Difference between revisions of "Part:BBa K1448001"
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<partinfo>BBa_K1448001 short</partinfo> | <partinfo>BBa_K1448001 short</partinfo> | ||
This device contains the region of the TEF1 yeast constitutive promoter, GST tag, ɤ-terpinene synthase coding site(BBa_K1448000), and CYC1 terminator. This device can constitutively express ɤ-terpinene synthase in yeast (introducing this device makes yeast cells express ɤ-terpinene synthase constitutively). | This device contains the region of the TEF1 yeast constitutive promoter, GST tag, ɤ-terpinene synthase coding site(BBa_K1448000), and CYC1 terminator. This device can constitutively express ɤ-terpinene synthase in yeast (introducing this device makes yeast cells express ɤ-terpinene synthase constitutively). | ||
+ | |||
+ | |||
+ | == '''Characterization''' == | ||
+ | |||
+ | |||
+ | |||
+ | === Western-bloting analysis === | ||
+ | |||
+ | |||
+ | [[File:westernblot of terpinene.png|thumb|500px|right|(Fig.2 Western-bloting analysis of GST and d-limonene synthase (Lane2), ɤ-terpinene synthase (Lane3) and β-pinene synthase(Lane4) fusion proteins expressed in yeast. (A) The gel picture of SDS-PAGE and western-blot; Lanes: 1, p427-TEF; 2, p427-TEF-LS; 3, p427-TEF-ɤTPNS; 4, p427-TEF-βPINS. (B) vector map of plasmids. (C) The names and molecular weights of each mono-terpene synthase.)]] | ||
+ | |||
+ | Yeast cells carrying plasmids of p427-TEF-GST-monoterpenoid synthase were cultivated overnight in SD medium (0.67% yeast nitrogen base and 2% glucose with G418) 50mL at 28°C. Cells were collected and kept at -30˚C. Western blot analysis was carried out using anti-GST antibody and 12.5% polyacrylamide gel with SDS. | ||
+ | |||
+ | '''<Results>''' | ||
+ | |||
+ | Western blot data (Fig.2) showed clear bands around 90kDa in the lanes of p427-TEF- monoterpenoid synthase but not in the lane of empty vector. | ||
+ | <br><br><br><br><br><br> | ||
+ | <br><br><br><br><br><br> | ||
+ | <br><br><br><br><br><br> | ||
+ | <br><br><br><br><br><br> | ||
+ | <br><br><br><br><br><br> | ||
+ | |||
+ | ---- | ||
+ | |||
+ | === In vivo detection of ɤ-terpinene using GC === | ||
+ | |||
+ | |||
+ | '''Introduction''' | ||
+ | |||
+ | Since ɤ-terpinene is a volatile material, we expected that an amount of ɤ-terpinene were detected at the headspace of the culture in which the transformant with ɤ-terpinene generator is cultivated and tried the detection by GC analysis. | ||
+ | |||
+ | |||
+ | [[File:detection of ɤ-terpinene using GC-MS.png|thumb|700px|Fig.4 GC-analysis data]] | ||
+ | |||
+ | ===GC analysis ofɤ-terpinene in the culture=== | ||
+ | |||
+ | |||
+ | '''Materials and Methods''' | ||
+ | |||
+ | We cultivated the transformants containing ɤ-terpinene generator in p427-TEF or only empty p427-TEF in 50 mL SD medium (0.67% yeast nitrogen base and 2% glucose with G418) at 29˚C, 120 rpm for 24 h. | ||
+ | |||
+ | Analytic instrument: Shimadzu GC14A | ||
+ | |||
+ | Column: GLScience InertCap1 (15m Length,0.25 mm I.D., 0.25mm film thickness) | ||
+ | |||
+ | Injection port 200C, Detector port 210 C | ||
+ | |||
+ | Detector: Flame Ionization Detector | ||
+ | |||
+ | Column Oven Temperature 40C/5min- 10C/min-200C/5min | ||
+ | |||
+ | Carrier Gas: Helium | ||
+ | |||
+ | |||
+ | |||
+ | '''Result''' | ||
+ | |||
+ | |||
+ | we uesd SPME (SOLID PHASE MICROEXTRACTION) to catch ɤ-terpinene at the headspace of the culture. However, any amounts of ɤ-terpinene were not detected at the headspace of the culture (the exposure time is 30min). | ||
+ | |||
+ | Next we tried to catch ɤ-terpinene in the liquid phase of the culture using monotrap (the exposure time is 1h) and an amount of ɤ-terpinene was detected in the liquid phase of the culture. | ||
+ | |||
+ | ---- | ||
+ | ===Growth curve=== | ||
+ | <br> | ||
+ | [[File:Growth curve of yeast cells containing ɤ-terpinene generator.png|thumb|500px|right|Fig.4 transformant: BY4742 containing ɤ-terpinene generator inserted in p427-TEF, control: BY4742 containing empty p427-TEF ]] | ||
