Cong T. Trinh:Cell free preparation

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Cell free preparation

Day 0: Preparation of buffer, medium, equipment and cell innoculum

Equipment

  • 1L bottle from Beckman Coulter Inc.,
  • 0.5L and 50mL bottles from Nalgene Inc. that fit with the rotors JS5.3, JLA8100, JA10, and JA20, respectively should be cleaned with Millipore water and autoclaved to eliminate DNAse, RNAse, protease, etc.
  • Prepare 20-40L autoclaved Millipore water in 40 carboys.

Medium

Composition of 2x YTPG (yeast plus tryptone plus phosphate buffer) in 10L

Chemicals Company Amount
2xYT      Difco    310 g
NaH2PO4    ----    26.4 g (or 22 mM)
Na2HPO4    ----    56.8 g (or 40 mM)
Glucose    ----    100 mM

IPTG induction with a working concentration of 1 mM (as OD600nm reaches 0.5 or 0.25 g/L).

Option: arabinose (used for inducible promoter), chloramphenicol (30 ug/mL) or other appropriate antibiotics.

All chemicals are autoclaved except sugars and antibiotics.

Cells

BL21star(DE3) pGroE. The plasmid pGroE encodes GroEL/ES and works with either BL21(DE) or BL21star(DE3).

Buffer

S30 buffer contains two types A and B.

  • Type A is used for washing cells while Type B is used for disrupting cells.
Type A S30 buffer contains:
10 mM Tris-acetate buffer (pH 8.2)
14 mM magnesium acetate
60 mM potassium acetate
1 mM dithiothreitol (DTT)
0.05% (v/v) 2-mercaptoethanol (2-ME).

Note: This buffer is used to wash cells after cell cultivation.

  • Type B S30 buffer is similar to Type A buffer without 2-ME.

Note: This buffer is used to re-suspend, disrupt cells by the French Press, and dialysis the cell free extract. We use 10 g of wet cells for 12.7 mL of buffer B. S30 buffer should be stored at 4°C.

Pre-incubation solution contains:

293.3 mM Tris-acetate pH 8.2
2 mM magnesium acetate
10.4 mM ATP
200 mM creatine phosphate
4.4 mM DTT, 0.04 mM amino acids
26.7 ug/mL creatine kinase

Note: Creatine phosphate and kinase are used to exhaust the endogeneous mRNA and supply ATP as a continuous energy generation system.

Comments:

  • Stock 1M Tris-acetate pH8.2 (pH adjusted with acetic acid), 1.4M magnesium acetate, 6M potassium acetate are prepared separately.
  • Note: Tris-acetate functions as a buffer to maintain pH. Mg2+ and K+ ions are important for ribosome assembly.
  • DTT must be added on the date of using the buffer because it is not stable. DTT is supplied in the form of powder and stored at 4oC. DTT is an antioxidant used to stabilize enzymes and other proteins containing sulfhydryl groups.
  • Milipore water should be autoclaved and used for preparing stock solutions and buffers.
  • 2-ME (Sigma M3148-100mL, U.S.A.) is provided in the form of liquid.
For instance, to prepare 4L of the Type A S30 buffer, add 10 mL of 1M Tris-acetate buffer (pH 8.2), 10 mL of 1.4M magnesium acetate, 6M potassium acetate, 0.61 g DTT, and 2 mL of 2-ME.
  • Buffers that contain DTT should be freshly prepared.
  • Stock 100 mM ATP is prepared from the salt powder in autoclaved Millipore water. The initial pH must be adjusted to 7.0.
  • Stock 40 mM amino acids are prepared in autoclaved Millipore water. All powder should be dissolved in 20 mM DTT.
  • Stock 1M creatine phosphate is prepared in the autoclaved Millipore water.
  • Stock 10 mg/mL creatine kinase is prepared in 50% glycerol.

Day 1: Cell cultivation and harvesting

Cell inoculum preparation

Overnight grown culture is transferred to fresh medium to prepare seed culture. iAs OD600nm of inoculum reaches 0.8-1, transfer 500 mL of cell culture to the bioreactor containing 10L medium (1:20).

Base operating conditions for fermentation

  • 14L in-situ Bioengineering fermenter is used with a working volume of 10L.
pH7.0, 37°C, %DO controlled at 10%, air flow rate 10 L/min, initial agitation 300 rpm.
Cascade control for %DO
As the %DO reaches 10% (the master output signal is 10%), the cascade controller is turned on. *The controller for agitation is set between 300-600 rpm with the output signal of 40%. Maximum agitation rate is 1500 rpm for the output signal of 100%.

pH controller

  • Proportional controller is set 50% and deadband at 0.1.

Cell harvesting and processing

  • After OD600nm reaches ~6.0 (3g cdw/L), cell cultivation is stopped.
  • Cell culture is collected on ice in 1L bottles.
  • Centrifuge cells at 4°C at 3500-5000 rpm for 10 minutes by using the JLA 8100 rotor.
  • Remove cell supernatant, suspend cells in 500 mL of the Type A S30 buffer, and transfer them into 0.5L bottles.

