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.
When we thought of making this biosensor, we went through the database and searched for the useful genes for this project. However, to our regret, we couldn't find any useful genes. What is more, we learned that almost nothing is known about how the theanine is degraded by bacteria!!! Therefore, we decided to start this project from identifying the "theanine responsive genes" which works as a sonsor.
The question is where we can find them? At first, 2 strains of E.coli and three strains of B.subtilis were easily accessible to us. So we carried out a growth test to see whether the bacteria could grow using theanine as a sole nitrogen source. We found that B.subtilis NCIB 3610 could grow much better than others with theanine. Then our journey started. Our first job was to analyze the gene expression in B.subtilis NCIB 3610 to identify the theanine responsive genes.
(Please visit our experiment page to see why we chose this strain.)
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 are strongly induced in the presence of L-theanine.
After the identification, lacZ was integrated into the chromosome to be fused to nasA, amtB, yrbD respectively. The principle of the sensor is quite simple. If certain amount of L-theanine is contained in the green tea we add to the sensor, the β-galactosidase should be expressed, resulting in the formation of blue insoluble pigment.
Finally, we carried out Beta-galactosidase (lacZ) assay to measure the expression of the theanine responsive genes and discussed the feasibility of our theanine sensor.