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rdf:type |
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lifeskim:mentions |
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pubmed:issue |
12
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pubmed:dateCreated |
2008-6-16
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pubmed:abstractText |
A simple, sensitive, and rapid cell-free assay system was developed for detection of N-acyl homoserine lactone (AHL) autoinducers involved in bacterial quorum sensing (QS). The present approach improves upon previous whole-cell biosensor-based approaches in its utilization of a cell-free assay approach to conduct bioassays. The cell-free assay was derived from the AHL biosensor bacterium Agrobacterium tumefaciens NTL4(pCF218)(pCF372), allowing the expression of beta-galactosidase upon addition of exogenous AHLs. We have shown that beta-galactosidase expression is possible in cell-free solution [lysate from Agrobacterium tumefaciens NTL4(pCF218)(pCF372) culture]. Assay detection limits with the use of chromogenic substrate X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) ranged from approximately 100 nM to 300 nM depending on the specific AHL. Replacement (of X-Gal) with the luminescent substrate Beta-Glo increased sensitivity to AHLs by 10-fold. A major advantage of the cell-free assay system is elimination of time-consuming steps for biosensor cell culture conditioning, which are required prior to whole-cell bioassays. This significantly reduced assay times from greater than 24 h to less than 3 h, while maintaining high sensitivity. Assay lysate may be prepared in bulk and stored (-80 degrees C) over 6 months for future use. Finally, the present protocol may be adapted for use with other biosensor strains and be used in high-throughput AHL screening of bacteria or metagenomic libraries.
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pubmed:commentsCorrections |
http://linkedlifedata.com/resource/pubmed/commentcorrection/18424536-10648535,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18424536-10713083,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18424536-11157219,
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http://linkedlifedata.com/resource/pubmed/commentcorrection/18424536-9177164,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18424536-942051,
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http://linkedlifedata.com/resource/pubmed/commentcorrection/18424536-9673021
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pubmed:language |
eng
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pubmed:journal |
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pubmed:citationSubset |
IM
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pubmed:chemical |
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pubmed:status |
MEDLINE
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pubmed:month |
Jun
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pubmed:issn |
1098-5336
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pubmed:author |
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pubmed:issnType |
Electronic
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pubmed:volume |
74
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
3667-71
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pubmed:dateRevised |
2011-11-17
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pubmed:meshHeading |
pubmed-meshheading:18424536-Acyl-Butyrolactones,
pubmed-meshheading:18424536-Agrobacterium tumefaciens,
pubmed-meshheading:18424536-Biosensing Techniques,
pubmed-meshheading:18424536-Cell-Free System,
pubmed-meshheading:18424536-Densitometry,
pubmed-meshheading:18424536-Galactosides,
pubmed-meshheading:18424536-Indoles,
pubmed-meshheading:18424536-Luminescent Agents,
pubmed-meshheading:18424536-Luminescent Measurements,
pubmed-meshheading:18424536-Quorum Sensing,
pubmed-meshheading:18424536-Sensitivity and Specificity,
pubmed-meshheading:18424536-Time Factors,
pubmed-meshheading:18424536-beta-Galactosidase
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pubmed:year |
2008
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pubmed:articleTitle |
Rapid screening of quorum-sensing signal N-acyl homoserine lactones by an in vitro cell-free assay.
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pubmed:affiliation |
Department of Environmental Health Sciences, Arnold School of Public Health, University of South Carolina, Columbia, SC 29208, USA. kawaguch@gwm.sc.edu
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pubmed:publicationType |
Journal Article,
Comparative Study,
Research Support, U.S. Gov't, Non-P.H.S.
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