Team:Freiburg/Protocols/prepLysate

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Protocol Cell-free expression

Preparation of a Lysate for Cell-free exxpression

material:
duration: Preparation 1 week

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Buffer A:

Component	final concentration
TRIS-acetate (pH 8	10 mM
Potassium acetate	60 mM
Magnesium acetate	14 mM
DTT	1 mM
ß-mercaptoethanol	7 mM

• Buffer B is the same as A, but without the ß-mercaptoethanol
• Buffer C is the same as B, but without DTT

Preincubation Buffer

Component	stock concentration	endconcentration [µl]
Tris Ac, pH 7.6	-	300mM
Mg(OAc)2	-	10 mM
ATP	at least 217 ng/µl	10mM
phosphoenol pyruvate (PEP)	-	80mM
Amino Acid mix	-	5mM
pyruvate kinase	at least 217 ng/µl	3250 - 5000 ng
DEPC-H2O	-	up to 50 µl

Implementation

• incubate 2-4 hrs at 37 °C in autoclaved tubes or 96 well plate (low bind)

Growth of Bacteria

Harvesting, washing and lysis

• cells were grown in a 2 litre chicanery flask containing 1 litre of Terrific Broth medium until reaching OD600 = 0,8
• The expression of T7 RNA polymerase then was induced by adding 0.5 ml of 1 M IPTG to the medium
• further growth until OD600 = 6.5, then cells were put on ice
• cells were pelleted at 6000 g at 4°C for 30 minutes in a fixed-angle rotor
• supernatant was decanted and the cells were washed two times in buffer A
• Again, the cells were pelleted at 5000 g for 20 min at 4°C and after-wards resuspended in 1.3 ml of buffer B
• feed: Using a french press with a 1“ diameter piston, the cells were lysed at 1000 psi (≈ 6.9 MPa)
• About 7.5 ml of lysate was obtained after pressing.

Harvesting, washing and lysis

• obtained lysate is pelleted via ultracentrifugation at 30,000 g for 30 min at 4°C
• The expression of T7 RNA polymerase then was induced by adding 0.5 ml of 1 M IPTG to the medium
• The resulting extract is mixed with the preincubation buffer in a ratio of 10:1 (lysate:buffer)
• preincubated lysate is then transferred to a 3.5 kDa dialysis tubing and desalted by dialysing it against buffer C at 4°C for 90 minutes
• after renewing the buffer, once more over night at slow stirring
• the lysate is ready for use and, after shock-freezing in liquid N2, can be stored in small ali-quots at - 80°C.

Remarks

• The Preincubation step takes care of what is called the „run off“ step, a step imple-mented in order to eliminate endogenous cellular mRNA. The removal of endog-enous mRNA bound to ribosomes/polysomes is accomplished by finishing their synthesis via the added PEP, pyruvate kinase and ATP at 37°C. The residual endogenous mRNA will then subsequently become degraded due to the high RNase content of the extract. This step is precautionally performed in the dark, because some components are slightly light sensitive.
• The lysate can be frozen again after use, but looses effectivity

Reaction is adapted from the European Molecular Biology Laboratory (EMBL), Heidelberg, Germany

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