Utilizing RPE Rubisco to enhance carbon dioxide fixation in transgenic corn

Design Brief

Arjun Chandra1, Yash Kalwani1, Kevin Lee1, Sadie Rooney1, Annie Song1, Mahathi Veluri1, and Megan Zhang1

1 Andover High School, Andover, Massachusetts, United States; 2 BioBuilder Club, Boston, Massachusetts, United States; 3 Pfizer BioPharma, Andover, Massachusetts, United States

Reviewed on 7 May 2022; Accepted on 25 June 2022; Published on 15 October 2022

With help from the 2022 BioTreks Production Team.

AbstractPDFCopyright

Excessive greenhouse gas emissions play a significant role in the global climate crisis. Carbon dioxide (CO2) is the principal greenhouse gas emitted by human activities that contributes to the greenhouse effect, resulting in rising global temperatures. Lowering atmospheric CO2 levels is critical to slowing the rate of global warming and preventing the catastrophic impact of climate change. A possible solution is to enhance plants’ intrinsic ability to absorb CO2 through photosynthesis. Several groups have introduced cyanobacterial CO2-concentrating mechanisms (CCMs) into plant chloroplasts as a strategy to enhance crop photosynthesis and yield. However, the rate of photosynthetic carbon fixation in CCM-enhanced plants is still limited by the ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) enzyme. Using recombinant DNA technology, we propose to create CCM-enhanced transgenic yellow dent corn (Zea mays var. indentata) that utilizes the RPE Rubisco protein, found in the endosymbiont of the deep-sea tubeworm Riftia pachyptila, which was determined to have a higher carboxylation efficiency and ready-to-deploy enzyme capacity that does not require chaperones. CCM-enhanced corn has the potential to concentrate CO2, while RPE-Rubisco may increase corn’s ability of CO2 fixation. In effect, the CCM-RPE-Rubisco-enhanced corn can be tested for large-scale agricultural production as a product that contributes to decreasing atmospheric CO2, alleviating global warming while producing food for general consumption.

Keywords: RPE-Rubisco, carbon dioxide, Agrobacterium, transgenic plant, climate change

Authors are listed in alphabetical order. Lindsey L’Ecuyer2 and Barbara Tevelev3 mentored the group. Please direct all correspondence to sadie.rooney@comcast.net, llecuyer@k12.andoverma.us, and meganzhangbiology@gmail.com.

This is an Open Access article, which was copyrighted by the authors and published by BioTreks in 2022. It is distributed under the terms of the Creative Commons Attribution License, which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background 

The greenhouse effect (Figure 1) describes the process by which solar infrared energy passes through the atmosphere and reaches the planet, where it warms the surface before being reradiated into the atmosphere. Greenhouse gases, like carbon dioxide, water vapor, nitrous oxide, and methane, form an atmospheric layer that acts as an insulator, reducing the amount of heat that escapes back into space and thus keeping the planet warm. This is especially essential at night so the Earth can maintain a safe and stable temperature suitable for life (Denchak, 2019).

Figure 1: Greenhouse effect. Ultraviolet radiation from the sun loses energy as it enters the Earth’s atmosphere, releasing infrared radiation (heat). The earth’s surface absorbs part of the radiation and reflects the rest. The layer of greenhouse gases stops most of the reflected heat from returning to space, thus insulating the planet and maintaining a stable temperature.

Human activity has increased the greenhouse gases released into the atmosphere, thus disrupting the carefully created “balance” that keeps the planet from growing too hot or too cold to support life. In addition to CO2, gases with higher Global Warming Potential (GWP) than CO2 have also been added to the atmosphere (table 1). 1 ton of CO2 is the base unit for GWP, meaning it has a GWP of 1. Nitrous oxide and methane, though found in smaller quantities, have GWPs of 265 and 28, respectively, and therefore are also threats due to their increased capacity to trap heat in the atmosphere (IPCC, 2014). Despite its lower GWP, the high atmospheric concentration (412.5 ppm) of CO2 distinguishes it as a greater threat, as its atmospheric concentration creeps past 400 ppm in recent years. Other greenhouse gases, such as water vapor and fluorinated gases, are also emitted from varieties of manufacturing and industrial processes. As more and more countries industrialize, carbon dioxide (CO2), emitted through the burning of fossil fuels, has become one of the biggest contributors to the greenhouse effect. Higher levels of atmospheric CO2 not only result in a higher global average temperature but also causes several significant changes in the planet’s climate. The changing weather patterns alter and devastate ecosystems worldwide. Warming trends force species to migrate to higher latitudes or higher elevations where temperatures are more conducive to their survival. Similarly, as sea levels rise, saltwater intrusions force key species to relocate, adapt, or die. The rapid rate of change makes adaptation nearly impossible, thus removing critical organisms from the food chain (McKie, 2013). In addition, flooding and droughts threaten the safety of both human settlements and natural habitats. The melting of glaciers results in an unreliable water supply to human settlements that rely on spring thaw (Denchak, 2019). 

