Inserted fragments and destination vector are prepared 3:1 (0.075pmol and 0.025pmol) and/or 5:1 (0.125pmol and 0.025pmol) in mole number. Calculate volume of each component based on their concentration and length. Type IIS restriction enzymes (e.g. BpiI, BsaI, and BsmbI) are used to create overhangs between fragments and vector without leaving scar. Restriction enzymes, T4 ligase, and ligase buffer are from NEB®.
Add components for Golden Gate assembly in the following order:
___________________________________________________
Step Temperature Time
___________________________________________________
Initial melting 98°C 30 sec
___________________________________________________
35 cycles:
Melting 98°C 10 sec
Annealing based on Tm of primers 30 sec
Extension 72°C 30s/kbp
___________________________________________________
Final extension 72°C 10 min
___________________________________________________
Holding 16°C ∞
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E. coli transformation
The competent cells used in this protocol is E. coli DH5α cells from NEB®.
- Add 7.5 μl of your DNA assembly reaction into a 25 μl aliquot of E. coli DH5α cells
- Place tube containing cells mixed with assembled DNA on ice for 10 min.
- Time-sensitive step: Heat shock cells by placing them into 42°C water bath for 30 sec.
- Time-sensitive step: To allow cells to recover from heat, place tube on ice for 5 min.
- Pipette 950 µl of room temperature SOC into the mixture.
- Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
- Warm selection plates to 37°C.
- Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.
- Spread 50-100 µl of each dilution onto a selection plate and incubate overnight at 37°C.
Colony PCR
After overnight growth, selection plates are expected to have colonies that can be picked.
- Use sterile tips or toothstick to pick as many colonies as possible in sterile condition
- Use the tip/toothstick with colony biomass to re-streak it on a new selection plate (with the same antibiotic)
- Put the tip/toothstick with remaining colony biomass into strips of microtubes with the following components:
Components
|
Volume
|
ddH2O
|
3μL
|
10μM forward verification primer
|
1μL
|
10μM reverse verification primer
|
1μL
|
2X Phire plant direct master mix (from Thermo ScientificTM)
|
5μL
|
Total: 10μL
Use agarose gel electrophoresis to verify the colony containing correctly assembled plasmids. Phire master mix contains loading dye so loading dye is not required for loading the gel.
cPCR Thermocycler Program:
Step Temperature Time
___________________________________________________
Initial melting 98°C 5 min
___________________________________________________
40 cycles:
Melting 98°C 5 sec
Annealing based on Tm of primers 5 sec
Extension 72°C 20s/kbp
___________________________________________________
Final extension 72°C 10 min
___________________________________________________
Holding 16°C ∞
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Inoculation
Colonies containing correct clones are inoculated in liquid culture. Use sterile tips/toothsticks to pick the correct colony and directly put into sterile culture tube with 4mL of liquid LB (with antibiotic). Incubate overnight at 37°C.
Plasmid Extraction "Miniprep"
NEB® Monarch® Plasmid DNA miniprep kit is used to extract plasmids from liquid culture.
- Pellet 1–5mL bacterial culture (not to exceed 15 OD units) by centrifugation at 13,000 rpm for 30 seconds. Discard supernatant.
- Resuspend pellet in 200μL Plasmid Resuspension Buffer (B1) (pink). Vortex or pipet to ensure cells are completely resuspended. There should be no visible clumps.
- Lyse cells by adding 200μL Plasmid Lysis Buffer (B2) (blue/green). Invert tube immediately and gently 5–6 times until color changes to dark pink and the solution is clear and viscous. Do not vortex. Incubate for one minute.
- Neutralize the lysate by adding 400μL of Plasmid Neutralization Buffer (B3) (yellow). Gently invert tube until color is uniformly yellow and a precipitate forms. Incubate for 2 minutes.
- Clarify the lysate by spinning for 10 minutes at 13,000 rpm.
- Carefully transfer supernatant to the spin column and centrifuge for 1 minute. Discard flow-through.
- Re-insert column in the collection tube and add 200μL of Plasmid Wash Buffer 1. Centrifuge for 1 minute at 13,000 rpm.
- Add 400μL of Plasmid Wash Buffer 2 and centrifuge for 1 minute at 13,000 rpm. Transfer column to a clean 1.5 ml microfuge tube. Add ≥ 30μL DNA Elution Buffer to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA .
- Blank Nanodrop with elution buffer.
- Measure the quality and concentration of the extracted DNA with Nanodrop.
Restriction Digestion Analysis
Before sending plasmids for sequencing, a restriction analysis can be carried to verify the miniprepped plasmids. Selection of enzymes should be determined by the cut sites on plasmids.
