E. coli Producing Propane

Our propane pathway is based on the research done by Dr. Pauli Kallio et al and Menon et al [9, 10]. Right after we decided our topic we got in touch with Dr. Pauli Kallio from the University of Turku. He was very excited about our project and eager to help. We were able to get Kallio’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, Escherichia coli BL21 (DE3), was chosen because it’s a strain that produces the 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), Dr. Kallio 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 Kallio’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 their own rbs and a T7 terminator. We chose to use the same RBS's and terminator sequences as Kallio's group, as they had already tested the system. Our construct's 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 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 2000 bp each and included 30 bp 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 constraint 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. 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.

Degrading Cellulose

To further develop the idea of using propane as a biofuel, feedstock alternatives were sought as glucose itself interferes with food production. At first, we planned to use cyanobacteria as a host organism to produce carbohydrates to the propane pathway but with the current time interval the project would become too challenging to be accomplished. However, non-edible carbon sources like cellulose were already investigated by previous iGEM teams and cellulose hydrolysis by bacteria wasn’t connected to the propane pathway yet. Therefore, we decided to create an application that could use the utilization of cellulosic waste in order to generate a more ethical life cycle for our biofuel, which could be easily integrated into the original plan.

Because we wanted to create the abilities of degrading cellulose and producing propane into the same strain, genes expressing cellulases needed to be in the same backbone. Two plasmids already contained propane pathway genes and the approximated limit of the insert size in the plasmid is about 10 kb. The number of different degrading enzymes was limited to three cellulases in order to limit the plasmid size and the stress of the organism. To convert cellulose polymers into glucose units, the proper hydrolysis requires enzymes endoglucanase, exoglucanase and β-glucosidase. Endoglucanases hydrolyze covalent bonds from the middle of polysaccharide and exoglucanases cleave oligosaccharides from the ends of the chain. β-glucosidases convert oligosaccharides into glucose which are utilized for metabolic reactions.

Usually, fungal strains Trichoderma reesei and Saccharomyces cerevisiae produce two different types of endoglucanases and exoglucanases belonging to different enzyme families. Due to our enzyme limit, only Type I cellulases (CenA, accession M15823 and Cex, accession M15824) from Cellulomonas firmi were chosen to be expressed. We found only a couple of Biobricks which could be used for our project from the iGEM registry but those parts had complicated status. Fortunately, the sequences were available on the registry for parts BBa_K392006 and BBa_K392007 and Distribution Kit 2015 contained the gene responsible for β-glucosidase BBa_K118028 from Cytophaga hutchinsonii. Therefore, we decided to synthesize endoglucanase and exoglucanase expressing genes utilizing the sequences previously mentioned and take β-glucosidase from the kit. These three sequences will be amplified with PCR and connected with the homology ends of genes created by primers. The connections will also be made with Gibson Assembly method. The cellulase genes are positioned downstream to the same T7 promoter induced by IPTG.

E.coli strains do not naturally contain any secretion systems for endoglucanases and exoglucanases so if the bacteria has previously been researched for cellulose degradation, usually the enzymes are separated from cytoplasm with overflow. However, BL21 strain which we were using naturally contained β-glucosidase gene in the genome but because the expression levels and enzyme activities were poorly documented in databases, we decided to add the gene Osaka team was using. For cenA and cex secretion, pelB-secretion tag sequence BBa_J32015 made by 2010 Duke team was synthesized before the genes to be fused with the cellulases which would be moved into cell’s periplasmic space.

Cellulase producing capacity will be first investigated with carboxymethyl cellulose (CMC) plates which are labeled with Congo Red assay. Congo red dye is the sodium salt of 3,3'-([1,1'-biphenyl]-4,4'-diyl)bis(4-aminonaphthalene-1-sulfonic acid) which has a strong affinity to cellulose fibers. If the cellulose polymers are digested by enzymes on the plate, the spot will be changed into colorless and a halo will appear around the bacterial colony. However, the method will not tell whether the glucose is produced or not.

For glucose analysis, 3,5-dinitrosalicylic acid will be utilized. It is a compound which reacts with reductive sugars like glucose forming 3-amino-5-nitrosalicylic acid. The product absorbs light with the wavelength of 540 nm. Three different controls are needed when analyzing the produced glucose concentration because the cultivation liquid with CMC already contains sugar: one without any strain, one with E.coli without cellulase producing capacity and one with cellulose hydrolyzing.

Micelle Fusions Enhancing the Production

Based on the previous studies about this pathway [9, 10], we knew the propane yields weren’t very high. We thought about trying to enhance the system by searching for homologs for the enzymes, but thought this would be too time-consuming and also not very innovative. We then ran into a research article by Huber et al [11]. The group had designed a synthetic amphiphilic protein that spontaneously formed membrane-like structures inside the cell. These proteins were 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 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 and 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 our models suggesting 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, 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.

Constructs

Assembly Methods

Both two of our propane plasmids and our cellulose plasmid were constructed with homologous pairing. Our main approach was Gibson Assembly which is based on > 25 bp homologous regions between each piece that we want to combine. The Gibson Assembly master mix includes the enzymes exonuclease, DNA polymerase and ligase. The method functions so that the exonuclease starts degrading the parts 5’ ends and reveals the homologous regions as single stranded DNA. The exonuclease then inactivates because of the reaction temperature, and homologous pairing takes place. After this DNA polymerase will fill in any gaps that were produced by the exonuclease, and ligase ligates the pieces together. The image below describes the system when one insert is combined with a backbone. With Gibson, you can simultaneously combine up to at least 6 fragments. Our Propane Plasmid 1 and Cellulose Plasmid needed a four fragment assembly and Propane Plasmid 2 a five fragment assembly. The principle is the same regardless of how many pieces you wish to combine.

