Team:Stony Brook/Description

Description

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The Problem

Humans have been pumping carbon dioxide into the air at an increasing rate ever since the coal-powered steam engine was invented in the 1750s [13]. In 2017 alone, humans released ~32.5 gigatons of carbon dioxide into the atmosphere [12]. The increased rate of carbon dioxide entering our atmosphere (ranging in the gigatons per year) has led to a number of environmental effects that have direct impacts on human health and safety [13].

Figure 1. Greenhouse gases in the atmosphere [13]

Introducing Climate Change and Ocean Acidification

The increasing levels of carbon dioxide have numerous detrimental effects on the environment. Firstly, by trapping heat causes increases in global temperatures. These temperatures then power more intense weather events and melt ice reserves. Secondly, the ocean absorbs some of the excess carbon dioxide, leading to increasing acidity. Specifically, humans have caused oceans to become 30% more acidic since the Industrial Revolution [17], damaging coral reefs and other marine habitats in the process.

Unfortunately, the rate at which carbon dioxide is pumped into the atmosphere by human pollution far exceeds the rate that it is absorbed by plants and other carbon sinks (natural sources that capture carbon dioxide), as evidenced by increasing levels of carbon dioxide in the atmosphere [14]. According to many climate models, climate change is already irreversible [15]. A large percentage of the carbon dioxide that we are pumping into the atmosphere will likely remain for millenia, continuously trapping heat, unless we figure out a way to capture emissions faster than we produce them.

Current Solutions Just Not Good Enough

Even if humankind stops emitting carbon dioxide today and uses only renewable energy, the effects of carbon dioxide will still exist for thousands of years.

Currently, the best carbon capture technology costs $94 to $232 per ton of carbon dioxide captured [11]. In order for the world to go carbon neutral (not even carbon sink), ~37 billion tons of carbon dioxide would need to be sunk each year [12]. The cost of sinking that much carbon (at a modest estimate) would cost ~3.5 trillion dollars per year. Unless a major and highly coordinated global government action is taken, it will never be economically feasible to stop, much less reverse the effects of global warming.

The cost per ton of carbon dioxide captured must more become economically feasible or mass carbon capture will never happen.

Looking to the Past

Luckily for us, nearly 2.4 billion years ago, cyanobacteria, bacteria capable of photosynthesis, changed the composition of the atmosphere from containing large amounts of carbon dioxide and virtually no oxygen, to being nearly as oxygen rich as our atmosphere is today [16]. This event is known as the “Great Oxidation Event.”

Cyanobacteria are one of the only organisms that have a historical precedent for changing the entire world’s climate and atmospheric composition. When trying to solve our carbon dioxide problems today, it makes sense to look back on nature’s example. Therefore, for our project, we chose to genetically engineer a well-studied and moderately fast-growing strain of cyanobacteria, Synechococcus elongatus PCC 7942 (S. elongatus), for the purpose of carbon capture. Specifically, we will engineer our cyanobacteria to efficiently produce and secrete a sugar called sucrose, which can be used in industrial fermentation to produce certain plastics and ethanol biofuel. By using cyanobacteria-derived sucrose as opposed to corn-derived sucrose, food prices for developing nations are not driven up, and unsustainable amounts of fertilizers and fossil fuels are not required [1]. By using cyanobacteria-derived sucrose over sugarcane-derived sucrose, deforestation and disruption of the natural ecosystems in the Amazon rainforest are reduced [2].

Photosynthetic cyanobacteria strains such as S. elongatus can produce sucrose more efficiently than both corn and sugarcane. Previously engineered S. elongatus has consumed carbon dioxide and secreted roughly 80% of that carbon intake as sucrose [3]. Crops such as sugarcane only allocate 15% of their carbon as sucrose, and large amounts of land and fertilizer are required for their production [3]. Because of the high efficiencies involved, sucrose production from cyanobacteria may be an effective method for carbon capture by converting carbon dioxide into sugars that can be used to make stable, high-value products such as bioplastics and biofuels.

Figure 2. Photosynthesis consumes carbon dioxide. In our system, photosynthesis is designed to produce sucrose, a useful industrial product.

Our Project

Ducat et al. and Xuefeng et al. both successfully engineered S. elongatus to produce and secrete sucrose in large quantities; however, the process has not been commercialized because of harvesting costs and inefficient photobioreactor design [4].

Our goal was to make cyanobacteria carbon sequestration industrially viable. Our approach was to characterize genes for sucrose production and secretion in S. elongatus under BioBrick standards. Additionally, we aimed to characterize various novel nutrient-dependent and light-dependent promoters in the same strain. Finally, the capstone of this characterization involved controlling the sucrose genes with the novel promoters to improve efficiency and control in industrial settings.

