Results & Discussion

Results

1. Growth test

Fig.1 shows the result of growth test of each strain. In every picture, the plate on the bottom contains L-theanine, the plate on the right contains NH₄Cl (positive control), and the plate on the left contains nothing as a nitrogen source (negative control).

As a consequence, it turned out that B.subtilis 168 and B.subtilis NCIB 3610 could grow well in the media containing L-theanine as a sole nitrogen source.

B.subtilis 168

B.subtilis NCIB 3610

B.subtilis var. natto

E.coli K-12 C600

E.coli BL21 5 strains of bacteria were cultured on minimum media for 6 days at 37℃. In every picture, the plate on the bottom contains L-theanine, the plate on the right contains NH₄Cl, and the plate on the left contains nothing as a nitrogen source. These pictures show that B.subtilis strain NCIB 3610 and 168 could grow well using L-theanine as a sole nitrogen source.

2. RNA extraction

Fig.2 shows bacterial growth and time when we added each component and sampled each medium. We extracted RNA from these media we sampled.

Fig. 2 Bacterial growth
When the OD reached 0.15, L-theanine (final concentration 18.7mM), glutamate (final concentration 1.1 mM), L-theanine(18.7 mM)+glutamate(1.1 mM) ,and water were added to each flask. When the OD 600 reached 0.3, 1 ml of each medium was transferred to a fresh Eppendorf tube.

Fig.3 shows the results of electrophoresis of RNA samples. It indicates that RNA extraction was succeeded, as there were two clear bands derived from ribosomal RNA.

(M: Marker,　G: Glutamate,　T: Theanine)

(M: Marker, G+T: Glutamate+ Theanine, W: Water)

3. RNA-seq

We have selected possible candidate genes based on the RPKM value.

Below are the conditions we set to discover genes that are induced stronger in the presence of L-theanine than glutamate and water.

• RPKM
  RPKM（theanine）>100
• Gene Expression
  theanine/water>3, glutamate/water<3

In this analysis, we were able to narrow down the number of candidate genes to 31. Below are the data of these genes.

Fig. 4 Rough selection of candidate genes by RNA-seq

Moreover, we narrowed down these 31 genes to 8 by picking up the genes that were induced well in the presence of L-theanine but not induced much in the presence of glutamate

Fig. 5 More strict selection of candidate genes by RNA-seq.

Finally, we selected 8 genes (nasD, nasA, iseA, yrbD, pucR, amtB, glnK, and ureA)

4. RT-qPCR

To measure the expression of these 8 genes more accurately, we carried out RT-qPCR. Fig. 6 is the result of qPCR analysis.

Finally, we selected three genes (nasA, yrbD, and amtB), and designed our Biobrick parts so that our parts would be inserted into these three genes. (If you want to know how we designed these parts, please visit our design page.)

5. Transformation

Our Biobricks BBa_K2233000(target for nasA), BBa_K2233001(target for yrbD), and BBa_K2233002(target for amtB) were taken up into different B.subtilis NCIB 3610 respectively. After the transformation, DNA sequences of nasA, yrbD, and amtB were amplified by PCR. We verified that our Biobrick parts were successfully inserted into the chromosome of B.subtilis NCIB 3610.

6. Beta-galactosidase assay

Fig. 8 shows the result of beta-galactosidase assay test. It was revealed that both yrbD and amtB were induced much more strongly in the presence of theanine than glutamate and water four hours after adding each component.

In order to measure the concentration of theanine in an easy way, we carried out X-gal plate assay. Below is the result of X-gal plate assay with B.subtilis NCIB 3610 whose yrbD region was fused to BBa_K2233001

It indicates that beta-galactosidase was not expressed well when we added glutamate, and it was expressed well when we added theanine as we anticipated. However, it was also expressed well when water was added.

Discussion and future work

In our experiments, we proved that our Biobrick parts(BBa_K2233000, BBa_K2233001, and BBa_K2233002) were successfully inserted at the target site by homologous recombination as we had expected.

By using our Biobrick parts, we could analyze the expression of yrbD and amtB in the derivative of M9 media containing L-theanine, glutamate, or water respectively. We revealed that yrbD and amtB fused to our Biobrick parts were expressed much more with L-theanine than glutamate and water. Therefore, it is estimated that our parts BBa_K2233001(target for yrbD) and BBa_K2233002(target for amtB) have a potential to change our chassis B.subtilis NCIB 3610 into a biosensor for L-theanine.

In order to prove whether our parts are useful for L-theanine sensor, we believe that next step we should take is to confirm how the expression of our fused genes will change according to the concentration of L-theanine.

As for BBa_K2233000(target for nasA), we gave up to use this part due to a lack of time.

Although we have successfully verified that two of our parts were induced much more by L-theanine, we couldn’t see clear difference of L-theanine and water in X-gal plate. We estimated that it was due to inappropriate setting of the content of X-gal in the plate, the amounts of bacteria we add, the time we observe the plate, or simple difference of liquid culture and solid culture. We need more research to analyze the concentration of L-theanine easily with our Biobrick parts.