Table 1. Relative atmospheric abundance and GWP of greenhouse gasses

GasAtmospheric abundanceGWP
Carbon dioxide400 ppm1
Methane1800 ppb28
Nitrous oxide325 ppb265

Furthermore, CO2 reacts with water molecules (H2O) to form carbonic acid, and the increased acidity makes aquatic environments uninhabitable to many species (Lindsey, 2021). CO2 levels are higher than they have ever been in at least 800,000 years, at approximately 412.5 parts per million (ppm) (Lindsey, 2021). Even if the global temperature rises one degree (1ºC), there could be catastrophic effects. Thus, it is vital to reduce current atmospheric quantities of CO2

Currently, climate change is countered in a variety of ways. On the consumer level, making educated choices, such as using fluorescent light bulbs rather than regular light bulbs, takes small steps towards sustainability. On a larger scale, an industrial push towards utilizing renewable energy reduces fossil fuel combustion and CO2 emissions. These methods, though capable of making change, are currently ineffective because the acts of consumers alone are unable to counteract the significant carbon footprint of companies, and there has been significant pushback in the US to proposals to switch to renewable energy.

On the other hand, a viable solution is capitalizing on plants’ ability to consume CO2. Plants already regulate the amount of CO2 in the atmosphere through photosynthesis, the process by which plants and other organisms convert light energy, CO2, and water into chemical energy for growth and reproduction. With a total of 7,000 billion tons of CO2 in the atmosphere, plant photosynthesis decreases this amount by more than 100 billion tons annually (Baslam et al., 2020). Different elements of the photosynthetic cycle are potential targets for human alteration to improve photosynthetic traits, increase CO2 consumption, and benefit agriculture. 

Plants and algae absorb CO2 and produce the oxygen that we use to breathe through their process of photosynthesis (figure 2) (Baslam et al., 2020). Photosynthesis begins in the thylakoids, flattened sacs inside the chloroplast. In the thylakoids, the chlorophyll absorbs sunlight and transfers electrons to the photosystems, releasing ATP and NADPH (chemical energy), and splitting H2O to release O2 gas. ATP and NADPH then travel into the stroma (aqueous fluid within the chloroplast), where the Calvin cycle reactions take place. In the Calvin Cycle, CO2 joins to RuBP (ribulose-1,5-bisphosphate, a 5-carbon molecule) with the help of an enzyme called Rubisco, converting atmospheric carbon into a usable carbon source, creating two molecules of 3-phosphoglyceric acid (3-PGA). The energy from NADPH and ATP then converts these 3-PGA molecules into a three-carbon sugar, G3P. Some of these G3P molecules go on to make glucose, and the rest go on to “regenerate” RuBP for the next cycle (Gunther, n.d.). This process can be made more efficient through the alternation of Rubisco and thus increase CO2 consumption.

Figure 2: Photosynthesis at the macro level. In photosynthesis, light reactions first occur in the thylakoids of the plant cell. Sunlight, through light reactions, releases oxygen gas and produces ATP and NADPH. After exiting the thylakoid membrane, ATP and NADPH enter the stroma (inner space of the chloroplasts) and join with carbon dioxide gas from the environment. These molecules then go through the Calvin cycle, producing sugars in the form of glucose.

As previously described, the Rubisco enzyme is a key part of photosynthesis. Rubisco is an enzyme found in most photoautotrophs allowing them to fix carbon dioxide in the Calvin-Benson-Bassham (CBB) cycle. However, Form I Rubisco enzymes from a variety of plants, algae, and cyanobacteria all have low rates of carboxylation, with significantly lower efficiency than most enzymes (Zhang et al., 2021). Additionally, high levels of atmospheric O2 in the environments of many photoautotrophs facilitate the photorespiration process, which consumes energy and releases CO2 (Cai et al., 2014). Therefore, plants alone are incapable of reducing atmospheric CO2 levels at a rate high enough to counter climate change. Thus, countering deforestation alone is not enough to offset climate change. Though plants store high quantities of carbon, it is not reasonable to plant enough trees to completely offset atmospheric CO2. Thus, further action is needed. 

Previous attempts to improve the efficiency of Form I Rubisco have had limited success. Instead, a higher carbon-fixation efficiency can be found in the Form II Rubisco enzyme from bacteria that live in endosymbiosis with the deep-sea tubeworm Riftia pachyptila, known as RPE Rubisco. The higher carboxylation efficiency of this enzyme allows the bacteria to provide R. pachyptila, which lives near CO2-rich hydrothermal vents, with enough energy to support its extremely fast growth rate (Zhang et al., 2021). Thus, if common plants such as corn employ RPE Rubisco, the rate of carbon fixation by plants can be greatly improved. 

