LIGHTNING-ASSISTED ENVIRONMENTAL GENETIC THERAPY
Résumé
Remediation of polluted environmental media presents an ongoing challenge, with each contaminated site requiring a thorough description to choose an appropriate remediation technology. One popular remediation technique is monitored natural attenuation (MNA), which relies on the ability of indigenous microbes to degrade or sequester contaminants. However, if the indigenous population lacks the biochemical makeup or genes to tackle the pollutants, which is common with xenobiotic compounds, then other - often more costly - remediation efforts are necessary. In mammals, the idea of gene therapy involves the introduction of a gene into cells to correct a disease state resulting from improper gene function. Our research proposes using the same idea to augment the function of a soil community via gene delivery to facilitate degradation of a contaminant. Previous research has successfully used microbes containing degradation genes to inoculate indigenous microbes via horizontal gene transfer. However, direct addition of the genes using naked DNA in lieu of a microbial vector might make the process more efficient and circumvent the issue of adding genetically modified organisms (GMOs) to the existing community. In this research, we added a lindane-degrading gene (linA) in a broad-host range plasmid, resulting in the plasmid pBLN, to a microbial soil community that lacked the ability to degrade lindane. We attempted to increase the rate of transformation by electroporation in liquid media or application of simulated lightning in a soil medium. In the first round of experiments, we extracted bacteria from soil, mixed the bacteria with pBLN, and electroporated the liquid mixture. We then incubated the electroporated cells in a liquid medium with lindane, and monitored lindane degradation via an increase in chloride concentration. Soil microbes, either electroporated with pBLN or simply incubated for a 2 hour period with pBLN, displayed an increased ability to degrade lindane as compared to a negative control. The presence of linA in the samples with increased degradation was verified using PCR. In the second phase of this work, the bacterial transformation in a soil matrix, using a lightning generator to provide the electric shock to the soil bacteria in situ, simulates a potential mechanism for site treatment. This research demonstrates the feasibility of environmental gene therapy to remediate xenobiotic-contaminated sites.