A destabilized bacterial luciferase for dynamic gene expression studies

Michael S. Allen, John R. Wilgus, Christopher S. Chewning, Gary S. Sayler, Michael L. Simpson

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

Fusions of genetic regulatory elements with reporter genes have long been used as tools for monitoring gene expression and have become a major component in synthetic gene circuit implementation. A major limitation of many of these systems is the relatively long half-life of the reporter protein(s), which prevents monitoring both the initiation and the termination of transcription in real-time. Furthermore, when used as components in synthetic gene circuits, the long time constants associated with reporter protein decay may significantly degrade circuit performance. In this study, short half-life variants of LuxA and LuxB from Photorhabdus luminescens were constructed in Escherichia coli by inclusion of an 11-amino acid carboxy-terminal tag that is recognized by endogenous tail-specific proteases. Results indicated that the addition of the C-terminal tag affected the functional half-life of the holoenzyme when the tag was added to luxA or to both luxA and luxB, but modification of luxB alone did not have a significant effect. In addition, it was also found that alteration of the terminal three amino acid residues of the carboxy-terminal tag fused to LuxA generated variants with half-lives of intermediate length in a manner similar to that reported for GFP. This report is the first instance of the C-terminal tagging approach for the regulation of protein half-life to be applied to an enzyme or monomer of a multi-subunit enzyme complex and will extend the utility of the bacterial luciferase reporter genes for the monitoring of dynamic changes in gene expression.

Original languageEnglish
Pages (from-to)3-9
Number of pages7
JournalSystems and Synthetic Biology
Volume1
Issue number1
DOIs
StatePublished - 1 Mar 2007

Fingerprint

Bacterial Luciferases
Gene expression
Half-Life
Genes
Synthetic Genes
Gene Expression
Gene Regulatory Networks
Proteins
Reporter Genes
Networks (circuits)
Amino acids
Monitoring
Photorhabdus
Enzymes
Amino Acids
Holoenzymes
Gene Expression Profiling
Transcription
Escherichia coli
Fusion reactions

Keywords

  • Destabilized protein
  • Gene expression
  • Luciferase
  • Lux
  • Photorhabdus luminescens
  • Reporter genes
  • ssrA

Cite this

Allen, Michael S. ; Wilgus, John R. ; Chewning, Christopher S. ; Sayler, Gary S. ; Simpson, Michael L. / A destabilized bacterial luciferase for dynamic gene expression studies. In: Systems and Synthetic Biology. 2007 ; Vol. 1, No. 1. pp. 3-9.
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A destabilized bacterial luciferase for dynamic gene expression studies. / Allen, Michael S.; Wilgus, John R.; Chewning, Christopher S.; Sayler, Gary S.; Simpson, Michael L.

In: Systems and Synthetic Biology, Vol. 1, No. 1, 01.03.2007, p. 3-9.

Research output: Contribution to journalArticle

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