We propose to engineer transgenic corn plants that incorporate the Rubisco gene from the tube worm through agrobacterium-mediated transformation, the most common method of engineering plant genomes. The transgenic corn plants will have increased capacity for CO2 consumption—approximately 3 times more in vivo and 19 times more in vitro (Zhang et al., 2021). Fields of this RPE Rubisco-enhanced crop can be planted to replace current corn crops. Additionally, they can be planted around factories, cities, or other large CO2 producers to offset emissions. The food grown can be fed to humans or livestock and provides significant agricultural benefits. If successful in corn, similar enhancements could be applied to plants more suited for growth in urban settings (parks, gardens, etc.) or perhaps even house plants for filtering CO2. Overall, we hope to engineer an efficient crop to offset CO2 emissions with limited need for resources and resistance to drought.

Systems level

In normal plants, the Rubisco enzyme is a limiting factor in CO2 consumption. While most enzymes can work on thousands of molecules per second, an individual Form I Rubisco enzyme processes only three CO2 molecules per second (Goodsell, 2000). If RPE Rubisco was used instead, we could increase the molecules of CO2 processed per second, thus increasing the overall amount of CO2 adsorbed. To increase the efficiency of fixation, we propose to genetically engineer yellow dent corn by inserting the RPE Rubisco gene via Agrobacterium-mediated transformation (figure 3).

Figure 3: Overall system. A Ti plasmid containing the RPE Rubisco gene will be expressed in CCMs-enhanced corn cells via Agrobacterium-mediated transformation, leading to corn plants with enhanced CO2 consumption that also create consumable products.

RPE Rubisco has been found to be vastly more efficient than other forms of Rubisco. It is 19 times more efficient in vitro and 3.6 times more efficient in vivo than 7002 Rubisco, another high-functioning expression of the enzyme (Zhang et al., 2021). Applying this increase in efficiency to real-world farms, we reason that our genetically modified corn would increase CO2 consumption by around 3.6 times. We will need to perform experiments with expressing the enzyme in corn to assure a 3.6 times increase in efficiency, as it may not perform as well outside of its native context. 

Further, it is important to note that implementing this gene may not translate directly to 3.6 times consumption, as farming and transportation equipment currently used in corn agriculture also emit CO2. Nevertheless, these agricultural processes are already used, meaning that this modified corn would be more efficient in carbon consumption than currently grown yellow dent corn.

One such community to which our corn could be applied is Michigan, a state which emits 162 million metric tons of CO2 each year (Rankings: Total Carbon Dioxide Emissions, n.d.). According to the Michigan State University, a typical Michigan corn field absorbs 16.33 metric tons of CO2 per 4 thousand square meters each year (Thelen, 2007). Michigan corn fields typically grow field corn (Zea mays), of which yellow dent corn is a derivative (Field Corn, n.d.). However, if they made the switch to our genetically modified corn, and CO2 capturing increased to the predicted 3.6 times, each field would capture 58.79 metric tons of CO2 per 4 thousand square meters each year. 

Applying this on a larger scale, the United States grows an average of 350 billion square meters of corn each year (United States Department of Agriculture, 2022). This translates to 15 billion bushels nationwide each year. On average, one acre of corn in the US absorbs 7.26 metric tons of CO2 yearly (Bennett & Cornett, 2018). Thus, as it currently stands, the corn annually grown in the U.S. captures 620 million metric tons of CO2. At the same time, it takes 371 grams per square kilometer of CO2 to grow corn in the US due to fossil fuels in farming equipment, transport, and even soil fertilizers (Stecker, 2013). Thus, the nation’s corn currently has a net CO2 capture rate of 492 million metric tons per year.

Instead, if our modified corn is used, which is 3.6 times more efficient, the amount of CO2 captured would increase to 2.2 billion metric tons a year. When again factoring in the amount of CO2 produced in the farming process, the nation’s corn would have a net CO2 capture rate of 2 thousand metric tons per year. Since the nation emits around 36 billion tons of CO2 each year, we would need around 16 times the current land used for corn to make the nation completely carbon neutral, around 5.5 billion square kilometers (IEA, 2022). As the nation already devotes 3.6 billion square kilometers to crops but once used 3.8 billion square kilometers in 2000, this is not unimaginable but may require partnering with other nations to utilize more fertile land area (Shahbandeh, 2022).

RPE Rubisco has the potential to significantly increase the ability of plants to sequester CO2. However, while Rubisco catalyzes the CO2 fixation involved in the plant’s growth, it also catalyzes an energy wasteful photorespiration process that consumes oxygen. Therefore, for our design to work, our corn must be enhanced by CO2-concentrating mechanisms (CCMs) to ensure that carboxylation is favored over oxygenation. During oxygenic photosynthesis, light energy transfers water taken up by plant roots to carbon dioxide to produce carbohydrates and oxygen. Several groups have successfully demonstrated the introduction of cyanobacterial CCMs into the chloroplasts of rice and tobacco (Iñiguez et al., 2021). There is precedent for porting microbial enzymes and machinery focused on carbon fixation into other organisms. 

