In vitro transcription-translation using bacterial genome as a template to reconstitute intracellular profile

Kei Fujiwara, Tsunehito Sawamura, Tatsuya Niwa, Tatsuki Deyama, Shin Ichiro M. Nomura, Hideki Taguchi, Nobuhide Doi

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

In vitro transcription-translation systems (TX-TL) can synthesize most of individual genes encoded in genomes by using strong promoters and translation initiation sequences. This fact raises a possibility that TX-TL using genome as a template can reconstitute the profile of RNA and proteins in living cells. By using cell extracts and genome prepared from different organisms, here we developed a system for in vitro genome transcription-translation (iGeTT) using bacterial genome and cell extracts, and surveyed de novo synthesis of RNA and proteins. Two-dimensional electrophoresis and nano LC-MS/MS showed that proteins were actually expressed by iGeTT. Quantitation of transcription levels of 50 genes for intracellular homeostasis revealed that the levels of RNA synthesis by iGeTT are highly correlated with those in growth phase cells. Furthermore, activity of iGeTT was influenced by transcription derived from genome structure and gene location in genome. These results suggest that intracellular profiles and characters of genome can be emulated by TX-TL using genome as a template.

Original languageEnglish
Pages (from-to)11449-11458
Number of pages10
JournalNucleic Acids Research
Volume45
Issue number19
DOIs
Publication statusPublished - 2017 Nov 2

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Bacterial Genomes
Genome
RNA
Cell Extracts
In Vitro Techniques
Genes
Proteins
Electrophoresis
Homeostasis

ASJC Scopus subject areas

  • Genetics

Cite this

In vitro transcription-translation using bacterial genome as a template to reconstitute intracellular profile. / Fujiwara, Kei; Sawamura, Tsunehito; Niwa, Tatsuya; Deyama, Tatsuki; Nomura, Shin Ichiro M.; Taguchi, Hideki; Doi, Nobuhide.

In: Nucleic Acids Research, Vol. 45, No. 19, 02.11.2017, p. 11449-11458.

Research output: Contribution to journalArticle

Fujiwara, Kei ; Sawamura, Tsunehito ; Niwa, Tatsuya ; Deyama, Tatsuki ; Nomura, Shin Ichiro M. ; Taguchi, Hideki ; Doi, Nobuhide. / In vitro transcription-translation using bacterial genome as a template to reconstitute intracellular profile. In: Nucleic Acids Research. 2017 ; Vol. 45, No. 19. pp. 11449-11458.
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