CONSTRUCTION OF MICROARRAYS FOR E. COLI O157:H7

• Sponsoring Institution: Agricultural Research Service/USDA
• Project Status: COMPLETE
• Funding Source: USDA COOPERATIVE AGREEMENT
• Reporting Frequency: Annual
• Accession No.: 0405828
• Project Start Date: Aug 19, 2002
• Project End Date: Jul 14, 2004
• Program Code: [(N/A)]- (N/A)

Recipient Organization
IOWA STATE UNIVERSITY
2229 Lincoln Way
AMES,IA 50011

Performing Department
VETERINARY MEDICAL RESEARCH INSTITUTE

Non Technical Summary
(N/A)

Animal Health Component

30%

Research Effort Categories

Basic

40%

Applied

30%

Developmental

30%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3310	1100	10%
311	3410	1100	10%
712	3310	1100	30%
712	3410	1100	20%
712	4010	1100	30%

Knowledge Area
311 - Animal Diseases; 712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
3310 - Beef cattle, live animal; 4010 - Bacteria; 3410 - Dairy cattle, live animal;

Field Of Science
1100 - Bacteriology;

Keywords

escherichia coli

food safety

bacteria

bacterial genetics

genes

gene expression

cattle

colonization

Goals / Objectives
The objectives of this cooperative research project are to construct gene microarrays for E. coli O157:H7 strain EDL933. These microarrays will be used to identify differences in gene expression of E. coli O157:H7 that are grown or exposed to different environmental conditions both in vivo and in vitro. E. coli O157:H7 genes that are preferentially expressed in vivo will be candidates for further evaluation to determine their specific role in colonization, survival, and persistence in cattle.

Project Methods
The published genomic sequence of E. coli O157:H7 will be used to design primers for PCR amplification of the 3574 open reading frame (ORFs) that are common to both O157 and K-12 strains, the 1387 O157 unique ORFs, the 528 K-12 unique ORFs, and the 100 genes that are carried on the pO157 plasmid present in O157:H7 E. coli. The PCR products will be spotted in a microarray and used in hybridization assays. RNA isolated from O157:H7 E. coli grown or exposed to different in vivo and in vitro conditions will be used to produce labeled cDNA for hybridization. This will be used to identify genes that are differentially expressed under a variety of different conditions both in vitro and in vivo.

Progress 08/19/02 to 07/14/04

Outputs
4. What were the most significant accomplishments this past year? D. Progress Report: This report serves to document research conducted under a specific cooperative agreement between ARS and Iowa State University. Additional details of research can be found in the report for the parent project 3635-32000-051-00D, Prevention of Losses from Colibacillosis and E. coli O157:H7 in Cattle and Swine. This project is funded by the National Alliance for Food Safety. The published genomic sequences of E. coli O157:H7 and E. coli K-12 have been used to design DNA primers for polymerase chain reaction (PCR) amplification of all of the 3,574 genes that are common to both O157 and K-12, the 1,387 genes that are unique to O157, the 528 genes that are unique to K-12 and the 100 genes on the large plasmid present in O157. These primers have been evaluated for amplification of specific PCR products and are being used to print microarrays containing representative sequences for all of the E. coli O157 and K-12 genes. These microarrays will be used to support the parent project objective in experiments to identify genes that are important for growth and survival of E. coli O157:H7 under different environmental conditions and in cattle. The objectives of this Specific Cooperative Agreement directly complement the objectives of the parent project by providing microarrays that can be used as tools to identify the genes and mechanisms that are important for colonization by E. coli O157:H7 in cattle.

Impacts
(N/A)

Publications