Cyanobacterial CCMs enable relatively rapid CO2 fixation by elevating intracellular inorganic carbon as bicarbonate, then concentrating it as CO2 around the Rubisco enzyme in specialized protein micro-compartments called carboxysomes. A simplified carboxysome from Cyanobium has already been created within the tobacco chloroplasts. The endogenous Rubisco large subunit gene was replaced with cyanobacterial Form-1A Rubisco large and small subunit genes, along with genes for two key α-carboxysome structural proteins. This minimal gene set produces carboxysomes, which encapsulates the introduced Rubisco and enables autotrophic growth at elevated CO2 consumption. Photosynthetic and yield improvements have already been observed when manipulating Rubisco’s degree of activation along with advanced CCMs in crops. Further engineering Rubisco carboxylation capacity continues to be a promising target for improving photosynthesis and yield (Long et al., 2018).

Device level

We chose yellow dent corn as our chassis because it is the most common variety of corn grown in the United States. This corn is widely used in agriculture and can be used for human and animal consumption (Yellow Dent Corn, 2015). Thus, it would be easiest to implement on a large scale, as farmers already understand how to grow it and the public is comfortable eating it. On a genetic level, yellow dent corn is the most commonly genetically modified corn (Schaufler et al., 2018). Thus, there is precedent and abundant information on how to best edit it.

[table “e202205t04” not found /]

We specifically chose Agrobacterium tumefaciens because this bacterium is an important tool in plant biotechnology due to its natural ability to transfer DNA into the genomes of host plants. Genetic manipulations of A. tumefaciens have yielded considerable advances in increasing transformational efficiency in several plant species and cultivars. Modulating the expression of various mediators of A. tumefaciens virulence can lead to more successful plant transformation; thus, applying synthetic biology to enable targeted engineering of the bacterium may enable new opportunities for advancing plant biotechnology.

We plan to apply Agrobacterium-mediated genetic transformation using binary vectors (figure 4). This is a widely used method for generating transgenic plants. This system utilizes the ability of the bacteria Agrobacterium tumefaciens to insert foreign DNA into the corn genome. The Agrobacterium binary vector system is derived from natural tumor-inducing (Ti) plasmids. Agrobacterium transfers a region of the Ti-plasmid known as the transfer DNA (T-DNA) into corn, where it is integrated into the host genome. The T-DNA is delineated by flanking 25 bp T-DNA border repeat sequences in direct orientation with one another. Typically, in the binary vector system, all tumor-associated intervening T-DNA sequences are removed, leaving the T-DNA border repeats, which flank and direct host integration of the user’s sequence of interest (Hwang et al., 2017).

The complete binary vector system consists of two parts. The first is the T-DNA binary vector that contains two T-DNA repeats bracketing the DNA sequence, which will be inserted into the corn host. The RPE Rubisco is cloned into this portion of the binary vector. The second plasmid, referred to as the vir helper plasmid, encodes components necessary for integration of the region flanked by the T-DNA repeats into the genome of plant cells. Prior to transforming the target plant cells, these two plasmids are brought together in A. tumefaciens by co-transformation, co-electroporation, or conjugation. When the binary vector and the vir helper plasmid are both present in the same Agrobacterium cell, proteins encoded by the vir helper plasmid act in trans on the T-DNA border repeat elements to mediate processing, secretion, and host genome integration of the sequence between the left and right border repeat elements (Kroemer, n.d.). 

To make sure that we infect the plant with the desired soil bacterium, we propose to use an A. tumefaciens strain, AGL-1, as it allows for Agrobacterium-mediated genetic transformation of maize. We will insert a T-DNA binary vector that has a kanamycin gene, allowing for the Agrobacterium to be resistant to kanamycin. This antibiotic can be used as a selective marker to kill or prevent the growth of bacteria that do not have resistance. Knowing that our strain will have this resistance, we can use the antibiotic to eliminate the unwanted A. tumefaciens strains, leaving us with our preferred strain, AGL-1 (Gold Biotechnology, n.d.). 

Parts level

We created a T-DNA binary vector by modifying an already known Agrobacterium vector to enhance the expression of carbon dioxide fixation (figure 5). Corn are monocots, possessing one cotyledon: an embryonic leaf. ZmUbi is known to be a strong promoter and drives high-level gene expression in monocots, which increases the rate of transcription (Zou, 2014). By modifying this Agrobacterium, more of the RPE Rubisco gene will be expressed. We will likewise add Kana, a selectable marker that allows plants to be resistant to kanamycin (table 2). This ensures that the plant’s bacteria are not killed or inhibited from growing. 

