Notebook

2023/07/08 (JST)

18:00 (JST) - 21:00 (JST)

Participants

Yuta Yamagishi
Jun Sakai
Keisuke Nishimoto
Kaisei Otake

Methods

none

Results

none

Notes

Lab Guide
Wi-Fi connection Printer connection
Pipetting practice using ATP absorbance

2023/07/14 (JST)

15:00 (JST) - 18:40 (JST)

Participants

Yuta Yamagishi
Keisuke Nishimoto
Kaisei Otake
Hinami Kadono

Methods

Overlap-PCR of pgm fragments (fr1~fr3)
PCR of pET21a(pgm),pgm(after overlap-PCR)
Preparation of culture media
Agar medium preparation

Results

None

Notes

2023/07/17 (JST)

15:00 (JST) - 21:00 (JST)

Participants

Yuta Yamagishi
Jun Sakai
Kaisei Otake
Keisuke Nishimoto
Keisuke Taira

Methods

Results

Electrophoresis resulted in an extra band 3.5-4.0 kbp in size in addition to the band of interest.
No pgm bands occurred.

Notes

Overlap-PCR and PCR of pgm fragments (fr1~fr3)
PCR for UGT709
PCR for pET21a(UGT)
Electrophoresis of pET21a(pgm), pgm
pET21a(pgm), dpn1 processing of pgm
Electrophoresis of pET21a(UGT), UGT709
dpn1 processing of pET21a(UGT), UGT709
PCR for pET(UGT) (redo)

2023/07/19 (JST)

15:00 (JST) - 21:00 (JST)

Participants

Yuta Yamagishi
Kaisei Otake
Keisuke Taira
Jun Sakai

Methods

dpn1 processing

Results

PCR was done at pgm1~pgm3. PCR was done at 60°C, but the annealing temperature was wrongly set at 55°C.
I made a mistake in the amount I put in before PCR and had to start over.
Electrophoresis showed that in addition to the target band, an extra band of 3.5 to 4.0 kbp in size was generated. It is likely that the primers were amplified by binding to another low tm-value region during the annealing stage, in addition to the targeted band.
The pgm band did not occur, possibly because the PCR did not work, so we redid the PCR.
A possible solution is to reduce the amount of template, but this time we changed the annealing temperature: 1.56°C (control) 2.58°C 3.60°C 4.68°C (same temperature as the extension, so 2steppcr) Please check the bands the next time you do a swim.

Notes

Overlap-PCR, PCR of pgm fragment (fr1~fr3)
PCR for UGT709
PCR for pET21a(UGT)
Electrophoresis of pET21a(pgm), pgm
pET21a(pgm), dpn1 processing of pgm
Electrophoresis of pET21a(UGT), UGT709
pET21a(UGT), dpn1 processing of UGT709
PCR for pET(UGT) (redo)

2023/07/21 (JST)

10:10 (JST) - 19:30 (JST)

Participants

Yuta Yamagishi
Keisuke Taira
Keisuke Nishimoto
Jun Sakai

Methods

Results

Absorbance
UGT74_up: 230.8nm
UGT74_down：184.3nm
UGT94_up：97.6nm
UGT94_down：126.7nm
Longer DNA was amplified by increasing the time of extension.
The pgm3 band occurred normally, but both overlap1 and 3 had an extra band around 500b in addition to the target band.

Notes

Refinement of UGT74_up, UGT74_down, UGT94_up, UGT94_down
PCR of pCDF-Duet, pgm3 Overlap-PCR of pgm1, 2
Electrophoresis of pCDF-Duet
pCDF-Duet 再PCR 3step＋エクステンション 100s
pCDF-Duet 2step+ extension 90s electrophoresis dpn1 treatment
electrophoresis of pgm1, 2 overlap, pgm3
pET21a-UGT709G1　In-Fusion
Transformation of BL21(DE3) pRK
Transformation of DH5α pRK
Transformation of pet21a-UGT709G1
LB aga culture preparation

2023/07/24 (JST)

10:00 (JST) - 22:10 (JST)

Participants

Yuta Yamagishi
Keisuke Nishimoto

Methods

Results

Confirmation of growth on agar medium
BL21
pRK had no colonies growing.
Do the pRK incubation temperatures again at 20°C, 30°C, and 37°C.
DH5a
pRK and pET21a-UGT709 showed colonies.
In electrophoresis, I tried 4 different ways, including extension and 2-step, using overlap and PCR, respectively, but extra bands were produced.
I thought there was a problem with the primers, so I redesigned them and ordered them.
I made primers for fr1 and fr2 with overlap, fr3 with separate PCR, and fr1 with separate PCR, fr2 and fr3 with overlap.
I measured the concentration of DNA purified by nanodrop, which is 85.9 ng/µl for pgm fr3 and 141.8 ng/µl for pCDF-Duet, so DNA is present at a normal value.

