Team:Kobe/Experiments


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.


Our project consists of 5 main steps.
  1. Growth test
  2. RNA extraction
  3. RNA-seq
  4. RT-qPCR
  5. Transformation
  6. Beta-galactosidase assay

Growth test

As stated previously, our project is based on the chassis genome character, which means it depends on whether the chassis have theanine inducible genes. Roughly predicted, if bacteria can assimilate L-theanine, at least one of their genes related to the digestion of L-theanine must be strongly induced by L-theanine.

Therefore, we carried out the simple screening test to see whether the strains could use L-theanine as sole nitrogen source.

We arranged 2 strains of E.coli and 3 strains of B.subtilis and cultivated these bacteria on the M9 agar medium (without NH4Cl).

As a result, B.subtilis 168 and B.subtilis NCIB 3610 could grow using L-theanine as sole nitrogen source. Finally, we decided to analyze the genes expression of B.subtilis NCIB 3610 and identified the genes which could be active only in the presence of L-theanine. (Please visit our result page for more information)


RNA extraction

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 sources(L-theanine, glutamate, water, L-theanine+glutamate). 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. After setting the condition, we cultivated our bacteria in each media and extracted RNA from each sample.

Protocols

  1. 500ml of following solution was prepared in 500 ml of Erlenmeyer flask. (10×M9、0.5％glucose, 0.0001M CaCl 2, 0.002M MgSO 4, Trace element, 0.05g /L NH 4Cl, 0.02% Yeast Extract, water) Cells were inoculated at OD 600 of 0.02. and grown at 37℃, 180 rpm.
  2. 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.
  3. Take the medium as a reference. When the OD 600 reached 0.3, 1 ml of culture was transferred to a fresh Eppendorf tube.
  4. 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.


RNA－seq

What is RNA-seq?

RNA-seq is a powerful technique to analyze the gene expression in different conditions (different tissues or different stage of developments).
RNA-seq uses next-generation sequence technology to read the sequences of transcripts. The reads obtained from this sequencing can then be aligned to refence genome 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 depending on different nitrogen sources(L-theanine, glutamate, water). What we want to find out is the gene which is more expressed in the presence of L-theanine than in the preesence of 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. Mapping the reads to the genome was also done by our instructors. Based on the experts advice, we have roughly selected around 10 promising sensor genes for further analysis in qPCR section.


Quantitative Real Time PCR (RT-qPCR)

What is RT-qPCR?

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

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. At this point, DNA binding dye is added in the PCR mixture. This dye intercalates with any double strand DNA, causing the fluorescence, which allows 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 levels of gene expression.

Why RT-qPCR?

In the previous RNA-seq, we selected some promising gene for sensor. We carried out RT-qPCR to measure the each of the gene expression depending on 5 different nitrogen source (L-theanine, glutamate, L-theanine+glutamate, water, sample without as a reference) We trie to get quantitatively more acurate information of genes.

Protocols

  1. After mRNA samples were 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 SystemⅡ was used for the analysis. The expression level of rpsJ (30S ribosomal protein S10 inB.subtilis) was also analyzed and as a 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
    cDNA 5 µl
    milliQ up to 20µl
    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

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, has 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.

Protocols

    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℃.


Beta-galactosidase assay

In order to measure the expression of nasA, amtB, and yrbD, we used the Beta-galactosidase assay test. The recombinant B.subtilis was cultured in 50 ml of 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 levels of target gene expression.

Protocols

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 into the media at OD600 of 0.02. Start cultivation (10 ml) with vigorous aeration.
  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 appropriate amount of Z-buffer
  2. Incubate the cells 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.

Finally, we did X-gal plates test. First, we prepared the following X-gal agar plates(10×M9 salts、0.5％glucose, 0.0001M CaCl2, 0.002M MgSO4, Trace element, 0.05g /L NH4Cl, 0.02% Yeast Extract, 1.5% agar, 0.016% X-gal, water) and covered ut with semi-solid agar mixed with pre-culture media at the OD600 of around 0.4. Cups were put on the top of the overlay, and nitrogen source was added with various concentrations.