Difference between revisions of "Part:BBa K1049002"

 
 
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<partinfo>BBa_K1049002 short</partinfo>
 
<partinfo>BBa_K1049002 short</partinfo>
  
 
This device converts isoamyl alcohol to the odor isoamyl acetate.
 
This device converts isoamyl alcohol to the odor isoamyl acetate.
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 +
== 1,SDS-PAGE ==
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We constructed ATF2 generator. (BBa_K1049002) Our team KIT-Kyoto 2013 constructed this part for the purpose of measurement. T7 promoter is an IPTG-inducible promoter. We added 20µL IPTG (100mM) to our genetically modified E.coli after cultivation at 28 and 37 ˚C. 2 hours after, we extracted soluble proteins from it by FastBreak™ Cell Lysis Reagent and did SDS-polyacrylamide gel electrophoresis.
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 +
[[File:KIT2013SDS.png|500px]]
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 +
ATF2 gene encodes AATase, which is about 62kDa. The consumption of protein marker is like this.
 +
Myosin 200kDa β‐Galactosidase 120kDa Bovine Serum Albumin 95kDa Glutamine dehydrogenase 68kDa Ovalbumin 50kDa Carbonic Anhydrase 36kDa Myoglobin 27kDa Lysozyme 20kDa Aprotinin 10kDa
 +
You can find the band at lanes which are added IPTG just beneath the band of 68kDa.
 +
 +
 +
 +
 +
== 2,Growth curves ==
 +
We measured the turbidities of the transformants every 2 hour and made the growth curves. We transferred the transformant prepared to 100 mL flasks and add 660uL IPTG and 108uL isoamyl alcohol to them after 2 hours of the start. We measured the turbidities every 2 hour. The measurements were carried out for 12 hours.
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 +
LB　100mL
 +
 +
ampicillin　150uL
 +
 +
sample 50uL
 +
 +
IPTG 660uL
 +
 +
isoamyl alcohol 108uL
 +
 +
37°C, 125/min
 +
 +
[[File:Growth atf2.png|500px]]
 +
 +
In addition, we measured the turbidities of the transformants without adding IPTG and isoamyl alcohol too. We measured 4 samples; ATF2-pET-15b, ATF2-pET-15b + IPTG, empty pET-15b, empty pET-15b + IPTG.
 +
As the result, ATF2-pET-15b transformants grow well relative to control(transformants having empty pET-15b vector). We propose the folllowing hypothesis. Isoamyl acetate indicates a stimulatory effect of growth on E.coli cells when compared to isoamyl alcohol so that transformants having ATF2-pET-15b grow well relative to control.
 +
And ATF2-pET-15b without IPTG grow well relative to ATF2-pET-15b add IPTG. It is explained by the following hypothesis. When we add IPTG to the transformant having ATF2-pET-15b, ATF2 protein (AATase) is expessed in bulk in the transformant. A massive amount of AATase has a potential to surpress the growth.
 +
 +
 +
 +
 +
== 3, Bioassay using Drosophila ==
 +
Next, to confirm the activity of AATase, we added isoamyl alcohol after IPTG induction and cultivated for about 2 hours. We used E. coli cells carrying the empty vector (pET-15b) as a control and compare it with the E.coli cells carrying pET15b-ATF2 after addition of isoamyl alcohol. To compare the production of isoamyl acetate, we carried out a bioassay using Drosophila. Because Drosophila favors the fruit odor like isoamyl acetate. [1]
 +
After the addition of IPTG and isoamyl acetate, the culture was impregnated into the filter and placed in a case containing 10 Drosophilas. We monitored the behavior of Drosophila. This is the result. For 1 hour, 7 flies gathered to the ATF2. These results clearly indicate that ATF2 produces isoamyl acetate from isoamyl alcohol.
 +
 +
[[File:KIT_flies.png|500px]]
 +
 +
This is the result. For 1 hour, 7 flies gathered to the ATF2. These results clearly indicate that ATF2 produces isoamyl acetate from isoamyl alcohol.
 +
 +
 +
 +
== 4,The comparing with the ability of ATF1 and ATF2 ==
 +
 +
 +
In addition, according to the previous study [2], the ability of ATF2 protein to produce isoamyl acetate in yeast is higher than that of ATF1 protein.
 +
It is known that both ATF1 and ATF2 protein are involved in producing isoamyl acetate.
 +
 +
[[File:Earlystudy.png|500px]]
 +
 +
 +
In 2006, MIT iGEM team submitted ATF1 coding sequence. (BBa_J45006) Our new part, ATF2 coding sequence, fall under the category of the improvement of function existing BioBrick Part, BBa_J45006. Herewith, our team, KIT-Kyoto 2013 iGEM team, meets the additional requirements for a Gold Medal.
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----
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== Reference ==
 +
 +
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[1]Dong H Cha et al. "A four-component synthetic attractant for Drosophila suzukii (Diptera: Drosophilidae) isolated from fermented bait headspace",
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 +
[2] Yoshimoto Hiroyuki et al. "Mechanisms of Acetate Ester Production and Control in Yeasts -Monograph-", Seibutsu-Kogaku Kaishi 79(2), 33-40, 2001-02-25
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 03:23, 28 September 2013

Isoamyl acetate generator

This device converts isoamyl alcohol to the odor isoamyl acetate.