Notes

Overlap-PCR of pgm1 and 2
Electrophoresis of overlap-PCR fragments of pgm1, 2
Purification of pgm fr3 and pCDF-Duet
In-Fusion of pCDF-Duet, UGT74, 94
Transformation of pCDF-Duet-1-UGT74F8-UGT94E13 and pCDF-Duet-1-UGT94E13-UGT74F8 into DH5α
Preparation of LB medium

2023/07/25 (JST)

15:00 (JST) - 20:00 (JST)

Participants

Yuta Yamagishi
Koei Naito
Jun Sakai

Methods

Results

none

Notes

Transformation of BL21 (pRK)
Dilution of primer

2023/07/28 (JST)

10:00 (JST) - 21:30 (JST)

Participants

Yuta Yamagishi
Kaisei Otake
Jun Sakai

Methods

Results

pgm fr1: no band
pgm fr1-2: no band
pgm fr2-3: showed band
pgm fr2 (2step,3step(extension:30sec/kb))electrophoresis: no band

Notes

PCR of pgm fr1-2,pgm fr2-3,pgm fr1,pgm fr2
Electrophoresis of pgm fr1-2,pgm fr2-3,pgm fr1
Electrophoresis of pgm fr2 (2step,3step(extension:30sec/kb))
Preparation of media containing TET12.5
Liquid culture of pRK,74-94,94-74

2023/07/29 (JST)

10:00 (JST) - 21:30 (JST)

Participants

Yuta yamagishi
Keisuke Taira
Keisuke Nishimoto
Kaisei Otake
Shota Aso
Jun Sakai

Methods

Electrophoresis of fr2 showed a band at the desired position.

Results

[Absorbance
1 ugt709 1: 54.4
2 ugt709 2: 22.5
3 7494 1: 21.4
4 7494 2: 157.0
5 9474 1: 203.1
6 9474 2: 16.2

Notes

pcr of pgmfr2
Electrophoresis of pgmfr2
Purification of pgmfr1,fr2
Plasmid extraction of UGT709,74-94,94-74
UGT709, 74-94, 94-74, transformation of pgm fr1~fr3 into BL21(DE3)
Transformation of pgm-galU into DH5α
Centrifuge liquid culture of RIKEN plasmid

2023/07/30 (JST)

10:30 (JST) - 11:00 (JST)

Participants

Yuta Yamagishi

Methods

None

Results

Confirmation of growth on agar medium
BL21
pRK had no colonies growing. Since pCDF and pET21a-UGT709 showed colonies, perhaps the problem is low efficiency due to the large DNA size.
DH5a
pET21a-pgm-galU also showed no colonies. This may be due to low efficiency of InFusion.
Measurement of pRK concentration
I measured the concentration of pRK with Quantus, which is more accurate than nanodrop, and found only 7 ng/ul. It may be possible to extract plasmids from DH5a and use a higher concentration.

Notes

Confirmation of growth on agar medium
Measurement of pRK concentration

2023/07/31 (JST)

16:00 (JST) - 20:40 (JST)

Participants

Yuta Yamagishi
Shota Aso
Kaisei Otake

Methods

None

Results

None

Notes

Inoculation of pRK(DH5α) into 5ml of LB medium

2023/08/01 (JST)

12:00 (JST) - 22:00 (JST)

Participants

Yuta Yamagishi
Kaisei Otake
Shota Aso
Yuta Shimamoto

Methods

Results

None

Notes

2023/08/02 (JST)

15:00 (JST) - 22:00 (JST)

Participants

Yuta Yamagishi
Keisuke Taira

Methods

Results

pet21a Purification, absorbance measurement
127.1ng/μl
The band was showing in both conditions, but it was thin, so I added 5 more cycles in the 3step one.

