2024/7/24 (Wed)

13:00-20:30 (JST)

Experiment Supervisor:
Koichi Yano

Participants:
・Shiori Kajikawa (all)
・Shoya Inoue (all)
・Kei Hato (-15:00)

Experiment:
・Lab safety guide
・gDNA purification of B. subtilis natto BEST195
・planting 8 different strains of B. subtilis, B. subtilis subsp. Natto, E. Coli

Results:
・Participants learned the safety rules in Sue’tsugu lab.
・gDNA precipitation was visually confirmed

Additional Notes:
・Participants must make sure to take note of, and share cautions within the lab with other members

2024/07/25 (Thu)

10:00-18:30 (JST)

Experiment Supervisor:
Koichi Yano

Participants:
・Mizuho Sakai (11:30-)
・Rikuto Fukushima (-16:30)
・Shiori Kajikawa (-13:00)
・Kei Hato (11:30-15:00)

Experiment
・PCR of aprE fragment
・TAE-EtBr agarose gel creation (2% and 0.8%)
・PCR of extracted gDNA
・Moving bacteria to freezing glycerol stock

Results
None

2024/07/26 (Fri)

12:30-18:30 (JST)

Experiment Supervisor:
Koichi Yano

Participants:
・Mizuho Sakai (all)
・Rikuto Fukushima(13:00-)
・Shiori Kajikawa (15:50-)

Experiment:
・Autoclaving effluent
・Electrophoresis yesterday’s PCR product
・PCR of DNA extracted yesterday through different cycle setting
・Researching about BioBrick RFC10

Results:
・No bands except lane 10 (plasmid) or lane 1/12(ladder)showed.
・Upon troubleshooting, we found that the PCR setup, conducted yesterday, was done in 2-steps instead of 3

Additional Notes:

2024/07/27 (Sat)

10:30-18:00 (JST)

Experiment Supervisor:
Kazuyuki Fujimitsu

Participants:
・Rikuto Fukushima(-15:30)
・Shiori Kajikawa (11:30-)
・Tatsuhiko Akiyama (17:00-)
・Yukiya Horiba (17:00-)

Experiment:
・Electrophoresis of yesterday’s PCR product
・Troubleshooting
・Extraction of gDNA from B. subtilis natto BEST195 (This time, use Wizard kit)
・PCR and electrophoresis (3rd time) of PCR product
・Pipette training session for Tatsuhiko and Yukiya

Results:

・No bands except ladders were seen in electrophoresis.
・Made alterations to PCR= increase extension time, lower annealing temperature, increase the addition of primers and DNA sample, reconducted DNA extraction which may have failed first time

2024/07/29 (Mon)

10:30- 19:30 (JST)

Experiment Supervisor:
Koichi Yano

Participants:
・Rikuto Fukushima (all)
・Shiori Kajikawa (-14:30)
・Kei Hato (-17:00)
・Saki Tsuchiya (13:30-18:00)
・Mizuho Sakai (14:30-15:30)

Experiment:
<Wet lab>
・Clarification of plans for this week (increased production of NK and fibrin plate assay)
・Ordering necessary material for mainly this week’s experiments (fibrin, thrombin)
・PCR and electrophoresis of vector plasmid

<Education>
・PCR trial for lab session on Aug 4th

Results:
<Wet lab>
・Experiment was planned for this week
・PCR confirmed via electrophoresis

<Education>
・PCR & electrophoresis committed 3 times, all negative results. Resume trial tomorrow, but adding water and heating in microwave when breaking down the soybeans

2024/07/30 (Tue)

12:30-17:00 (JST)

Experiment Supervisor:
Koichi Yano

Participants:
・Shiori Kajikawa (all)
・Saki Tsuchiya (13:00-17:00)
・Mizuho Sakai (13:00-)
・Kei Hato (13:30-)
・Shoya Inoue (14:00-)

Experiment:
<Wet lab>
・Creation of culture medium for B. subtilis, B. subtilis Natto, and preculture of E. Coli C600
・Ordering primers for colony PCR fragments

<Education>
PCR trial for experiment session on Aug. 4

Results:
<Wet lab>
None

<Education>
・PCR failed. No bands except the ladder.

