Part:BBa_K395602:Experience

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Applications of BBa_K395602

We performed gas chromatography to confirm the production of the esters, and the results revealed that MpAAT1 successfully converted alcohol and Acetyl CoA into our target esters (Fig. 1).

Fig. 1. Gas chromatography analysis of apple fragrance expression device.
(1) MpAAT1 + BuOH, (2) No MpAAT1 + BuOAc, (3) No MpAAT1 + BuOH, (4) MpAAT1 + 2-MeBuOH, (5) No MpAAT1 + 2-MeBuOAc, (6) No MpAAT1 + 2-MeBuOH, (7) MpAAT1 - 2-MeBuOH.
This work is done by Toshitaka Matsubara.

====Strains of E. coli====
E. coli BL21 (DE3)

====Varieties of plasmid====
MpAAT1 on pSB6A1 Trx on pACYC184

====Substrate====
Butanol (final 0.4%) 2-methyl butanol (final 0.2%)

====Inducer====
100 mM IPTG (final 100 μM) 20% arabinose (final 0.1%)

====Internal standard solution====
Undecane solution: undecane 10 μL + ether 990 μL

====MpAAT1 expression vector====
The coding sequence of MpAAT1 gene was synthesized and optimized sequence by Mr.Gene. This artificial gene was ligated into vector pSB6A1 as MpAAT1 expression vector. Moreover, we introduced pTrx6 into this expression plasmid to stabilize the MpAAT1 gene product.

====MpAAT1 over expression conditions====
Artificial gene has T7 promoter on the upstream of MpAAT1. This promoter works by taking over T7 RNA polymerase from E. coli. Therefore we utilized E. coli BL21 (DE3) which has T7 RNA polymerase. Furthermore, arabinose was added in culture to induce Trx which has arabinose-induced promoter.

====Cultivation ====
In order to express MpAAT1 and Trx in E. coli BL21 (DE3), we added 3 μL of 100 mM IPTG and 15 μL of 20% arabinose in 3ml LB culture.

Expression of MpAAT1 recombinant protein in E. coli

The cells were grown over night. The microbial solution was diluted 100-folds and grown for 2 hours in a fresh culture. After grown until the O.D. becomes 0.1, substrate and antibiotic, and inducer were added into the culture. This was grown overnight again. After the incubation, we centrifuged the solution (7000 × g, 3 min) and collected the supernatant. Then, ether was used to separate oil through liquid-liquid solution. (shake supernatant solution 0.5 mL, with ether 0.5 mL and undecane solution). Finally, the oil layer was collected and analyzed by gas chromatography.

====Gas Chromatography analysis====
Gas Chromatography : SHIMADZU GAS CHROMATOGRAPH GC-14B Column: J&W SCIENTIFIC, DB-17, Film thickness 0.25 μm, Column Dimensions 15 m × 0.320 mm, Temperature Limits 40°C to 280°C (300°C Program) Conditions: column temperature 35°C, injector temperature 180°C, detector temperature 180°C Sample was injected 5 μL.

(→[http://2010.igem.org/Team:Tokyo_Tech/Project/Apple_Reporter2 our wiki])

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