Our Project

Background

Climate change is argued to be one of the greatest challenges faced by mankind. The current climate change is mainly caused by us humans as we have been using the Earth’s precious fossil fuel stocks without returning the emitted gases into the natural carbon cycle. According to the Intergovernmental Panel on Climate Change (IPCC), even if we could stop all the emissions right now, the Earth’s average temperature would rise 0.6°C. [1] This means we must act now. To fight climate change we have come up with a solution which would tackle the emissions made by the road transportation. These emissions make up a considerable 11% of the world’s greenhouse gas emissions. [2]

While power generation accounts for about a quarter of the world’s greenhouse gas emissions, 11% might seem like a small number. [2] What makes emissions made by transportation significant, though, is that there are currently no good alternatives for gasoline. Electric cars are emerging, but they still have quite a way to go before reaching the price-range suitable for middle class. [3] Propane is already widely used as a replacement of gasoline. In South Korea, 2.2 million vehicles [4] run on propane and in Turkey, 37% of passenger cars use it. On a large scale though, only 1.2% of vehicles worldwide are suitable to run on propane. [4] This may seem discouraging, but in reality converting a gasoline motor into a propane one is quite simple and inexpensive. DIY converter kits are sold online for less than $500. In Canada, depending on the vehicle type, conversion done by a third party costs somewhere around 2500-6500 dollars. [5]

On top of its use as a transportation fuel, propane is also a popular cooking fuel in developing countries. It’s more commonly used in urban areas, as access to propane can be difficult in rural areas. Nevertheless, according to the International Energy Association (IEA), about 20% of household in the rural areas of Botswana use propane as their source of energy. In the urban areas the usage is around 60%. Expansive infrastructure is in place for example in Brazil, where 98% of households have access to propane due to government funded efforts. [6] One major goal of promoting propane is to replace currently used biomass fuels, including wood. Wood consumption is often unsustainable and threatens the local ecosystems. Additionally, the traditional fuels (biomass and coal) produce high emissions of carbon monoxide, hydrocarbons and particulate matter. IEA suggests that these impurities are responsible for more premature deaths than Malaria in developing countries. [6]

Propane can be considered a clean fuel, as it emits less CO2 than gasoline and ethanol and, when compared with traditional biomasses, has significantly smaller emission factors based on both mass and energy delivery. [7] Thus, propane is a viable option for replacing traditional biomasses as cooking fuel, although currently the price is not competitive without government subsidies.

However, the propane we are currently using is produced as a side product of the petrochemical industry. This initially means that propane is a fossil fuel, emitting stored CO2 into the atmosphere without a way of returning to the natural carbon cycle. This contributes not only to climate change but also ocean acidification. This endangers the livelihoods of hundreds of millions of people directly or indirectly dependent on the marine ecosystems. [8]

We want to make the use of propane as a fuel sustainable. We want to design an Eschericia coli capable of producing propane from cellulose.

Degrading Cellulose

E. coli Producing Propane

Our propane pathway is based on the research done by Pauli Kallio et al [1] and Menon et al [2]. Right in the beginning we got in touch with Pauli Kallio from the University of Turku. He was very excited about our project and eager to help. We were able to get Pauli’s groups plasmid maps from him and decided to use these as our starting material. As they had already tested this pathway, we could be sure that their genes were functional in E. coli.

Our chassis organism, E. coli BL21 (DE3) was chosen because it’s a strain that produces T7 promoter when induced with IPTG. This is the strain that was used in Kallio’s research and was available at our lab. In addition to the regular BL21 (DE3), Pauli was kind to send us a BL21(DE3) with YjgB and YqhD knocked out when we realized that producing some knock outs ourselves would be too expensive and time consuming. YjgB and YghD are E. coli’s endogenous genes which produce butyraldehyde consuming enzymes. This means that they compete with our pathway’s final enzyme, ADO which uses butyraldehyde as its substrate.

We had been warned about the pathway’s vulnerabilities: it consisted of 10 different enzymes and in earlier research, it had been built with a minimum of three different plasmids. The stress for the bacteria was high. We thought that one way of reducing this stress was to use just two plasmids for the propane pathway. We then moved forward to design our complete constructs. We used Pauli’s group’s construct as a basis, and arranged the genes similarly. The arrangement of our first plasmid is the same as the original one. It starts with a T7 promoter and an additional lac operator. The T7 polymerase is IPTG inducible in our BL21 (DE3) strain, but we wanted to make sure there were no leaks in our system before the actual induction. That’s why the promoters adjacent to the genes are induced by IPTG. Following the promoter, we have the YciA, sfp and CAR each with a separate rbs (Lähde???) and a T7 terminator. We chose to use the same RBS's and terminator sequences as Pauli Kallio's group, as they had already tested the system. Our constructs terminator sequence is used in common cloning vectors (such as pET and pDF). Our second plasmid includes the same promoter, rbs’s, and terminator, but the genes AtoB, hbd, crt and ter, which is a similar construct as the original one made by Kallio's group. We decided to add an another promoter to the plasmid to create two operon systems. We did this to ensure that all the genes would be transcribed by the polymerase. The last genes in our second construct, ADO, petF and fpr were arranged quite randomly and function under the same promoter and induction systems as all the other genes.