Figure 5: Vector. Modified T-DNA binary vector containing ZmUbi, EGFP, and Neo/Kana

Additionally, we will utilize EGFP. EGFP is a stabilizer that provides green fluorescence, brightness, pH resistance, and environmental protection. Using DNA recombinant technology, we can combine the EGFP gene into the RPE Rubisco gene and insert it into the yellow dent plant cells (Zou, 2014). If the cell produces green fluorescence, we know that the plant cell will express the target gene. This will be used primarily in proof of principle experiments and may be removed from the final product in order to increase market acceptability.

Table 2. T-DNA Binary Vector Components

NamePositionSize (bp)TypeDescriptionApplication Notes
ZmUbi22-20141993PromoterMaize ubiquitin promoterStrong constitutive promoter, drives high-level gene expression in monocots.
Kozak2039-20446MiscellaneousKozak translation initiation sequenceFacilitates translation initiation of ATG start codon downstream of the Kozak sequence.
EGFP2045-2764720ORFEnhanced green fluorescent protein; codon optimized based on a variant of wild type GFP from the jellyfish Aequorea victoriaCommonly used green fluorescent protein; ranked high in brightness, photostability and pH stability among all fluorescent proteins.
Nos pA2805-3058254PolyA_signalNopaline synthase polyadenylation signalAllows transcription termination and polyadenylation of mRNA transcribed by RNA polymerase II.
RB T-DNA repeat3084-310825MiscellaneousRight border repeat of T-DNAUpon recognized by Ti plasmid in Agrobacterium, it transfers the region between T-DNA border repeats to plant cells.
pVS1 StaA4409-5038630ORFStability protein from pVS1 plasmidEssential for plasmid segregational stability in Agrobacterium.
pVS1 RepA5467-65401074ORFReplication protein from pVS1 plasmidPermits replication of low-copy plasmid in Agrobacterium.
pVS1 oriV6606-6800195Replication_originOrigin of replication from pVS1 plasmidPermits replication of low-copy plasmid in Agrobacterium.
pBR322 oriComplement (7470-8058)589Replication_originpBR322 origin of replicationFacilitates plasmid replication in E. coli; regulates low-copy plasmid number when Rop protein is present (15-20) and medium-copy plasmid number when Rop is absent (100-300).
KanamycinComplement (8145-8939)795ORFAminoglycoside 3’- phosphotransferase gene.Allows E. coli and Agrobacterium to be resistant to kanamycin.
LB T-DNA repeat9364-938825MiscellaneousLeft border repeat of T-DNAUpon recognition by Ti plasmid in Agrobacterium, it transfers the region between T-DNA border repeats to plant cells.
CaMV 35S pAComplement (9479-9653)175PolyA_signalCauliflower mosaic virus 35S polyadenylation signalAllows transcription termination and polyadenylation of mRNA transcribed by RNA polymerase II.
KanaComplement (9710-10507)798ORFNeomycin phosphotransferase II geneAllows plants to be resistant to G418 or Kanamycin.
CaMV35S (enhanced)Complement (10583-11336)754PromoterA chimeric promoter with the doubled CaMV 35S enhancer elementStrong promoter.

Safety

Although we are not at the stage where we are directly handling RPE Rubisco, when we begin working with RPE Rubisco and the corn cells, we will follow lab safety guidelines and wear gloves, goggles, and all necessary PPE (personal protective equipment) to protect ourselves and minimize contamination to our experiment. The specific bacteria we are using is A. tumefaciens which has Plant Biosafety Level 1 (BSL-1P). These microbes present minimal potential hazard to laboratorians and the environment. Organisms with BSL-1P are also not known to consistently cause disease in healthy adults.

The transgenic corn does not pose any biosafety hazards to the general public because we are only changing the speed at which the corn utilizes carbon dioxide to create glucose—the primary makeup of the corn itself is unchanged. Before growing our corn in an outdoor environment and feeding it to livestock, we will first grow it in a lab under controlled conditions and monitor it to see if it will release any unexpected chemicals into the air or soil. Furthermore, we can test the safety of the corn crop by feeding it to a controlled population of lab mice or cows, allowing us to monitor the effect that ingesting the corn has. This allows us to check for any adverse effects of eating the corn before feeding it to a large population of cows. 

Another important safety issue is the potential for our crop to spread as an invasive species. To combat this, we propose to grow our crop in isolated fields. Further, since we chose corn as a host, our crop needs to be replanted yearly. Thus, any stray crops would die, allowing local ecosystems to maintain balance. If there is a fear of insects spreading the crop in a particular ecosystem, we could opt to grow the corn in greenhouses. At the same time, the process of planting and maintaining the crop could create more jobs to benefit surrounding communities.

