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Multigene expression in vivo: Supremacy of large versus small terminators for T7 RNA polymerase
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
2012 (English)In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 109, no 4, 1043-1050 p.Article in journal (Refereed) Published
Abstract [en]

Designing and building multigene constructs is commonplace in synthetic biology. Yet functional successes at first attempts are rare because the genetic parts are not fully modular. In order to improve the modularity of transcription, we previously showed that transcription termination in vitro by bacteriophage T7 RNA polymerase could be made more efficient by substituting the standard, single, TF large (class I) terminator with adjacent copies of the vesicular stomatitis virus (VSV) small (class II) terminator. However, in vitro termination at the downstream VSV terminator was less efficient than at the upstream VSV terminator, and multigene overexpression in vivo was complicated by unexpectedly inefficient VSV termination within Escherichia coli cells. Here, we address hypotheses raised in that study by showing that VSV or preproparathyroid hormone (PTH) small terminators spaced further apart can work independently (i.e., more efficiently) in vitro, and that VSV and PTH terminations are severely inhibited in vivo. Surprisingly, the difference between class II terminator function in vivo versus in vitro is not due to differences in plasmid supercoiling, as supercoiling had a minimal effect on termination in vitro. We therefore turned to TF terminators for BioBrick synthesis of a pentameric gene construct suitable for overexpression in vivo. This indeed enabled coordinated overexpression and copurification of five His-tagged proteins using the first construct attempted, indicating that this strategy is more modular than other strategies. An application of this multigene overexpression and protein copurification method is demonstrated by supplying five of the six E. coli translation factors required for reconstitution of translation from a single cell line via copurification, greatly simplifying the reconstitution.

Place, publisher, year, edition, pages
2012. Vol. 109, no 4, 1043-1050 p.
Keyword [en]
synthetic biology, gene expression, T7 RNA polymerase, transcription terminator, translation
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-171398DOI: 10.1002/bit.24379ISI: 000300692700021OAI: oai:DiVA.org:uu-171398DiVA: diva2:511137
Available from: 2012-03-20 Created: 2012-03-19 Last updated: 2012-03-20Bibliographically approved

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