Arlene A Wise

US Patent:

7303894, Dec 4, 2007

Filed:

Sep 18, 2003

Appl. No.:

10/665455

Inventors:

Arlene A. Wise - Philadelphia PA, US
Cheryl R. Kuske - Los Alamos NM, US
Thomas C. Terwilliger - Santa Fe NM, US

Assignee:

Los Alamos National Security - Los Alamos NM

International Classification:

C12Q 1/02
C12N 1/21
C12N 15/00
C12N 15/74
C12N 15/87
C12Q 1/00
C12Q 1/66
C12P 19/34

US Classification:

435 29, 4352523, 43525233, 43525234, 435440, 435 912, 435471, 435463, 435 8, 435 4, 530350, 536 231

Abstract:

Detection of phenols using engineered bacteria. A biosensor can be created by placing a reporter gene under control of an inducible promoter. The reporter gene produces a signal when a cognate transcriptional activator senses the inducing chemical. Creation of bacterial biosensors is currently restricted by limited knowledge of the genetic systems of bacteria that catabolize xenobiotics. By using mutagenic PCR to change the chemical specificity of the species CF600 DmpR protein, the potential for engineering novel biosensors for detection of phenols has been demonstrated. DmpR, a well-characterized transcriptional activator of the P. CF600's dmp operon mediates growth on simple phenols. Transcription from Po, the promoter heading the dmp operon, is activated when the sensor domain of DmpR interacts with phenol and mono-substituted phenols. By altering the sensor domain of the DmpR, a group of DmpR derivatives that activate transcription of a Po-lacZ fusion in response to eight of the EPA's eleven priority pollutant phenols has been created. The assays and the sensor domain mutations that alter the chemical specificity of DmpR is described.