Lab 8 - Lab 8 Assay of Isobutanol Production Using...

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Lab 8: Assay of Isobutanol Production Using Metabolically-Engineered, Biofuel-Producing E.coli Strains. Introduction The purpose of this lab was to determine the concentration of isobutanol produced in culture by metabolically-engineered, biofuel-producing E.coli strains enzymatically. In this lab, the isobutanol concentration in the culture supertanant from three different strains was compared. All the three strains were engineered to produce isobutyraldehyde through the overexpression of the genes encoding acetolactate synthase (alsS), acetohydroxy acid isomeroreductase (ilvC), dihydroxy-acid dehydrogenase (ilvD), and 2-ketoacid decarboxylase (kivd). In addition, the each stains overexpressed a specific alcohol dehydrogenase which were either yqhD (from E.Coli), ADH2 (from Saccharomyces cerevisiae), or adhA (from Lactcoccus lactis) as table below. These stains were controlled by the E.coli lactose operon repressor (LacI). However, expression was induced by the addition of IPTG because IPTG was not decomposed of ether bond (-O-) by LacI. Strain 1 SA542/pSA69 (p15A ori; Kan R ; P l lacO 1 : alsS-ilvCD)/ pSA138 (ColE1 ori; Amp R ; P l lacO 1 : kivd-yqhD) Strain 2 SA542/pSA69 (p15A ori; Kan R ; P l lacO 1 : alsS-ilvCD)/ pSA55 (ColE1 ori; Amp R ; P l lacO 1 : kivd-ADH2) Strain 3 SA542/pSA69 (p15A ori; Kan R ; P l lacO 1 : alsS-ilvCD)/ pSA138 (ColE1 ori; Amp R ; P l lacO 1 : kivd-adhA) Table 1: The relevant genotypes of three strains. In this lab, isobutyraldehyde was produced by Colorimetric Enzyme-based Detection. We could measure the Alcohol dehydrogenase by measuring NADH production from spectrophotometer in 340 nm. In this reaction, ADH2 catalyzed the isobutanol to isobutyraldehye with reduction of NAD + to NADH. By the NADH, 1-methoxyphenzine methosulfate (MTT) would be reduced. Then, NADH formation was coupled to the reduction of 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (yellow) to its formazan derivative (MTT formazan) (purple) in 570 nm.
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Figure 1: Colorimetric enzyme-based detection of isobutanol with coupled assay.
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  • Winter '14
  • stevenkim
  • Enzyme, isobutanol

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