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 Scopus citations

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 - Mar 2007

Keywords

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

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