Presented by Team KAIT_Japan 2020
Eri Tashiro, Chikaho Suzuki
iGEM Student Team Member, iGEM Team Mentor, iGEM Team Primary PI,
Sponsor, Kanagawa Institute Of Technology, Cosmo Bio Co.,LTD, Promega Corporation, IDT(INTEGRATED DNA TECHNOLOGIES)
Abstract
We've all suffered from the unpleasant odor of culturing E.coli.Therefore, we wanted to make unpleasant odors comfortable by development of E.coli that biosynthesize aromatic components. We decided on the sweet-smelling vanillin as the aromatic substance.
In this research, the metabolic pathway to biosynthesize vanillin, we selected a pathway that metabolizes glucose to L-phenylalanine and introduces an enzyme gene to metabolize it to vanillin. Making the E.coli smell like vanilla may help alleviate unpleasant odors during the experiment and make it more enjoyable, allowing you to be more creative with your experiments.
Team:KAIT Japan/Poster
Poster: KAIT_Japan
E. coli that Create a Creative Environment
Introduction
Our goal is development of “E. coli that create a creative environment”
Idea
We have been bothered for a long time by the unpleasant odor generated when we cultivate E.coli.
Therefore, we wondered if it would be possible to make the experiment more pleasant by development of E. coli biosynthesize aromatic component.
In terms of what kind of aroma we wanted to give it, we chose the vanilla scent because we all are familiar with it in puddings and ice cream.
We researched vanilla fragrance and found that a compound called vanillin is the main aromatic component.
Therefore, we wondered if it would be possible to make the experiment more pleasant by development of E. coli biosynthesize aromatic component.
In terms of what kind of aroma we wanted to give it, we chose the vanilla scent because we all are familiar with it in puddings and ice cream.
We researched vanilla fragrance and found that a compound called vanillin is the main aromatic component.
Motivation
If you can experiment with a pleasant scent, you may be able to create more fun and creative ideas while taking care of stress.
Engineering
To get E.coli to synthesize vanillin, we had to decide what to use as a precursor.
Three candidates were identified: ferulic acid, amino acids such as phenylalanine and tyrosine, and glucose.
Ferulic acid, phenylalanine, and tyrosine are expensive.
On top of that E.coli dose not have many of the genes needed for the pathway from phenylalanine and tyrosine to vanillin.
We chose to start from glucose because we found that if we started with glucose, the raw materials could be synthesized at a lower cost and using the shikimic acid pathway(shown below).
The enzyme gene to be incorporated is shown in the figure below.
Genetic modification is used for the biosynthesis of vanillin, but it is confirmed by electrophoresis whether it is actually incorporated.
Incubate after transformation and check for vanillin production by GCMS.
Three candidates were identified: ferulic acid, amino acids such as phenylalanine and tyrosine, and glucose.
Ferulic acid, phenylalanine, and tyrosine are expensive.
On top of that E.coli dose not have many of the genes needed for the pathway from phenylalanine and tyrosine to vanillin.
We chose to start from glucose because we found that if we started with glucose, the raw materials could be synthesized at a lower cost and using the shikimic acid pathway(shown below).
The enzyme gene to be incorporated is shown in the figure below.
Genetic modification is used for the biosynthesis of vanillin, but it is confirmed by electrophoresis whether it is actually incorporated.
Incubate after transformation and check for vanillin production by GCMS.
Section 1
First, we conducted an experiment to confirm the compatibility of the scents of E. coli and vanilla.
40 µL of vanilla essence was added to the liquid medium in which E. coli was precultured to prepare a dilution series.
40 µL of vanilla essence was added to the liquid medium in which E. coli was precultured to prepare a dilution series.
Section 2
Second, We tried construction of IDT ’s parts and transformation to E.coli.
We designed enzyme genes have 4 reaction enzyme sites. These enzymes have EcoR1, Xba1, Spe1, Pst1.
Enzyme1 is long (2kbp), so we divided it into two parts.
In this experiment (construction of IDT ’s parts), two parts of enzyme 1 were connected to BamH1.
Next, we tried transformation. We used 3 iGEM parts. These parts are from 2019 to 2015.
Next, we tried transformation. We used 3 iGEM parts. These parts are from 2019 to 2015.
Results
The first experimental result.
The figure shows the dilution series and its odor.
〇 is the one that was hard to feel the smell of E. coli, △ is the one that felt the smell of E.coli a little, and × is the one that only felt the smell of E. coli.
As a result, the odor of E.coli was suppressed by the fragrance of vanilla in the 1 to 10-fold diluted liquid media. Also, the vanilla fragrance was emited stronger when autoclaved. Its scent was like a pudding.
Next, second experimental result.
We succeeded in connecting enzyme 1 divided into two parts with BamH1.
But, transformation was unsuccessful. Now, We thinking about Why unsuccessful.
As a result, the odor of E.coli was suppressed by the fragrance of vanilla in the 1 to 10-fold diluted liquid media. Also, the vanilla fragrance was emited stronger when autoclaved. Its scent was like a pudding.
Next, second experimental result.
We succeeded in connecting enzyme 1 divided into two parts with BamH1.
But, transformation was unsuccessful. Now, We thinking about Why unsuccessful.
References and Acknowledgements
We would be grateful who have supported our work.
We are "Supporters"
Members of Iida lab, Yasuhiro Iida, Yuuichi Mannnami, Misato Izutu, Sayaka Narishige, Hiroyuki Takahashi of Leave a Nest Co., Ltd.
We are "Sponsors"
Kanagawa Institute of Technology, Integrated DNA Technologies, Cosmo Bio Co., Ltd, Promega Corporation
Our team could not participate in this contest without assistance from these companies. They supported our project expenses such as experimental costs, registration and participation fees.
Thanks to all the following sponsors !!
We are "Supporters"
Members of Iida lab, Yasuhiro Iida, Yuuichi Mannnami, Misato Izutu, Sayaka Narishige, Hiroyuki Takahashi of Leave a Nest Co., Ltd.
We are "Sponsors"
Kanagawa Institute of Technology, Integrated DNA Technologies, Cosmo Bio Co., Ltd, Promega Corporation
Our team could not participate in this contest without assistance from these companies. They supported our project expenses such as experimental costs, registration and participation fees.
Thanks to all the following sponsors !!