2024/07/31 (Wed)

11:00-18:00 (JST)

Experiment Supervisor:
Koichi Yano

Participants:
・Shiori Kajikawa (all)
・Mizuho Sakai (11:30-)
・Rikuto Fukushima (14:00-)

Experiment:
・Preparation of E. coli C600 competent cell
・Creation of LB medium for transformation

Results:
・At the end, we realized that the “E. coli” cells we were using was actually B. subtilis NEST115.
・Thus, we made a glycerol stock of competent B. subtilis NEST115 cells.
・We will re-conduct the same experiment using E. coli C600

Additional Notes:

2024/08/01 (Thu)

10:00-17:00 (JST)

Experiment Supervisor:
Koichi Yano

Participants:
・Shiori Kajikawa (all)
・Kei Hato (all)
・Rikuto Fukushima (13:00-)
・Ryuzo Kijima (16:00-)

Experiment:
・Preparation of E. Coli C600 competent cell glycerol stock

Results:
・Made a glycerol stock of competent E. Coli C600 competent cell

Additional Notes:
・Creation of phosphate buffer

2024/08/02 (Fri)

11:00-15:00 (JST)

Experiment Supervisor:
Koichi Yano

Participants:
・Shiori Kajikawa (all)
・Shoya Inoue (-13:00)
・Rikuto Fukushima (13:30-)
・Ryuzo Kijima (14:00-)

Experiment:
・DNA Column purification of aprE and plasmid fragment, DNA quantity determination using Nanodrop and Quantus

Results:
・NanoDrop DNA concentration measurement was 168.8ng/μl, 141.8ng/μl, 137.8 ng/μl, and 103.5 ng/μl for tubes A, B, C, D respectively, for the PCR samples from 7/27 and 7/29
・Quantus DNA concentration measurement was 139.7nM, 140.5nM, 134.8nM, 22.6nM for tubes A, B, C, D respectively

2024/08/05 (Mon)

10:00-21:00 (JST)

Experiment Supervisor:
Koichi Yano

Participants:
・Kei Hato (-13:00)
・Misaki Ozawa (11:00-18:30)
・Ryuzo Kijima (13:30-18:30)
・Shiori Kajikawa (13:30-)

Experiment:
・infusion cloning of aprE fragments with pNK1
・transformation into E. coli DH5α
・Competence measurement of E. coli C600 with transformation

Results:
None

2024/08/06 (Tue)

11:00-19:30 (JST)

Experiment Supervisor:
Koichi Yano

Participants:
・Shiori Kajikawa (-14:00)
・Yukiya Horiba (13:30-)

Experiment:
・Colony PCR of transformed E. Coli DH5α/pNK1 and electrophoresis

・Inoculation of successful colonies to liquid medium

Results:
・Colonies were observed on the transformation plate
・All colonies were estimated to be about the targeted length (7.5kbp)

2024/08/07 (Wed)

10:00-17:30 (JST)

Experiment Supervisor:
Koichi Yano

Participants:
・Kei Hato (all)
・Misaki Ozawa (11:00-17:00)
・Rikuto Fukushima (13:00-15:00)

Experiment:
・Trial of creating milk plate/fibrin plate assay

Results:
None

2024/08/08 (Thu)

10:00-20:00 (JST)

Experiment Supervisor:
Koichi Yano

Participants:
・Kei Hato (10:00-17:30)
・Rikuto Fukushima
・Misaki Ozawa (14:00-20:00)

Experiment:
・Creation of milk plate/fibrin plate assay
・PCR and electrophoresis for miniprep pNK1(DH5α)

Results:

Additional Notes:

2024/08/09 (Fri)

10:00-18:00 (JST)

Experiment Supervisor:
Koichi Yano

Participants:
・Shiori Kajikawa (all)

Experiment:
・Creation of competent cells with B. subtilis 168 and B. subtilis subsp. Natto BEST195
・Column purification and NanoDrop
・Observing results of yesterday’s fibrin/milk plate assay

Results:
・NanoDrop DNA concentration measurement for the purified PCR column was 141.8ng/μL, 97.7ng/μL, 135.8ng/μL, 133.1ng/μL for tubes 1, 2, 3, and 4 respectively
・We have determined an optimal recipe/protocol for plate creation, as well as how the strains would look depending on whether it is grown on LB medium or soybeans.

