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Engineering uroporphyrinogen III methyltransferase as a red fluorescent reporter in E. coli

Paper ID Volume ID Publish Year Pages File Format Full-Text
17009 42632 2014 6 PDF Available
Title
Engineering uroporphyrinogen III methyltransferase as a red fluorescent reporter in E. coli
Abstract

•The evolved barley UMT variant as a red fluorescent reporter by error-prone PCR conferred the cells with increased fluorescence.•Under different promoters, induction conditions and in the different E. coli strains, the evolved UMT emitted more cell fluorescence than the wild type UMT.•The evolved UMT displayed better performance than the wild type UMT on monitoring function in vivo cleavage by E. coli clpXP protease.•Before they are exported to the periplasm, both proteins catalyze the substrate in the cytoplasm and emit cell fluorescence.

Uroporphyrinogen III methyltransferase (UMT) is a novel reporter owing to the catalytic products in the cells that emit strong red fluorescence under UV light. Here, we engineered the gene encoding the functional barley UMT (bUMT) by error-prone PCR and broadened the application UMT as a red fluorescent reporter in Escherichia coli. A variant, termed mbUMT, was selected and emitted stronger cell fluorescence than the wild type bUMT expressed in different E. coli strains, under different promoters and induction conditions respectively. The constructed mbUMT with a C-terminal ssrA tag was degraded in cells by the protease ClpXP encoded by E. coli chromosome, whereas the bUMT was expressed as active aggregates. Before they are exported to the periplasm, both proteins catalyze the substrate in the cytoplasm and emit cell fluorescence. The results suggested that the evolved bUMT is a better candidate to monitor in vivo degradation by E. coli ClpXP.

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Keywords
UMT, uroporphyrinogen III methyltransferase; Uro’gen, uroporphyrinogen; SAM, S-adenosyl-l-methionine; SAH, S-adenosyl-l-homocysteine; GFP, green fluorescent proteinUroporphyrinogen III methyltransferase; Red fluorescent reporter; Engineering; Fluorescence
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Engineering uroporphyrinogen III methyltransferase as a red fluorescent reporter in E. coli
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volumes 61–62, July–August 2014, Pages 1–6
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