Discussion

By inserting the RPE Rubisco present in the symbiote of deep sea tube worms into plants, we will be able to increase the carbon dioxide intake of plants by several times, thereby removing more CO2 from the atmosphere and more efficiently converting it into usable energy for the plant. Corn would be a good vessel for the RPE Rubisco because of how commonly it is grown in the United States, maximizing the carbon dioxide intake. Yellow dent corn is the main type of corn that goes to feed our livestock; especially when nearly half of our corn grown in the United States goes to feed animals, it would be extremely beneficial to the environment to take CO2 from the atmosphere and allow us to potentially yield more crop while using less land (United States Department of Agriculture, 2015). One of the main causes of deforestation is the need for more farmland. As the human population continues to increase, we will need more food to sufficiently feed everyone, and if we can successfully utilize RPE Rubisco in corn plants, it can help address that issue.

Rubisco is the most abundant enzyme on the planet, and by altering such an important enzyme, we risk impacting other aspects of the plant (Bar-On & Milo, 2019). RPE Rubisco in a corn plant should make the plant more efficient at taking in CO2 and, therefore, more efficient at creating glucose for itself. RPE Rubisco was found to increase glycolate production 14-fold (Yang et al., 2021). However, at this stage of development, it is difficult to tell if it would create a more nutritious or different type of corn or if it will just cause the corn plant to grow quicker. In theory, this practice could be copied on a global scale to decrease CO2 levels and combat world hunger. Other possible uses could be space exploration or to greatly counteract CO2 emissions in urban center green roof farms. 

Another potential uncertainty is taking into account cellular respiration in plants in general. During plant respiration, rather than taking in CO2 from the atmosphere, the plant takes in O2 and releases CO2 into the atmosphere (Gillespie, 2018). We are certain that RPE Rubisco would accelerate the rate at which CO2 is converted into glucose, but it is difficult to determine if the plant would release more CO2 at night due to the RPE Rubisco. 

Additionally, the EGFP stabilizer that we use to provide green fluorescence is optional and can be used for proof of principle experiments. When the high capacity of the modified corn to remove CO2 is proven, the fluorescence gene will be removed, so the corn will maintain its quality, marketability, and safety.

We predict the public would be concerned about the safety of transgenic corn. Genetically modified organisms (GMOs) are safe for human and animal consumption. The FDA states that genetic material found in GMOs does not transfer to consumers, and there is no difference between organic and GMO foods on consumer health (U.S. Food and Drug Administration [FDA], 2022). GMOs have no evidence of harmful side effects(Norris, 2015). Already, Bacillus thuringiensis (Bt) corn, which is engineered to decrease pests, is prominent in the US to feed both humans and livestock. 92% of corn planted in the United States in 2018 was genetically modified. Further, over 95% of meat and dairy animals eat GMO crops (FDA, 2022). 

Nevertheless, Pew Research data reveals that 51% of the American public believes GMOs are worse for human health than organic foods. Further, 88% of the public believes GMOs result in health problems (Funk, 2020). Their data additionally suggested that approval for GMOs amongst the public was trending downwards, with more and more growing wary of adverse effects. To combat this, we hope to educate the public about the benefits, specifically by relating the need for our crop to the growing issue of climate change. Further studies on our specific crop’s safety would help to assure the public that it is fit for consumption. If our efforts to sway the public about the safety of our crop fail, our crop can be used as feed specifically for livestock. This would fill another purpose while increasing public support.

Next steps

To continue, we will conduct an experiment as proof of principle. In a container, we will insert RPE Rubisco into yellow dent corn and leave a second container unmodified as a control group. In the same stable conditions, we expose these two groups to sunlight. Each day, we will monitor CO2 consumption in parts per million by using a CO2 meter to test and track CO2 levels in each container. Moving forward, we will monitor CO2 consumption, growth quality, and nutritional value first in a contained greenhouse and later in a contained field.

Another future step we hope to take is to decrease the amount of water required to grow our crop. This would make the corn cheaper and easier to grow and thus increase the likelihood that farmers would switch to our product. This can be achieved through further genetic motivation or through selective breeding. There are several genes that can be introduced to corn to improve water efficiency and increase drought resistance (McFadden et al., 2019). One example is the GmDREB1 gene. Transgenic wheat carrying this gene from soybean showed drought tolerance under a ubiquitin promoter (Shiqing et al., 2005). Another example is the HaHB4 gene. Transgenic wheat transformed with a mutated transcription factor (HaHB4) had increased water use efficiency and yield (Fernanda et al., 2019). These are two potential examples we may target to further improve the corn with enhanced CO2 consumption. If we choose to only grow seeds from plants that require less water, over time, we will produce a plant that requires very little water to carry out necessary life processes while having no impact to yield.

Furthermore, making corn more nutritious will allow people to stay healthier while still enjoying food. Corn is used in many things, including food products, beverages, alcohol, and fuel. Products made with corn rather than food with processed flour can increase or maintain gut health and help lower the chances of getting diseases such as heart disease and type two diabetes. Eating carbohydrates in corn, such as fiber, also lead to staying full for longer periods and between meals. Recently, scientists have found that inserting a bacterial gene causing corn to produce methionine, a key amino acid that corn lacks, can enhance the nutritional value of corn without affecting plant growth. Methionine is needed for growth and tissue repair, improving the tone and flexibility of skin and hair (Rutgers University, 2017). Implementing this with our other experiments and future steps could create more nutritious CO2-consuming transgenic corn requiring less water without affecting the plant’s growth.