- Use the enzymes manufacturer to identify buffer to use
- Add following components in a PCR tube:
Components
|
Amount
|
ddH2O
|
? μL (Add up to 10 μL)
|
DNA
|
500ng
|
Buffer
|
2μL
|
Enzyme A
|
0.5μL
|
Enzyme B
|
0.5μL
|
- Incubate at the optimum temperature of the selected enzymes with suggested incubation time.
- Use agarose gel electrophoresis to verify the colony containing correctly assembled plasmids.
- Load samples to the gel with 6X loading dye.
Glycerol Storage of Culture Samples
- Take 2 screw down cryo-tubes with green and red caps.
- Pipette 900μL of sterile 50% Glycerol into each tube in sterile condition.
- Pipette 900μL of liquid culture into each tube.
- Vortex for 5-10s to mix.
- Put sample into the respective box and correct position. Note that the red tubes are taken in the -80°C freezer.
Error-Prone PCR
Add the following components to set up error-prone PCR:
Components
|
Concentration
|
Plasmid containing region of interest-adjust
|
0.5ng/μL
|
Respective Primers
|
0.4μM
|
dATP & dGTP
|
0.2mM
|
dCTP & dTTP
|
0.2mM
|
MgCl2
|
5mM
|
MnCl2
|
0.2mM
|
NEB® Taq Polymerase
|
0.05U/μL
|
Adjust the system with wanted total volume
epPCR Thermocycler Program:
___________________________________________________
Step Temperature Time
___________________________________________________
Initial melting 94°C 2 min
___________________________________________________
40 cycles:
Melting 94°C 30 sec
Annealing based on Tm of primers 30 sec
Extension 68°C 1min/kbp
___________________________________________________
Final extension 68°C 5 min
___________________________________________________
Holding 16°C ∞
___________________________________________________
B. subtilis Genome Experiments
B. subtilis Genome Extraction
Prepare TEN stock
Components
|
Concentration
|
Tris/HCl at pH 8.0
|
10mM
|
EDTA
|
10mM
|
| NaCl |
150mM |
Prepare TEN* stock
Components
|
Concentration
|
Tris/HCl at pH 8.0
|
10mM
|
EDTA
|
1mM
|
| NaCl |
50mM |
- Inoculate 10mL LB medium from a fresh overnight culture and incubate at 37°C in a shaker.
- At OD600 of 0.8-1.0 harvest cells by centrifugation for 10 min at 5,000 rpm.
- Resuspend cell pellet in 400µL TEN and transfer the solution to a 2 mL eppendorf tube.
- Add 20µL of 20 mg/mL lysozyme and incubate for 20 min at 37°C.
- Add 2µL of20 mg/mL RNase and incubate for 3 min at 65°C.
- Add 40µL of 10% (w/v) SDS, small amount (covering the tip of a spatula) of proteinase K and 550µL TEN*, mix and incubate for 2 hours at 60°C.
- Add 900 µl phenol (equilibrated with TE buffer, pH 7.5-8.0) and mix well by inverting the tube.
- Centrifuge for 5 min at 13,000 rpm, RT and transfer the upper phase into a new 1.5mL Eppendorf tube.
- Repeat the extraction once with phenol and twice with chloroform:isoamylalcohol (24:1).
- Transfer the aqueous phase to 10 ml -20°C cold ethanol in a test tube • Coil up the precipitated DNA with the bended tip of a Pasteur pipette.
- Air dry the DNA.
- Dissolve DNA in 100µL TEN* overnight at 4°C.
- For PCR, dilute the chromosomal DNA 1:50.
B. subtilis Transformation
B. subtilis 168 transformation
- Plate the day before B. subtilis 168.
- Pick a colony and inoculate 3mL of MC medium.
- Incubate at 37ºC and 200 rpm until OD600 1.1 - 1.5 (4-5h). (Final log phase / early stationary phase).
- Transfer of DNA or 10µL of plasmid (0.5-2 ug) to a sterile 1.5mL tube 20µL and 1 ml of the culture. Vortex for 5 seconds and incubate at 37ºC and 200rpm 40 min.
- Seed on one plate 200µL of the culture with selective medium. Centrifuge the rest, resuspend at 75µL and seed on another plate.
- Incubate at 37 °C overnight, pick colonies and reseed in selective medium.
Super-competent B. subtilis SCK6 transformation
B. subtilis SCK6 contain anti-erythromycin gene integrated in the genome.
SCK6 storage preparation
- Prepare autoclaved LB with 1μm/mL erythromycin.
- Prepare autoclaved LB with 1μm/mL erythromycin and 1% w/v xylose
- Pick a colony of SCK6 cells and inoculate into 3ml of LB.
- Incubate overnight (~12hrs) at 37 °C at 200 rpm.
- Dilute with LB containing 1% w/v xylose to 1.0 OD at A600.
- Incubate at 37 °C at 200 rpm for 2 hrs.
- Take glycerol stocks of competent cells with 10% v/v glycerol and place them at -80 °C. Or use them directly
Transformation
- Make M9 minimal media with 5μg ml-1 chloramphenicol.
