1997 Progress Report: Microbial Monitoring With Artificial Stable RNAs

EPA 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 Period Covered by this Report: January 1, 1997 through December 31, 1998
Project Amount: $335,701
RFA: Exploratory Research - Environmental Biology (1996) RFA Text |  Recipients Lists
Research Category: Biology/Life Sciences , Ecosystems

Objective:

The long term goal of this project is to establish a stable RNA approach for labeling and tracking microorganisms in complex ecosystems. In brief, a unique identifier sequence is inserted into a fragment of a 5S rRNA gene carried by the target bacterium. The resulting chimera gene expresses an RNA which has stability that is comparable to that of naturally occurring 5S rRNA and therefore accumulates in cells in large amounts. Standard detection systems for ribosomal RNA, as well as PCR based methods, can then be used to determine the presence or absence of the target organism. It is envisioned that this technology will facilitate studies of risk associated with the release of both naturally occurring and genetically engineered organisms into the environment. We are conducting the exploratory research needed to establish the utility of the approach.

Progress Summary:

The immediate project goals are to (1) establish the utility of the approach in a variety of bacteria, (2) to integrate stable RNA identifiers into bacterial genomes, (3) to determine the extent to which identifiers can be changed, (4) to establish soil based assay systems for use with stable RNA identifiers. Several project milestones have been achieved. Refinements to the Escherichia coli construct were made and we now have succeeded in developing a plasmid based expression system for Pseudomonas putida. The identifier RNA is expressed in Pseudomonas putida and accumulates in approximately a 1:5 ratio to the wild type 5S rRNA. This demonstrates that the identifier system can be used in bacteria that are not closely related to E. coli. However the expression level of 15-20% is less than the 35-50% that is seen in E. coli. This may reflect more rapid degradation in Pseudomonas or loss of efficiency of the promoter. A second significant milestone was the incorporation of the E. coli construct into the E. coli genome. In this case, the identifier accumulates in the expected 1:6 ratio with the naturally occurring 5S rRNA. We have also begun efforts to characterize the variety of inserts that can be used as identifiers. We have constructed two families of identifier inserts, one consisting of random 13-mers and the other random 50-mers. A large number of these random inserts were successfully incorporated into monitoring system. RNA blots were used to examine thirty six of these constructs for RNA expression. In each case, an RNA of the expected size was found. These results suggest that there will be no difficulty in obtaining many unique identifiers. Initial results with inserts that produce mRNAs for directly detectable products, (e.g. the green fluorescence protein) were not repeatable and this avenue of research is no longer being pursued.

Significant progress has been made towards the development of assay procedures for stable RNAs in the environment. The E. coli genomic construct is being used extensively in our efforts to develop assay procedures suitable for environmental studies. The focus to date has been development of field compatible methods for recovery of the RNA. A number of published methods have been attempted and in our hands the method of Tsai et al. (Appl. Env. Microbiol. 57: 765-768, 1991) seems most effective. Particular attention has focused on efforts to simply methods as much as possible. In this regard we have attempted several immobilized affinity chromatography method for final RNA purification which could in principle be done in one step. These include anion exchange and boronate. Although both of these approaches show some promise, we believe we may have made a breakthrough recently with our observation that metal affinity chromatography is especially effective with our target RNAs.

Future Activities:

In the future, we will continue the efforts described above. In terms of the plasmid based system, our immediate goal is to determine if it can be successfully transferred to a number of bacteria. In principle, this should work due to the broad host range of the underlying plasmid. Assuming, effective transformation procedures can be developed, the key question is whether or not the RNA product accumulates to adequate levels (10-15% of total 5S rRNA) in a variety of organisms. We initially plan to do these studies in Rhodococcus sphaeroides, Alcaligenes faecalis and Rhizobium meliloti. In view of our demonstration that the existing identifier accumulates in Pseudomonas putida, another important objective will be to incorporate the identifier in the genome as has already been done with E. coli. We expect to continue our studies of which types of identifier sequences are likely to work best; e.g. are certain structural elements or primary sequence segments important? In the area of sample preparation and assay development we will be doing significant follow up studies on the effectiveness of metal affinity chromatography for rapid RNA purification. We are now at a stage where there is enough understanding of separation and purification of the identifier RNA that we can begin work on the assay procedure itself. We will examine several methods, including the hybrid capture assay originally proposed as well as an emerging technology; "molecular beacons" ( intra-molecular fluorescence quenching).


Journal Articles on this Report : 2 Displayed | Download in RIS Format

Other project views: All 43 publications 10 publications in selected types All 9 journal articles
Type Citation Project Document Sources
Journal Article Pitulle C, Dsouza L, Fox GE. A low molecular weight artificial RNA of unique size with multiple probe target regions. Systematic and Applied Microbiology 1997;20(1):133-136. R825354 (1997)
R825354 (Final)
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  • Journal Article Singh N, Willson RC. Boronate affinity adsorption of RNA: possible role of conformational changes. Journal of Chromatography A 1999;840(2):205-213. R825354 (1997)
    R825354 (1999)
    R825354 (Final)
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  • Supplemental Keywords:

    biotechnology, monitoring, soil, water, genetics, PCR., Scientific Discipline, Ecosystem Protection/Environmental Exposure & Risk, Genetics, Environmental Chemistry, Chemistry, Monitoring/Modeling, Engineering, artificial stable RNA, microbial monitoring, bacteria monitoring, DNA vector, microorganism, bioluminescence, green flourescent protein, RNA gel profile

    Progress and Final Reports:

    Original Abstract
  • 1998
  • Final Report