Notes

pcr (3step and 2step) electrophoresis of pet21a for gibson assembly (from left to right: ① ladder ➁ 3step ③ 2step)
pet21a Purification, absorbance measurement
pet21a pgmgalU Gibson Assembly
After pRK transformation, spread on agar medium

2023/08/03 (JST)

11:00 (JST) - 20:30 (JST)

Participants

Shota Aso
Kaisei Otake
Yuta Yamagishi

Methods

Results

none

Notes

Performed gibson assembly.
Transformation to DH5-a
TB media preparation

2023/08/04 (JST)

8:15 (JST) - 22:30 (JST)

Participants

Shota Aso
Keisuke Taira
Yuta Yamagishi

Methods

Results

none

Notes

OD Determination 11:36 14:36 17:08 20:36 22:36
Start of main culture 8:36
TB media preparation

2023/08/05 (JST)

10:00 (JST) - 19:42 (JST)

Participants

Haruto Amano
Jun Sakai

Methods

Results

Miniprep was performed and pgm1 and pgm2 were obtained at concentrations of 34.9 and 36.3, respectively.

Notes

OD measurement after main culture
Plasmid extraction of pgm
Making pgm glycerol stock

2023/08/06 (JST)

10:00 (JST) - 20:00 (JST)

Participants

Yuta Yamagishi
Kaisei Otake

Methods

Transformation of BL21

Results

None

Notes

Sampling on Time
OD value measurement in conjunction with the time
Creation of pRK and pET21a-pgm-galU coexisting strain

2023/08/07 (JST)

10:00 (JST) - 23:00 (JST)

Participants

Yuta Yamagishi
Kaisei Otake
Keisuke Nishimoto

Methods

Results

Confirmation of growth on agar medium
BL21
Coexistence of pRK and pET21a-pgm-galU had no colonies growing.
I measured the concentration of DNA purified by nanodrop, which is 223.5 ng/µl for pET21a-pgm-galU_1, 108.1 ng/µl for pET21a-pgm-galU_2, 180.0 ng/µl for pET21a-UGT709G1_1, and 207.1 ng/µl for pET21a-UGT709G1_2, so DNA is present at a normal value.

Notes

OD measurements of those sampled at 19:36 on 23/08/06
Sampling & OD measurement (1-4) according to time
Preincubation at 37°C, 180 rpm in test tubes
PCR for sequencing of pET21a-pgm-galU, pET21a-UGT709G1 and their electrophoresis
Purification of pET21a-pgm-galU, pET21a-UGT709G1
Inoculation of DH5α with pgm-galU into 5ml of LB, and preparation of 4 test tubes with OD values of 0.09, 0.03, 0.01 and 0.003.
Prepare tet agar medium & spread with OD600 3.0.

2023/08/08 (JST)

10:00 (JST) -

Participants

Yuta Yamagishi
Kaisei Otake
Keisuke Nishimoto

Methods

Results

None

Notes

Sampling and OD measurement
OD measurement (every hour, 4 test tubes)
Sequencing of pET21a-pgm-galU_1, 2, pET21a-UGT709G1_1, 2
Extraction of crocetin from culture medium

2023/08/09 (JST)

10:10 (JST) - 23:40 (JST)

Participants

Yuta Yamagishi
Keisuke Nishimoto
Kaisei Otake

Methods

Results

Sequence Confirmation
G389 in pET21a-pgm-galU_ 2 was set to S.
Since there was no problem with pET21a-pgm-galU_ 1, I will use it as is.
pET21a-UGT709G1_1 and pET21a-UGT709G1_2 had the correct sequence as well, so I will use them as they are.
I measured the concentration of DNA purified by nanodrop, which is 41.8 ng/µl for pET21a-pgm-galU_1_1, 40.7 ng/µl for pET21a-pgm-galU_1_2, 40.5 ng/µl for pET21a-pgm-galU_1_3, and 37.8 ng/µl for pET21a-pgm-galU_1_4, so DNA is present at a normal value
Crocetin detected
The molecular weight of crocetin is 328.4 g/mol, and the MS result is 329.175, an error that can be explained by the addition of protons. The molecular weight of the intermediate carotenoids up to the crocetin is completely different, so it can be said that crocetin could be produced.

Notes

Sampling, OD measurement
Sequence confirmation of pET21a-pgm-galU_1, pET21a-pgm-galU_ 2, pET21a-UGT709G1_1, pET21a-UGT709G1_2
Plasmid extraction of pET21a-pgm-galU
Cultivation of pRK strain (37°C)
Detection of crocetin
Start pre-culture

2023/08/10 (JST)

10:15 (JST) - 22:10 (JST)

Participants

Keisuke Taira
Jun Sakai
Yuta Yamagishi

Methods

Results

Sampling from chloroform is tough because the cabinet dissolves.