Author contributions

All authors worked on general research, writing the abstract, and general editing. A.C., A.S., M.V., M.Z., and S.R. worked on background information. A.C. created the main diagram and formatted citations. A.C. and S.R. to detail how the Ti plasmid containing the RPE Rubisco gene will be expressed. S.R. and A.S. wrote about lab, community, and environmental safety and future development. S.R. and M.Z. wrote about the next steps for the project. Y.K. and G.L. worked on describing Agrobacterium and the specifics of the T-DNA binary vector. M.Z. and M.V. created figures and captions. 

Acknowledgments

We would like to thank our advisors and mentors, Ms. Lindsey L’Ecuyer and Ms. Barbara Tevelev, for their endless support and encouragement. With their extensive feedback, knowledge, and resources, we have been able to further understand the scientific process and hopefully integrate our ideas to enhance carbon dioxide fixation in corn. With the help of the BioBuilder Educational Foundation, we have been able to apply our research and knowledge to solve critical issues of the 21st century.

We would also like to express appreciation to Dr. Robert Wilson, Dr. Yin Li, and Dr. Junli Zhang for their feedback and for providing us with the tools and resources to continue our research.

This project was accomplished through participation in the BioBuilderClub, a program organized by BioBuilder Educational Foundation. BioBuilderClub engages high school teams around the world to combine engineering approaches and scientific know-how to design/build/test our own project ideas using synthetic biology. 

References 

Bar-On, Y. M., & Milo, R. (2019, March 5). The global mass and average rate of Rubisco. PNAS, 116(10), 4738-4743. https://doi.org/10.1073/pnas.1816654116 

Baslam M, Mitsui T, Hodges M, Priesack E, Herritt MT, Aranjuelo I and Sanz-Sáez Á (2020). Photosynthesis in a Changing Global Climate: Scaling Up and Scaling Down in Crops. Front. Plant Sci. 11:882. https://doi.org/10.3389/fpls.2020.00882 

Bennett, C., & Cornett, S. (2018, June 4). Corn’s Carbon Cowboy Busts Outstanding Yields. AgWeb. Retrieved from https://www.agweb.com/news/crops/crop-production/corns-carbon-cowboy-busts-outstanding-yields 

Cai, Z., Liu, G., Zhang, J. et al. (2014). Development of an activity-directed selection system enabled significant improvement of the carboxylation efficiency of Rubisco. Protein Cell 5, 552–562. https://doi.org/10.1007/s13238-014-0072-x 

Denchak, M. (2019, July 16). Greenhouse Effect 101. NRDC. Retrieved from https://www.nrdc.org/stories/greenhouse-effect-101 

Field Corn. (n.d.). Michigan Grown. Retrieved from https://michigangrown.org/field-corn/ 

Funk, C. (2020, March 18). About half of U.S. adults are wary of health effects of genetically modified foods, but many also see advantages. Pew Research Center. Retrieved from https://www.pewresearch.org/fact-tank/2020/03/18/about-half-of-u-s-adults-are-wary-of-health-effects-of-genetically-modified-foods-but-many-also-see-advantages/ 

Gillespie, C. (2018, April 25). Cellular Respiration in Plants. Sciencing. Retrieved April 27, 2022, from https://sciencing.com/cellular-respiration-plants-6513740.html 

Gold Biotechnology. (n.d.). AGL-1 Agrobacterium Electrocompetent Cells. GoldBio. Retrieved from https://www.goldbio.com/product/14434/agl-1-agrobacterium-electrocompetent-cells

Goodsell, D. (2000, November). Molecule of the Month: Rubisco. PDB-101. Retrieved from https://pdb101.rcsb.org/motm/11 

Gunther, T. Calvin Cycle. National Geographic Society. Retrieved from https://www.nationalgeographic.org/media/calvincycle/ 

Hwang, H.-H., Yu, M., & Lai, E. M. (2017, October 20). Agrobacterium-Mediated Plant Transformation: Biology and Applications. The Arabidopsis Book, 2017(15). https://doi.org/10.1199/tab.0186 

IEA. (2022, March 8). Global CO2 emissions rebounded to their highest level in history in 2021. IEA. Retrieved from https://www.iea.org/news/global-co2-emissions-rebounded-to-their-highest-level-in-history-in-2021 

Iñiguez, C., Aguiló-Nicolau, P., & Galmés, J. (2021). Improving photosynthesis through the enhancement of Rubisco carboxylation capacity. Biochemical Society transactions, 49(5), 2007–2019. https://doi.org/10.1042/BST20201056

