Peachchild (Talk | contribs) |
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2. RNA extraction | 2. RNA extraction | ||
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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 shows bacterial growth and time when we added each component and sampled each medium. We extracted RNA from these media we sampled. | ||
</p> | </p> | ||
− | <img src="https://static.igem.org/mediawiki/2017/6/67/T--Kobe--result6.jpg" /> | + | <img src="https://static.igem.org/mediawiki/2017/6/67/T--Kobe--result6.jpg" width="400" /> |
− | + | <img src="https://static.igem.org/mediawiki/2017/0/09/T--Kobe--result7.png" width="400" /> | |
− | <img src="https://static.igem.org/mediawiki/2017/ | + | <br /> |
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<small> | <small> | ||
Fig. 2 Bacterial growth<br /> | Fig. 2 Bacterial growth<br /> | ||
− | When the OD reached 0.15, L-theanine (final concentration 18.7mM), glutamate (final | + | When the OD reached 0.15, L-theanine (final concentration 18.7mM), glutamate (final concentraton 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. |
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3. RNA-seq | 3. RNA-seq | ||
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</p> | </p> | ||
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+ | RPKM stands for “Reads Per Kilobase of exon per Million mapped fragments”. This value indicates the gene expression from RNA sequencing data by normalizing the total read length and the number of sequence reads. | ||
+ | </p> | ||
<p> | <p> | ||
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− | Gene Expression | + | Gene Expression |
theanine/water>3, glutamate/water<3 | theanine/water>3, glutamate/water<3 | ||
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− | + | As a result, we were able to narrow down the number of genes to 31. Below are the data of these genes. | |
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<br /> | <br /> | ||
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− | Moreover, we narrowed down these 31 genes to 8 by picking up | + | Moreover, we narrowed down these 31 genes to 8 by picking up genes that were induced well in the presence of L-theanine and not induced well in the presence of glutamate |
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4. RT-qPCR | 4. RT-qPCR | ||
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<figure> | <figure> | ||
<img src="https://static.igem.org/mediawiki/2017/2/20/T--Kobe--result11.jpg" /> | <img src="https://static.igem.org/mediawiki/2017/2/20/T--Kobe--result11.jpg" /> | ||
− | <figcaption> Fig. 6 | + | <figcaption> Fig.6</figcaption> |
</figure> | </figure> | ||
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+ | Fig. 6 Relative expression of 8 genes compared with the expression level of rpsJ (reference gene). rpsJ is one of the housekeeping genes, which codes for 30S ribosomal protein S10 in <i>B.subtilis</i> . | ||
+ | </p> | ||
<p> | <p> | ||
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 <a href="https://2017.igem.org/Team:Kobe/Design">our design page</a>.) | 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 <a href="https://2017.igem.org/Team:Kobe/Design">our design page</a>.) | ||
</p> | </p> | ||
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5. Transformation | 5. Transformation | ||
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<img src="https://static.igem.org/mediawiki/2017/0/05/T--Kobe--result12.jpg" /> | <img src="https://static.igem.org/mediawiki/2017/0/05/T--Kobe--result12.jpg" /> | ||
− | <figcaption>Fig. 7 Transformation check using electrophoresis of PCR products | + | <figcaption>Fig. 7 Transformation check using electrophoresis of PCR products</figcaption> |
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− | </figcaption> | + | |
</figure> | </figure> | ||
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+ | (marker, nasA 1-1, nasA, 1-2, nasA 2-1, nasA 2-2, nasA (negative control), amtB 1, amtB 2, amtB 3, amtB (negative control), yrbD 1-1, yrbD 1-2, yrbD 3-1, yrbD (negative control)) | ||
+ | Two of four nasA, three of three amtB, and three of three yrbD had longer sequences (about 5.7 kbp) than negative control (about 1.4 kbp). This indicated that our Biobrick parts were successfully fused to these genes. | ||
+ | |||
+ | </p> | ||
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6. Beta-galactosidase assay | 6. Beta-galactosidase assay | ||
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− | + | We could analyze the expression of yrbD and amtB in M9 derivative media containing L-theanine, glutamate, or water respectively by using our Biobrick parts. 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 <i>B.subtilis</i> NCIB 3610 into a biosensor for L-theanine. | |
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− | As for BBa_K2233000(target for nasA), we | + | As for BBa_K2233000(target for nasA), we couldn’t evaluate its expression due to a lack of time. |
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Revision as of 02:38, 2 November 2017
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.
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 concentraton 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.
3. RNA-seq
We have selected possible candidate genes based on the RPKM value.
RPKM stands for “Reads Per Kilobase of exon per Million mapped fragments”. This value indicates the gene expression from RNA sequencing data by normalizing the total read length and the number of sequence reads.
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
As a result, we were able to narrow down the number of 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 genes that were induced well in the presence of L-theanine and not induced well 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.
Fig. 6 Relative expression of 8 genes compared with the expression level of rpsJ (reference gene). rpsJ is one of the housekeeping genes, which codes for 30S ribosomal protein S10 in B.subtilis .
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.
(marker, nasA 1-1, nasA, 1-2, nasA 2-1, nasA 2-2, nasA (negative control), amtB 1, amtB 2, amtB 3, amtB (negative control), yrbD 1-1, yrbD 1-2, yrbD 3-1, yrbD (negative control)) Two of four nasA, three of three amtB, and three of three yrbD had longer sequences (about 5.7 kbp) than negative control (about 1.4 kbp). This indicated that our Biobrick parts were successfully fused to these genes.
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.
We could analyze the expression of yrbD and amtB in M9 derivative media containing L-theanine, glutamate, or water respectively by using our Biobrick parts. 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 couldn’t evaluate its expression due to a lack of time.