2018 Stony Brook iGEM
The Stony Brook iGEM Team is proud to present to you their sweet and energy filled project! Made with love <3
Contacts
Email: igem.sbu@gmail.com
The Earth absorbs a certain percentage of radiation from the sun and therefore heats up. It reflects the rest of the radiation back into space. Gases such as carbon dioxide and water vapor absorb much of the radiation that is normally reflected out of the atmosphere. As a result, more of the sun’s energy is trapped as heat. That extra heat energy is used to power more intense weather events, and can raise the overall temperature of the planet, leading to melting ice reserves and rising sea levels.
Additionally, the ocean absorbs some of the excess carbon dioxide as carbonic acid, causing the oceans to grow more acidic. As a result of human activity, the ocean has become 30% more acidic since the start of the Industrial Revolution [17]. This increase in acidity has caused a significant decline in the amount of coral reefs as their calcified structures dissolve in acidic environments.
Unfortunately, carbon dioxide stays in the atmosphere much longer than gases such as water vapor because carbon dioxide does not liquify or solidify like water vapor does at the temperatures that are normally found in Earth’s atmosphere. The rate at which carbon dioxide is pumped into the atmosphere by human pollution also far exceeds the rate that it is absorbed by plants and other carbon sinks (natural sources that absorb carbon dioxide), as evidenced by increasing levels of carbon dioxide in the atmosphere. As a result, the percent of carbon dioxide in our atmosphere has increased from 285.2 ppm in 1850 to 391.15 ppm at present [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 determine a way to capture emissions faster than we produce them.
Even if humankind stops emitting carbon dioxide today and uses only renewable energy, the effects of climate change 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.
Luckily for us, nearly 2.4 billion years ago, nature came up with the solution to high atmospheric carbon dioxide levels [16]. This solution is known as the “Great Oxidation Event.” Cyanobacteria, the first oxygen-releasing photosynthetic organisms, 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.
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, for the purpose of carbon sequestration. Specifically, we will engineer our cyanobacteria to efficiently produce and secrete sucrose, which can serve as a feedstock in industrial fermentation to produce PLA plastic 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 fixed carbon dioxide and secreted roughly 80% of fixed carbon as sucrose without the need for cell-harvesting [3]. Crops such as sugarcane only allocate 15% of their fixed 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 sequestration by converting carbon dioxide into sugars that can be used to make stable, high-value products such as bioplastics and biofuels.
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.
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.
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.
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!
1. Pimentel, D. (2003). Ethanol Fuels: Energy Balance, Economics, and Environmental Impacts Are Negative. Natural Resources Research
The Stony Brook iGEM Team is proud to present to you their sweet and energy filled project! Made with love <3
Email: igem.sbu@gmail.com