Our backup plans for Gibson Assembly were ELIC and OE-PCR. Of these, ELIC is a method that’s only been published in March 2015 and is thus poorly understood. Basically linear pieces of DNA, which contain homology to one another are transformed into a cell, and the cell’s endogenous homologous recombination system assembles these into a plasmid. OE-PCR also takes advantage of the homologous regions, but uses these as primers. The PCR program follows the regular guidelines, but the annealing temperature is based on the homologous region. As the strands of DNA detach from each other, the homologous area functions as a primer that allows the polymerization of the whole construct.

Backbones

As we ordered our constructs as gBlocks, they needed to be divided into up to 2000 bp pieces. We added 30 bp overlaps to each of our piece to enable the Gibson Assembly for our pieces. Due to a design mistake though, we didn’t design a 30 bp overlap between our Brick and the backbone: we thought we would be able to construct the bricks as linear DNA and ligate them into the backbone with the help of restriction enzymes. We did however have the prefix on all of our first brick-to-be pieces and had added the suffix with PCR. We hoped that the homologous area created by the prefix and suffix (about 15 bp) would be enough to attach our construct to the BioBrick backbone pSB1C3.

The situation became a bit more complicated when it came to attaching the Propane Plasmid 2 and the Cellulose Plasmid into the commercial pACYCDuet-1 and pCDFDuet-1 vectors. As these were cloning vectors, they already contained the T7 promoter, RBS and terminator. Better yet, since they are Duet plasmids, they contain all of these for two different genes. To avoid the risk of our plasmid producing an unwanted product from another vector promoter, we restricted the second expression system out completely. As our Propane Plasmid 2 construct was based on Kallio’s plasmid maps, which were also built into Novagen’s vectors, our T7 and the first RBS were the same as in the vectors. Thus, after our construct is in the biobrick backbone, we will use new primers which will remove the prefix of our constructs 5’ end and add a homologous region to the vector in the 3’ end (31 bp). With the Cellulose Plasmid, the T7 and RBS sequences differed from Novagen’s vectors, so we needed to design a forward primer as well which created a homologous region for the vector in our constructs 5’ end. The picture on top left describes the primer design to transfer the Cellulose Brick into a commercial backbone and the picture below the transfer of a propane plasmid to a commercial backbone.

Amphiphilic constructs

To add the amphiphilic proteins, we used PCR to remove the terminator and the suffix from the end of our Propane Plasmid 1 and Propane Plasmid 2 Bricks. Our primers added a region to the 3’ end of our Propane bricks to create homology with the amphiphilic protein and its linker. The amphiphilic protein was synthesized including one linker, and for CAR we added a second one with a primer. A suffix had been added to the 3’ end of the synthesized amphiphilic protein with PCR primers.

These same methods were supposed to be used to create the Violacein construct with and without amphiphilic fusions, but due to the time constraints, we weren’t able to assemble any of our Violacein Plasmids.

Continuous Production

The project is based on research done in the University of Turku [9] by Pauli Kallio and others, who were kind to send us their E. coli strain BL21 (DE3 ΔyjgB ΔyqhD, pET-TPC4 + pCDF-cAD + pACYC-Fdx-Fpr) which produces propane with the reaction pathway utilizing the intermediates of fatty acid biosynthesis and enzymes like FASII and Tes4. Onwards from the butyrate, the reaction pathway’s enzymes are similar to our reactions'. The strain has already been cultivated as a bench scale using an Erlenmeyer flask. The authors of this research proposed that the propane producing capacity could be also tested with continuous production where the cells are grown until the steady state is achieved. The steady state will be maintained with the regular feed of fresh media into a chemostat while the outline will continuously remove the same amount of media with products. Thus, the growth rate can be readily controlled by changing the diluting speed of media.

Because the strain BL21 (DE3 ΔyjgB ΔyqhD, pET-TPC4 + pCDF-cAD + pACYC-Fdx-Fpr) produces propane only by inducing with IPTG instead of constitutive production, certain limitations are met when designing the chemostat conditions. Cultivating microbes with a maximum capacity would consume too much IPTG which is quite a valuable reagent as a diluting rate needs to be high. Therefore, the chemostat will be used for investigating how the environmental conditions affect the product formation. The easiest way is to wait until the steady state without any induction and add after that IPTG. Because the process conditions are changed, OD600 is measured all the time.

The overall media volume of chemostat will be 500 ml due to the limit of the IPTG amount. The steady state is approximated to take 10 hours but it may vary radically. Diluting rate is kept to 0.5 1/h and glucose feed 10 g/L. When OD600 reaches its balance, the IPTG concentration for media is increased to 0.5 mM. Induction will take another 10 hours so the overall process time will be probably few days. Gas products will be measured with GC-MS analysator which will be connected with the outlet stream of the chemostat. Because the reactor size is limited (2 L), only few sensors can be attached to the same unit. Probably we are going to measure the cell density and pH-levels.