Part:BBa_K395602

Apple fragrance generator

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])

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1765
  Illegal NotI site found at 1666
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 195
  Illegal BamHI site found at 1635
  Illegal XhoI site found at 1675
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 205
  Illegal AgeI site found at 1419
  
• 1000
  COMPATIBLE WITH RFC[1000]