Sucrose BioBricks

S. elongatus naturally produces sucrose in saltwater to balance osmotic pressure [5]. We intended to modify S. elongatus to produce and export sucrose under freshwater conditions in order to reduce harvesting costs associated with salt-removal. We aimed to construct two BioBricks: one which produces sucrose, and one which exports it.

The protein responsible for the rate-determining, or slowest, step in S. elongatus sucrose production is sucrose phosphate synthase (sps), but it requires sodium ions to function [6]. We instead codon optimized the sps gene from another cyanobacteria strain, Synechocystis sp. PCC 6803, which shows sucrose production in the absence of sodium ions [6]. Unfortunately, issues in gene synthesis did not allow us to transform with and characterize sps.

The sucrose permease protein (cscB), derived from E. coli, is a membrane protein that can pump sucrose out of S. elongatus [3]. There is currently a BioBrick for cscB, but it does not meet RFC10 standards due to illegal cut-sites. We aimed to codon optimize cscB for our strain and make it BioBrick compatible. Incorporating cscB into our system eases sucrose harvest from extracellular media without needing to kill the bacteria.

In order to initially characterize the cscB BioBrick, we cloned it into Dr. Susan Golden’s IPTG-inducible vector pAM2991 (Addgene plasmid # 40248). We used a sucrose/d-glucose assay to compare the effects of cscB on extracellular sucrose concentrations against the wild-type when sucrose production is induced by saltwater.

Figure 3. CscB (sucrose symporter) pumps sucrose across the cell membrane using a proton gradient.

Promoter Biobricks

S. elongatus enters stationary phase, stopping replication, upon producing and secreting sucrose because carbon is redirected from growth [3]. Because this phenotype is unfavorable and easily selected out of our cyanobacteria, we aimed to induce sucrose production and secretion once S. elongatus has reached maximum cell density. Researchers such as Ducat et al. have used IPTG-inducible promoters; however, such promoters are leaky, and using IPTG is not cost-effective or easy to toggle in an industrial setting [3]. Additionally, despite S. elongatus being a model photosynthetic bacterium, there is a lack of characterized promoters in the registry.

We intended to characterize a variety of promoters in S. elongatus as BioBricks. These promoters will be valuable tools for the rest of the cyanobacteria synthetic biology community. We characterized the ferric ion repressible promoter associated with the idiA gene [7,8], the high-light inducible promoter of psbA2 [9], and strong constitutive promoters of cpcB native to S. elongatus and Synechocystis sp. PCC 6803 [10]. We cloned these promoters into Dr. Golden’s promoterless luxAB vector pAM1414 (Addgene plasmid # 40237). Then, a luciferase assay was used to measure levels of expression.

Figure 4. Our regulatory promoters

Application of Novel Promoters

After characterizing the promoters as BioBricks, we intended to use them to induce sucrose production and secretion by combining them with our cscB and sps BioBricks. Using some of the aforementioned promoters in an industrial setting has numerous advantages. The ferric ion repressible promoters initiate transcription with high on-off ratios once iron concentrations fall below millimolar thresholds [7,8]. Since iron is a micronutrient in our cyanobacteria’s BG-11 media, this is easily tunable. We can adjust the concentration of the media such that iron is completely exhausted upon reaching maximum cell density, which would trigger sucrose production.

Alternatively, the psbA2 promoter would make sucrose production easy to toggle. It is only expressed when S. elongatus is exposed to ¼ peak sunlight intensity or greater (roughly 500 µmoles photons/s/m^2) [9]. We can grow S. elongatus under low light until it reaches stationary phase. Upon achieving maximum cell density, we can increase light levels, triggering sucrose production. Using this promoter also keeps sucrose production in phase with light intensity, which is desirable as sucrose acts as an electron acceptor and should mitigate oxidative stress under intense light.

Unfortunately, we did not have enough time to collect data on this aspect of our project, but hopefully, other iGEM teams can carry on our work!

Lab Automation, Directed Evolution, and Open-Source Protocols

Unfortunately, we encountered many roadblocks in our project. We originally wanted to automate protocols that used UV-based random mutagenesis and artificial selection for different phenotypes such as high growth rate and high sucrose production, as a proof of concept for directed evolution of our strain. However, we were not able to pursue all of our goals due to DNA synthesis issues and contamination (we did not have sugar-producing cyanobacteria to use directed evolution on). We did collaborate with the Queens Canada iGEM Team to make a video explaining directed evolution.

This year, our team had the honor of winning an Opentrons OT-2 robot that we used for lab automation. We programmed several protocols with the Opentrons, including InterLab protocols and well plate setups.