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A destabilized bacterial luciferase for dynamic gene expression studies.

Allen MS, Wilgus JR, Chewning CS, Sayler GS, Simpson ML - Syst Synth Biol (2007)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Molecular-Scale Engineering and Nanoscale Technologies (MENT) Research Group, Oak Ridge National Laboratory, P.O. Box 2008, Building 3500, MS 6006, Oak Ridge, TN, 37931-6006, USA.

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.

No MeSH data available.


Related in: MedlinePlus

Relative bioluminescence over time for luxA variants contained within the entire lux cassette following removal of the IPTG inducer
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Fig2: Relative bioluminescence over time for luxA variants contained within the entire lux cassette following removal of the IPTG inducer

Mentions: Variants of luxA were subsequently cloned into a vector containing the entire lux operon by restriction digestion interior to each of the luxA and B genes and replacement of the wild type fragment with fragments containing the modified luxA gene. Results of experiments on luminescent decay following the removal of inducer (isopropyl-β-d-thiogalactopyranoside; IPTG) are shown in Fig. 2.Fig. 2


A destabilized bacterial luciferase for dynamic gene expression studies.

Allen MS, Wilgus JR, Chewning CS, Sayler GS, Simpson ML - Syst Synth Biol (2007)

Relative bioluminescence over time for luxA variants contained within the entire lux cassette following removal of the IPTG inducer
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2533149&req=5

Fig2: Relative bioluminescence over time for luxA variants contained within the entire lux cassette following removal of the IPTG inducer
Mentions: Variants of luxA were subsequently cloned into a vector containing the entire lux operon by restriction digestion interior to each of the luxA and B genes and replacement of the wild type fragment with fragments containing the modified luxA gene. Results of experiments on luminescent decay following the removal of inducer (isopropyl-β-d-thiogalactopyranoside; IPTG) are shown in Fig. 2.Fig. 2

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Molecular-Scale Engineering and Nanoscale Technologies (MENT) Research Group, Oak Ridge National Laboratory, P.O. Box 2008, Building 3500, MS 6006, Oak Ridge, TN, 37931-6006, USA.

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.

No MeSH data available.


Related in: MedlinePlus