1,SDS-PAGE

We constructed ATF2 generator. (BBa_K1049002) Our team KIT-Kyoto 2013 constructed this part for the purpose of measurement. T7 promoter is an IPTG-inducible promoter. We added 20µL IPTG (100mM) to our genetically modified E.coli after cultivation at 28 and 37 ˚C. 2 hours after, we extracted soluble proteins from it by FastBreak™ Cell Lysis Reagent and did SDS-polyacrylamide gel electrophoresis.

KIT2013SDS.png

ATF2 gene encodes AATase, which is about 62kDa. The consumption of protein marker is like this. Myosin 200kDa β‐Galactosidase 120kDa Bovine Serum Albumin 95kDa Glutamine dehydrogenase 68kDa Ovalbumin 50kDa Carbonic Anhydrase 36kDa Myoglobin 27kDa Lysozyme 20kDa Aprotinin 10kDa You can find the band at lanes which are added IPTG just beneath the band of 68kDa.



2,Growth curves

We measured the turbidities of the transformants every 2 hour and made the growth curves. We transferred the transformant prepared to 100 mL flasks and add 660uL IPTG and 108uL isoamyl alcohol to them after 2 hours of the start. We measured the turbidities every 2 hour. The measurements were carried out for 12 hours.

LB　100mL

ampicillin　150uL

sample 50uL

IPTG 660uL

isoamyl alcohol 108uL

37°C, 125/min

Growth atf2.png

In addition, we measured the turbidities of the transformants without adding IPTG and isoamyl alcohol too. We measured 4 samples; ATF2-pET-15b, ATF2-pET-15b + IPTG, empty pET-15b, empty pET-15b + IPTG. As the result, ATF2-pET-15b transformants grow well relative to control(transformants having empty pET-15b vector). We propose the folllowing hypothesis. Isoamyl acetate indicates a stimulatory effect of growth on E.coli cells when compared to isoamyl alcohol so that transformants having ATF2-pET-15b grow well relative to control. And ATF2-pET-15b without IPTG grow well relative to ATF2-pET-15b add IPTG. It is explained by the following hypothesis. When we add IPTG to the transformant having ATF2-pET-15b, ATF2 protein (AATase) is expessed in bulk in the transformant. A massive amount of AATase has a potential to surpress the growth.



3, Bioassay using Drosophila

Next, to confirm the activity of AATase, we added isoamyl alcohol after IPTG induction and cultivated for about 2 hours. We used E. coli cells carrying the empty vector (pET-15b) as a control and compare it with the E.coli cells carrying pET15b-ATF2 after addition of isoamyl alcohol. To compare the production of isoamyl acetate, we carried out a bioassay using Drosophila. Because Drosophila favors the fruit odor like isoamyl acetate. [1] After the addition of IPTG and isoamyl acetate, the culture was impregnated into the filter and placed in a case containing 10 Drosophilas. We monitored the behavior of Drosophila. This is the result. For 1 hour, 7 flies gathered to the ATF2. These results clearly indicate that ATF2 produces isoamyl acetate from isoamyl alcohol.

KIT flies.png

This is the result. For 1 hour, 7 flies gathered to the ATF2. These results clearly indicate that ATF2 produces isoamyl acetate from isoamyl alcohol.


4,The comparing with the ability of ATF1 and ATF2

In addition, according to the previous study [2], the ability of ATF2 protein to produce isoamyl acetate in yeast is higher than that of ATF1 protein. It is known that both ATF1 and ATF2 protein are involved in producing isoamyl acetate.

Earlystudy.png


In 2006, MIT iGEM team submitted ATF1 coding sequence. (BBa_J45006) Our new part, ATF2 coding sequence, fall under the category of the improvement of function existing BioBrick Part, BBa_J45006. Herewith, our team, KIT-Kyoto 2013 iGEM team, meets the additional requirements for a Gold Medal.

Reference

[1]Dong H Cha et al. "A four-component synthetic attractant for Drosophila suzukii (Diptera: Drosophilidae) isolated from fermented bait headspace",

[2] Yoshimoto Hiroyuki et al. "Mechanisms of Acetate Ester Production and Control in Yeasts -Monograph-", Seibutsu-Kogaku Kaishi 79(2), 33-40, 2001-02-25

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 444
    Illegal XbaI site found at 67
    Illegal PstI site found at 1555
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 444
    Illegal PstI site found at 1555
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 444
    Illegal BglII site found at 1
    Illegal BamHI site found at 1721
    Illegal XhoI site found at 1152
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 444
    Illegal XbaI site found at 67
    Illegal PstI site found at 1555
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 444
    Illegal XbaI site found at 67
    Illegal PstI site found at 1555
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 1682