Difference between revisions of "Part:BBa K1653025"
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<partinfo>BBa_K1653025 short</partinfo>
 
<partinfo>BBa_K1653025 short</partinfo>
  
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FOH is probably generated through FPP hydrolysis by endogenous phosphatases, which are induced by an increased intracellular FPP level. Analogously, we hypothesized that ''E. coli'' could produce FOH under cellular conditions of an increased intracellular FPP level through metabolic engineering. A MEP pathway has been shown to synthesize IPP and DMAPP efficiently in ''E. coli''.
  
  
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FOH is probably generated through FPP hydrolysis by endogenous phosphatases, which are induced by an increased intracellular FPP level. Analogously, we hypothesized that ''E. coli'' could produce FOH under cellular conditions of an increased intracellular FPP level through metabolic engineering. A MEP pathway has been shown to synthesize IPP and DMAPP efficiently in ''E. coli''. Because of its high hydrophobicity and low volatility, decane was chosen to extract and solubilize FOH from culture broth. The decane overlay in the two-phase culture did not affect growth, and FOH could be solubilized in the decane phase with negligible volatile loss. We adopt 1 mL of decane overlaid to 5 mL of culture broth. Two-phase culture of ''E. coli'' JM109 (BBa_K1653025) was carried out in 2YT medium containing  1% glycerol at 29°C for 48 h. The decane phase of the two-phase culture was collected to analyze the FOH content by GC-MS. In the GC-MS analysis (Fig. 4A-G), there was a main peak at 8.5 min in the collected decane phase sample, which corresponded to the reference solution of the standard FOH compound dissolved in decane. Mass spectrometry confirmed that the peak at 8.5 min was FOH (Fig. 4-A). However, the peak was not observed in two-phase culture without introducing BBa_K165025. The formation of FOH from FPP was further confirmed by blocking FPP synthesis. In the GC-MS, the FOH peak was observed in ''E. coli'' JM109 (BBa_K1653025)  culture, whereas no peak was observed with transformed ''E. coli'' JM109. It was found that FOH need not only ispA([https://parts.igem.org/Part:BBa_K1653018 BBa_K1653018]) but also Terpene precursor mass-production device([https://parts.igem.org/Part:BBa_K1653024 BBa_K1653024]) in ''E. coli''.　We submit new part(BBa_K1653025)  as producing FOH.
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Because of its high hydrophobicity and low volatility, decane was chosen to extract and solubilize FOH from culture broth. The decane overlay in the two-phase culture did not affect growth, and FOH could be solubilized in the decane phase with negligible volatile loss. We adopt 1 mL of decane overlaid to 5 mL of culture broth. Two-phase culture of ''E. coli'' JM109 (BBa_K1653025) was carried out in 2YT medium containing  1% glycerol at 29°C for 48 h. The decane phase of the two-phase culture was collected to analyze the FOH content by GC-MS. In the GC-MS analysis (Fig. 4A-G), there was a main peak at 8.5 min in the collected decane phase sample, which corresponded to the reference solution of the standard FOH compound dissolved in decane. Mass spectrometry confirmed that the peak at 8.5 min was FOH (Fig. 4-A). However, the peak was not observed in two-phase culture without introducing BBa_K165025. The formation of FOH from FPP was further confirmed by blocking FPP synthesis. In the GC-MS, the FOH peak was observed in ''E. coli'' JM109 (BBa_K1653025)  culture, whereas no peak was observed with transformed ''E. coli'' JM109. It was found that FOH need not only ispA([https://parts.igem.org/Part:BBa_K1653018 BBa_K1653018]) but also Terpene precursor mass-production device([https://parts.igem.org/Part:BBa_K1653024 BBa_K1653024]) in ''E. coli''.　We submit new part(BBa_K1653025)  as producing FOH.
 
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<br>
 
[[http://2015.igem.org/Team:Nagahama/Medal_Parts#Farnesol_.28FOH.29_production_device.28BBa_K1653025.29 here] is our result page.]
 
[[http://2015.igem.org/Team:Nagahama/Medal_Parts#Farnesol_.28FOH.29_production_device.28BBa_K1653025.29 here] is our result page.]
  
 
[[File:ispA+MEP_dev3.jpg|thumb|center|600px|Fig4:The FOH standard solution (Ref) was used as a control. The peak corresponding to the FOH standard at 8.5 min is indicated by an arrow. The peak at 8.5 min was applied to GC/MS. The FOH standard solution (Ref) was used as a control. ''E. coli'' JM109(BBa_K165025) were compared with respect to FOH formation using GC-MS.]]
 
[[File:ispA+MEP_dev3.jpg|thumb|center|600px|Fig4:The FOH standard solution (Ref) was used as a control. The peak corresponding to the FOH standard at 8.5 min is indicated by an arrow. The peak at 8.5 min was applied to GC/MS. The FOH standard solution (Ref) was used as a control. ''E. coli'' JM109(BBa_K165025) were compared with respect to FOH formation using GC-MS.]]

Revision as of 11:44, 17 September 2015

Farnesol (FOH) production device

FOH is probably generated through FPP hydrolysis by endogenous phosphatases, which are induced by an increased intracellular FPP level. Analogously, we hypothesized that E. coli could produce FOH under cellular conditions of an increased intracellular FPP level through metabolic engineering. A MEP pathway has been shown to synthesize IPP and DMAPP efficiently in E. coli.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 948
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 4735
    Illegal BamHI site found at 4063
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 244
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1937


Because of its high hydrophobicity and low volatility, decane was chosen to extract and solubilize FOH from culture broth. The decane overlay in the two-phase culture did not affect growth, and FOH could be solubilized in the decane phase with negligible volatile loss. We adopt 1 mL of decane overlaid to 5 mL of culture broth. Two-phase culture of E. coli JM109 (BBa_K1653025) was carried out in 2YT medium containing 1% glycerol at 29°C for 48 h. The decane phase of the two-phase culture was collected to analyze the FOH content by GC-MS. In the GC-MS analysis (Fig. 4A-G), there was a main peak at 8.5 min in the collected decane phase sample, which corresponded to the reference solution of the standard FOH compound dissolved in decane. Mass spectrometry confirmed that the peak at 8.5 min was FOH (Fig. 4-A). However, the peak was not observed in two-phase culture without introducing BBa_K165025. The formation of FOH from FPP was further confirmed by blocking FPP synthesis. In the GC-MS, the FOH peak was observed in E. coli JM109 (BBa_K1653025) culture, whereas no peak was observed with transformed E. coli JM109. It was found that FOH need not only ispA(BBa_K1653018) but also Terpene precursor mass-production device(BBa_K1653024) in E. coli.　We submit new part(BBa_K1653025) as producing FOH.
[[http://2015.igem.org/Team:Nagahama/Medal_Parts#Farnesol_.28FOH.29_production_device.28BBa_K1653025.29 here] is our result page.]

Fig4:The FOH standard solution (Ref) was used as a control. The peak corresponding to the FOH standard at 8.5 min is indicated by an arrow. The peak at 8.5 min was applied to GC/MS. The FOH standard solution (Ref) was used as a control. E. coli JM109(BBa_K165025) were compared with respect to FOH formation using GC-MS.