We first planned on creating a biobrick from each of our genes and assembling the plasmids with the three antibiotics assembly method, but soon realized it would take too much time. We were then introduced to the Gibson Assembly system, and decided to give it a try. This is also when we gave up on the idea of creating a separate brick of each of our genes, but rather wanted to provide whole plasmids that would allow easy production of propane.

So, we divided our whole plasmids into pieces of about 2000bp each and included 30bp overlaps into our gBlocks-to-be. This is when we also realized, that synthesis isn’t as simple as it sounds: we had to optimize more than half of our sequences to be fit for gBlock synthesis. We will then proceed with NEBuilder kit, overlapping PCR (OE-PCR) and/or ELIC to combine our constructs. We used multiple methods because of the time-constrait and the fact that we didn't know which one would work the best. Due to a construct design error, our assembly pieces contained the BioBrick prefix in the very first part, but the last part did not include the suffix. The suffix was added by PCR after synthesis and the prefix & suffix areas functioned as the homologous overlap areas between our brick and the backbone.

In the assembly phase, we also had to take into account the plasmids we were to use. As our whole system would require three plasmids altogether if we were to add the cellulose hydrolysing enzymes to the same bacteria, we had to be careful about the plasmids’ compatibility groups. Our plasmids needed different antibiotic resistances and intercompatible origins of replication. We ended up using one plasmid backbone from the iGEM distribution kit. Our second and third plasmids are commercial Novagen plasmids pACYCDuet-1 and pCDFDuet-1. Needs explanation of how we got rid of the extra T7 promoters!! Based on our pathway kinetics model, we assmbled our constructs so that the most rate-limiting enzyme, ADO was in the highest copy-number plasmid and second bottleneck CAR in the backbone with the next highest copy-number. Unfortunately the construct design process was too far at this point for us to assemble both CAR and ADO into the high copy-number plasmid. After our constructs were successfully assembled they were sent for sequencing to check that everything worked as expected. We then transformed them into competent E. coli BL21(DE3) ΔyjgB ΔyqhD strain with chemical transformation and screened the transformants with double-antibiotic plates. These cells should be able to produce propane. All we would need to do is induce the production with IPTG and identify the propane by gas chromatography.

Micelle Fusions Enhancing the Production

Based on the previous studies about this pathway [1, 2], we knew the propane yields weren’t very high. We thought about trying to enhance the system by looking for homologs for the enzymes, but thought this would be too time-consuming but also not very innovative. We then ran into a research article by Huber et al [3]. The group had designed a synthetic amphiphilic protein that spontaneously formed membrane-like structures inside the cell. These proteins we’re designed quite like membrane lipids: there is a hydrophilic and a hydrophobic end. According to the energy minimum principle, the proteins’ hydrophilic ends will face the liquid phase of the cell and the hydrophobic ends will pack together. This way the proteins will be able to form either a double layer (similar to the double lipid layer) or a micelle.

To both enhance the knowledge of these amphiphilic proteins and to gain a better yield of propane, we thought of fusing enzymes to these proteins. This would bring the enzymes close together and possibly enhance their productivity. Because our system is so big, we needed to make some compromises though - we didn’t think it was possible to attach all 10 of our pathway’s enzymes into these amphiphilic proteins, but decided to do it to two of the last enzymes: CAR & ADO. We chose these enzymes for two reasons, the first one being more significant. The product of CAR (and the substrate of ADO) is butyraldehyde, which is toxic to the cell. Therefore, if too much butyraldehyde is built up in the cell, we will lose our cell line. Also, because of the toxicity, cells have many endogenous enzymes that consume butyraldehyde, thus reducing the amount of available butyraldehyde to be converted into propane by ADO. Our second reason has to do with this. Our models suggest that ADO is a bottleneck in our system. Therefore we would like to try to keep its substrate concentration high in the close proximity of ADO, so that it can function as efficiently as possible.

In the situation where CAR and ADO are fused with the amphiphilic proteins, we expect the amphiphilic proteins to form micelles rather than membranes, as the enzymes will most probably repulse each other. This is why we are calling the system amphiphilic micelles, but in reality it doesn’t matter to us whether the formations are micelles or membranes because the enzymes will nevertheless be closer together in both formations.

We wanted to test our hypotheses with something we could easily detect. We found the Violacein pathway [4], which could by the use of three enzymes produce a detectable green color. We believe, that if the violacein green color production could be enhanced by fusing these enzymes with the amphiphilic proteins, we could have an idea of whether the propane production could as well be enhanced in a similar way. There are obviously differences between these systems, but it would give us a rough estimate of whether these types of constructs would be possible to build in the first place.

Continuous Production