Grantee Research Project Results
A comprehensive methodology to track genetically recoded organisms and assess their impacts on freshwater microbial communities
EPA Grant Number: R840203Title: A comprehensive methodology to track genetically recoded organisms and assess their impacts on freshwater microbial communities
Investigators: Konstantinidis, Konstantinos (Kostas) T. , Hatt, Janet K , Vereen, Ethell
Institution: Georgia Institute of Technology - Main Campus (School of Civil & Environmental Engineering)
Current Institution: Georgia Institute of Technology - Main Campus (School of Civil & Environmental Engineering) , Morehouse College
EPA Project Officer: Callan, Richard
Project Period: July 1, 2021 through June 30, 2024
Project Amount: $759,980
RFA: Assessment Tools for Biotechnology Products (2020) RFA Text | Recipients Lists
Research Category: Chemical Safety for Sustainability , Safer Chemicals
Description:
Nucleic acid based approaches for identifying and tracking microbes through the environment, including those that are products of synthetic biology (synbio), promise high levels of sensitivity and throughput. Further, these approaches are well suited to assess the ecological effects of synbio organisms on the natural microbial communities, including whether or not such organisms are capable of passing their genes to indigenous populations. However, nucleic acid based approaches are not without challenges. For instance, while several bioinformatics methods have recently been proposed to determine in situ abundance of a target organism or gene and determine presence or absence (i.e., limit of detection), these methods have not yet been standardized, and it is not clear how the data from different approaches can be compared. Further, detecting a target organism in a background of a complex microbial community such as those that are typically found in environmental samples remains challenging because the frequency of false-positives remains essentially unknown. False positive signal may result from inadequate identification of regions of the genome that are either too highly conserved to be diagnostic (e.g., rRNA genes), or prone to frequent horizontal genetic exchange (e.g., mobile elements). To overcome the false-positive signal, a large library of sequence data representing different pollution inputs (e.g., sewage vs. animal feces vs. synbio organisms) need to be built and curated. Such a library will, in addition, help to advance the highly-related field of Microbial Source Tracking (or MST) (i.e., identification of the sources of pollution), which is central to the mission of environmental compliance agencies.
Objective:
- Perform dialysis bag incubations with a synthetic E. coli expressing recoded enzymes vs. E. coli expressing the wildtype proteins in order to assess the effects of the former on the natural microbial community relative to the latter.
- Develop biomarkers representing different sources of pollution and synbio organisms by examining large metagenomic/genomic libraries from around the world.
- Make the underlying gene and genome biomarkers searchable online as part of a “MST webserver” to help users identify the source(s) of pollution in their samples and presence of synbio organisms by simply uploading their metagenomic dataset(s) to the webserver.
- Using the best performing biomarkers identified under #2 above, to quantify the relative contributions of different the sources of pollution of impaired creeks in the Atlanta metro area.
The overarching hypothesis is that the modified E. coli will not pass its functional genes to indigenous microbes due to inability of these genes to function in non-modified cells.
Approach:
We will address our objectives by using well-controlled laboratory incubations spiked with synbio organisms, and a comprehensive bioinformatics analysis of the resulting, time-series metagenomic samples from the incubations that will identify novel synbio- and MST-specific biomarkers and the effect of the spiked organisms on the autochthonous microbial communities in the inocula. The biomarkers will be validated against a plethora of available deep-coverage metagenomic datasets in the public database as well as frequent sampling of our field sites.
Expected Results:
Outputs: i) An advanced, dialysis-bag based system to study the fate of synbio organisms and their effects on indigenous microbes; ii) a comprehensive wet-lab methodology and associated bioinformatics pipeline to assess the relative in situ abundance of target organism(s) in environmental samples; iii) new, robust biomarkers of different sources of pollution (e.g., animal vs. human feces vs. sewage vs. synbio organisms); iv) a “MST webserver” to allow identification of the sources of pollution and their relative importance based on sequence datasets.
Outcomes: i) An improved understanding of the ecological effects of synbio organisms on indigenous freshwater microbial communities; ii) easy-to-use, online tools for evaluation and monitoring of the risk to human health and the environment from the use of synbio organisms.
Publications and Presentations:
Publications have been submitted on this project: View all 4 publications for this projectJournal Articles:
Journal Articles have been submitted on this project: View all 4 journal articles for this projectSupplemental Keywords:
recoded genomes, amber mutations, genome detection, bioinformatics, metagenomics.Progress and Final Reports:
The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.