Cell-free protein synthesis in a microfabricated reactor

Abstract

Microbiochemical reactors having two inlet ports and one outlet port were fabricated on a silicon wafer by means of anisotropic etching in order to develop a parallel and automatic experimental system for cell-free translation. Using cell-free extract prepared from Escherichia coli, we tested the reactor for the translation of polyuridylic acid and MS2 phage RNA, and found that polypeptide and protein syntheses could be proceeded according to the genetic codes on the mRNAs. It indicates that the microfabricated reactor is useful for enzymatic reactions including complicated ones like cell-free translation. We also discuss the possibility of microsystems as advanced experimental tools for not only cell-free translation but also other various biochemical and biological research ®elds.

Determination of the Termination Efficiency of the Transcription Terminator Using Different Fluorescent Profiles in Green Fluorescent Protein Mutants

“…The next step is to combine the method with a cell-free translation system, especially in a microfabricated device. 14 We have reported in vitro translation in microreactors, 15,16 and have already succeeded in the cell-free translation of GFP in a microfabricated device equipped with a fluorescence microscope, and the in situ detection of the expressed GFP. 17 Further studies are underway to attain this goal.…”

Cell‐Free Protein Synthesis in Microfluidic Array Devices

“…IVT has been implemented in miniaturized devices or a microwell format (23)(24)(25)(26)(27)(28). However, one of the major problems with the microplate is its short reaction time because of the fast depletion of reactants and inhibition from the reaction products, thus significantly reducing protein expression yield.…”

Nano‐enabled synthetic biology

“…For example, small volume reaction containers are being considered for high‐throughput screening, (Grosvenor et al , 2000; Angenendt et al , 2005), single molecule enzymology (Rondelez et al , 2005; Rissin and Walt, 2006), and analyses of single cells (Cooper, 1999; Johannessen et al , 2002). These reaction containers are also being developed for the cell‐free synthesis of proteins (Nojima et al , 2000; Tabuchi et al , 2002; Yamamoto et al , 2002; Angenendt et al , 2004; Kinpara et al , 2004; Mei et al , 2005). In addition to creating a new approach to the parallel production of various proteins, these structures are permitting novel functional assays (Angenendt et al , 2005; Mei et al , 2005).…”

“…In addition to creating a new approach to the parallel production of various proteins, these structures are permitting novel functional assays (Angenendt et al , 2005; Mei et al , 2005). The use of microfabricated structures allows for the controllable exchange and mixing of reagents (Nojima et al , 2000; Wang et al , 2005) and the integration of sensitive techniques for sampling and analysis of reaction products.…”