The tube was centrifuged for another 1 min to make sure the bacteria is not in the solution, which may affect measurement of absorbance.
5. Absorbance at 605 nm was measured and recorded.
6. The production curve was measured over 72 h.
Dopaxanthin & betacyanin (T7-4,5-DODA)
1. The bacterial strains with the inducible promoter was cultured at 37℃, 220 rpm until OD600=1.0.
2. 0.1 mM IPTG was added in both flasks. The flasks were cultured at 37℃, 220 rpm.
3. After 16 h, the bacterial solution was centrifuged at 5000 ×g for 10 min. The supernatant was discarded and the cell pellet was resuspended in 50 mL sterilized ddH2O.
4. In the flask for dopaxanthin, 9 g/L L-DOPA and 2.64 g/L Vitamin C (sodium L-ascorbate) was added. In the flask for indoline-betacyanin, 1.5 g/L L-DOPA, 0.0452 g/L indoline, and 2.64 g/L Vitamin C was added. The substrates were added at 0 h.
5. The flasks were cultured at 20℃, 120 rpm. Every 12 h, 2 mL bacterial solution was moved to a EP tube, the tube was centrifuged, and absorbance at 415 nm (dopaxanthin) and 525 nm (indoline-betacyanin) was measured and recorded.
6. The production curve was measured over 96 h.
IPTG Induction
After overnight culture in shaking tubes, the bacterial solution was added 1:100 into a matching culture medium and cultured at 37℃, 220 rpm for ~3 h (or until OD600~1.0). For T7 promoters, 0.1 mM IPTG was added. For pTac promoters, 1 mM IPTG was added.
References
1. Retrieved July 2020, from https://www.protocols.io/view/growth-media-for-v-natriegens-u3ceyiw
2. Retrieved July 2020, from http://cshprotocols.cshlp.org/content/2010/8/pdb.rec12295.short
3. T. Tschirhart et al., "Synthetic Biology Tools for the Fast-Growing Marine Bacterium Vibrio natriegens", ACS Synthetic Biology, vol. 8, no. 9, pp. 2069-2079, 2019. Available: 10.1021/acssynbio.9b00176.