Team:KIT-Kyoto/Parts

From 2013.igem.org




Parts_kit2013.JPG


Parts

We designed and constructed these parts originally.

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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

This 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.


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 20 µL IPTG (100 mM) to our genetically modified E.coli after cultivation at 28˚C 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 62 kDa. The consumption of protein marker is like this.

Myosin 200 kDa β‐Galactosidase 120 kDa Bovine Serum Albumin 95 kDa Glutamine dehydrogenase 68 kDa Ovalbumin 50 kDa Carbonic Anhydrase 36 kDa Myoglobin 27 kDa Lysozyme 20 kDa Aprotinin 10 kDa

You can find the band at lanes which are added IPTG just beneath the band of 68 kDa.


2,Growth curve

We measured the turbidities of the transformants every 2 hour and made the growth curves. We transferred the transformant to 200 mL flasks and add 660 µL IPTG and 108 µL isoamyl alcohol to them after 2 hours of the start. We measured the turbidities every 2 hours. The measurements were carried out for 10 hours.

LB 100 mL

ampicillin 150 µL

sample 50 µL

IPTG 660 µL

isoamyl alcohol 108 µL

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 pET-15b-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.

              ATF2result_htm_m7be19f37.png
              The_number_of_fly.jpg

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



4,The comparison of 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.


[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