Additional Notes:
・Sent column samples for sequencing analysis

2024/08/10 (Sat)

13:00-18:00 (JST)

Experiment Supervisor:
Kazuyuki Fujimitsu

Participants:
・Shiori Kajikawa (all)

Experiment:
・LB agar plate creation
・NanoDrop of miniprep plasmid
・Transformation of pNK1 #4, 8, 14, 19 to E. coli C600

Results:
・NanoDrop DNA concentration measurement of the miniprep plasmid was 31.6ng/μL, 31,3ng/μL, 32.9ng/μL, 45ng/μL for GTstr13, 14, 15, 16 respectively.

2024/08/12 (Mon)

13:30-17:30 (JST)

Experiment Supervisor:
Fujimitsu Kazuyuki

Participants:
・Rikuto Fukushima (all)
・Misaki Ozawa (-16:00)
・Lee Doria (-17:00)

Experiment:
・Inoculation of E. coli C600/<05><110><α1>
・PCR of pNK1 plasmid to create pNK2 and pNK3

Results:
・Bands were found near 1.6kbp, when it is supposed to be near 6 kbp….
・After troubleshooting, we found that the primer GT10 had another binding site, other than the one that we anticipated

Additional Notes:
・Reconstructed primers and ordered them (GT34). Will redo this experiment on 8/21

2024/08/19 (Mon)

10:30-17:00 (JST)

Experiment Supervisor:
Koichi Yano

Participants:
・Shiori Kajikawa (all)
・Mizuho Sakai (15:00-)

Experiment:
・Picture record of inoculation plates from 08/10
・LB plate creation (spectinomycin, chloramphenicol included)

・Inoculation of E. coli C600 with various plasmids (<05>, <110>, <α1>, pNK #4, 8, 14, 19)

Results:
None

Additional Notes:

2024/08/20 (Tue)

10:00-19:30 (JST)

Experiment Supervisor:
Koichi Yano

Participants:
・Doria Lee (-12:30)
・Shiori Kajikawa (11:30-)

Experiment:
・Miniprep of E. coli strains that were inoculated yesterday
・NanoDrop of strains
・gDNA purification of B. subtilis (stock YAN17632)
・Creation of minimal medium for conjugational transfer
・Checking Sequence analysis of E. coli C600/pNK1 #4

Results:
・NanoDrop DNA concentration was 58.3ng/μL, 55.4ng/μL, 54.8ng/μL, 70.6ng/μL, 75.6ng/μL, 70.8ng/μL, 93.6ng/μL for strains GTstr17, GTstr18, GTstr19, GTstr20, GTstr21, GTstr22, and GTstr23, respectively.
・NanoDrop DNA concentration for the gDNA purification was 116.1ng/μL

Additional Notes:

2024/08/21 (Wed)

12:00-19:30 (JST)

Experiment Supervisor:
Koichi Yano

Participants:
・Shiori Kajikawa (all)
・Saki Tsuchiya (all)
・Mizuho Sakai (14:00-16:00)

Experiment:
・Electrophoresis of miniprep plasmids/B. subtilis_YAN17632 gDNA
・Nanodrop of gDNA_YAN17632_240820
・PCR of pNK1 to create pNK2 and pNK3, electrophoresis
・Transformation of dimer plasmid into B. subtilis 168/pLS20cat and B. subtilis natto BEST195 (continued from 240809)

Results:

Each miniprep plasmid is the same length. It was divided into a trimer(9.42kbp), a dimer(5.81kbp), and a monomer(3.52kbp). The length of gDNA was 23.13kbp.
Nanodrop DNA concentration measurement gDNA_YAN17632_240820 was 75.3ng/μL

The length of the PCR product was 5.65kbp.

Additional Notes:
When measuring DNA concentration with NanoDrop, we used TE buffer near NanoDrop, and it decreased a lot from yesterdy.
Therefore, we redid with TE buffer of our lot.