+ | '''Introduction''' : | ||
+ | Various mono-terpenoids are known to have an anti-microbial property. In order to evaluate the anti-microbial property of -terpinene, we made the growth curve of yeast cells containing -terpinene generator and compare to that of yeast cells without -terpinene generator. | ||
+ | <br><br> | ||
+ | '''Method''' : | ||
+ | We cultivated the transformants containing -terpinene generator in p427-TEF or only empty p427-TEF in 50 mL SD medium (0.67% yeast nitrogen base and 2% glucose with G418) at 29˚C, 120 rpm for 24 h. OD600 was measured every 2 h. | ||
+ | <br><br> | ||
+ | '''Result''' : | ||
+ | Yeast cells containing ɤ-terpinene generator showed slower growth than cells containing empty p427-TEF, indicating that γ-terpinene inhibits the growth of yeast cells. | ||
+ | <br> | ||
+ | <br><br> | ||
+ | <br><br><br><br><br><br> | ||
+ | <br><br> | ||
+ | |||
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Latest revision as of 02:39, 18 October 2014
Yeast ɤ-terpinene generator
This device contains the region of the TEF1 yeast constitutive promoter, GST tag, ɤ-terpinene synthase coding site(BBa_K1448000), and CYC1 terminator. This device can constitutively express ɤ-terpinene synthase in yeast (introducing this device makes yeast cells express ɤ-terpinene synthase constitutively).
Characterization
Western-bloting analysis
Yeast cells carrying plasmids of p427-TEF-GST-monoterpenoid synthase were cultivated overnight in SD medium (0.67% yeast nitrogen base and 2% glucose with G418) 50mL at 28°C. Cells were collected and kept at -30˚C. Western blot analysis was carried out using anti-GST antibody and 12.5% polyacrylamide gel with SDS.
<Results>
Western blot data (Fig.2) showed clear bands around 90kDa in the lanes of p427-TEF- monoterpenoid synthase but not in the lane of empty vector.
In vivo detection of ɤ-terpinene using GC
Introduction
Since ɤ-terpinene is a volatile material, we expected that an amount of ɤ-terpinene were detected at the headspace of the culture in which the transformant with ɤ-terpinene generator is cultivated and tried the detection by GC analysis.
GC analysis ofɤ-terpinene in the culture
Materials and Methods
We cultivated the transformants containing ɤ-terpinene generator in p427-TEF or only empty p427-TEF in 50 mL SD medium (0.67% yeast nitrogen base and 2% glucose with G418) at 29˚C, 120 rpm for 24 h.
Analytic instrument: Shimadzu GC14A
Column: GLScience InertCap1 (15m Length,0.25 mm I.D., 0.25mm film thickness)
Injection port 200C, Detector port 210 C
Detector: Flame Ionization Detector
Column Oven Temperature 40C/5min- 10C/min-200C/5min
Carrier Gas: Helium
Result
we uesd SPME (SOLID PHASE MICROEXTRACTION) to catch ɤ-terpinene at the headspace of the culture. However, any amounts of ɤ-terpinene were not detected at the headspace of the culture (the exposure time is 30min).
Next we tried to catch ɤ-terpinene in the liquid phase of the culture using monotrap (the exposure time is 1h) and an amount of ɤ-terpinene was detected in the liquid phase of the culture.
Growth curve
Introduction :
Various mono-terpenoids are known to have an anti-microbial property. In order to evaluate the anti-microbial property of -terpinene, we made the growth curve of yeast cells containing -terpinene generator and compare to that of yeast cells without -terpinene generator.
Method :
We cultivated the transformants containing -terpinene generator in p427-TEF or only empty p427-TEF in 50 mL SD medium (0.67% yeast nitrogen base and 2% glucose with G418) at 29˚C, 120 rpm for 24 h. OD600 was measured every 2 h.
Result :
Yeast cells containing ɤ-terpinene generator showed slower growth than cells containing empty p427-TEF, indicating that γ-terpinene inhibits the growth of yeast cells.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 204
Illegal XhoI site found at 715 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]