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Wet Lab: Protocols: Preparation of E. coli S30 Extract

Day 1

Equipment

• 37°C shaking incubator
• 1L conical flasks x 3
• Pipette fillers + pipettes (5ml, 10ml and 25ml)
• Spectrometer + cuvettes
• Weighing scale
• Centrifuge + 150ml centrifuge tubes
• Pipettes + pipette tips (20µl, 200µl and 1000µl)

Reagent

• 2xYT medium
• IPTG
• Buffer A
  • 10mM Tris-acetate (pH 8.2)
  • 14mM Mg-acetate
  • 60mM K-glutamate
  • 1mM dithiothreitol (DTT)
  • 0.05% (v/v) 2-mercaptoethanol (2-ME)

Procedure

Growing the cells

1. Grow E. coli strain BL21 (DE3) cells at 37°C in 3L of 2xYT medium till O.D.600 = 0.6.Ensure vigorous agitation and aeration.
2. Add 1mM IPTG to cell culture to express T7 RNA polymerase.
3. Harvest cells when O.D.600 = 4.5. At this point, cells are at mid-log phase.
4. Wash cells three times by suspending them in 20ml of buffer A per gram of wet cells.
5. Centrifuge and weigh the wet cell pellets before storing them at -80°C.

(Note: The cells may take more than 1 day to grow to O.D.600 = 4.5.)

Day 2

Equipment

• Pipette filler + pipettes (5ml, 10ml and 25ml)
• Weighing scale
• French press + French press cell
• Centrifuge + 50ml centrifuge tubes
• Pipette + pipette tips (20µl, 200µl, 1000µl)
• 37°C shaking incubator
• Dialysis membrane with molecular weight cut-off of 10,000
• Magnetic stirrer
• 4°C cold room

Reagent

• Buffer B
  • 10mM Tris-acetate (pH 8.2)
  • 14mM Mg-acetate
  • 60mM K-glutamate
  • 1mM DTT
• Pre-incubation solution
  • 293.3mM Tris-acetate (pH 8.2)
  • 2mM Mg-acetate
  • 10.4mM ATP
  • 4.4mM DTT
  • 0.04mM amino acids
  • 16.9mM phosphoenolpyruvate
  • 0.77U/ml pyruvate kinase

(Note: For the ATP regenerating system in the pre-incubation solution, phosphoenolpyruvate and pyruvate kinase are used instead of creatine phosphate and creatine kinase. This is due to cost considerations.)

Procedure

Lysing the cells

1. Suspend thawed cells in 12.7ml of buffer B per 10g of wet cells.
2. Disrupt cells in a French press cell at a constant pressure of 20,000psi.This is about 140,000kPa.

Retaining the cell extract

1. Centrifuge the crude lysate at 30,000RCF for 30min at 4°C.
2. Carefully remove the top layer of the supernatant (lipid layer) and the pellet and centrifuge again.
3. Shake the final supernatant at 100rpm.
4. Gradually add 3ml of the pre-incubation solution to 10ml of the supernatant.
5. Pre-incubate the supernatant with gentle shaking at 37°C for 80min. This degrades endogenous genetic content (DNA and mRNA).
6. Dialyze the pre-incubated sample for 45min each at 4°C against 50 volumes of buffer B using a membrane with molecular weight cut-off of 10,000. Repeat the dialysis step three times.
7. Centrifuge the retained extract at 4000RCF for 10min at 4°C to obtain the supernatant.
8. Divide resulting S30 extract into small aliquots and store at -80°C.

(Note: Protease inhibitors are added to pre-incubation solution to prevent degradation of proteins required for gene expression.)

Notes

• Total time required: ~ 3 days.
• The protocol is based on [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T3C-4K242VK-1&_user=10&_coverDate=12%2F01%2F2006&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=fbf36c742708c667b5c4481856ca22b5 Simple procedures for the construction of a robust and cost-effective cell-free protein synthesis system] by Kim DM et al.
• Modifications to protocol:
  • The original protocol uses creatine phosphate and creatine kinase instead of phosphoenolpyruvate and pyruvate kinase.
  • The original protocol does not use protease inhibitors in the pre-incubation solution.