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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

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
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Fig1: 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

Mentions: Bioluminescence measurements of cells harboring plasmids containing the wild type luxAB genes of P. luminescens behind a constitutive promoter were compared to those of luxA and luxB variants containing carboxy-terminal tags designed to accelerate degradation of the protein(s) within the cell. Assays using constructs containing only luxA and luxB (i.e. which lack luxC, D, and E and hence are unable to produce the substrate) were conducted by adding antibiotics to block transcription and translation (rifampicin and tetracycline, respectively), followed by monitoring bioluminescence with the addition of aldehyde over time. As shown in Fig. 1, modification of the LuxB protein did not result in a decrease of bioluminescence. Bioluminescence in this variant was found to remain relatively constant similar to the wild type over the course of the experiments. Addition of the same -aav tag (encoding AANDENYAAAV) to the luxA gene resulted in a rapid decrease in bioluminescence after time t0. An even more dramatic decrease in bioluminescence was observed when both LuxA and LuxB included the -aav tag. Interestingly, the clone containing luxABaav (wild type luxA with modified luxB) appeared to produce more light than did the wild-type luxAB. Since this result was reproducible within the experimental parameters (Fig. 1) yet inconsistent with those generated in the absence of antibiotics (Table 3), it is believed that this result is an experimental artifact.Fig. 1


A destabilized bacterial luciferase for dynamic gene expression studies.

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

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
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2533149&req=5

Fig1: 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
Mentions: Bioluminescence measurements of cells harboring plasmids containing the wild type luxAB genes of P. luminescens behind a constitutive promoter were compared to those of luxA and luxB variants containing carboxy-terminal tags designed to accelerate degradation of the protein(s) within the cell. Assays using constructs containing only luxA and luxB (i.e. which lack luxC, D, and E and hence are unable to produce the substrate) were conducted by adding antibiotics to block transcription and translation (rifampicin and tetracycline, respectively), followed by monitoring bioluminescence with the addition of aldehyde over time. As shown in Fig. 1, modification of the LuxB protein did not result in a decrease of bioluminescence. Bioluminescence in this variant was found to remain relatively constant similar to the wild type over the course of the experiments. Addition of the same -aav tag (encoding AANDENYAAAV) to the luxA gene resulted in a rapid decrease in bioluminescence after time t0. An even more dramatic decrease in bioluminescence was observed when both LuxA and LuxB included the -aav tag. Interestingly, the clone containing luxABaav (wild type luxA with modified luxB) appeared to produce more light than did the wild-type luxAB. Since this result was reproducible within the experimental parameters (Fig. 1) yet inconsistent with those generated in the absence of antibiotics (Table 3), it is believed that this result is an experimental artifact.Fig. 1

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