Team:KIT-Kyoto/Parts

Parts

We designed and constructed these parts originally.

<groupparts>iGEM013 KIT-Kyoto</groupparts>

BBa_K1049000

This gene encodes an enzyme involved in the production of aromatic odor in yeast cells. we submitted a new BioBrick part that encodes an aldehyde degradative enzyme.

BBa_K1049001

It is the gene encoding aldehyde dehydrogenase of yeast. We have submitted a new BioBrick part, which was the ATF2 gene encoding the enzyme that produces isoamyl acetate from isoamyl alcohol.

BBa_K1049000

This device converts isoamyl alcohol to the odor isoamyl acetate.Sequence and Features.

We constructed ATF2 generator; BBa_K1049002 in order to measure the quantitative data. 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.

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.

https://2013.igem.org/File:KIT_flie.png

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.

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.

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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