Notes

Shaking culture of prk 11:05～
Creation of coexisting stocks in TSS
crocetin extraction
Preparation of spec100 medium
crocetinのUV vis測定

2023/08/11 (JST)

13:10 (JST) - 20:00 (JST)

Participants

Masayuki Su'etsugu
Kaisei Otake
Keisuke Nishimoto

Methods

Results

0.3188 g of crocetin was obtained.

Notes

Pre-culture of pCDF-Duet-74-94
Dried crocetin chloroform solution
Pre-culture of pgm-galU
Transformation of pCDF-94-74

2023/08/12 (JST)

8:00 (JST) - 21:00 (JST)

Participants

Yuta Yamagishi
Kaisei Otake
Keisuke Taira

Methods

Results

none

Notes

This is the start of the nutritional program (10:20).
Start of prenatal care (13:00)
Plasmid extraction of pgm-galU
Extraction of sample 4 8/5 10:00~8/8 18:36 total
Start of pre-culture of pCDF9474 and 7494

2023/08/13 (JST)

10:05 (JST) - 20:05 (JST)

Participants

Keisuke Nishimoto
Keisuke Taira
Yuta Shimamoto

Methods

Results

Notes

2023/08/14 (JST)

12:00 (JST) - 21:00 (JST)

Participants

Kaisei Otake

Methods

Results

I measured the concentration of DNA purified by nanodrop, which is 90.7 ng/µl for pRK, 77.5 ng/µl for pgm, 51.5 ng/µl for UGT709, 124.0 ng/µl for 7494, and 11.1 ng/µl for 9474, so DNA is present at a normal value

Notes

pgm+7494 to NEB competent cell, plate: cb50 + spec100
pgm+9474 to NEB competent cell, plate: cb50 + spec100
pgm + 7494 + pRK to NEB competent cell, plate: cb50 + spec100 + tet12.5
pgm + 9474 + pRK to NEB competent cell, plate: cb50 + spec100 + tet12.5
pRK + UGT709 to BL21 gold, plate: tet12.5 + cb50
Start pre-culture of pRK, 7494, 9474, 709
Measure absorbance of pRK, pgm, UGT709, 7494, 9474

2023/08/15 (JST)

10:00 (JST) - 20:00 (JST)

Participants

KaiseiOtake
HarutoNakamura
SyotaAso
ArisaImazu
JunSakai

Methods

none

Results

none

Notes

OD measurement
TSS of pRK+7494/9474
Starting main culture of pRK

2023/08/16 (JST)

14:20 (JST) - 22:00 (JST)

Participants

Yuta Yamagishi
Kaisei Otake
Keisuke Nishimoto

Methods

Results

Confirmation of growth on agar medium
Only pRK + UGT709 had about 3 colonies growing
No other colonies.
I measured the concentration of DNA purified by nanodrop, which is 19.3 ng/µl for pRK.

Notes

Confirmation of coexistence
The results of the culture are as follows: pgm + 7494, pgm + 9474 from colonies and from liquid culture, respectively, and concussion culture.
Mini prep and absorbance measurement of pRK
pRK (* 10 pcs) culture (for plasmid extraction)
Liquid culture of pgm + 7494, pgm + 9474, pRK + pgm, pRK + UGT709

2023/08/17 (JST)

13:00 (JST) - 0:00 (JST)

Participants

Yuta Yamagishi
Kaisei Otake
Shota Aso

Methods

Results

None

Notes

Miniprep and electrophoresis of pRK, pgm7494, and pgm9474
Start of liquid shaking culture of pgm-pRK
Incubate pRK+7494+pgm, pRK+9474+pgm, and positive control on plates after electrophoresis.

