Difference between revisions of "Team:Amsterdam/Project/Overview"

 
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<h2>The Problem</h2>
 
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                                                Our economy still depends largely on fossil resources. For decades, this has fueled the incredible progress of our world, but not without the massive cost of geopolitical instability and global warming - costs we’re now facing more than ever. With the current consequences of climate change visible around the world and global energy demands that are expected to double in 2050, the quest for clean, renewable energy is one of mankind’s most important modern challenges.
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                                              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.
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<h3>The Bio-economy</h3>
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<p>A bio-based economy - one which we use renewable biomass to produce the things our society needs - is often posed as a promising alternative to the burning of fossil fuels. But using sugar crops means competing with scarce arable land, while lignocellulosic biomass is still difficult to utilize efficiently. Cyanobacteria, which require mostly CO2 and light, hold great potential for sustainable production, but - compared to chemotrophs - are vexed by low productivity and often overlooked issues of genetic instability.</p>
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<p>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.
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<p>The problem is that cyanobacteria don’t like to allocate their carbon resources to biofuel molecules - they would rather use them to grow instead. Consequently, novel gene insertions quickly accumulate mutations that render them inactive, giving these mutants a growth advantage that filters out the producing strain. As a result, photobioreactor runs often face sudden declines in productivity, a massive problem when it comes to large-scale implementation of cyanobacterial bioproduction.</p>
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<p>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 <i>Escherichia coli</i> 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.
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<p >Combining the best of cyanobacterial sustainability and chemotroph productivity</p>
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<p>Rather than mimic the <i>modus operandi</i> 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 <i>E. coli</i>) to turn CO<sub>2</sub> into useful carbon product.  Such photosynthetic romance is perhaps not the ultimate solution (imagine a single organism with both the productive potential of <i>E. coli</i> 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.</p>
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<p> the idea is simple: we engineer cyanobacteria to produce simple carbon compounds using CO2 and sunlight in ways that are genetically stable. These molecules are shared with a chemotroph like <i>E. coli</i>, which uses them to produce a desired end-product, like commodity chemicals, pharmaceuticals, or biofuels.</p>
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The basic premise of our consortium’s functionality is simple: a phototroph (<i>Synechocystis</i> PCC 6803 in the case of our prototype consortium) fixates CO<sub>2</sub> and converts it to relatively simple carbon compounds. These compounds serve as fuel for a chemoheterotroph (<i>E. coli</i> in the same prototype case), which uses the compounds to produce a desired end-product. Since the CO2-fixating, carbon-sharing <i>Synechocystis</i> 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 <i>E. coli</i> for bioproduction!
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<p>In our proof-of-principle consortium, <i>E. coli</i> produces isobutanol (an important biofuel) to highlight its sustainable production potential. But our engineered <i>Synechocystis</i> strains are essentially fully modular cyanobacterial production engines, and can be coupled to any biotechnological production process to remove dependencies on crops or petrochemicals and make it truly sustainable.
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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 <i>Synechocystis</i> 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 <i>Synechocystis</i> in a way that would remain stable over time.  
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<p>The workhorse of synthetic biology, <i>E. coli</i> benefits from the availability of a well-categorized genetic toolbox, making it the ideal chemotroph candidate for our consortium. <b>We aim to engineer biofuel production in <i>E. coli</i> for use in our protoype consortium.</b> Moreover, we intend to engineer interactions with <i>Synechocystis</i> in order to create a genetic safety switch that ensures our consortium only functions when its members are together.</p>
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<p>Also known as the ‘Green <i>E. coli</i>’, <i>Synechocystis</i> is a model organism that growns on CO2 and light. We want to engineer <i>Synechocystis</i> to produce and share carbon compounds in ways that are genetically stable by coupling this production to its own growth. This essentially results in a modular phototrophic production egine. By then connecting <i>Synechocystis</i> to <i>E. coli</i> in our consortium, the latter would receive a continuous supply of sustainably-produced compounds required to produce virtually any desired end-product.</p>
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<p>Such an endeavour requires several tasks. The two organisms have to be genetically modified to synthesize the desired molecules, preferably in an stable fashion. We also need to characterize how these new activities influence the physiological parameters of each bacteria, i.e. how fast they grow, what is the production rate, how stable are these rates, etc. These parameters can be used to build models that allow us to understand the dynamics of the system. Last but not least, the production in the resulting consortium has to be compared with other consortia.</p>
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<p>Our modelling efforts will generate simulations of consortium development and will allow us to assess and use optimal organism ratios for the production of a desired end-product. Moreover, we intend to leverage genome-scale metabolic models to create a suite of open-source software tools that can be used to construct genetically stable and safe synthetic consortia in which a phototrophic species functions as the carbon provider for a product-producing chemotroph.
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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: </p>    
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<p><i>Synechocystis</i> and <i>E. coli</i> work well together, but are far from the only possible members of a synthetic consortium. Yeast, for instance, could be of great interest to the biotech community when coupled to our cyanobacterial production module. Alongside our main proof-of-principle consortium, we will therefore develop a micro-droplet screening protocol to rapidly assess the viability of novel consortia. This would greatly reduce trial-and-error time in the lab and allow users to identify consortia that serve their biotech requirements.
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<li>On the one hand, we’ve used models and simulations to engineer a rationally-designed, proof-of-principle consortium in which a phototroph fixates CO<sub>2</sub> 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 <i>Synechocystis</i> and <i>E. coli</i>. At the same time, algorithms based on the <i>Synechocystis</i> genome-scale metabolic map informed us on how to implement stable carbon production, which guided our engineering efforts. Together with an engineered <i>E. coli</i>-dependency in <i>Synechocystis</i>, 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.
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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.  
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Using the isobutanol pathway biobrick made by <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Results/Pathway">Bielefeld’s iGEM team</a> 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?</p>
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<h4 align="center"><a href = "https://2015.igem.org/Team:Amsterdam/Project/Eng_rom"> ENGINEERING  ROMANCE</a></h4>
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<h4><a href = "https://2015.igem.org/Team:Amsterdam/Project/Stability"> stable ROMANCE</a></h4>
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Latest revision as of 12:02, 6 October 2015

iGEM Amsterdam 2015

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.

Consortium

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:

  1. 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.
  2. 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?

The project

ENGINEERING ROMANCE

Simulating ROMANCE

stable ROMANCE

TESTING ROMANCE

evolving ROMANCE