A destabilized bacterial luciferase for dynamic gene expression studies

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.

Fig. 1

Total bioluminescence (arbitrary units) of clones containing wild type luxAB, and luxABaav (left axis), and luxAaavB, luxAaavBaav and the negative control (right axis) over time following the addition of tetracycline and rifampicin (t = 0). Measurements were made following the addition of N-decanal as described in the “Materials and methods”. Values are averages of triplicate samples with error bars representing standard deviations among the replicates. The negative control contains the parent plasmid without the luciferase genes

Fig. 2

Relative bioluminescence over time for luxA variants contained within the entire lux cassette following removal of the IPTG inducer