2023/08/18 (JST)

12:00 (JST) - 23:30 (JST)

Participants

Yuta Yamagishi
Kaisei Otake
Arisa Imazu

Methods

https://static.igem.wiki/teams/4955/wiki/protocols/culture-for-substance-production.pdf https://static.igem.wiki/teams/4955/wiki/protocols/od-measurement.pdf

Results

All colonies were formed.
pRK, pgm, UGT709, 7494, 9474 were diluted, but no bands were observed, so they were diluted again.
Other genes were clearly visible.
709:0.235,7494:0.692,9474:1.324
All the bacteria looked strange, so I assumed the reason was because I did not poke the single cell, spread the single cell on the plate & inoculated 5 ml of LB. (23:30)

Notes

Confirmation of coexisting stocks Electrophoresis of plasmid used pRK+709,pRK+7494+pgm,pRK+9474+pgm Start of pre-culture (17:00-)(OD600 0.09)
measurement (19:30) due to unusually good growth
Started culture of 7494,9474 at x1/100
OD measurement of 709,1/100 7494,1/100 9474 at 22:30

2023/08/19 (JST)

12:10 (JST) - 21:00 (JST)

Participants

Yuta Yamagishi
Kaisei Otake

Methods

Results

None

Notes

OD measurement after pre-culture
Induction & glucose+: glucose is added.
Started main culture for glu+.
TSS of pRK + UGT709
Start culture of pRK strain
Start of main culture
Glycerol stock is prepared.

2023/08/20 (JST)

14:00 (JST) - 21:00 (JST)

Participants

Yuta Yamagishi
Kaisei Otake

Methods

Culture for substance production

Results

None

Notes

Start of pRK+UGT709 pre-culture (14:30)
Incubate pRK+UGT709 plate at 37°C
∙ 21:00 200 μl sampling from glu+,- of pRK+7494+pgm and pRK+9474+pgm (24 hrs elapsed)

2023/08/21 (JST)

9:50 (JST) - 21:00 (JST)

Participants

Yuta Yamagishi
Kaisei Otake

Methods

Results

OD value was 2.901 after pre-pRK culture o/n before and after 709 + pRK.

Notes

200 µl sampling from glu+,- of pRK+7494+pgm and pRK+9474+pgm (36 hrs elapsed)
Measurement of OD value after o/n pre-culture of 709+pRK
Start of 709+pRK main culture (10:40)
Dilution of the sample that started the main culture at 10:40 today to get OD600=10 at 10:00 on 8/22 (doubling time was 2 hours, and diluted with TB to get OD0.08).
Pre-culture of streaked colonies from a single colony of 709+ pRK was started (20:12)
21:00 200 µl sampling from glu+,- of pRK+7494+pgm and pRK+9474+pgm (48 hours elapsed)

2023/08/22 (JST)

10:30 (JST) - 21:00 (JST)

Participants

Keisuke Taira
Kaisei Otake

Methods

Results

prk 709 od Determination 10:40
１　4.932
２　5.002
prk 709 Re-od Determination 13:30
１　8.01
２　8.70
Extraction of crocin
Ethanol does not yellow.

Notes

prk 709 od Determination 10:40
Sampling of prk pgm 7494 glu-, prk pgm 7494 glu+, prk pgm 9474 glu-, prk pgm 9474 glu+ 11:00
prk 709 Starting of this cultivation.
prk 709 Re-od Determination 13:30
prk 709 induction + glu addition ( 13:30)
Extraction of crocin

2023/08/23 (JST)

12:00 (JST) - 21:40 (JST)

Participants

Keisuke Nishimoto
Kaisei Otake
Jun Sakai

Methods

Results

PCR for pRK
The fr3 band did not occur.
Purification of fr1,2
The concentration of fr2 was low.

Notes

Dilution of pRK5~8 primers
PCR for pRK
Electrophoresis of pRK
Transformation of ΔREIpRK into DH5α
Purification of fr1,2
Gibson Assembly of ΔREpRK

2023/08/24 (JST)

11:00 (JST) - 20:00 (JST)

Participants

YutaYamagishi
KeisukeNisimoto
JunSakai
KaiseiOotake
HarutoNakamura

Methods

MINIPREP

Results

refining pRK
→117.7ng/ul

Notes

refining pRK
cultivate pRK
Mini prep ,nano drop and electrophoresis of pRK+7494 and pRK+9474
Sampling pRK-UGT709
Checking whether a pRK-UGT709 plate has smells of except escherichia coli

2023/08/25 (JST)

11:00 (JST) - 18:10 (JST)

Participants

Shota Suzuki
Keisuke Nishimoto

Methods

Results

None

Notes

Miniprep and nanodrop of pRK(ΔRE)
Restriction enzyme treatment and electrophoresis of pRK and pRK(ΔRE)
Culture of pRK+pgm+7494, pRK+pgm+9474

