Performing Department
(N/A)
Non Technical Summary
Recently, this team experimentally demonstrated, for the first time, that horizontal gene transfer (HGT) events can indeed occur from genetically engineered (GE) cyanobacteria to a common laboratory strain of E. coli. The next important step is to assess whether HGT could occur from GE cyanobacteria to other microorganisms including soil-environmental related bacteria such as Serratia marcescens, Pseudomonas putida, Bacillus licheniformis and Risk Group-2 organisms, such as Staphylococcus aureus, Campylobacter jejuni, Salmonella Sp, and Pseudomonas aeruginosa, which are widespread in nature and could pose a serious health risk to humans and animals if they were to obtain antibiotic resistance through HGT from GE organisms. Therefore, in the proposed project, this team will measure the rates of HGT from GE cyanobacteria to soil-environmental bacteria/Risk Group-2 microorganisms. The team will also characterize and elucidate the HGT mechanisms, such as to whether the HGT is through a direct cell-to-cell interaction or by an indirect route of cell-to-liquid environment (eDNA)-to-cell process, using a newly developed protocol for measuring HGT rates from GE algae to non-photosynthetic organisms. The team will further assess the fate/stability of the transgenes and the survivability of GE cyanobacteria in comparison with their related wild-type organisms. The team will achieve experimental assessment on the survivability and HGT of GE cyanobacteria with the native microorganisms in soil-pond environmental water samples. The proposed project will directly support the Biotechnology Risk Assessment Research Grants (BRAG) Program in the program area "3. Gene Transfer between Genetically Engineered Animals, Plants, and Microorganisms and Related Wild and Agricultural Organisms." This project will yield quantitative rates of HGT from GE cyanobacteria to soil-environmental bacteria/Risk Group-2 microorganisms and better understanding of possible HGT routes that will be highly valuable to the federal regulatory agencies related to biotechnology to make scientific evidence-based policy.
Animal Health Component
20%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
0%
Goals / Objectives
The novel protocol to measure HGT from algae to non-photosynthetic organisms illustrated in Figure 1, with Eq. (1) for the HGT rates [3], will be used to quantitively measure the rates of HGT from GE cyanobacteria to soil-environmental bacteria (Objective 1). It will also be used to characterize the mechanisms whether the HGT is through a direct cell-to-cell interaction or by an indirect cell-to-liquid environment (eDNA)-to-cell process (Objective 2). In addition, the stability of the transgenes and the survivability of GE cyanobacteria in comparison with their related wild-type organisms will be assessed (Objective 3).
Project Methods
The ability for foreign genes, especially promoters and genes that confer antibiotic resistance to horizontally transfer to other microorganisms, poses a bio-risk to animals and humans. This project willextends the mothods of the previous USDA BRAG project work done with a typical laboratory E. coli strain DH5α to more relevant natural bacterial strains found in the soil environment as well as strains that could compromise animal and human welfare if they acquire antibiotic resistance. Through use of our demonstrated protocol [1, 3] with the HGT frequency calculation (Eq. 1), this project will yield quantitative data on the rates of HGT from GE cyanobacteria to soil-environmental bacteria and better understanding on the mechanisms of HGT on the question of whether the HGT is through a direct cell-to-cell interaction and/or by an indirect route of cell-to-liquid environment (eDNA)-to-cell process. The experimental methods and results of thisproject will be highly valuable to the biotechnology federal regulatory agencies to make scientific evidence-based policy. For examples, if the study confirms that the rate of HGT from the genomic-integrated GE cyanobacteria is much slower than that from plasmid-based GE cyanobacteria, regulatory agencies may encourage the use of genomic-integrated GE cyanobacteria as a preferable practice in the developing field for photobiological production of advanced biofuels and bioproducts. If it is shown that HGT requires a direct cell-to-cell interaction, regulatory agencies and the USDA BRAG program may consider developing a biocontainment strategy, such as using the CRISPR/Cas9 technique to knockout potential cell-to-cell mating genes responsible for HGT to limit its potential biosafety risks. If the project results show that HGT can occur through an indirect route of cell-to-liquid environment (eDNA)-to-cell process, regulatory agencies could consider the potential biosafety risk of the "environmental DNA (eDNA)"in relation to HGT. There are several potential sources of eDNA to be indirectly transferred to soil-environmental bacteria, such as compost of GE crop plants and manures of farmed animals having GE forages.