Conclusions
We have decided to tackle an imposing ecological problem of oil consumption. The fact is that fossil fuel consumption has increased in the 21st century and that we depend on oil in almost all areas of our life. But fossil fuels are a finite source of energy, so we have decided to try to find an alternative for this in our iGEM project. Oil reserves are going to be emptied in a few decades and in that time we have to find an acceptable biofuel for our civilization to be able to survive. We have therefore decided to try to create a system of transforming waste butanoic acid into butanol, similar enough to oil that it acts as a biofuel without any modifications for internal combustion engines. Butanoic acid is a side-product in bioethanol production and represents a waste that seems to be useless. The amount of butanoic acid produced is as much as 3 g/L. To accomplish our goal, we needed three genes. We could not have imagined at the beginning of the project how much effort our work was going to take, but at the end we are satisfied with our results.
We have managed to ligate the ordered three gene fragments into empty pSB1C3 vector, and then ligate into that vector a double terminator and a promoter and RBS as well. So it was 3 cloning steps for each gene. Through this process we have created nine new functioning BioBricks, which we have characterized, documented and submitted to the Registry.
We were able to confirm that our genes are functional and active when promoter is added with SDS- PAGE and Western blot to determine that the proteins were indeed our specific protein, which we have planned with a His-tag. The proteins are expressed in the insoluble fraction of the lysate, most likely due to the use of a strong promoter and a strong RBS. The cells were normally grown 37 °C, but after realising the proteins are expressed in the insoluble fraction, we also tried to grow them at lower temperatures. We tried growing them at 20 °C and 16 °C, at which point, quite some of the protein was finally expressed in the soluble fraction. This needs more optimization thought, and perhaps the use of promoter and RBS with less strength will produce the protein in the soluble fraction. Ideally, the three constructs (containing CtfA, CtfB and BdhB) would be ligated together so that the bacteria are able to perform the conversion from butanoic acid to butanol after only one transformation.
As a source of carbon in our bioreactor experiments we used waste from which we produced biohydrogen, which can be used as a source of energy. The other by products involve bacterial biomass, which could prove useful as a fertilizer in agriculture, and butanoic acid, which we use as a source of carbon for the next reaction, in which butanoic acid is converted into butanol. In addition to the butanoic acid we added glycerol, which enabled the final production of butanoic acid to butanol.
One of the biggest steps of testing the growth conditions for E. coli was optimization, which was troublesome due to slow growth of bacteria and the strain of maintain of anaerobic conditions, in which the production of butanol is performed. Since E. coli is a facultative anaerobic organism, it can grow in anaerobic conditions; however the growth is much slower in comparison to the growth in aerobic conditions. Another issue with optimization was testing different ratios of substrates in such a way, that the least glycerol and as much butanoic acid as possible would be used, while still retaining the amount of substrates low enough, so that it wouldn’t be toxic for our bacteria. We have done our best to replicate the different factors that could influence the conversion could be preformed and optimize conditions for that conversion. Applying this in practice after the bacteria is successfully transformed with a single plasmid carrying all three genes is the next stage of our project. When put to a larger scale, such production would lower the need for fossil fuels in everyday life as well as offer new jobs. Butanol spills are also easily biodegraded to non-toxic concentrations in comparison with gasoline. Produced biobutanol could be used as fuel for various vehicles and transport, such as cars, public transport and even farm tractors. Additionally, it would prove as a source of energy for electric generators and central heating in houses and large buildings, like schools and hospitals.
Besides completing our project satisfactory we are also proud to have done some other work as well. We have validated a BioBrick created by the Aalto-Helsinki team and characterized three already existing BioBricks.
We also successfully presented iGEM and synthetic biology to the public, engaging and educating at the same time, to children, students and adults. We engaged in debates and contemplated on the ethics of synthetic biology and expanded our horizons concerning science, the natural world and the whole of a human experience.
