S30 extract

Translation extracts Rabbit reticulocyte lysates (RRL) (Promega), wheat germ (WG) extract (Promega), bacterial S30 extract (Promega). Extracts from Krebs-2 cells were prepared as described (Svitkin and Sonenberg, 2004). 

At both ends of the mRNA, the ribosome display construct should include stemloops, 5 - and 3 -stemloops are known to stabilize mRNA against RNases in vivo as well as in vitro. The presence of stemloops is important, especially in the E. coli ribosome display system, because the extract used for in vitro translation contains high RNase activities. To date, five of twenty E. coli RNases have been shown to contribute to mRNA degradation (Hajnsdorf et al., 1996), and they are probably all present in the S30 extract. The efficiency of ribosome display was in-... 

The protocol for IVTT was described previously [7]. Briefly, an amino acid mix containing all the amino acids for protein synthesis was mixed with the nucleic acid mix. This nucleic acid mix consisted of the plasmid pET14b which contained the DNA sequence for the enzyme OpdA, E. coli S30 extract, nucleotides, and all other necessary ingredients. The amino acid mix and the nucleic acid mix were delivered through separate ports into the microchannel and fused to form microdroplets. 

Wu N, Zhu Y, Brown S, Oakeshott J, Peat TS, Surjadi R, Easton C, Leech PW, Sexton BA (2009) A PMMA microfluidic droplet platform for in vitro protein expression using crude E. coli S30 extract. Lab Chip 9 3391-3398... 

High quality cellular extracts should be prepared by cultivating cells in fermenters with good aeration. First, a growth curve should be determined and an efficient chilling technique of the fermenter broth should be established. Cells must be harvested in mid-log phase and should be chilled down to 10-14°C before centrifugation. Several strains and modifications for S30 extract preparations are described (5). We describe a basic protocol for the preparation of high quality S30 extracts for the CF expression of both soluble and membrane proteins. 

The final total protein concentration of the S30 extract should be between 20 and 40 mg/mL. Each new batch of extract should be adjusted to its optimal concentration of Mg + (12-25 mM) and 1C (250-350 mM) ions. Perform Mg concentration screens in 2 mM steps and IC concentration screens in 20 mM steps while monitoring GFP expression. 

Master mix RFM Pyruvat kinase tRNA ( . colt) T7RNAP Ribolock DNA template E. colt S30 extract MilliQ water... 

Proteins from the S30 extract may co-precipitate with the MP. Those impurities can be reduced by washing the pellet with mild detergents and/or increased NaCl and DTP concentrations. 

A number of in vitro transcription/translation systems are commercially available, including rabbit reticulocyte, wheat germ, and E. coli S30 extracts (18). There is also a variety of rabbit reticulocyte sj tems, including nuclease-treated, non-nuclease-treated, DTT-deficient, and coupled transcription/translation. We... 

A cell-free extract (S30) from E. coli K12 has been developed as an efficient coupled transcription/translation system which performs protein synthesis in vitro from the genes cloned in plasmids under the T7, T3, or SP6 promoter. Capping of the mRNA for eukaryotic proteins is apparently unnecessary with the S30 system. The coupled transcription/translation system, which was originally developed as the S3 0 translation system (122), can be used in a batchwise or continuous-flow mode (123,124). Use of the ribosome fraction collected from the S30 extracts is reported to improve the yield and efficacy further and more advantageously with nonlinearized plasmids than with linearized plasmids (125). 

Fig. 8. Salt dependence of enzyme synthesis in an S30 system. (Experiment together with H. J. Rahmsdorf.) 50 ml of an exponentially growing culture of strain XA 7007 (suA) in rich medium were harvested at O.D.goo = 0.4. After washing once with TMA buffer, the cells were suspended in 0.4 ml TMA buffer and lysed by ultrasonication. The debris were removed. The S30 incubation mixture contained, in addition to 20 (d of the S30 extract, the following components in a final volume of 0.05 ml tris HCl pH 8.0 51 mM K-acetate 50 mM amino acids 0.2 mM each ATP 2mM UTP, CTP, GTP 0.5 mM each PEP 20 mM dithiothreitol 2.5 mM tRNA 500 (xg/ml T3 DNA 50 xg/ml MgClg 11 mM (the S30 extract adds to 15 mM MgClg in toto) varying concentrations of NH4CI. After 40 minutes at 37°, aliquots were tested for lysozyme (o-------o)...

An S30 extract (a cell extract after removal of the cell debris) supports enzyme synthesis under the direction of added DNA. Similar to the crude system, the S30 contains the salt and substrates from the cell sap and, because of the dilution during cell disruption, additional salts and substrates are needed (Fig. 8). 

ZuBAY and coworkers (Zubay and Chambers, 1969) have established a modification of the S30 system of Matthaei and Nirenberg (I96I), a preincubated S30 system. This system found wide application for the study of synthesis of E, coli enzymes. The S30 is mixed with a small volume of a solution containing all the components needed for translation (amino acids, ATP, ATP-regenerating system) and preincubated at 37° for 80 minutes. The S30 extract is then dialyzed against a desired buffer at 0°. Table 2 summarized the preparation of the S30 system and lists the constituents of the incubation mixture. 

In an S30 extract from thermophilic bacteria, the endogenous DNA can be efficiently digested by incubation with pancreatic DNase at low temperature (G. Bauer, W. Siegert and P. H. Hofschneider, personal communication). The DNase is then destroyed by preincubation at the growth temperature of the bacteria. The system has been used for the synthesis of coat proteins under the direction of 0(x-4 phage DNA (Rabussay et al., I969), the products being detected by gel electrophoresis and immunoprecipitation (Bauer et al., personal communication). 

---