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Protocol for E. Coli Crude Extract Preparation, 3X

Time needed: 3 days

Materials

2xYT:

• 2xYT-broth powder (MP biomedicals)
• Cat #: 3012-032

Bacto-Agar:

• Bacto-agar (BD)
• Product number: 214010

Bead:

• 0.1mm diameter glass beads (Biospec products)
• Catalog #: 11079101

Bead beater:

• Mini-Beadbeater-1 (Biospec products)
• Catalog #: 3110BX

Bead beating tube:

• Polypropylene microvials (Biospec products)
• Catalog #: 522S

Bead filter:

• Micro bio-spin chromatography columns (Bio-Rad)
• Product number: 732-6204

Centrifuge (big size):

• Allegra 64R high-speed refrigerated benchtop centrifuge (Beckman Coulter)

Centrifuge (ultra size):

• Beckman J2-M1 (Beckman Coulter)

Centrifuge bottle (50ml):

• Polyallomer bottle with screw-on cap (Beckman Coulter)
• Part number: 357003

Centrifuge bottle (500ml):

• Centrifuge bottle assembly polycarbonate (Beckman Coulter)
• Part number: 355607

Chloramphenicol:

• Chloramphenicol powder (Sigma-Aldrich)
• Product number: C1919

Cuvette:

• Fisherbrand Disposable Cuvettes
• Cat#: 14-955-127

Dialysis cassette:

• Slide-A-Lyzer Dialysis Cassette, 10MWCO (Thermo Scientific), maximum capacity: 3mL
• Product number: 66380

E. coli strain:

• BL21 Rosetta 2 competent cell (Novagen)
• Product number: 71402

Electrode:

• Electrode, 3-in-1 for Φ 400, 500 series pH meter, 12x 150 mm, Epoxy
• Product number: A57198

Erlenmeyer glass flask:

• KIMAX erlenmeyer glass flask
• Product number: 26500 (4000ml)

Floater:

• Float Buoys: comes with Slide-A-Lyzer Dialysis cassette

Incubator shaker:

• Incubator shaker (New Brunswick)
• Innova 4300

K-glutamate:

• L-Glutamic acid potassium salt monohydrate (Sigma Aldrich)
• Product number: G1149

Liquid nitrogen

Magnetic stirrer:

• Thermolyne Nuova II
• Fisher Scienetific Isotemp Digital Magnetic Stirrer

Mg-glutamate:

• L-Glutamic acid Hemimagnesium salt tetrahydrate (Sigma Aldrich)
• Product number: 49605

PH meter:

• pH meter, Φ 410 pH/mV meter, waterproof, handheld
• Product number: A58734

Potassium phosphate monobasic solution:

• Potassium phosphate monobasic solution (Sigma Aldrich)
• Product number: P8709

Potassium phosphate dibasic solution:

• Potassium phosphate dibasic solution (Sigma Aldrich)
• Product number: P8584

Protein assay:

• Bio-rad Protein Assay Dye Reagent concentrate (Bio-Rad)
• Product number: 500-0006

Spectrometer:

• Genesys 10 uv (Thermo Electron corporation)

Table top centrifuge:

• Microcentrifuge model 5415D (Eppendorf)

Table top vortex:

• Analog vortex mixer (VWR)
• Catalog number: 58816-121

Tris:

• Tris base, white crystals or crystalline powder, molecular biology (Fisher Scientific)
• Catalog number: BP 1 52 1

Ingredients

2xYT broth:

• Concentration of 2xYT: 31g/L
• Dissolve 62g of 2xYT powder with 1.8L of water in total volume → the rest of volume will be filled by phosphate and water later

2xYT + Phosphate + Chloramphenicol plate: (2xYT+P.P.+Ch.)

