Coupled transcription-translation in chloroplast lysates

This chapter describes the methods for coupled transcription-translation in chloroplast lysates. A heterologous Escherichia coli lysate is the major in vitro system used for studying chloroplast gene expression by coupled transcription-translation experiments. However, transcriptional and translational regulatory factors unique to chloroplasts may not be present in E. coli extracts, and chloroplast gene expression may not be optimal in heterologous environment. As an alternative to the heterologous E. coli system, a homologous, DNA template-dependent, in vitro coupled transcription-translation system using chloroplast lysates from higher plants is developed. This system is comparable to E. coli lysates in terms of ability to transcribe and translate E. coli chromosomal DNA. The chloroplast lysates are as active as E. coli lysates at equivalent protein concentrations. The chloroplast lysate, in vitro coupled transcription-translation system expresses the genes present in total chloroplast DNA, vector plasmids, plasmids containing cloned fragments of chloroplast DNA, and E. coli chromosomal DNA. The lysate retains full transcriptional and translational activity when freeze-dried and reconstituted to original volumes.