- Add 1μL of PCR product with plasmid library to 100μL of super-competent cells.
- Grow at 37 °C at 200 rpm for 90 mins.
- Dilute in 10mL of M9.
- Grow at 37 °C at 200 rpm for 14hrs.
- Dilute the cells 1:20 in M9.
- Grow at 37 °C at 200 rpm for 8 hrs.
- Serial dilute and add to LB plates.
Sporulation and Spore Purification
Method follows the iGEM Freiburg team technique, which in turn is derived from the Nicholson paper (1990) and validated in a bunch of other papers (with tiny modifications), so it should work fine.
1X PBS (Phosphate-Buffered-Saline, 1L recipe) preparation
- Prepare 800 mL of distilled water in a suitable container.
- Add 8g of Sodium chloride to the solution.
- Add 0.2g of Potassium Chloride to the solution.
- Add 1.44g of Sodium Phosphate Dibasic to the solution.
- Add 0.245g of Potassium Phosphate Monobasic to the solution.
- Adjust solution to desired pH (typically pH ≈ 7.4).
- Add distilled water until the volume is 1L.
DSM (Difco sporulation media, 1L recipe) preparation
- Add 8g of Nutrient Broth n4, 1g of KCL, 1mM MgSO4.
- Autoclave the mixture.
- Add 1mM Ca(NO3)2, 1uM FeSO4, 10uM MnCl2 (all filter-sterilised of course!).
Important: always use fresh media since the FeSO4 can rust out quickly, spoiling it
Overnight B. subtilis culture
- Inoculate your strain into 4ml LB medium.
- Grow overnight, 37°C at 200rpm.
- Measure OD600, backdilute to 0.1-0.2/mL in 10ml LB medium.
- Let cells grow until OD600 of 0.8/mL, 37C at 200rpm (B. subtilis duplicates every 30mins, so this step should take roughly an hour: be careful with times).
Sporulation induction
- Centrifuge samples at 13,000rpm for 1min.
- Wash pellet with 1X PBS.
- Resuspend in 5mL of DSM.
- Incubate for 24h, 37°C at 200rpm.
Spore purification
Lysozyme method
- Add 15mg/mL of lysozyme to the samples (1:6 dilution).
- Incubate for 1h at room temperature.
- Wash 6 times with PBS
Heat shock method
- Heat up samples at 80°C for 10min to kill off vegetative bacteria.
- Wash two times with PBS and resuspend in sterile milliQ water.
Notes: The final step is to maximise the amount of spores in the samples, killing off the germinated spores. The Freiburg team showed that their method increases spore concentration in the purified samples from 75% to 91%. Joaquin says that it should be enough to warm them up to kill off cells but keep the spores. Efficacy of both methods could be tested in parallel, or even in a combinatorial way (heat shock after lysozyme treatment).
Antifungal Assay
Screening System
Preparation
- Creation of the mutant library via error prone PCR
- Transform the library into B. subtilis.
- Sporulate the strains using sporulation protocol (Sporulation and Spore Purification).
- Treat spores with lysozyme to lyse germinated cells and be left only with spores.
- Inactivate the lysozyme via heat denaturation (15min at 100°C).
Negative selection
- Transfer spores into minimal growth media and incubate.
- Treat spores with lysozyme to lyse spores that might have germinated for random GerA mutations.
- Inactivate the lysozyme via heat denaturation (15mis at 100°C).
Positive selection
- Add N-acetyl-glucosamine to minimal media.
- Separate germinated cells from spores (use the PEG-potassium phosphate protocol) .
- If spores have germinated, it means that the receptor has been successfully engineered to respond to N-acetyl-glucosamine.
- Plate the successful cells onto rich medium.
- Pick some of them and send for sequencing.
Fungal Killing Assay
Preparation
- 10-day culture of fungi
- PDA plates (1.5%)
- Optical microscope to observe
- B. subtilis liquid culture
- A spectrophotometer
Dual Plate Assay:
- Use a metal straw to pick 10-day-old culture agar discs of fungal pathogens disposed at the centre of Petri dishes - Measure the concentration of the B. subtilis stock liquid culture by a spectrophotometer
- Perform serial dilution using the B. subtilis stock liquid culture (With concentration of 0.8 OD, 0.4 OD, and 0.2 OD) - Place 10 µL of the original and diluted B. subtilis liquid cultures in a square form around the agar disc at 2 cm distance from the centre and incubated at 28℃ until mycelial growth fill plates
- Calculate the growth inhibition. Growth inhibition (%) = (R1 − R2/R1) × 100 (Trivedi et al., 2008)
- R1 = radial growth of control
- R2 = radial growth of the fungus in dual culture
- Observe the morphological change of fungal mycelium. Studied by using optical microscope (X40 magnification)