• Concentration of 2x YT: 31g/L
• Concentration of chloramphenicol: 34mg/ml
• Concentration of phosphate: 62ml of phosphate/ 1L of total volume
• Weigh 1.24g of 2x YT, 0.44g of Bacto-Agar
• Transfer 2xYT and Bactor-Agar in a bottle, add water up to 40ml and then autoclave it
• When temperature is about 50°C after autoclaving, add 2.5ml of phosphate and 60μl of chloramphenicol at 34mg/ml (proper quantity is 40μl, but I add 50% more because it is still hot (~50°C))
• Mix well and pure 25ml of mixture on petri dish (100X15mm)
• Cure it on room temperature for 1 hour

Chloramphenicol:

• Concentration of chloramphenicol stock: 34mg/ml
• FW: 323.13
• Dissolve 6.8mg of chloramphenicol powder with 200μl of alcohol in total volume

Phosphate:

• 100ml of potassium phosphate dibasic solution + 55ml of potassium phosphate monobasic solution

S30 buffer A (2 liters) :

• Final concentration and pH: 14mM of Mg-glutamate, 60mM of K-glutamate, pH7.7
• Weigh 10.88g of Mg-glutamate and 24.4g of K-glutamate
• Transfer powders into 2L beaker with magnetic stirrer
• Add water up to 1.9L and place it on magnetic stir plate
• Run plate, start to measure pH of solution and keep running plate and measuring pH
• Add 50mM of Tris: 50ml of Tris at 2M
• Add Acetic Acid up to reach ph7.7
• Add water to fill up to 2L
• Add DTT to 2mM final concentration just before use.

S30 buffer B (2 liters):

• Final concentration and pH: 14mM of Mg-glutamate, 60mM of K-glutamate, pH8.2
• Weigh 10.88g of Mg-glutamate and 24.4g of K-glutamate
• Transfer powders into 2L beaker with magnetic stirrer
• Add water up to 1.9L and place it on magnetic stir plate
• Run plate, start to measure pH of solution and keep running plate and measuring pH
• Add Tris up to reach pH8.2
• Add water to fill up to 2L
• Add DTT to 1mM final concentration just before use.

Tris:

• Concentration of Tris: 2M
• FW: 121.14
• Dissolve 60.57g of powder with 250ml of water in total volume

Pre-Preparation

Clean all materials:

• 4L Erlenmeyer glass flask (erlens)
• 500ml centrifuge bottles
• 50ml centrifuge bottles
• Funnel
• Beaker
• Magnetic stirrer
• Floater
• Rinse all with dH2O
• Rinse floater and magnetic stirrer with isopropanol
• Burn at 110°C except floater

Day 1: Transformation

Start to prepare 2xYT+P.P.+Ch. plate at around noon (follow the ingredients protocol)

At around 5pm, start transformation:

• Take E. coli strain stock (33% glycerol) out from -80°C
• Pick some of stock and dissolve it into 500ml of 2xYT broth (Immediately place back E. coli strain stock into -80°C)
• Do plating with 5~10µl of dissolved E. coli stock and place it in 37°C incubator
• Check DTT at -40°C: it is aliquot by 1ml at 1M.
• 6 tubes will be used: 4 for S30 buffer A and 2 for S30 buffer B

Day 2: Miniculture

Start the 1st miniculture at noon:

• Start to warming up at 11:30
• 4ml of 2xYT, 0.27ml of phosphate and 4µl of chloramphenicol
• Pick one colony from plate and start miniculture at noon

Prepare 2xYT, phosphate and S30 buffer A and B (follow the ingredients protocol)

Start the 2nd miniculture at 8:30:

• Start to warming up at 8:00
• 50ml of 2x YT, 3.3ml of phosphates, 50μl of chloramphenicol and 100μl of miniculture

When warming up at 8:00, prepare other materials for tomorrow morning:

• 6 of 4L erlens: wrap their opening up with aluminum foil and place them on bench
• 6 of rubber caps: wrap them up with aluminum foil and place them on bench
• 2 of 1.8L 2xYT bottles: place them on bench
• Phosphate bottle: place it on bench
• Water (autoclaved): place it on bench
• 4 of 500ml centrifuge bottles: place them on bench
• 4 of 50ml centrifuge bottles: place them on bench
• Funnel: wrap it with aluminum foil and place it on bench
• S30 buffer A and B bottles: place them in chromato
• Glass bead bottle: put it into 110°C oven
• Two rotors with lids for J2-M1: place them in chromato

Day 3

TIme:

Warm up nutrition:

• Add 124ml of phosphate into 1.8mL of 2xYT and add water to fill up to 2L → 2 bottles
• Transfer 660mL of nutrition into 4L erlen → 6 erlens
• Incubator shaker at 220rpm at 37°C

Induce 6.6ml of the 2nd culture (last night) into each 4L erlen 30min after warm-up