2023/08/26 (JST)

10:00 (JST) -

Participants

Haruto Amano
Kaisei Otake

Methods

MINIPREP

Results

The order of electrophoresis:
MK3
1kbladder
Restriction enzyme-pRK (without restriction enzyme treatment)
pRK (without restriction enzyme treatment)
Restriction enzyme-pRK (with restriction enzyme treatment)
pRK (with restriction enzyme treatment)
pRK+pgm+7494 (without restriction enzyme treatment)
pRK + pgm + 9474 (without restriction enzyme treatment)
pRK + pgm + 7494 (with restriction enzyme treatment)
pRK + pgm + 9474 (with restriction enzyme treatment)

Notes

Miniprep of pRK + pgm + 7494/9474
Restriction enzyme-pRK, restriction enzyme treatment of pRK with Restriction enzyme and Ecor1 & pgm + pRK + 7494/9474. As per the protocol, we used a larger amount of final.
Restriction enzyme treated above and electrophoresis for comparison.
Streak pRK + pgm + 7494/9474, pRK, 7494 + pgm on the plate

2023/08/27 (JST)

10:00 (JST) -

Participants

Haruto Amano
Keisuke Taira
Kaisei Otake

Methods

Results

From left to right: ① 1kb ladder, ② SC ladder, ③ pRK + pgm + 7494 (5ng), ④ pRK + pgm + 9474 (5ng), ⑤ pRK + pgm + 7494 Ecor I treated (5ng), ⑥ pRK + pgm + 9474 Ecor I treated (5ng), ⑦ none, ⑧~11 were performed without dilution without changing conditions from previous phases of ③~⑥.

Notes

Electrophoresis of pRK+pgm+7494 and pRK+pgm+9474
Introduction of 7494/9474 into pRK by TSS, respectively

2023/08/28 (JST)

10:00 (JST) - 22:20 (JST)

Participants

Masayuki Su'etsugu
Kaisei Otake

Methods

Results

Measurement of pRK+709 product (GC/MS) should be done by LC/MS

Notes

Start of pRK (positive control), pgm + 7494 (negative control), pRK + pgm + 7494 (objective) culture
Preparation of TB medium
Started pRK culture in case pRK+7494/9474 did not grow.
Dodecane extraction from pRK+709
Measurements of pRK+709 product (GC/MS)
Transformation of pRK(ΔRE) into BL21(DE3)
TSS of pRK+7494/9474 again
Induction & glucose addition (20:30)

2023/08/29 (JST)

12:00 (JST) - 22:30 (JST)

Participants

Arisa Imazu
Kaisei Otake

Methods

Sampling from 4 targeted pRK+pgm+7494 (12:17)
ΔREpRK plate confirmed, culture started (12:46)
Culture for substance production
Confirmation of pRK7494,pRK9474 plates and start of culture
Culture for substance production
RE,709(posicon) introduced into ΔREpRK by TSS
TSS
21:10 200 μl sampling from 4 flasks of the target (pRK+pgm+7494) in shaking culture at 25°C
Started culture of ΔREpRK (BL21) in 5ml of LB (for making elepos of ΔREpRK +RE/7494/9474)
Culture for substance production
Start culture of ΔREpRK(DH5α) in 5ml of LB (for grist preparation & restriction enzyme treatment)
Culture for substance production
Initiation of culture of 9474(DH5α) in 5ml of LB (10 pcs)
Culture for substance production
Autoclave of 10% glycerol (for elepocompilation)

Results

Inoculation may not have been successful (amount of bacteria in suspension was low)
OD after 1 hour = 0.135 (13:53)
OD after 2 hours = 0.030 (14:56)
(I re-measured and it was 0.050 (15:02), so it is possible that the measurement after 1 hour was incorrect.)
→Dr. Suetsugu looked at the inoculum and found that it did not seem to be inoculated properly, so he started another culture (15:54).
Only 7494 made on 8/27 had colonies (many yellow on the outside of the plate, one white on the inside).
Since there is a possibility that the transformation was not successful, the bacteria from this plate were not used for inoculation, and inoculated from the pRK plate made on 8/26 and started culture (14:55)

Notes

2023/08/30 (JST)

11:00 (JST) - 22:30 (JST)

