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Biology subjects

French, K. E.

Publications and source records attributed to French, K. E..

3 recordsLinked to original sources

Conjugation-dependent \"gene drives\" harness indigenous bacteria for bioremediation

Engineering bacteria to clean-up oil spills is rapidly advancing but faces regulatory hurdles and environmental concerns. Here, we develop a new technology to harness indigenous soil microbial communities for bioremediation by flooding local populations with catabolic genes for petroleum hydrocarbon degradation. Overexpressing three enzymes (almA, xylE, p450cam) in E.coli led to degradation rates of 60-99% of target hydrocarbon substrates. Mating experiments, fluorescence microscopy and TEM revealed indigenous bacteria could obtain these vectors from E.coli through several mechanisms of horizontal gene transfer (HGT), including conjugation and cytoplasmic exchange through nanotubes. Inoculating petroleum-polluted sediments with E.coli carrying the vector pSF-OXB15-p450camfusion showed that the E.coli die after five days but a variety of bacteria received and carried the vector for over 60 days after inoculation. Within 60 days, the total petroleum hydrocarbon content of the polluted soil was reduced by 46%. Pilot experiments show that vectors only persist in indigenous populations when "useful," disappearing when this carbon source is removed. This approach to remediation could prime indigenous bacteria for degrading pollutants while providing minimal ecosystem disturbance.

synthetic biology↗

Nanoparticle-mediated transformation expands horizon of organism engineering

Our ability to engineer organisms will unlock new discoveries in how cellular processes work and enable new advances in biotechnology. However, many organisms are recalcitrant to traditional transformation methods. Here, we describe a new nanoparticle-based method of transformation which can be used to transform bacteria, plants, algae and diatoms.

synthetic biology↗

A high-throughput fluorescence-based assay for the detection of petroleum hydrocarbon degrading bacteria

Over the past 100 years, oil spills and long-term waste deposition from oil refineries have significantly polluted the environment. These contaminants have widespread negative effects on human health and ecosystem functioning. Natural attenuation of long chain and polyaromatic hydrocarbons is slow and often incomplete. Bioaugmentation of polluted soils with indigenous bacteria that naturally consume petroleum hydrocarbons could speed up this process. However, the characterization of bacterial crude oil degradation efficiency--which often relies upon expensive, highly specialized gas-chromatography mass spectrometry analyses--can present a substantial bottleneck in developing and implementing these bioremediation strategies. Here, we develop a low-cost, rapid, high-throughput fluorescence-based assay for identifying wild-type bacteria that degrade crude oil using the dye Nile Red. We show that Nile Red fluoresces when in contact with crude oil and developed a robust linear model to calculate crude oil content in liquid cell cultures based on fluorescence intensity (FI). To test whether this assay could identify bacteria with enhanced metabolic capacities to break down crude oil, we screened bacteria isolated from a former Shell Oil refinery in Bay Point, CA and identified one strain (Cupriavidus sp. OPK) with superior crude oil depletion efficiencies (up to 83%) in only three days. We further illustrate that this assay can be combined with fluorescence microscopy to study how bacteria interact with crude oil and the strategies they use to degrade this complex substance. We show for the first time that bacteria use three key strategies for degrading crude oil: biofilm formation, direct adherence to oil droplets, and vesicle encapsulation of oil. We propose that the quantitative and qualitative data from this assay can be used to develop new bioremediation strategies based on bioaugmentation and/or biomimetic materials that imitate the natural ability of bacteria to degrade crude oil.

microbiology↗