Experiments and Protocols

Preparation of Component E. Coli Cells

Grow cells to an OD600 of approximately 0.6.
Pour 50 mL of culture into a conical tube.
Centrifuge for 10 minutes at 3000 rpm at 4C.
Decant all media.
Resuspend pellet in 10 mL cold 0.1 M calcium chloride.
Incubate for 10 minutes on ice.
Centrifuge again for 10 minutes at 3000 rpm at 4C.
Decant the calcium chloride solution.
Resuspend the pellet in 5 mL cold 0.1 M calcium chloride/15% glycerol.
Aliquot around 200 ul of cells into microcentrifuge tubes.
Store at -80C immediately.

Transformation Protocol

Thaw tubes of cells on ice; have one tube designated as the control.
Mix DNA with cells (or diH2O if control) and incubate for 30 minutes on ice.
Heat shock at 42℃ for 30 seconds and on ice for 2 minutes.
Shake cells in SOC broth for 1 hour at 37℃, bring agar plates out of cold room to adjust to room temperature.
Deposit 200μL of each culture onto a plate, spread with glass spreader sterilized in ethanol, which is burned off. Use a flame to aid with sterility.
Leave plates for 10 minutes for culture to dry, then leave inverted in 37℃ incubator overnight.

Plasmid Isolation

Minipreps were performed using MoBio UltraClean® Standard Mini Plasmid Prep Kit.

LB Broth

Mix 5 g tryptone, 2.5 g yeast extract, and 5 g NaCl in a microwaveable flask.
Fill with diH2O to 500 mL.
Autoclave in a tray that is covered in a thin layer of water.

SOC Broth

In one microwaveable flask, mix 10 g tryptone, 2.5 g yeast extract, 0.25 g NaCl, and 0.09 g KCl.
Fill with diH2O to 500 mL.
In a small container, mix 0.48 g MgCl2 and 1.23 g MgSO4 heptahydrate. Add a small amount of diH2O (<10mL).
In another small container, mix 1.80 g glucose with a small amount of diH2O (<10 mL).
Autoclave all containers in a tray that is covered with a thin layer of water.
Add the concentrated glucose and magnesium solutions to the 500 mL mixture.

LB Agar Plates

Mix 5 g tryptone, 2.5 g yeast extract, 5 g NaCl, and 7.5 g agar.
Fill with diH2O to 500 mL.
Autoclave in a tray that is covered in a layer of water. Store LB Agar until needed.
To prepare to make plates, microwave bottle of LB Agar until dissolved, then wait until bottle is cool enough to handle.
Add all necessary antibiotics in appropriate concentrations (100 mg/mL for ampicillin, 25 mg/mL for chloramphenicol, 50 mg/mL for kanamycin, 10 mg/mL for tetracycline).
Quickly pour mixture into as many plates as possible in a layer that covers the bottom. Use a nearby flame for sterility.
Cover plates and turn off flame when evaporation slows down.
Label and seal plates when the gel is solid, store in cold room.

Polymerase Chain Reaction

Mix 12.5 μL of Q5 Master Mix, 1.25 μL each of necessary forward and reverse primer, 2 μL of DNA sample, and 8 μL of diH2O in a PCR tube.
Run PCR program in thermal cycler, with annealing temperature and potential touchdown protocol set as needed.

Gibson Assembly Cloning

Gibson primers are used to amplify fragments and install 20bp overhangs.
NEB Assembly Mastermix used to assemble parts in 2 piece fragments, incubated at 50C for 1 hour.

Agarose Gel

Mix TAE buffer with 1% agarose (1 g per 100 mL) in microwaveable flask and microwave until dissolved.
Wait five minutes to cool. Add ethidium bromide solution needed for 0.2 μg/mL concentration.
NEB 2-log ladder is used as a reference in all gels.

SDS PAGE

NuPAGE Bis-Tris Mini Gels, 6.5mL of protein pellet resuspended in PBS, 2.5μL of NuPAGE LDS Sample Buffer, 1 μL of reducing agent, 8-12% Bis-Tris Gradient.

