Team:Kobe/Overview

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Overview

As the first iGEM team from Kobe University, we focused on creating the L-theanine biosensor, which can measure the concentration of L-theanine in green tea. Our approach is quite simple. We first identify the "theanine sensor genes", which are the genes strongly induced in the presence of L-theanine. We identified 3 candidate sensor genes in B.subtilis NCIB 3610. and integrated the lacZ gene into the chromosome to fuse to those sensor genes.

Here is the story.

When we thought of making this biosensor, we went through the database and searched for the "theanine sensor genes", which could be strongly induced in the presence of L-theanine.However, to our regret, we couldn't find the useful genes available to us. Therefore, we decided to start this project from identifying the "theanine sonsor genes". The question is where we can find them? First we had two candidates, E.coli and B.subtilis. After screening of the bacteria by a simple growth test, we found that B.subtilis NCIB 3610 could grow using L-theanine as a sole nitrogen source. We considered that that this bacteria must have the theanine sensor genes. Then our journey started. Our first job was to analyze the gene expression in B.subtilis NCIB 3610 to identify the theanine sensor genes.
(Please visit our experiment page to see why we chose this strain.)

The problem is that L-theanine is not the only component in green tea, which means that our sensor may be inhibited by other factors. It is too complicated to take into account all these unfavorable effect. Thus, we made a rough assumption first that the amino-acids included in green tea are almost exclusively L-theanine and glutamate. L-theanine is formed from glutamate and ethylamine. To improve the theanine specificity of our biosensor, we had to search for the genes which could be more induced in the presence of L-theanine than glutamate.To put it simply,the goal of this project is to create the biosensor which is more sensitive to L-theanine than to glutamate, and to test it with real green tea.

During the first half of the project, we worked hard at analyzing the gene expression in B.subtilis NCIB 3610 depending on different nitrogen sources(L-theanine, Glutamate, water). Through a lot of RNA-seq and RT-qPCR work, we identified 3 chromosomal genes (nasA, amtB, yrbD) which may possibly be induced in the presence of L-theanine.

Once those genes were identified, we designed the biobricks(lacZ gene sequence) and insert the lacZ gene into the chromosome.

After the transformation, B.subtilis were cultured on X-gal plates, where some green tea is dropped. In this context, if certain amount of L-theanine is contained in the green tea we add, the β-galactosidase should be expressed, resulting in the formation of blue insoluble pigment.

Through a Beta-galactosidase (lacZ) assay, we measured the expression of theanine inducible gene and discussed the feasibility of our theanine sensor.