Sintezna biologija za proizvodnjo biobutanola

Abstract

In 2015, the first-ever Slovenian high school team competed at the iGEM (international Genetically Engineered Machine) competition in synthetic biology. The team was carefully selected from a list of candidates proposed by their high school teachers of chemistry and biology. Composed of eight students from 7 high schools from across the country, the team split into two groups, working in two institutions but with regular common meetings. The group of four students who worked at the National Institute of Chemistry focused on biotechnological production and the second group was preparing genetic constructs at the University of Ljubljana Faculty of Chemistry and Chemical Technology. The central problem that students tangled was the conversion of C-4 acids that are side-products of anaerobic waste degradation, into the corresponding alcohol. In the biotechnological part of the project students tested butanoic acid production in an anaerobic fermentation broth using a 15-channel computer-controlled bioreactor system, configured to allow online analysis of gaseous products (by GC) and frequent analysis of dissolved compounds (by HPLC). Next, at optimal butanoic acid production conditions (c = 1 g/L butanoic acid) growth of E. coli was examined in terms of medium composition, temperature and pH in order to obtain optimal operational parameters for biotechnological transformation of butanoic acid to butanol. Overall, E. coli growth was analysed in 72 different growth media in microtiter plates. Students followed inhibition of E. coli growth by butanol, acetone and isobutanol in the presence of butanoic acid and glycerol. The synthetic biology group started with PCR amplification of three genes ( ctf A and ctf B encoding two polypeptide chains of Co-A transferase, and bdh B encoding butyraldehide dehydrogenase for a two.stage conversion of butyril CoA to butanol) from anaerobic microbial community. Amplification failed, probably due to contaminants in the broth and a low number of bacteria harbouring these genes. Consequently, synthetic genes were designed with appended sequences for easier cloning, for a hexahistidine tag for detection of recombinant proteins using specific antibodies. Synthetic genes were inserted into pSB1C3 vectors and for each of them a strong promoter- ribosome binding site region was inserted in front of the enzyme coding region. Such combined constructs were further transferred into vectors with double terminators of transcription. In total, 9 different DNA constructs were prepared and deposited in the Registry of biological parts. All final expression constructs efficiently directed production of recombinant enzymes in E. coli DH5α under aerobic conditions. The giant jamboree of competing teams took place in Boston, USA, from September 24 to 28 with more than 260 teams from around the globe. In the high school track the Slovenian team presented the project entitled “From waste to fuel: Reprogrammed E. coli for sustainable production of biobutanol from butanoic acid”. The project received high recognition, as it was awarded a gold medal for completed tasks and was nominated among five best teams in categories Best Integrated Human Practices, Best Wiki, Best Presentation and Best High School Project.