Participants

Kaisei Otake

Methods

Results

None

Notes

11:30 Sampling 200 µl from 4 flasks of the target (pRK + pgm + 7494) in shaking culture at 25°C.
12:00 Dilute 1/100 of pRK(BL21)(ΔRE) and start culture at 25℃, 30℃, 37℃. culture is 100ml, shake in 300ml flask at
Electroporation
Inoculate 5ml of LB with pRK(DH5α)(ΔRE).
Autoclave
22:10 200 µl sampling from 4 of the flasks of interest (pRK + pgm + 7494) in shaking culture at 25°C

2023/08/31 (JST)

12:00 (JST) - 23:30 (JST)

Participants

Kaisei Otake
Arisa Imazu
Akane Shoji

Methods

Sampling of 4 of the targeted pRK+pgm+7494 in shaking culture at 25°C (12:06)
Inoculated from ΔREpRK+re plate and started pre-culture with 5ml of LB
Culture for fluorescence microscopy
Inoculated from ΔREpRK+7494 and ΔREpRK+9474 plates and started pre-culture in 5ml of LB
Culture for fluorescence microscopy
Confirmation of necessary tools for the main culture to be done tomorrow
Preparation of plates containing re inducers and antibiotic
Preparation of plates containing re inducers and antibiotics (re-made)
Sampling of 4 of the targeted pRK+pgm+7494 in shaking culture at 25°C (20:30)
Streak ΔREpRK + re and Δre (negacon for zombification by induction) on a plate containing re inducers and antibiotics
Restriction enzyme treatment of ΔREpRK (EcorI and RE) and confirmation by electrophoresis & fusion

Results

Inoculated from plate #2 of 4 plates and started incubation at 13:10.
Inoculated from both plates with 50 µl of Eleppo and started incubation at 13:10.
ATC 5 µl and gent7100 µl were added to 100 ml of LB aga.
The rest were dispensed in 5ml only and stored at -80°C
Concentration is too thin to see. Redo tomorrow.

Notes

2023/09/01 (JST)

12:00 (JST) - 22:00 (JST)

Participants

Arisa Imazu
Kaisei Otake

Methods

100ml flask autoclave
Confirmation of colony inhibition on aTc plate of ΔREpRK +RE
Redo pre-culture of ΔRE+RE, ΔRE+7494, ΔRE+9474, and start main culture
Culture for fluorescence microscopy
Agarose gel preparation
Measurement of OD value at 2 hours after the start of main culture
OD measurement
Restriction enzyme treatment of ΔREpRK (EcorI and RE) and confirmation by electrophoresis & fusion

Results

No signs of inhibition were observed when we checked at 12:43 p.m., but when we checked again at 3:40 p.m., we confirmed that one micro colony was formed on each plate.
As a result of the pre-culture started yesterday, the culture medium was almost clear, so the pre-culture was started again. (14:38) At the same time, the main culture was started using the culture medium that had been pre-cultured since yesterday. (14:22)
ΔRE+RE 0.178→0.028
ΔRE+7494 0.188→0.038
ΔRE+9474 0.154→0.004
The TB was 0.15 when measured because the blank was taken in an empty cuvette.
Concentration is too thin to see. Redo tomorrow.

Notes

2023/09/02 (JST)

13:00 (JST) - 22:00 (JST)

Participants

Towako Ikegami
Jun Sakai
Keisuke Nishimoto
Kaisei Otake
Keisuke Taira
Haruto Nakamura

Methods

Results

ΔREpRK (with restriction enzyme treatment) showed no bands
From left to right: mk3,15kb, pRK (without restriction enzyme treatment), pRK (with restriction enzyme treatment), ΔRERK (without restriction enzyme treatment), ΔREpRK (with restriction enzyme treatment), 1kb

Notes

OD measurement
Electrophoresis & fusion of ΔRE + pRK

2023/09/03 (JST)

10:30 (JST) -

Participants

Yuta Yamagishi
Keisuke Taira
Keisuke Nishimoto
Jun Sakai

Methods

none

Results

Plate confirmation of ΔREpRK + re
It was not growing. I brought the plate back to 37 degrees Celsius once more, just in case.
Electrophoresis & FUSION of restriction enzyme-treated ΔREpRK
Re-electrophoresis of ΔREpRK
The band didn't show up.
Since the band was not seen at the desired location, we will try a different limiting enzyme next time.

