Experiments

In this page, we are going to describe the overview of the research and experiments during the iGEM activity. Please go through our Notebook for the detailed protocols.

• 1.Growth Test
• 2.RNA-Seq
• 3.RT-qPCR
• 4.Transformation
• 5.Beta-galactosidase assay

1. Developing Growth condition and extracting RNA

In order to identify the "theanine sensor genes", we decided to compare the whole gene expression in B.subtilis NCIB 3610 depending on different nitrogen source(L-theanine, glutamate, water, L-theanine+glutamate, reference) When comparing the gene expression between bacteria samples, all sample bacteria growth should follow the similar time course so that the number of cells does not affect the mRNA expression pattern. Before we extracted RNA samples from the cells, we worked hard at developing the medium for RNA sampling. This medium is a derivative of M9 minimum media with a little amount of Yeast Extract as a nitrogen source.

Protocols

50 ml of following solution was prepared in 500 ml of Erlenmeyer flask. (10×M9、0.5％glucose, 0.0001M CaCl₂, 0.002M MgSO₄, Trace element, 0.05g /L NH₄Cl, 0.02% Yeast Extract, water) Cells were inoculated at OD₆₀₀ of 0.02. and grown at 37℃, 180 rpm. 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. Take the medium as a reference. When the OD₆₀₀ reached 0.3, 1 ml of culture was transferred to a fresh Eppendorf tube. The cells were collected by centrifugation for 10 min (4℃, 6000 rpm)
mRNA was extracted according to the protocols as described in the Protocol page.

2. RNA－Seq

What is RNA-seq?

RNA-seq is a powerful technique to analyse the gene expression in different conditions (such as different tissues or different stage of developments)
RNA-seq use next-generation sequence technology to read the sequence of all mRNA. The reads obtained from this can then be aligned to refence genome in order to make a map of whole-genome transcriptome map. The number of mRNA reflects the difference of gene expression, giving us new insight about the regulation of the whole genes in the genome.

Why RNA-seq?

In our project, RNA-Seq was used to compare the gene expression in B.subtilis NCIB 3610 deppending on differnet nitrogen source(L-theanine, glutamate, water).The genes we wanted should be over-expressed in L-theanine against glutamate and water.

In this part of the project, we were tremendously helped by our instructor, Dr.Tanaka. Although we, students prepared RNA samples for the analysis, Dr. Tanaka generously carried out RNA-seq. He also mapped the reads to the genome and presented the results to us. Based on the experts advice, we have roughly selected some hopeful sensor genes for further analysis in qPCR section.

3. Quantitative Real Time PCR (RT-qPCR)

What is RT-qPCR?

RT-qPCR is a technique to measure the expression of each genes.

In RT-qPCR analysis, RNA (mRNA) is first transcribed into complementary DNA (cDNA) by reverse transcriptase. cDNA is then used as a DNA template in conventional PCR. DNA binding dye is added in this PCR mixture. This dye intercalates with any double strand DNA, causing the fluorescence, which allowed to detect the number of PCR products. By tracking the progress of PCR reaction through fluorescent measurement, we can accurately deduce the initial numbers of cDNA, which is proportionate to the level of gene expression.

Why RT-qPCR?

We analysed the gene expression of NCIB 3610 strain depending on 5 different nitrogen source (L-theanine, as a Reference

Protocols

1. After RNA samplesvwere treated with DNaseI (sample preparation), concentration of RNA was measured using NanoVue.

   	ng/µl
   sample T	412
   sample G	332
   sample T+G	232
   sample W	610
   reference	118

   (T: theanine, G:glutamate, T+G: theanine+glutamate, W: water, reference:)
2. Reverse transcription was carried out according to ReverTra Ace® qPCR RT Kit from TOYOBO.
3. qPCR was carried out according to THUNDERBIRD® SYBR® qPCR Mix. ThemalDice (TaKaRa) Real Time SystemⅡwas used for the analysis. The expression level of rpsJ (30S ribosomal protein S10 inB.subtilis) was also analyzed and used as the reference gene. Make the serial dilution of cDNA. Prepare the mixture below.

   Forward primer	0.3 µM
   Reverse primer	0.3 µM
   ThunderBird	10 µｌ
   milliQ	2.6 µl
   DNA	5µl
   total	20 µl

In this step, we finally identified three genes (nasA, amtB, yrbD) strongly induced in the presence of L-theanine. After identifying those genes, we designed our BioBrick parts as described in the design page. (BBa_K2233000, BBa_K2233001, BBa_K2233002)

4. Transformation of B.subtilis

After we identified the possible theanine sensor genes, we designed our BioBrick parts and transformed the B.subtilis strain NCIB 3610 and strain 168.
But there was a problem with transformation. The problem is that strain NCIB 3610, which is a wild-type natural isolate of B.subtilis, have low transformation efficiency compared to B.subtilis strain 168, which is a laboratory strain widely used for research. What we did was to transform the B.subtilis strain 168 first and then transformed the B.subtilis strain NCIB 3610 using the extracted strain 168 genome with the same condition.

protocol

Cells were pre-cultured overnight on LB agar plates at 37℃. The cells were inoculated into 10 ml MDCH liquid media at OD₆₀₀ of 0.3. The cells were grown at 37℃,180 rpm until the OD₆₀₀ reached 1.5, and then the same volume (10 ml) of MD medium was added. After 1 hour of incubation at 37℃ with shaking at 180 rpm, 1.0 ml of the culture was transferred to a fresh conical tube, where 100-1000 ng of DNA was added. After 2hours of further incubation at 37℃, the cells were spread onto LB agar plates with chloramphenicol (final concentration: 5 µg/ml) and grown overnight at 37℃.

5. Beta-galactosidase assay

In order to measure the expression of nasA, amtB, and yrbD, we used the lacZ assay. The recombinant B.subtilis was cultured in the media (the same media we used for RNA-seq) and cells were collected at different time points. Cells are exposed to lysozyme, which breaks down cell membrane and extracts the Beta-galactosidase inside the cell. The amount of Beta-galactosidase is proportionate to the level of target gene expression.

Day1

1. Prepare the 5 ml media in each test tube.
2. Make the serial dilution of bacteria (Take 500 µl of media and transfer to the next one) and grow the cells overnight at 37℃, 180 rpm.

Day2

1. The cells were inoculated in the medium at OD₆₀₀ of 0.02. Start cultivation (10 ml) with vigorous aeration in our media
2. When the OD₆₀₀ reached 0.15, nitrogen source (water, L-theanine, Glutamate, theanine+Glutamate) was added to each flask.
3. The culture was taken at each time point 0h, 1h, 2h, 3h, 4h. The cells were collected by centrifugation for 10 min at 150 rpm, and stored at -20℃.

Day3

1. Resuspend the cells in 0.3 ml (0h, 1h, 2h) or 0.4 ml( 3h, 4h) of Z-buffer
2. Incubate the suspension at 37℃ for 30 min
3. Spin down the cells at 15000 rpm for 5 min and collect the supernatant.
4. Dilute the supernatant 10 times. Measure the concentration of protein. Pierce™ BCA Protein Assay Kit was used to measure the concentration of protein.
5. Take another 100 µl of supernatant into another Eppendorf tube.
6. Incubate at 28℃ for 5 min.
7. Add 300 µl of 4mg/ml ONPJ
8. Leave for 10 min
9. Add 600 µl of 1M Na₂CO₃
10. Measure the OD₄₂₀
11. Standard curve was generated by diluting 100 mM O-Nitrophenol (in N,N-Dimethylformamide) with water.