Note: The suspension should ensure to dissolve all cell pellets by continuously shaking and vortexing. In general, 20 mL of the Type A S30 buffer for every gram of wet cells should be used. The re-suspension step is time-consuming and requires good care of handling. High-speed centrifugation is not critical, but the lower speed is preferred because cell pellets can be easily resuspended.

  • Centrifuge cells again at 4oC at 3500-5000 rpm for 10 minutes by using the JA 20 rotor.
  • Repeat the cell re-suspension and centrifugation for three more times.
  • The cell pellets should be weighed and stored at -80oC.
At this step, we need to figure out how much the Type B S30 buffer should be used for cell disruption. The protocol is to use 12.7 mL of the Type B S30 buffer for 10 g of wet cells.

Note: For 10L cell cultures, first 10 1L bottles of cells are collected. Then 10 1L bottles of cells are reconstituted in 6 0.5L bottles, 4 0.5L bottles, and 2 0.5 bottles. With this batch run on 10/15/2010, we yielded approximately 130-150 g/10L wet cells.

Day 2: Cell free preparation

Cell disruption and collection of cell free solution

  • Thaw cell pellets stored at -80°C from day 0 by placing bottles on the top of ice and then add 12.7 mL of the Type B S30 buffer for every 10g wet cells.
Alternatively, add the buffer directly into the bottles containing the cell pellets and gently shake them with the vortex in the 4°C cold room.
The thawed and re-suspended cells look as in Figure 1A, 1B. Note: cells should be processed within a day after -80°C storage to still maintain high quality of cell free preparation.
  • Disrupt cells by using the French Press

Note: Care should be taken as disrupting cells with the French Press. It is preferred to disrupt cells with a single flow through. Hence, it is very important to keep the gauge pressure constant when opening the valve. The gauge pressure is set at 1500 psig and the cell pressure is 21,000 psi. Note: Sample collection from the French Press should be placed on ice to obtain high quality cell free. The disrupted cells look very viscous with foam and have brown appearance.

  • After finishing the cell disruption, cells are transferred to the 50 mL bottles and centrifuged at 30,000xRCF at 4°C for 30 minutes.

Note: We should observe three different portions in the bottle.

The first portion contains the pellet at the bottom of the bottle.
  • Within the pellet, we should see the inner white layer which is the undisrupted cell and the outer brown layer which contains the cell walls and insoluble proteins. Centrifugation at the minimum speed of 12,000xRCF should separate the insoluble proteins from the soluble proteins.
The second portion is the soluble cell free that we want.
The top layer is the lipid layer.
  • Carefully use the pipette to collect the soluble fraction. It is also important NOT to disrupt the cell pellet since we do not want to break gDNA that is entangled in the cell wall and contaminates the cell free solution.
  • Re-centrifuge the soluble cell free fraction at 30,000xRCF at 4°C for 30 minutes. At this centrifugation, there will be still a small portion of cell pellet formed.

Preparation of S12 cell free solution

  • Wrap the bottle with aluminum foil and incubate for 90 min at 37°C and 100 rpm.
  • After 90 min, place the bottle immediately on ice.
  • Centrifuge cell free solution at 4,000 RCF at 4oC for 10 min.

Note: At this step, unstable proteins are precipitated and forms the pellet.

  • Make cell free stock by freezing cells in liquid nitrogen and store at -80oC.

Preparation of S12 cell free solution

  • Add 3 mL of the pre-incubation solution with 10mL of cell free solution.

Note: Since the chemical mixture in the pre-incubation solution is highly concentrated, it is recommended to gently shake the bottle containing cell free solution and slowly add the pre-incubation solution in the dropwise manner by using a pipette.

  • Incubate the cell free solution for 90 min at 37°C and 100 rpm.
  • Transfer the cell free solution in the SNAKE skin.
  • Tie both ends of the SNAKE skin with clippers and places them into the beaker containing the cold Type B S30 buffer.
The volume ratio of the buffer to the cell free solution is 50:1.
  • Place the beaker with the stir bar in the cold room and stir the solution for an hour.
  • Replace the fresh buffer three times.

Note: At this step, we will see white pellet at the bottom.

  • Transfer all cell free solution into the bottle and immediately place it on ice.
  • Centrifuge cell free solution at 4,000 RCF at 4oC for 10 min.
  • Make cell free stock by freezing cells in liquid nitrogen and store at -80oC.

Note: The names of S12 and S30 cell free solution come from how the cell free solution is prepared. S12 corresponds to centrifugation with the minimum speed of 12,000xRCF after cell disruption while S30 corresponds to centrifugation at 30,000xRCF. In addition, S30 cell free extract also requires the cell free solution treated with energy source and amino acids followed by dialysis.



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