Microbial Monitoring With Artificial Stable RNAsEPA Grant Number: R825354
Title: Microbial Monitoring With Artificial Stable RNAs
Investigators: Fox, George E. , Willson, Richard C.
Institution: University of Houston
EPA Project Officer: Hahn, Intaek
Project Period: January 1, 1997 through December 31, 1999
Project Amount: $335,701
RFA: Exploratory Research - Environmental Biology (1996) RFA Text | Recipients Lists
Research Category: Biology/Life Sciences , Ecosystems
Description:The purpose of this project is to establish a stable RNA-based approach for labeling and tracking microorganisms in complex ecosystems. This technology will be readily incorporated into studies of risk associated with the release of both naturally occurring and genetically engineered bacteria into the environment. The monitoring system will also be an extremely valuable tool for monitoring organism behavior in laboratory studies of microbial ecosystems.
Previously, we have developed a recombinant DNA vector which encodes a deletion mutant of 5S rRNA. This deletion RNA is expressed from the growth rate-regulated ribosomal RNA promoters and accumulates to high levels. The resulting system is in effect a miniature rRNA operon and is referred to as rrnMINI. Site-directed mutagenesis was used to create a restriction site in the deletion RNA gene in order to facilitate the addition of replacement sequences. By appropriate insertion of replacement sequences into this construct novel RNAs which carry highly unique sequence segments can be created. These artificial RNAs (aRNAs), are not incorporated into ribosomes but nevertheless accumulate to high levels in the cell. Three alternative approaches might be useful for detecting such aRNAs. These are: (1) the presence of a unique size band in a high resolution low molecular weight RNA gel profile, (2) presence of a unique target sequence in the aRNA that can be detected by any of several hybridization strategies, and (3)incorporation of a mRNA in the aRNA that encodes a readily detectable protein product, e.g., the Green Fluorescent Protein.
In contrast to more traditional methods, detection based on the rrnMINI system will not depend on recovery of the organisms of interest in viable/culturable form, and it does not confer new capabilities such as drug resistance on the labeled organism. The reliance on a growth rate regulated promoter is an advantage not enjoyed by amplification (PCR) and direct detection (e.g., bioluminescence), approaches that are currently in use. In addition, potential problems associated with selective target amplification are avoided.
Additional exploratory research is being conducted to increase the suitability of this promising rrnMINI technology for use in microbial monitoring and risk assessment. The current system is plasmid based in E. coli. In order to minimize undesired spread of recombinant genes in the environment it is anticipated that whenever possible one will either use large plasmids, e.g., Tol, or directly integrate recombinant genes into the main genome. Therefore it will be essential to integrate the rrnMINI expression system into potential host organisms. In the present study this will be undertaken for E. coli, P. putida and the Tol plasmid. These efforts will be facilitated by minor improvements in the existing aRNA identification system. A second major project goal will be to carefully characterize at least one approach to aRNA detection.