Dynamic response of naphthalene biodegradation in a continuous flow soil slurry reactor

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 halflife 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 multisubunit enzyme complex and will extend the utility of the bacterial luciferase reporter genes for the monitoring of dynamic changes in gene expression.

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“… 1990 ; DiGrazia et al. 1991 ), monitoring performance of synthetic gene circuits (Elowitz and Leibler 2000 ; Hasty et al. 2002 ; Gardner et al.…”

Section: Introductionmentioning

confidence: 99%

A destabilized bacterial luciferase for dynamic gene expression studies

et al. 2006

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“…Slurry phase conditions have been successfully applied in the bioremediation of petroleum hydrocarbon and aromatic hydrocarbon contaminated soils (DiGrazia et al 1991;Hwang & Cutright 2002;Nano et al 2003;Ramirez et al 2001). Monitoring bioreactor-type remediation systems is typically based on the determination of pollutant concentration (Nano et al 2003).…”

Section: Introductionmentioning

confidence: 99%

Monitoring of accelerated naphthalene-biodegradation in a bioaugmented soil slurry

et al. 2005

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The effect of microbial inoculation on the mineralization of naphthalene in a bioslurry treatment was evaluated in soil slurry microcosms. Inoculation by Pseudomonas putida G7 carrying the naphthalene dioxygenase (nahA) gene resulted in rapid mineralization of naphthalene, whereas indigenous microorganisms in the PAH-contaminated soil required a 28 h adaptation period before significant mineralization occurred. The number of nahA-like gene copies increased in both the inoculated and non-inoculated soil as mineralization proceeded, indicating selection towards naphthalene dioxygenase producing bacteria in the microbial community. In addition, 16S rRNA analysis by denaturing gradient gel electrophoresis (DGGE) analysis showed that significant selection occurred in the microbial community as a result of biodegradation. However, the indigenous soil bacteria were not able to compete with the P. putida G7 inoculum adapted to naphthalene biodegradation, even though the soil microbial community slightly suppressed naphthalene mineralization by P. putida G7.

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“…Further, continuous on-line monitoring is possible (e.g. DiGrazia et al, 1991;Heitzer et al, 1994;Heitzer et al, 1992;King et al, 1990). …”

Section: Introductionmentioning

confidence: 99%