Difference between revisions of "Part:BBa K1049002:Experience"

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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 
This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
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===Applications of BBa_K1049002===
 
===Applications of BBa_K1049002===
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Our team KIT-Kyoto 2013 constructed this part for the purpose of measurement. T7 promoter is an IPTG-inducible promoter. We added 20uL IPTG (100mM) to our genetically modified E.coli after cultivation at 28 and 37 degree C. 2 hours after, we extracted soluble proteins from it by using FastBreak™ Cell Lysis Reagent and did SDS-polyacrylamide gel electrophoresis.
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Our team KIT-Kyoto 2013 constructed this isoamyl acetate generator for the purpose of measurement.
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[[File:ATF2SDS2.png|500px|thumb|left|Figure.1 SDS-PAGE Starting from the left, marker, ATF2 at 28 degree C, ATF2 + IPTG at 28 degree C, marker, ATF2 at 37 degree C, ATF2  + IPTG at 37 degree C]]
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T7 promoter is an IPTG-inducible promoter. We added 20uL IPTG (100mM) to our genetically modified E.coli after cultivation at 28 and 37 degree C. 2 hours after, we extracted soluble proteins from it by using FastBreak™ Cell Lysis Reagent and did SDS-polyacrylamide gel electrophoresis.
 +
 
 +
[[File:KIT2013SDS.png|500px]]
 +
 
 
ATF2 gene encodes AATase, which is about 62kDa. The consumption of protein marker is like this.
 
ATF2 gene encodes AATase, which is about 62kDa. The consumption of protein marker is like this.
  
 
Myosin                                200kDa
 
Myosin                                200kDa
 +
 
β‐Galactosidase                  120kDa
 
β‐Galactosidase                  120kDa
 +
 
Bovine Serum Albumin        95kDa
 
Bovine Serum Albumin        95kDa
 +
 
Glutamine dehydrogenase    68kDa
 
Glutamine dehydrogenase    68kDa
 +
 
Ovalbumin                          50kDa
 
Ovalbumin                          50kDa
 +
 
Carbonic Anhydrase            36kDa
 
Carbonic Anhydrase            36kDa
 +
 
Myoglobin                            27kDa
 
Myoglobin                            27kDa
 +
 
Lysozyme                            20kDa
 
Lysozyme                            20kDa
 +
 
Aprotinin                              10kDa
 
Aprotinin                              10kDa
 +
 
   
 
   
 
You can find the band at lanes which are added IPTG just beneath the band of 68kDa.
 
You can find the band at lanes which are added IPTG just beneath the band of 68kDa.
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 +
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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]]
 +
 +
In addition, according to this previous study [2], the ability of ATF2 protein producing isoamyl acetate in yeast is higher than 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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[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
 +
  
 
===User Reviews===
 
===User Reviews===

Latest revision as of 02:24, 25 September 2013

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K1049002

Our team KIT-Kyoto 2013 constructed this isoamyl acetate generator for the purpose of measurement. T7 promoter is an IPTG-inducible promoter. We added 20uL IPTG (100mM) to our genetically modified E.coli after cultivation at 28 and 37 degree C. 2 hours after, we extracted soluble proteins from it by using 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.


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

In addition, according to this previous study [2], the ability of ATF2 protein producing isoamyl acetate in yeast is higher than 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.

[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


User Reviews

UNIQ2097673b3d313e6e-partinfo-00000000-QINU UNIQ2097673b3d313e6e-partinfo-00000001-QINU