2024/08/22 (Thu)

11:00-17:30 (JST)

Experiment Supervisor:
Koichi Yano

Participants:
・Shoya Inoue (all)

Experiment:
・Column purification of PCR samples from 08/21
・Electrophoresis of column purification sample
・Recording of plasmid transformation into B. subtilis/B. subtilis natto (photo&cell count)
・Colony PCR

Results:

Additional Notes:

2024/08/23 (Fri)

12:00-21:30 (JST)

Experiment Supervisor:
Koichi Yano

Participants:
・Shiori Kajikawa (all)

Experiment:
・0.8% TAE-EtBr agarose gel creation
・Electrophoresis of yesterday’s colony PCR
・Quantus Fluoromerter_240822 column

・Infusion cloning to create pNK2 and pNK3

・Inoculation for glycerol stock creation (for tomorrow)

Results:

Additional Notes:

2024/08/24 (Sat)

11:00-16:00 (JST)

Experiment Supervisor:
Kazuyuki Fujimitsu

Participants:
・Kei Hato (11:00-14:00)
・Misaki Ozawa (12:30-)

Experiment:
・glycerol stock preparation
・transformation (pNK2, pNK3 to DH5α)

Results:

Additional Notes:

2024/08/26 (Mon)

10:00- 19:00 (JST)

Experiment Supervisor:
Koichi Yano

Participants:
・Shiori Kajikawa (all)
・Doria Lee (-12:30)
・Misaki Ozawa (15:30-)

Experiment:
・LB agar plate creation
・transformation (pNK2, pNK3 to DH5α)

Results:
・No colonies were found from the transformation from yesterday

Additional Notes:
・As wrong tubes were used previously for the transformation, the experiment was redone (have to use 1.5mL tubes instead of 0.6mL tubes to fit into the block) can not be heated properly when transforming

2024/08/27 (Tue)

10:00- 12:30 (JST)

Experiment Supervisor:
Koichi Yano

Participants:
・Doria Lee (all)

Experiment:
・counting colonies from Transformation_pNK2 into E. coli DH5α 240824
・PCR of colonies from transformation with 2 types of primer to create pNK4

Results:

Additional Notes:

2024/08/28 (Wed)

10:30- 16:00 (JST)

Experiment Supervisor:
Kazuyuki Fujimitsu

Participants:
・Kei Hato (-14:00)
・Misaki Ozawa (11:00 - )

Experiment:
・Electrophoresis of yesterday’s colony PCR

・Inoculation of pNK3 from electrophoresis
・Planting colonies (BEST 195 and B. subtilis 168) to difco and star agar plates

Results:

Additional Notes:

2024/09/02 (Mon)

10:00- 13:00 (JST)

Experiment Supervisor:
Kazuyuki Fujimitsu

Participants:
・Kei Hato (all)

Experiment:
・Glycerol freeze stock of bacterial strain DH5a/pNK3 clone, miniprep
・Column purification of PCR sample from 8/27

Results:
None

Additional Notes:

2024/09/03 (Tue)

16:30-19:00 (JST)

Experiment Supervisor:
Koichi Yano

Participants:
・Wingdor Doria Lee (all)

Experiment:
・Quantus of Fluoromerter_240822 column
・In-Fusion of Fluoromerter_240822 column
・Miniprep of E. coli DH5α/pNK3 #6, inoculation into LB+Sp for glycerol stock creation

Results:
None

Additional Notes:

2024/09/04 (Wed)

16:30-19:00 (JST)

Experiment Supervisor:
Koichi Yano

Participants:
・Wingdor Doria Lee (all)

Experiment:
・LB agar plate creation
・Glycerol Freeze Stock of bacterial strains

Results:
None

Additional Notes:

2024/09/05 (Thu)

14:00-19:30 (JST)

Experiment Supervisor:
Koichi Yano

Participants:
・Mizuho Sakai (all)
・Wingdor Doria Lee (16:00-19:00)

Experiment:
・Transformation_pNK5 into E. coli DH5α 240904 *USING 1.5mL Tubes

・QIAprep Spin Miniprep Kit (pNK3 #6)
・Electrophoresis (pNK3 #1, 6, 9, 21, 24)

Results:

Additional Notes:

2024/09/08 (Sun)

11:00-18:30 (JST)

Experiment Supervisor:
Kazuyuki Fujimittsu

Participants:
・Misaki Ozawa (all)

Experiment:
・PCR and electrophoresis of colonies from DH5a/pNK5-infusion

Results:

Additional Notes:
・Used the wrong ladder, so redid electrophoresis a second time.

