Team:DeNovocastrians/Description
Inspiration
"MV Wakashio oil spill in Mauritius”, by International Maritime Organization, licensed under CC BY 2.0.
Environmental pollution is one of the major threats facing the world today (UN, 2018). Pollution has an impact on a range of ecosystems, and, the interconnectivity of ecosystems. Pollution is an inherent by-product of human activities, and disasters such as oil spills can have devastating effects. Recently, a crisis unfolded in the waters surrounding Mauritius. A ship filled with petrochemicals (chemicals derived from petroleum or natural gas) ran aground and leaked its fuel into the ocean, devastating sea life and causing catastrophic harm to the nearby reef ecosystem (ABC, 2020). The impact pollution has on nature can be seen on a world-scale but also at the local level. Ensuring proper management of our local environments can have a beneficial flow-on effect on the global ecosystem.
Our city of Newcastle in Australia has a strong industrial history, particularly for its coal mining and export. It has been found that our soils in Newcastle are significantly contaminated with heavy metals and hydrocarbons, these having a long-lasting impact on the people living here (Harvey et al., 2017). However, the city of Newcastle is also committed to protecting and restoring the environment. Numerous environmental management projects and legislations are in place to reduce the impact we have on the environment. Innovation and initiative for solving these important issues is needed for progress. For this reason, our goals for the 2020 iGEM competition were focused around an environmental aspiration.
A common pollutant of the industrial sector is the chemically-stable hydrocarbon, benzene. Benzene is a known carcinogen, found inside petrochemicals, industrial intermediates and consumer products (Liu et al., 2020). When oil spills occur, as we saw in Mauritius, benzene can leach into the environment and contaminate ecosystems. Cars and petrol stations are some of the largest sources of benzene in local environments, with residents living close to petrol stations being exposed to higher levels of benzene. Living in an industrial city, the exposure of benzene and other petrochemicals also increases. Benzene exposure is known to cause diseases such as non-Hodgkin’s lymphoma, leukaemia and multiple myeloma (W.H.O, 2010). As such, the management and removal of pollutants such as benzene is of international importance.
Aims
This project was designed with the aim of engineering microorganisms to efficiently detect, import and degrade the petrochemical benzene in the natural environment. To achieve this, it involved creating a benzene biosensor and the cloning of the benABCDE gene cluster into E. coli.
Project
benE Transporter
In order to degrade benzene, cells must have the ability to import it in the cell. Our project cloned the benzene transporter encoded by the benE gene, co-localised with the benABCD cluster. This transporter allows bacteria to import extracellular benzene/benzoate. We cloned the benE gene from a number of bacterial species and introduced them into the laboratory strain of E. coli. Functional assays were performed using gene knockouts of the benE gene as well as the benK gene also thought to be responsible for benzene/benzoate transportation.
benABCD Cluster
The degradation of benzene is encoded by the benABCD gene cluster and is a multi-step process that degrades benzene into catechol. The benAB genes form a complex encoding a benzoate dioxygenase, together with benC (encoding a reductase) converts the benzoate into benzoate diol. Next, benD (which encodes a dehydrogenase) will use benzoate diol and convert it into catechol (W Kitagawa 1 2001). Though there is some variation in the intermediary steps between species the process ultimately results in the conversion of benzene to catechol. Catechol can be degraded into acetyl-CoA and succinyl-CoA, which is fed into the citric acid cycle to be used for ATP production or formation of essential biomolecules. Several species of the genus Rhodococcus and Acinetobacter encode the benABCD cluster (Révész et al., 2019). By cloning the benABCD pathway from the Rhodococcus and Acinetobacter species we have identified, the cluster can be shuttled (via a plasmid vector) to transform the commonly used and model laboratory species Escherichia coli into a practically useful benzene degrader.
Microbial growth curves are used by scientists to study the differential effects of a given media, population genetics, and the effect of stressors on the growth of a microbial population (Tonner et al., 2017). We constructed growth curves to observe the pre-benABCDE insertion growth characteristics of different bacterial species in control media (Luria Broth and Mueller-Hinton Broth) and M9 minimal media added with benzene derivatives (Benzoate, Catechol) as a sole carbon source. Following insertion of our benABCDE gene cluster, bacterial growth curves were constructed using the control media and M9 media added with benzoate/catechol as sole carbon sources. The creation of these growth curves pre- and post- introduction of benzene degradation gene clusters allowed us to quantify the capability of our engineered microorganisms to degrade the hydrocarbon derivatives in the laboratory, acting as proxy for the environmental degradation of benzene.
