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.

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.

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.
Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.
Our project consists of 5 main sections.

The bacteria are

We considered that the organisms which can metabolize the L-theanine must have at least ,one theanine-inducible genes
In order to allow the cells(bacteria) to assimilate L-theanine, we prepared

1. Growth　Test for extracting RNA sample ( growth condition )

In B.subtilis, several compounds can be used as nitrogen source (NH4+, glutamate, glutamine, etc.) In order to identify the theanine-inducible genes, we investigated the expression of whole genes (mRNA) in B.subtilis NCIB 3610 utilizing different compounds as a source of nitrogen. When extracting mRNA for transcriptomic analysis, all target bacteria growth should follow the similar time course so that the number of cells do not affect the mRNA expression pattern.

Protocols

The cells were grown in 50 ml of following solution in (10×M9、50％ glucose, 0.1M CaCl2, 1M MgSO4, 100×Trace element, 10g/L NH4Cl, 10% Yeast Extract, water

Start the cultivation the 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), and water was added to each flask. When the OD reached 0.3, 1 ml of culture was transferred into a new Eppendorf tube. The cells were collected by centrifugation for 10 min (4℃, 6000 rpm)
mRNA was extracted according to the protocols se described in the Protocol page.

2. RNA－Seq

RNA-Sequencing (RNA-seq, next generation sequence) was carried out to reveal the short sequence reads derived from mRNA.
RNA-seq is one of the technologies called “Transcriptomics technologies”, which are used to study the total amount of RNA transcripts. The investigation for mRNA expression pattern (between different samples) is useful because information about the sum of each genes’ mRNA reflects the difference of gene expression, giving us new insight about the regulation of the whole genes in the genome.
(If you are interested in RNA-sequencing, please click here to learn more)

In our project, RNA-Seq was used to compare the expression level of all genes (B.subtilis NCIB 3610) in the presence of differnet nitrogen source, L-theanine, Glutame, and water. a comparison to approximate the theanine( glutamate can be also the umami factor in tea contains )as L B.subtilis NCIB 3610. We guessed that genes having high-expression level only in the L-theanine-added samples can be the theanine-inducible genes.

Although we, students prepared the RNA samples for analysis, Dr. Tanaka carried out RNA-seq for us. We selected several genes induced more in the presence of L-theanine. than other nutrients.

3. Quantitative Real Time PCR (RT-qPCR)

What is RT-qPCR? Quantitative Reverse transcription PCR (RT-qPCR) RT-qPCR is a technique that monitors the amplification of a targeted DNA molecule during the PCR. This technique is used when you want to measure the amount of DNA sample by measuring the amplification speed. Several methods were used in Real Time PCR. We used intercalation method. A DNA-binding dye binds to all double strand DNA during the PCR, causing fluorescence of the dye. An increase in DNA product during PCR therefore leads to an increase in fluorescence intensity. So we can roughly measure the amount of initial DNA (by comparing the number of Cq)
Fluorescent dye binds to or intercalate with any double-strand DNA(PCR products), and caused the fluorescence, which allowed to detect the number of The mRNA extracted was transcribed into cDNA, and then the cDNA was amplified by PCR. The amount of cDNA was
mRNA is first transcribe into complementary DNA (cDNA) by reverse transcriptase. Next, cDNA is used as the template for the qPCR. The more the first templates exist, the more the PCR fragments are amplified, thereby measuring the first

After identifying the genes that is induced in the presence of L-theanine, we designed our parts as described in the design page.

4. Transformation of B.subtilis

After we identified the possible theanine inducible promoter, we transformed the B.subtilis strain NCIB 3610 with our 3 biobricks.
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 first was to transform each of our 3 BioBrick parts into the B.subtilis strain 168 and then transformed the B.subtilis strain NCIB 3610 using the extracted strain 168 genome with the same condition.

protocol

The cells were inoculated into 10 ml MDCH liquid media at OD600 of 0.3. The bacteria 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 new 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 check the actual gene expression, beta-galactosidase assay was used.

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 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, different nitrogen source (water, L-theanine, Glutamate, theanine+Glutamate) was added to each flask.
  3. The culture was taken at each time point 0h(5 min), 1h (1 ml), 2h(1 ml), 3h(0.5 ml), 4h(0.5ml) the cells were collected by centrifugation for 10 min at 150 rpm. The cells were 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 exactly 20 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.