Part:BBa_K1049002
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.
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
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.
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.
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
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 444
Illegal XbaI site found at 67
Illegal PstI site found at 1555 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 444
Illegal PstI site found at 1555 - 21INCOMPATIBLE 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 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 444
Illegal XbaI site found at 67
Illegal PstI site found at 1555 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 444
Illegal XbaI site found at 67
Illegal PstI site found at 1555 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 1682
| n/a | Isoamyl acetate generator |