Benzene Biosensor
We created a biosensor to detect and measure environmental levels of benzene and catechol. Biosensors are an affordable, easy and sustainable way of detecting chemicals within a system. The biosensor we created uses transcription factors found upstream of the benABCD cluster and the catechol degradation cluster that recognize benzene and catechol respectively from Acinetobacter baylyi ADP1. These transcription factors were moved into a biosensor containing green fluorescent protein (GFP) and mCherry genes downstream of each of the two transcription factors. By utilising fluorescent proteins, we can visualise the fluorescent signal produced to show the amount of benzene prevalent in the environment, in addition, monitor the effectiveness of our engineered bacteria to degrade benzene.
Future Applications
Making a cell line capable of degrading benzene and other petrochemicals will be of huge benefit to society. Currently, cleaning up oil spills requires significant manpower and is very costly (Jernelöv, 2010). However, with our system, it will be possible to use the resources provided by nature. By releasing a pollutant-degrading microorganism out into a polluted area and the petrochemicals will be degraded. The data we have collected from the benzoate degradation and transporter assays can be used to further optimise degradation for bioremediation of local environments. These experiments help lay the foundations for the degradation of more complex poly-aromatic hydrocarbons.
However, before our engineered organisms can be released, ethical considerations need to be considered. The work done here can be further built upon to engineer benzene degrading organisms, for example, to contain a suicide switch whereby the microbes will die when all the benzene in the system has been degraded.
The biosensor could be easily and affordably used to assess an the levels of benzene pollution in an environment. The biosensor can be used for future teams to assess kinetics and collect data to further optimise benzene degradation genes from different organisms.
References
ABC news press. Ship leaking tons of oil off Mauritius has split apart. ABC news .
Ahamad, A., Pal, B.D., Ti-Wary, D., Mirsha K., Singh, P., Sing, V.K., Singh, R., Borhakur, A., Madhav, A. and Kumar, J. (2019). Abatement of Environmental Pollutants, 1-23.
Ernelov, A. (2010). The treats from oil spills: Now, Then, and in the Future. Ambio. 183, 56.
Harvey, P. J., Rouillon, M., Dong, C., Ettler, V., Handley, H. K., Taylor, M. P., Tyson, E., Tennant, P., Telfer, V. and Trinh, R. (2017). Geochemical sources, forms and phases of soil contamination in an industrial city. Sci. Total Environ. 584-585, 505-514.
IARC. A review of human carcinogens. F. Chemical agents and related occupations. Lyon, International Agency for Research on Cancer. A review of human carcinogens—Part F: Chemical agents and related occupations. The Lancet Oncology, 10(12) ,1143-114.
Kitagawa, W., Miyauchi, K., Masai, E. and Fukuda, M. . (2001). Cloning and characterization of benzoate catabolic genes in the gram-positive polychlorinated biphenyl degrader Rhodococcus sp. strain RHA1. J Bacteriol. 183 598-6606.
Liu, C., Huang, X. and Li, J. (2020). Outdoor benzene highly impacts indoor concentrations globally. Total Environ 720137640.
National research council Mexico (2013). An Ecosystem Services Approach to Assessing the Impacts of the Deepwater Horizon Oil Spill in the Gulf of. 2013. "An Ecosystem Services Approach to Assessing the Impacts of the Deepwater Horizon Oil Spill in the Gulf of Mexico." National Academies Press, 71 71-101.
Révész, F., Figueroa-Gonzalez, P.A., Probst, A.J., Kriszt, B., Banerjee, S., Szoboszlay, S., Maróti, G. and Táncsics, A. (2019). Microaerobic conditions caused the overwhelming dominance of Acinetobacter spp. and the marginalization of Rhodococcus spp. in diesel fuel/crude oil mixture-amended enrichment cultures. Archives of microbiology. 202, 329-342.
Tonner, P. D., Darnell, C. L., Engel, B. E. and Schmid, A. K. (2017). Detecting differential growth of microbial populations with Gaussian process regression. Genome Res. 27(2), 320-333.
W.H.O. (2010). https://www.who.int/ipcs/features/benzene.pdf . Geneva,
World Health Organization. 1-3.