Welcome to Slovenia HS's team Wiki!

ABOUT US

We are the first high school team from Slovenia to compete at iGEM and are very excited to face the challenges synthetic biology poses. The team is composed of eight high school students attending seven different secondary educational institutions across Slovenia. Our research is performed at the Laboratory for Environmental Sciences and Engineering, National Institute of Chemistry, Slovenia and at the Chair of Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana under supervision of knowledgeable and encouraging mentors.

         

WHAT WE DO

We have decided to tackle a very imposing ecological problem that is becoming more and more threatening due to fossil fuel depletion and a steady growth of energy consumption. Fossil fuels, such as oil and gasoline, have gained popularity in the beginning of the 20th century, following the industrial revolution. It is estimated that we have spent between 100 and 135 billion tons of oil since 1850 and the demands are still increasing. Fossil fuels are used in cars, airplanes and other vehicles, to power electricity plants, to heat our homes and to make many everyday products, such as medicines, cosmetics, plastics and synthetic fabrics. Our society is largely dependent on them, but fossil fuels take millions of years to form and are therefore non-renewable resources. According to some projections, we only have enough oil for the next 40 years, so it is becoming increasingly necessary to find an alternative method of obtaining fuels.

After a thorough discussion and literature survey, we have thus decided to make use of butanol. Research has already shown that because of its long chain and consequent nonpolarity, butanol can, amongst other uses, replace gasoline in internal combustion engines (it can be used in existing diesel engines without considerable modifications of the motor system).

In nature, many organisms have been proven to be able to produce butanol (n-butanol or 1-butanol) by converting glucose into acids and then converting acids into alcohols. This organisms, mostly bacteria of the Clostridium genus (Clostridium acetobutylicum, Clostridium beijerinckii and Clostridium saccharoperbutylacetonicum), however, have complex metabolism, slow conversion rate and are often hard to grow in laboratories or for industrious use. For these and other reasons, they are unsuitable for larger butanol production. On the other hand, bacteria E. coli have relatively simple and strikingly fast metabolism, already researched and utilized mechanisms for genetic manipulation and are relatively easy to grow and cultivate, making them the perfect laboratory and industrial organisms.

We have sought help from the Laboratory for Environmental Sciences and Engineering at the National Institute of Chemistry in Ljubljana, as they have been engaged in advancing processes that enable conversion of waste and other renewable raw materials into energy (e.g. biogas to syngas transformation, bio-oil production through pyrolysis of waste etc.) for a long time now, and have recently also developed a process for the biotechnological conversion of biodegradable waste into hydrogen by means of anaerobic mixed cultures grown at special fermentation conditions. During this process a series of intermediate products is produced, amongst others are high concentrations of butanoic acid. We had the idea of turning this organic acid into butanol, which is hydrophobic and can be separated from water medium by basic concentration and decanting processes.

That’s why we have set on the path to modify the E. coli in such a way, to enable transformation of butanoic acid to butanol. To aid in the process, we will also be using glycerol as a co-substrate, which can be readily obtained in a suitable form as a side product of biodiesel production processes.

We will thus genetically manipulate the E. coli bacteria into performing only the second phase of butanol production found in Clostridium bacteria (acids to alcohols conversion). By using biodegradable waste as substrate we intend to fully make use of all of the components involved in this processes (anaerobic microbial production of hydrogen), while subsequently (E. coli fermentation process) also producing the much-needed biofuel, thus enabling a simultaneous efficient and eco-friendly waste management and energy production process. Our aim is to achieve the highest possible yields with our genetically modified in an optimized bioreactor system.

In the first stage the biogas (H₂) will be isolated out of organic waste and suspended solids will be produced which may be used as a natural fertilizer. At the same time the butanoic acid formed will be redirected in another bioreactor (containing our modified E. coli) where it will be converted into bio-butanol.

Up to now, we have cloned three genes coding for CTFA, CTFB and BDHB proteins (in PSB1C3 vector), that are responsible for butyrate aldehyde to butanol in Clostridium acetobutylicum. We further intend to make composite constructs to include this genes in vectors with promotors and ribosomal binding sites to allow for expression of these genes in E. coli.

In the biotechnological laboratory we have performed an initial screening of growth conditions, such as butanoic acid concentration and butanoic acid / glycerol molar ratio, as well as comparing the growth in the presence and absence of oxygen. We are working with two negative controls, (i) E. coli strain Dh5 alpha and E. coli strain Dh5 alpha with PSB1C3 plasmid containing only the promotor and the ribosomal binding sites (i.e. with no genes included), to provide proof E. coli does not inherently convert butyric acid into butanol. We intend to perform similar tests with our final microorganism – modified E. coli and provide proof of concept for the said fermentation in a bench-scale laboratory bioreactor.

We are also looking for other teams with similar projects for possible collaborations, so if you are interested, we encourage you to contact us!