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A T7 RNA polymerase-based toolkit for the concerted expression of clustered genes

Paper ID Volume ID Publish Year Pages File Format Full-Text
23777 43472 2012 10 PDF Available
Title
A T7 RNA polymerase-based toolkit for the concerted expression of clustered genes
Abstract

Bacterial genes whose enzymes are either assembled into complex multi-domain proteins or form biosynthetic pathways are frequently organized within large chromosomal clusters. The functional expression of clustered genes, however, remains challenging since it generally requires an expression system that facilitates the coordinated transcription of numerous genes irrespective of their natural promoters and terminators.Here, we report on the development of a novel expression system that is particularly suitable for the homologous expression of multiple genes organized in a contiguous cluster. The new expression toolkit consists of an Ω interposon cassette carrying a T7 RNA polymerase specific promoter which is designed for promoter tagging of clustered genes and a small set of broad-host-range plasmids providing the respective polymerase in different bacteria. The uptake hydrogenase gene locus of the photosynthetic non-sulfur purple bacterium Rhodobacter capsulatus which consists of 16 genes was used as an example to demonstrate functional expression only by T7 RNA polymerase but not by bacterial RNA polymerase. Our findings clearly indicate that due to its unique properties T7 RNA polymerase can be applied for overexpression of large and complex bacterial gene regions.

► The concerted expression of a large hydrogenase gene cluster was achieved in Rhodobacter capsulatus. ► T7 RNA polymerase was unique in allowing full-length transcription of all hydrogenase genes. ► Quantitative transcript analysis revealed posttranscriptional processing of hydrogenase gene transcripts in R. capsulatus.

Keywords
Gm, gentamicin; IPTG, isopropyl-β-d-thiogalactopyranoside; Km, kanamycin; LB, Luria Broth; mob, mobilization site; PCR, polymerase chain reaction; qPCR, quantitative (real time) PCR; rpm, rounds per minute; bp, base pair; Sp, spectinomycinGene cluster; Ex
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A T7 RNA polymerase-based toolkit for the concerted expression of clustered genes
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 159, Issue 3, 15 June 2012, Pages 162–171
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us