Team:Freiburg/Protocols/prepLysate
Protocol Cell-free expression
Preparation of a Lysate for Cell-free expression
Material: see below
Preparation: 2-3 days
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 | 1M | 300mM |
| Mg(OAc)2 | 1M | 10 mM |
| ATP | 100mM | 10mM |
| phosphoenol pyruvate (PEP) | 1M | 80mM |
| Amino Acid mix | 5mM | 40µM |
| pyruvate kinase | 2U/µl | 8U/µl |
| DEPC-H2O | - | ad 750µl |
- 750µl of preincubation buffer are necessary for 1 l of culture
Initial remarks to the used Bacteria
- To achieve high levels of protein production, the BL21(DE3) strain was chosen, which carries the gene coding for a T7 RNA polymerase as a lambda prophage within its chromosome, which is inducible by addition of IPTG
- As the system is later to be used for expression of synthetic gene constructs of varying natural hosts, the bacteria need to be able to express so-called “rare codons”: AGA, AGG, AUA, CUA, GGA, CCC, and CGG are used less frequently in E. coli as they are e.g in human cells
- Cells were transformed with the pRARE2 plasmid, taken from Rosetta2 competent cells. The pRARE2 plasmid encodes for these rare codon - tRNAs
Growth of Bacteria
- Prepare an overnight preculture in 10 ml medium in a 100 ml Erlenmeyer-flask @ 37°C, 250 rpm
- inoculated in 1000 ml Terrific Broth in a 2000 ml chicanery-flask at 37°C, 150-180 rpm
- cells were grown 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 afterwards resuspended in 1.3 ml of buffer B
- 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.
Washing and lysis
- obtained lysate is pelleted via ultracentrifugation at 30,000 g for 30 min at 4°C
- 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