2024/09/10 (Mon)

16:00-18:00 (JST)

Experiment Supervisor:
Koichi Yano

Participants:
・Shoya Inoue (all)

Experiment:
・Combine DNA samples from 9/8 colony PCR

Results:
None

Additional Notes:

2024/09/11 (Wed)

17:00-20:00 (JST)

Experiment Supervisor:
Koichi Yano

Participants:
・Misaki Ozawa (all)

Experiment:
・Glycerol Freeze Stock of DH5a/pNK5-infusion
・Miniprep of DH5a/pNK5-infusion

Results:
None

Additional Notes:

2024/09/16 (Mon)

11:00-18:30 (JST)

Experiment Supervisor:
Kazuyuki Fujimitsu

Participants:
・Shoya Inoue (all)
・Mizuho Sakai (13:00-)

Experiment:
・Transformation_pNK2 into E. coli DH5α 240826
・NanoDrop DNA conc. measurement(240910_column)

Results:
・NanoDrop DNA concentration values for the four samples from 9/10 was 27.7ng/μL, 34.5ng/μL, 90.3ng/μL, and 79.5ng/μL
・When spreading on LB medium, mistakenly used an item that contained Cm.

Additional Notes:

2024/09/17 (Tue)

12:00-19:30 (JST)

Experiment Supervisor:
Koichi Yano

Participants:
・Saki Tsuchiya (all)
・Mizuho Sakai (16:30-）

Experiment:
・Sequence analysis

・Electrophoresis (100V, 25min, 2%agarose gel )
・Transformation_pNK2 into E. coli DH5α 240826

・Transformation of dimer plasmid into B. subtilis 168/pLS20cat (continued from 240809)

・Creating Natto with soybean and NK Ⅾay1

Results:
・In electrophoresis, all bands were seen. Only pnk5 3 was seen in different lengths.

Additional Notes:
We redid the ”Transformation_pNK2 into E. coli DH5α 240826” process that I made a mistake in yesterday.

2024/09/18 (Wed)

17:00-21:00 (JST)

Experiment Supervisor:
・Koichi Yano

Participants:
・Arisa Tani (all)

Experiment:
・Creating Natto with soybean and NK Day2
・Colony PCR (168/pLS20cat pNK5)

・Culture of transformed 168, (not transformed) BEST195 and S903 in preparation for conjugal Transmission

Results:
None

Additional Notes:

2024/09/19 (Thu)

16:00-20:30 (JST)

Experiment Supervisor:
・Koichi Yano

Participants:
・Rikuto Fukushima (-18:00)
・Arisa Tani (17:00-)

Experiment:
・Milk plate assay
・Fibrin plate assay
・Creating Natto with soybean and NK Day3
・Conjugation DAY 1
・Preparation of Glycerol Freeze Stock

Results:
Natto creation (sample) is completed.

2024/09/20 (Fri)

17:00-19:00 (JST)

Experiment Supervisor:
・Koichi Yano

Participants:
・Misaki Ozawa (all)

Experiment:
・Glycerol Freeze Stock of bacterial strains
・Conjugation Day2

Results:
None

Additional Notes:

2024/09/22 (Sun)

11:00-17:30 (JST)

Experiment Supervisor:
Kazuyuki Fujimitsu

Participants:
・Arisa Tani (all)

Experiment:
**・**S903, BEST195 fibrin plate assay preliminary experiment
・Colony PCR

・Electrophoresis of colony PCR product

Results:

Yesterday’s Conjugation

**・**The colony is clearly visible only on the plate that transmitted NK5. This is unknown because we did not take a negative control, but it may appear spontaneously. If it is spontaneous, the transmission of NK5 failed, while the other >2,000 colonies, pNK1 and <05, were successfully transmitted. The successful plate has the potential to have produced >2,000 colonies because too many were spread on the plate.

