Team:Amsterdam/Project/Overview
PROJECT OVERVIEW
Engineering romantic relationships to save the world
The Problem
The quest for sustainability
The grand problem
Oil. The most hated and loved liquid in mankind’s history. The sine qua non in all of the world’s remarkable progress - culturally, scientifically, economically - of the past hundred years. The black gold that still drives prosperity as we stand on the verge of drilling into arctic icesheets. But in contrast to the oil crazes that raged in the deserts of Texas whenever oil was struck, the fervor for drilling has turned into fear. It has become clear that there’s a cost to fossil fuels - a global tragedy of the commons that is finally bearing fruit. Climate change, geopolitical instability; they’re symptoms of an energy addiction that is here to stay. Without doubt then, the quest for clean, renewable energy is one of mankind’s most important and urgent modern challenges. And a challenge worthy of an iGEM project.
The ideal world
In a perfect universe, we would fulfil our carbon cravings by using atmospheric CO2 instead of drilling fossilised biomass from the earth. An eye for an eye approach to burning fuels that would prevent the buildup of greenhouse gasses. A bio-based economy - one which we use renewable, CO2-consuming biomass to produce the materials and fuels our societies depend upon - is the perfect description of exactly such system. But past approaches, using sugar crops that compete with arable land or lignocellulosic biomass that is often as difficult to digest for microbes as a car tire is for humans, have not fulfilled their promise. If the bio-based economy is to become more than just a distant dream, these obstacles must be overcome.
The green promise
Cyanobacteria, photoauxotrophic organisms that use CO2 and light to grow and that do not require arable land, may be the ideal green engines. But cyanobacteria are - compared to chemotrophs - vexed by low productivity and still limited in the range of products they can make: we simply do not have a similarly extensive metabolic engineering toolbox available for cyanobacteria that we do possess and exploit to turn organisms like Escherichia coli into high-yield cell factories. What we need is the ability to align the sustainability potential of cyanobacteria with the productive capacities of commonly used chemoheterotrophs, in a way that can be implemented with technologies that are available now.
Our solution
Combining the best of cyanobacterial sustainability and chemotroph productivity
Rather than mimic the modus operandi of the biotech industry to do so - focusing on using a single species in isolation to achieve a goal - we decided to mimic nature instead: to let microorganisms work together in a multi-species ecosystem - a synthetic consortium - and develop relationships that could make sustainable bioproduction a reality. Specifically, we aimed to engineer relationships between photoauxotrophic cyanobacteria and chemoheterotrophs (like E. coli) to turn CO2 into useful carbon product. Such photosynthetic romance is perhaps not the ultimate solution (imagine a single organism with both the productive potential of E. coli and the photosynthetic capacity of cyanobacteria), but as of yet, it may be the most feasible and effective way to couple sustainability to currently available bioproduction processes.
The basic premise of our consortium’s functionality is simple: a phototroph (Synechocystis PCC 6803 in the case of our prototype consortium) fixates CO2 and converts it to relatively simple carbon compounds. These compounds serve as fuel for a chemoheterotroph (E. coli in the same prototype case), which uses the compounds to produce a desired end-product. Since the CO2-fixating, carbon-sharing Synechocystis is essentially modular, it can be coupled to a multitude of biotechnological production processes to make these processes sustainable, including the dozens of iGEM projects that use E. coli for bioproduction!
Our approach
Much like in real life though, developing productive and loving relationships between organisms can be a tough nut to crack - a fact we were confronted with early on and that defined the focus of our project. At the start of our efforts, we noticed that our first carbon-sharing Synechocystis strains (producing lactate), stopped sharing after only a brief period of time - hardly a foundation for a thriving relationship. What we demonstrated was the importance of genetic stability: the need to engineer carbon production in Synechocystis in a way that would remain stable over time.
Determined not to allow the relationship that would hold our consortium together to fail or flourish according to the unpredictable nature of real-world love, we thus proceeded by grounding our desired photosynthetic romance in theory, developing an approach with experimental components driven by simulations and models. In short, our approach consists of two parallel strategies for designing microbial consortia:
- On the one hand, we’ve used models and simulations to engineer a rationally-designed, proof-of-principle consortium in which a phototroph fixates CO2 to drive, in a genetically stable way, product formation by a chemotroph. To run simulations that would reveal optimal organism ratios physiological parameters were obtained through physiology experiments for both Synechocystis and E. coli. At the same time, algorithms based on the Synechocystis genome-scale metabolic map informed us on how to implement stable carbon production, which guided our engineering efforts. Together with an engineered E. coli-dependency in Synechocystis, these interactions governed the way the members of our rationally-designed consortium would live together. This approach of engineering synthetic romance is much like an arranged marriage, albeit with slightly higher odds of love at first sight.
- We also developed a way of evolving romance more naturally, however, by creating an emulsion-based droplet protocol that can be used to continuously select for the co-culture with the most efficient metabolic relationship. We present this as a tool that can be used both to optimize the rationally-designed synthetic consortia, as well as to discover novel consortia from the diversity available in nature.
Using the isobutanol pathway biobrick made by Bielefeld’s iGEM team last year and a consortium with , we ultimately tested the viability of our approach both in terms of growth and product formation, putting our synthetic relationship to the ultimate test: would engineered microbial love last happily ever after while we could enjoy its fruits, or would cheaters remiss and ruin the romance?