Protein Induction

Cultures of B. megaterium were seeded into 100mL of LB media, and grown until OD600 of 0.5. Cultures were then induced with 1mM xylose, and grown for an additional hour. Pellets were harvested, and run on an SDS PAGE.

In Vitro Transcription of sgRNAs

Used NEB HiScribe T7 High Yield RNA Synthesis kit.

Dr. Sun Bacillus megaterium Protoplast Formation and Transformation

Materials Required:
1. SMMP: A = 2X AB3 medium, 7g Antibiotic Medium No. 3 in 200 ml dH2O, autoclave for 15 min. B = 2X SMM, 1M sucrose, 40 mM sodium maleic acid, 40 mM MgCl2, pH 6.5, autoclave for 12 min (should not turn brown). Mix equal volumes of A and B to get SMMP.
2. PEGP: 40% w/v PEG8000 in 1X SMM, autoclave for 12 min
3. CR5 Plates:
Part A = 51.1g sucrose, 3.25g MOPS, 0.33g NaOH, make up to 250 ml with dH2O, pH 7.3, autoclave 12 minutes in a 500 ml bottle.
Part B = 5.5g agar, 0.1g casamino acids, 5g yeast extract, make up to 142.5 ml with dH2O, autoclave for 20 min in 500 ml bottle.
Part C = 0.25g K2SO4, 10g MgCl2.6H2O, 0.05g KH2PO4, 2.2g CaCl2.2H2O OR 1.66g CaCl2, make up to 125 ml with dH2O, autoclave for 20 min.
Part D = 12% w/v proline, filter sterilise.
Part E = 20% w/v glucose, filter sterilise.
To make CR5: After autoclaving, warm all parts to 50C in water bath, then mix together using sterile technique by adding the following components into all of Part B: All Part A, 57.5 ml Part C, 25 ml Part D, 25 ml Part E.
Mix well and place back at 50C
Add desired antibiotic for plasmid selection
Aliquot 25 ml to each petri plate, allow to solidify, and store at 4C

Making B. megaterium Protoplasts
1. Scratch B. megaterium from -70C frozen stock into 5 ml AB3 medium. Incubate at 37C and 250 rpm overnight.
2. Add 1.0 ml from seed culture to 100 ml of prewarmed AB3 medium. Incubate at 37C and 250 rpm until A600nm is 1.0.
3. Centrifuge culture at 3500 x g for 10 min at room temperature and carefully discard supernatant.
4. Resuspend cell pellet in 5.0 ml of SMMP containing 2 mg/ml lysozyme (from a filter sterilised stock). Incubate for 90 min at 37C and 100 rpm. If desired, fine tune incubation time by monitoring protoplast formation by phase microscopy.
5. Centrifuge protoplasts 2000 x g for 10 min at 20C.
6. Resuspend protoplasts gently in 5.0 ml SMMP. Store 0.5 ml aliquots at -70C.

Transforming B. megaterium Protoplasts
1. In a sterile 15 ml Falcon tube, combine 500 ul of protoplast suspension and 500-1000 ng of plasmid containing transgene (for a standard miniprep, this should be 5 ul of plasmid to be safe). Plasmid methylation does not appear to be an issue for successful transformation.
2. Add 1.5 ml of PEGP. Incubate for 2 min at room temp with no mixing.
3. Add 5 ml of SMMP. Gently mix by inverting three times.
4. Centrifuge the sample 1550 x g for 10 min at room temp. Carefully pour off the supernatant.
5. Add 500 ul SMMP. Incubate at 37C and 100 rpm shaking for 90 min.
6. Plate 50200 ul of each incubation onto CR5 plates containing antibiotic for plasmid selection.
7. Incubate at 37C overnight.
9. Pick out colonies into liquid LB + antibiotic and grow overnight at 37C and 250 rpm shaking. Colonies on CR5 plates are often slimy; heavy growth may require streaking for isolated colonies on LB + antibiotic prior to picking into liquid medium.
10. Make stabilates in LB with a final glycerol % of 10%. Store at -70C.

Team:Tufts/Experiments