Team:KIT-Kyoto/Project

Project Overview

We are trying to construct a novel E.coli that has fruity flavor like Japanese rice wine (Japanese sake). In order to accomplish the purpose, yeast genes related with production of the Japanese sake fragrance were introduced into E. coli cells. We also tried to develop a way to eliminate bad smells of E. coli in parallel. Although we previously won a gold prize by the development of a novel pen (E. coli Pen) in 2010, its bad smells were weak points and must be improved. We will overcome this problem through the progress of our new project in 2013. So far, “smell” is not a popular keyword and not a major field in iGEM. However, we believe that our project will provide a new point of view to iGEM friends.

Results

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.

Future

Our main parts designed this time were these two, but we have an idea that we insert GFP, RFP, or CFP in these parts. By using fluorescence protein reporters, we can see the expression of the enzyme more exactly. However, there are many kinds of fluorescence proteins. Which protein is the best in our future projects? We examined how each fluorescence protein affects the growth of E. coli cells.

2013 KIT-Kyoto iGEM team assessed influence of fluorescence proteins on the growth of E.coli. We used BBa_I13521, BBa_I13522 and BBa_I13600 in order to measure the growth. We made growth curve of various transformants expressing reporter genes. The purple line is control. The red curve indicates E coli cells expressing mRFP. The green curve is E coli cells expressing GFP, The blue curve is E coli cells expressing eCFP. Growth of Escherichia coli is promoted with mRFP and eCFP. On the other hand, the cells with GFP showed the delay of growth. Therefore, it is thought that you should use mRFP or eCFP in the experiment to have to get more products earlier. Other teams will be able to choose appropriate parts in the future by our result that comparing these three parts and having made characterize

As you know, there are the three primary colors for light. In the case of light, various colors are expressed by a combination of blue, red,and green. We apply this way of thinking to smell. In fragrance companies, specific smells are made by blending several kinds of elements of various smells. For example, the smell of apples is consisted of these nine kinds of elements. According to this concept, we will create various elements of smell in E. coli cells and can produce about lots of fragrances by blending them. In addition, if we can reproduce the synthetic course of the element smell in the biosynthesis in E.coli perfection, we can create smells semipermanently. Imagine the world in which you can control all smells!!! We recommend the optimum methods for smell control. One of them is “ E.coli aromatherapy”. If the aroma diffuser contains several kinds of aroma oil produced by genetically modified E.coli, you can enjoy aromatherapy using fragrances you like.

We believe our approach help us make better environment in our lives!

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