Additional Notes:

2024/09/23 (Mon)

13:00-20:00 (JST)

Experiment Supervisor:
Kazuyuki Fujimitsu

Participants:
・Arisa Tani (all)

Experiment:
・Overnight culture creation
・Culture creation for PCR
・LB agar plates for filter membrane method
・Minimal medium agar plates

Results:
None

Additional Notes:

2024/09/24 (Tue)

13:30-22:00 (JST)

Experiment Supervisor:
Koichi Yano
Kazuyuki Fujimitsu

Participants:
・Mizuho Sakai (all)
・Saki Tsuchiya (17:00-20:00)

Experiment:
・Preparation of LB medium for Colony PCR
・Colony PCR

・Confirmation of the bacteria (Bacillus subtilis or Bacillus subtilis natto)
・Preparation of NB medium for fibrin assay

Results:
None

Additional Notes:

2024/09/25 (Wed)

17:00-22:00 (JST)

Experiment Supervisor:
Koichi Yano
Kazuyuki Fujimitsu

Participants:
・Arisa Tani (all)

Experiment:
・Conjugation Day2
・Inoculated on LB medium
・Electrophoresis (yesterday’s Colony PCR)

Results:
OD value measured before inoculating LB medium

・Electrophoresis (yesterday’s Colony PCR)

Additional Notes:

2024/09/26 (Thu)

17:00-22:00 (JST)

Experiment Supervisor:
Koichi Yano
Kazuyuki Fujimitsu

Participants:
・Arisa Tani (all)
・Mizuho Sakai (all)

Experiment:
・Preparation of 100mL of NB medium, fibrin assay

・Colony PCR, Colony count

・Electrophoresis of Colony PCR products
・fibrin plate assay

Results:
・Electrophoresis of Colony PCR products

Colony count

Conjugation results
BEST195

S903

Additional Notes:

2024/09/27 (Fri)

12:30-16:30, 17:30-22:00 (JST)

Experiment Supervisor:
Koichi Yano
Kazuyuki Fujimitsu

Participants:
・Arisa Tani (17:30-22:00)
・Mizuho Sakai (12:30-16:30)

Experiment:
・Confirmation of results of fibrin assay
・Preparation of NB medium
・Preparation of Conjugation

Results:
Results of yesterday’s fibrin plate assay

Additional Notes:
・The bacteria did not increase in the NB medium made in yesterday’s “Preparation of NB medium” process. The use of thin tubes may have resulted in a lack of oxygen due to the small surface area.

2024/09/28 (Sat)

13:00-21:30 (JST)

Experiment Supervisor:
Kazuyuki Fujimitsu

Participants:
・Mizuho Sakai (all)
・Arisa Tani (14:00-)

Experiment:
・Conjugation
・fibrin plates creation
・Preparation NB medium for fibrin assay, fibrin assay
・Fibrin plate assay
・Made some glycerol stocks

Results:
OD value of inoculated NB medium for fibrin assay

・Made some glycerol stocks

Additional Notes:

2024/09/29 (Sun)

11:00-22:00 (JST)

Experiment Supervisor:
Kazuyuki Fujimitsu

Participants:
・Arisa Tani (all)

Experiment:
・Creation of sodium phosphate buffer

・Fibrin plate creation
・Halo assay with natto suspension liquid and LB medium

Results:
・Yesterday’s results of fibrin plate assay
16h
BEST195×3

BEST195×4

S903×3

20h
BEST195×3

BEST195×4

S903×3

24h
BEST195×3

BEST195×4

S903×3

・OD value of natto solution in the process of “Halo assay with natto suspension liquid.

Additional Notes:

2024/09/30 (Mon)

18:00-22:00 (JST)

Experiment Supervisor:
Koichi Yano
Kazuyuki Fujimitsu

Participants:
・Mizuho Sakai (all)
・Arisa Tani (19:00-)

Experiment:
・Fibrin plate assay
・Liquid inoculated on LB medium
・Liquid suspended natto

Results:
None

2024/10/01 (Tue)

13:30-22:00 (JST)

Experiment Supervisor:
Koichi Yano
Kazuyuki Fujimitsu

Participants:
・Mizuho Sakai (all)
・Arisa Tani (19:00-)

Experiment:
・Confirmation of yesterday’s results of the fibrin assay
・Colony PCR

・Electrophoresis of Colony PCR products

Results:
・Yesterday’s results of fibrin assay
Liquid inoculated on LB medium
16h
BEST195

S903

20h
BEST195

S903

24h
BEST195

S903

Natto suspension liquid
16h
BEST195

S903

20h
BEST195

S903

24h
BEST195

S903

・OD values measured before Colony PCR

・Electrophoresis

Additional Notes:
There was a little water on the surface of the fibrin plate. We were able to confirm the halo, but need to keep a close eye on it.