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Team:ULB-Brussels/Introduction
From 2010.igem.org
Introduction
In this ever more energy-dependent world, where fossil fuel resources become scarce and raise environmental issues, the search for green energy sources is a growing concern in both civil and scientific communities. In this context, hydrogen turns out to be an interesting alternative. Indeed, hydrogen fuel cells do not affect the carbon footprint since their only side effect is a production of water. Although the hydrogen is the most abundant element on the planet, it is quite difficult to produce. Current hydrogen production relies mostly on chemical processes, such as petroleum cracking or water electrolysis, which require a lot of energy.
In order to develop greener and more energy-efficient processes, the use of micro-organisms as biocatalysts for hydrogen production has been studied for many years. A major concern for this approach is the use of dark fermentation, which attains very low yields, compared to other fermentative biofuel synthesis, e.g. methane or ethanol. However, while no industrial application has yet been achieved, scientific and technological advances allow further developments and opportunities in this field.
Turning wastewaters to substrate is convenient in several ways: wastewater treatment plants exist almost everywhere and E. coli is readily available for synthetic biology.
We propose to design a genetically engineered E. coli, Hydrocoli, with an improved natural hydrogen production pathway, using the organic compounds found in wastewaters as substrate. In addition, we planned to implement various features to enable Hydrocoli to perform other tasks related to wastewater treatment, such as signaling metallic contamination, eliminating nitrogen compounds, or hindering hydrogen consumption by methanogenic bacteria. We also plan to set up a programmed death module in order to prevent Hydrocoli proliferation outside the wastewater treatment plant.
This approach may result into a reduction of sludge which otherwise would need to be treated. Besides that, the use of genetically modified organism in wastewater treatment has never been achieved and could potentially increase the efficiency of the process. It could also show people that genetically modified organisms are tools able to improve our environment that should be considered.
The ethical and economical issues of our project and of synthetic biology in general will also be discussed.
In order to tackle this ambitious project in a relatively short time, we have decided to divide our approach in several modules:
- Hydrogen production
- Homologous recombination
- Bacteria programmed death module
- Detection of metal contamination
For each module, we developed to a computerized model in order to analyze the kinetics of reactions prior to the experiments.
Hydrogen production:
The main goal here is to direct the carbon flux through specific pathways, namely the mixed acid fermentation since this pathway leads to hydrogen production. In order to limit the flow through unwanted reactions, we plan to eliminate the enzyme catalyzing key reactions by deleting the corresponding genes. These genes are ldhA, ppc and focA.
E. coli also contains 2 hydrogenases, Hyab and Hybc, which might use the newly produced hydrogen. These two enzymes must obviously be inactivated.
We also plan to over-express several genes that are involved in enhancing the mixed acid fermentation: FNR, which is a global regulator of anaerobic growth, TdcE and PflB, coding for the two pyruvate formate lyase, which catalyze the transformation of pyruvate into formate a key reaction in mixed acid fermentation.
Homologous recombination:
In order to increase the production rate of hydrogen in our Hydrocoli, we plan to delete several genes. We plan to construct these deletions using a method based on the λ phage Red system developed by Datsenko and Wanner (2000).
We plan to develop homologous recombination tools that will be useful to the iGEM community The homologous recombination module focuses on the development of tools that would enable the deletion of any gene on known sequence, using the method mentioned above.
Quorum addiction module:
To avoid escape of Hydrocoli outside the wastewater treatment plant (or wherever they are confined), we plan to implement a system of quorum addiction. The system is composed of a toxin gene, parE, and its cognate antitoxin, parD. Expression of these genes is under the control of quorum sensing-dependent promoters. In high population density conditions (i.e. in the wastewater treatment plant), Hydrocoli is alive due to the ParD antitoxin production. If Hydrocoli escapes, the population density will drastically drop. These conditions favor the toxin expression and therefore, Hydrocoli will die.
In addition, we thought of possible improvements for Hydrocoli.
Since we considered its use in wastewater treatment, we wanted to equip it with a copper detection system, enabling a change of color of the bacteria when exposed to copper.
In the same perspective, we planned to implement a denitrification module in Hydrocoli. This would be very helpful, since this process is a very important step in wastewaters treatment.
Also, methanogenic bacteria are often found in wastewater treatment plant, and those are able to use hydrogen to form methane, which would obviously decrease the global hydrogen production. To avoid that, we thought of equipping Hydrocoli with adequate molecules to avoid the proliferation of those organism, such as bacteriocins. Bovicin HC5 from Streptococcus Bovis seemed an appropriated choice, since it has been shown to substantially decrease methane production in specific context (when mixed with rumenal bacteria). [1, 2]
[1] Mantovani et al - Bovicin HC5, a Lantibiotic Produced by Streptococcus bovis HC5,
Catalyzes the Efflux of Intracellular Potassium but Not ATP - Antimicrobial agents and chemotherapy - June 2008, p. 2247–2249 Vol. 52, No. 6
[2] Mantovani et Russel - Bovicin HC5, a bacteriocin from Streptococcus bovis HC5 - Microbiology - 2002, 148, 3347–3352