• It takes about 3 h ~ 3 h 20 min

Meanwhile, check other materials:

• Take glass bead bottle out from the oven
• Place a rotor for 500mL centrifuge bottle onto J2-M1 and turn the machine on
  • parameters: rotor number: 10, speed: 5000rpm, time: 12min, temperature: 4°C
• Place a rotor for 15mL falcon tube onto Allegra64R and turn the machine on
  • parameters: rotor number: F0850, speed: 7500rpm, time: 10min, temperature: 2°C
• Turn on the spectrometer
• Weigh 50ml centrifigue bottles: look at the table below
• Add 4mL of DTT at 1M into S30 buffer A

Check OD after 3 hours incubation of bacteria culture:

• Set the wavelength at 600nm
• Control: 1mL of nutrition
• Sample: 100µl of culture + 900µl of nutrition
• At OD600=1.5, stop growing and start pelleting of cell

Time:

Remember: keep bottles and tubes cool at all steps except 80min pre-incubation

Transfer bacteria culture to 500mL centrifuge bottles:

• Use 4 bottles, and all must be balanced to prevent damaging J2-M1, which is very powerful

Centrifuge it

• parameters: rotor number: 10, speed: 5000rpm, time: 12min, temperature: 4°C

Discard supernatant and repeat the previous step

Discard supernatant and add 200mL of S30 buffer A, and then resuspend the pellet cells in the bottle:

• make sure to shake vigorously by hand until no cell clusters remain

Centrifuge it with the same parameters and repeat the previous step

Prepare 37.5mL of S30 buffer A in 4 x 50mL falcon tubes

Discard supernatant and pour 37.5mL of S30 buffer A (prepared the previous step)

Resuspend the pellet cell gently (vortex by hand)

• it doesn’t need to be resuspended completely

Transfer pellet cell into 50mL centrifuge bottle through funnel and centrifuge it (change rotor)

• parameters: rotor number: 20, speed: 4000rpm, time: 8min, temperature: 4°C

Discard supernatant and centrifuge it again for 2 min, and then remove the residue of supernatant by pipetting

Time:

Weigh the mass of 50mL of centrifuge bottle (with pellet)

	unit	Bottle 1	Bottle 2	Bottle 3	Bottle 4
Empty bottle	mg
Bottle with pellet	mg
Mass of cell	g
Volume of S30 buffer A	mL
Mass of bead (mass of cell x 5.0)	g
Mass of bead (mass of cell x 0.05)	g
Mass of bead	g

• Volume of S30 buffer A: mass of cell * 0.9 [mL]
• Mass of bead: mass of cell * 5.0 [g] ← adjustable up to 5.1 depending on viscosity of cell

Add S30 buffer A and vortex

Add bead and vortex ←split bead quantity (mass of cell * 5.0) by 3. Whenever adding bead, vortex

Add bead more if necessary (depending on the viscosity) ← increment: mass of cell * 0.05

Transfer mixture of bead + cell into bead beating tubes:

• Do it as fast as possible
• Use 1mL pipet: set at 0.5mL and cut tip of pipet tip: mixture is viscous, so it has to be wide open end to make process faster.
• prepare bead beating tubes in advance in order to make them cool before adding mixture
• Transfer mixture in two tubes after two tubes, and so on
  • Fill them with mixture and spin quickly to remove air in the tubes
  • Add little bit of mixture on top of mixture in the tubes up to slight below of tube height
  • Add also little bit of mixture inside on cap
  • Close tubes tightly

Place tubes on ice for 30sec then run bead beater.

• parameter: speed: 46rpm, time: 30sec

Place tubes upside down on ice, wait 30sec, and then run bead beater again with same parameters

Remove screw cap on bead beating tube and put it into bead filter, and then place it on ice (bead + cell mixture should be in the ice side).

Once 8 structures (bead beating tube + bead filter) are ready, break the tip of filter, put it into 15mL falcon, and then centrifuge them.