IPCC, 2014: Climate Change 2014: Synthesis Report. Contribution of Working Groups I, II and III to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change [Core Writing Team, R.K. Pachauri and L.A. Meyer (eds.)]. IPCC, Geneva, Switzerland, 151 pp

Kroemer, T. (n.d.). A Quick Overview of Agrobacterium for Plant Transformation. GoldBio. Retrieved from https://www.goldbio.com/articles/article/a-quick-overview-of-agrobacterium-for-plant-transformation#_Toc61514150 

Lindsey, R. (2021, October 7). Climate Change: Atmospheric Carbon Dioxide. NOAA. Retrieved from https://www.climate.gov/news-features/understanding-climate/climate-change-atmospheric-carbon-dioxide 

Long, B.M. et al. (2018).Carboxysome encapsulation of the CO2-fixing enzyme Rubisco in tobacco chloroplasts. Nat Commun, 9, 3570. https://doi.org/10.1038/s41467-018-06044-0

McFadden, J., Smith, D., Wechsler, S., & Wallander, S. Development, Adoption, and Management of Drought-Tolerant Corn in the United States, EIB-204, U.S. Department of Agriculture, Economic Research Service, January 2019.

McKie, R. (2013, July 13). Climate change is happening too quickly for species to adapt. The Guardian. Retrieved from https://www.theguardian.com/environment/2013/jul/14/climate-change-evolution-species-adapt 

Norris, M. L. (2015, August 10). Will GMOs Hurt My Body? The Public’s Concerns and How Scientists Have Addressed Them – Science in the News. Science In The News, Harvard University. Retrieved from https://sitn.hms.harvard.edu/flash/2015/will-gmos-hurt-my-body/ 

Rankings: Total Carbon Dioxide Emissions, 2019. (n.d.). U.S. Energy Information Administration. Retrieved from https://www.eia.gov/state/rankings/?sid=MI#series/226

Rutgers University. (2017, October 9). Genetically boosting the nutritional value of corn could benefit millions: Scientists discover way to reduce animal feed and food production costs by increasing a key nutrient in corn. ScienceDaily. Retrieved from www.sciencedaily.com/releases/2017/10/171009154956.htm 

Schaufler, Karoline et al. (2018, August 14). GMO Feature: Corn: Living Non-GMO: The Non-GMO Project. GMO. Retrieved from https://livingnongmo.org/2018/08/14/gmo-feature-corn/

Shahbandeh, M. (2022, March 14). US farming: total land in farms 2021. Statista. Retrieved from https://www.statista.com/statistics/196104/total-area-of-land-in-farms-in-the-us-since-2000/ 

Shiqing, G., Huijun, X., Xianguo, C. et al. Improvement of wheat drought and salt tolerance by expression of a stress-inducible transcription factorGmDREB of soybean (Glycine max). Chin. Sci. Bull. 50, 2714–2723 (2005). https://doi.org/10.1007/BF02899641

Stecker, T. (2013, December 5). How Growing Corn Could Produce Less Pollution. Scientific American. Retrieved from https://www.scientificamerican.com/article/how-growing-corn-could-produce-less-pollution/

Thelen, K. (2007, June 7). Corn fields help clean up and protect the environment. Michigan State University. Retrieved from https://www.canr.msu.edu/news/corn_fields_help_clean_up_and_protect_the_environment 

United States Department of Agriculture. (2015, February). USDA Coexistence Factsheets – Corn. USDA. Retrieved from https://www.usda.gov/sites/default/files/documents/coexistence-corn-factsheet.pdf 

United States Department of Agriculture. (2022, January 12). Corn and soybean production up in 2021, USDA Reports Corn and soybean stocks up from year earlier, Winter Wheat Seedings up for 2022. National Agricultural Statistics Service. Retrieved from https://www.nass.usda.gov/Newsroom/2022/01-12-2022.php 

U.S. Food and Drug Administration. (2022, February 17). GMO Crops, Animal Food, and Beyond. FDA. Retrieved from https://www.fda.gov/food/agricultural-biotechnology/gmo-crops-animal-food-and-beyond 

Yang, F., Zhang, J., Cai, Z., Zhou, J., & Li, Y. (2021). Exploring the oxygenase function of Form II Rubisco for production of glycolate from CO2. AMB Express, 11(1), 65. https://doi.org/10.1186/s13568-021-01224-6

Yellow Dent Corn. (2015, February 18). Heartland Science. Retrieved from http://www.heartlandscience.org/agrifood/yelcorn

Zhang, J., et al. (2021). Discovery of a readily heterologously expressed Rubisco from the deep sea with potential for CO2 capture. Bioresour. Bioprocess. 8, 86. https://doi.org/10.1186/s40643-021-00439-6Zou, Y. (2014, June 11). Green Fluorescent Protein. The Embryo Project Encyclopedia. Retrieved from https://embryo.asu.edu/pages/green-fluorescent-protein