Conjugation-dependent "gene drives" harness indigenous bacteria for bioremediation
Summary (1 min read)
Horizontal ‘gene drives’
- Harness indigenous bacteria for bioremediation Katherine e. french1*, Zhongrui Zhou2 & norman terry1 engineering bacteria to clean-up oil spills is rapidly advancing but faces regulatory hurdles and environmental concerns.
- Current approaches to removing crude oil from the environment include chemical oxidation, soil removal, soil capping, incineration, and oil skimming (in marine contexts)7,8.
- Third, the environmental effects of engineered bacteria on native soil populations are unclear.
Results and discussion
- To compare the localization and activity of known petroleum hydrocarbon-degrading enzymes, the authors inserted five enzymes (alkB, almA, xylE, ndo, and p450cam) and required electron donors into the vector backbone pSF-OXB15 using Gibson Assembly23 (SI Fig. 1).
- OMVs are 50–250 nm in diameter45, much smaller than any of the vesicles produced by their strains.
- In their study, it is impossible to say definitively by which mechanism their vectors were transferred from E. coli DH5α to the wild-type bacteria and in reality multiple mechanisms of transfer may be possible.
- Instead, a number of diverse native soil bacteria now contained the plasmid, a trend which continued over the course of the experiment (Fig. 5).
- The authors pilot research has shown that transferring catabolic genes involved in petroleum degradation from E. coli DH5α to indigenous bacteria may be a viable solution.
- All vectors were constructed using the vector backbone pSF-OXB15 (Oxford Genetics) and the Gibson Assembly cloning method.
- The authors added 950 μl of SOC media to each Eppendorf and placed cultures into a shaking incubator set to 30 °C for one hour of vigorous shaking (250 rpm).
- Briefly, cell cultures containing the plasmids used in this study were grown overnight and 1 ml aliquots were lysed using Cellytic tablets (Sigma-Aldrich).
- To set up each assay, 96 well plates were filled with crude oil which had been complexed with Nile Red (10 ul of Nile Red per ml of crude oil).
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Frequently Asked Questions (16)
Q1. What are the contributions in "Horizontal ‘gene drives’ harness indigenous bacteria for bioremediation" ?
In this paper, the authors developed a new technology to harness indigenous soil microbial communities for bioremediation by flooding local populations with catabolic genes for petroleum hydrocarbon degradation.
Q2. What are the future works mentioned in the paper "Horizontal ‘gene drives’ harness indigenous bacteria for bioremediation" ?
Publicly available documentation and potentially even de-centralized approval of GM field trials ( e. g. through university Environmental Health and Safety offices ) could make field trials of GM bacteria more achievable in the near future. Future research is needed to determine ( 1 ) how long these plasmids are maintained under field conditions, ( 2 ) whether genetic mutations accumulate over time that might impact enzyme functioning, and ( 3 ) how vector-based gene drives harnessing natural processes of conjugation may affect local microbial community composition and soil metabolic functions.
Q3. What would be the way to eliminate the release of antibiotic resistance genes into the environment?
Replacing antibiotic selection markers with chromoprotein ones64 would eliminate the release of antibiotic resistance genes into the environment.
Q4. What is the way to use a GM organism to enhance the capacity of native soil?
E. coli DH5α engineered to carry plasmids containing genes involved in degradation of environmental toxins could be used to augment the capacity of native soil microbial communities to degrade pollutants of interest.
Q5. What are the current approaches to removing crude oil from the environment?
Current approaches to removing crude oil from the environment include chemical oxidation, soil removal, soil capping, incineration, and oil skimming (in marine contexts)7,8.
Q6. What is the way to remove the surface fraction of oil from the soil?
Practices such as skimming only remove the surface fraction of the oil while the water-soluble portion cannot be recovered, negatively affecting marine ecosystems12,13.
Q7. What is the reason for the low frequency of HGT between the donor and recipient bacteria in soil?
Previous studies show that frequencies of HGT between the donor and recipient bacteria in soil are low (e.g. 3 × 10–3 CFU transconjugants per gram of sterile soil), recipient cells come from only a few genera, and the spread of the catabolic vector through the microbial community does not always lead to enhanced degradation55–57.
Q8. What are the primary barriers to implementing this approach on current polluted industrial sites?
The primary barriers to implementing this approach on current polluted industrial sites are (1) lack of standardized procedures to test and ultimately allow the use of GM organisms for environmental applications and (2) the willingness of site managers to adopt this approach to remediation.
Q9. How did the authors determine the survival rate of engineered bacteria in contaminated soils?
To determine the survival rate of engineered bacteria in contaminated soils, the authors added E. coli containing the plasmid pSF-OXB15-p450camfusion to sediment taken from a former Shell refinery in Bay Point, CA.
Q10. How did Dubey and Ben-Yehuda show that gf?
Dubey and Ben-Yehuda52 show in their classic paper that gfp molecules, calcein, and plasmids could be transferred between B. subtilis cells.
Q11. How did the authors determine whether their engineered bacteria could transfer non-conjugated, synthetic?
To determine whether their engineered bacteria could transfer non-conjugative, synthetic vectors containing petroleum-degrading genes to indigenous soil and marine bacteria, the authors conducted a series of mating experiments.
Q12. What can be done to overcome the second barrier?
The second barrier can be overcome through public engagement with those working in the remediation sector (industry, site managers, and remediation consulting firms) and a shift in their approach to how the authors conduct remediation (favoring slower biological-based solutions that harness local ecological and chemical processes over faster processes such as oxidation and soil removal).
Q13. How did the authors determine whether the plasmids could be transferred to wild-type?
They also show that a non-integrative vector carrying a resistance marker from B. subtilis could be transferred to Staphylococcus aureus and E. coli (with recipient cells expressing antibiotic resistance).
Q14. How much dodecane did P. putida degrade in 10 days?
Solid-Phase MicroExtraction (SPME) GC/MS analysis of these cultures revealed that all three bacteria degraded 99% of dodecane in 10 days.
Q15. How did the authors determine whether HGT of the synthetic vectors to wild-type bacteria was achieved?
To determine whether HGT of the synthetic vectors to wild-type bacteria was achieved through mating, the authors conducted TEM of wild-type bacteria after exposure to E. coli DH5α carrying pSF-OXB15-p450camfusion.
Q16. What is the role of p450cam in the degradation of petroleum hydrocarbons?
Fluorescence microscopy also revealed for the first time the key role of extracellular enzymes in degradation of petroleum hydrocarbons.