• parameter (Allegra64R): rotor: F0850, speed: 7500rpm, time: 5min, temperature: 2°C

Transfer supernatant to new bead beating tubes: you may transfer turbid pellet, but try to transfer it as less as possible → use 1mL pipet

• number of tubes:

Centrifuge it

• parameter: rotor: F3602, speed: 12,000g, time: 20min, temperature: 2°C

Prepare same number of empty bead beating tubes as bead beating process

If time is allowed, start to clean materials: 4L erlens, 500mL and 50mL centrifuge bottles, and funnel

Transfer clear supernatant into the empty bead beating tubes: no turbid pellet

• use 1mL pipet to do it fast, but avoid pulling pellet
• Centrifuge dirty supernatant (because of pellet) on the table top centrifuge (parameter: 12000rpm for 5min) and transfer clear supernatant into the empty bead beating tubes: anyway total number of bead beating tubes of this step is same as number of bead beating tubes of bead beating process

Time: 11:45

Incubate them in the shaker at 220rpm at 37°C for 80min

• place bead beating tubes from the previous step into culture tube: remove screw cap

Prepare dialysis materials 70min after start of incubation

• Put two of 1L beakers and two of magnetic sticks into chromato to pre-cool
• Take a syringe (5mL), a needle and six of dialysis cassettes out and place them on bench
• Take out two tubes of DTT (each 1mL at 1M) from -40°C freezer

After 80min pre-incubation, transfer liquid (actually now it is extract, though) into micro-centrifuge tubes

Centrifuge it

• parameter: rotor: F3602, speed: 12,000g, time: 10min, temperature: 2°C

When 5min left, prepare dialysis setting

• Add two tubes of DTT into 2L of S30 buffer B and mix it
• Pour 900mL of S30 buffer B into 1L beaker: prepare two 1L beaker
• Clean hands with water with soap and then isopropanol
• Put dialysis cassette onto floater and place it on S30 buffer B in 1L beaker
• Place beakers on the magnetic stirrer in the chromato and run stirrer: dial at 8 or 300rpm

Transfer supernatant into two 15mL falcons

Mix it by inverting to make it homogeneous

Centrifuge it to recover extract as much as possible

• parameter: rotor: F0850, speed: 1000rpm, time: 1min, temperature: 2°C

Take two of 1L beaker

Transfer extract from two of 15mL falcons to six of dialysis cassettes (3mL maximum capacity) using syringe with needle

• transfer around 2.5mL of extract in one dialysis cassette
• insert extract and remove air with same syringe with same needle
• Place beakers on the magnetic stirrer in the chromate and run: dial at 8 or 300rpm

Do dialysis for 3 hours

Time:

Do Bradford:

• To check the density of protein of extract
• Measure OD at 595nm
• BSA standard provided by Bio-rad kit is at 1.42mg/ml: it is aliquot by 35.2µl and stored in -80°C
• Take BSA standard out
• Prepare BSA standard at 1mg/ml: 35.2µl of 1.42mg/ml + 14.8µl of water = 1mg/ml at 50µl
• Prepare BSA standard at 0.1mg/ml: 10µl of 1mg/ml + 90µl of water = 0.1mg/ml at 100µl
• Prepare sample at 20x: 2µl of sample + 38µl of water
• Prepare 7 cuvettes and add 800µl of water in each cuvette
• One cuvette for 0mg/ml sample
• One cuvette for 1mg/ml sample: add 10µl of 0.1mg/ml BSA standard
• One cuvette for 2mg/ml sample: add 20µl of 0.1mg/ml BSA standard
• One cuvette for 4mg/ml sample: add 4µl of 1mg/ml BSA standard
• One cuvette for 6mg/ml sample: add 6µl of 1mg/ml BSA standard
• One cuvette for 2µl of sample: add 2µl of sample
• One cuvette for 4µl of sample: add 4µl of sample
• Add 200µl of protein dying assay and mix well by pipetting
• Measure OD on spectrometer after 10min waiting
• OD595: mg/ml

Recover extract from dialysis cassettes to micro-centrifuge tubes by using new syringe and needle

• insert air by pushing syringe and recover extract

Aliquot it by 30µl ( it should be 27~30mg/ml) or adjust volume according to the standard (30µl at 27~30mg/ml): for example, 27µl at 33mg/ml)

Use liquid nitrogen to freeze immediately once aliquoting

Time:

From Shin J, Noireaux V: Efficient cell-free expression with the endogenous E. Coli RNA polymerase and sigma factor 70. Journal of Biological Engineering 2010, 4:8

http://www.jbioleng.org/content/4/1/8