E. coli S30 extracts

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... 

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... 

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-... 

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). 

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. 

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