Notes

Plate confirmation of ΔREpRK + re
Re-electrophoresis of ΔREpRK
Restriction Enzyme Treatment of ΔREpRK
Electrophoresis & FUSION of restriction enzyme-treated ΔREpRK

2023/09/04 (JST)

13:00 (JST) - 23:05 (JST)

Participants

Yuta Yamagishi
Jun Sakai
Kaisei Otake

Methods

Results

electrophoresis
From left to right:
1kb,15kb,ΔREpRK,ΔREpRK(cut),pRK,pRK(cut)

Notes

13:50 1/100 dilution of ΔREpRK(BL21), start culture at 25°C, 30°C, 37°C. culture is 100ml, shake in 300ml flask at,150rpm
Extraction of culture medium of pRK+pgm+7494,pRK+709
400 ml of 10%glycerol made
Restriction Enzyme Treatment of ΔREpRK and pRK
Introduction & inoculation of re into DH5α
electrophoresis
Dyeing & fusion
Competent cell preparation for electroporation of ΔREpRK & electroporation of re(CV)

2023/09/05 (JST)

13:00 (JST) - 14:30 (JST)

Participants

Yuta Yamagishi
Kaisei Otake

Methods

Results

No peaks could be detected for both crocin and picrocrocin.

Notes

Start pre-culture of pRK(ΔRE)+RE(CV) in 5ml of LB
Streak of plates of tet12.5+gent7 and tet12.5+gent7+CV
Measurement of samples that will be producing picrocrocin/crocin

2023/09/06 (JST)

12:00 (JST) - 20:30 (JST)

Participants

Yuta Yamagishi
Kaisei Otake

Methods

Results

None

Notes

Culture of pRK(ΔRE)+RE and OD measurement
14:24 Start of culture for zombie confirmation/fluorescence microscopy (diluted to OD 0.02), OD measurement
Start pre-culture of pRK(ΔRE)+RE(CV) in 5ml of LB.
Streak of tet12.5+gent7 and tet12.5+gent7+CV plates
Fluorescence microscopy

2023/09/07 (JST)

10:40 (JST) - 20:00 (JST)

Participants

Kaisei Otake
Jun Sakai
Keisuke Nishimoto

Methods

Results

Fluorescence microscopy
OD at start: 0.4

Notes

ΔRE + re Beginning of the current cultivation (11:00)
OD measurement
Start of fluorescence observation culture
Fluorescence microscopy
Start of pre-culture of pRK(DH5α),pRK(BL21),pRK+7494,7494
Electroporation of pETEc into competent cells for ΔREpRK electroporation

2023/09/08 (JST)

13:00 (JST) - 22:00 (JST)

Participants

Kaisei Otake

Methods

Results

None

Notes

Start of main culture of pRK(DH5α),pRK(BL21),pRK+7494,7494
OD measurement of pRK(ΔRE)+RE
Spread pRK, pRK+709, and pRK+pETCK on the same plate (tet12.5 + 50 μl IPTG).
Spread pRK(BL21), pRK(DH5α), pRK(ΔRE), pRK+pgm+7494, pRK+pgm+9474, pRK+709, pRK+7494, pRK+9474, 7494 (negacon) with IPTG+/-.
pRK(ΔRE) + RE induction of IPTG+, CV+/-.

2023/09/09 (JST)

13:20 (JST) - 19:00 (JST)

Participants

Kaisei Otake

Methods

Results

None

Notes

200μl sampling & OD measurement of CV+- of pRK(ΔRE) + RE (13:30)
OD measurement of pRK(DH5α),pRK(BL21),pRK+7494,7494 & dilution for induction at appropriate time.
Start pre-culture of pRK(ΔRE)+RE (14:30)
Streak pRK, pRK+709, pRK+pETCK again on plates with appropriate antibiotics

2023/09/10 (JST)

12:20 (JST) - 21:00 (JST)

Participants

Kaisei Otake
Yuta Yamagishi
Jun Sakai

Methods

Results

OD(12:30)
CV+ of ΔREpRK+re: 13.62
CV- of ΔREpRK+re: 14.8
pRK(DH5α): 7.77
pRK(BL21): 12.85
pRK + 7494: 12.81
7494: 15.57
14:30
OD of pre-culture solution is 1.7
19:00 ODx1/20
ΔREpRK + CV+ of re: 0.906
CV- of ΔREpRK + re: 0.760
pRK(DH5α)0.409
pRK(BL21): 0.444
pRK+7494: 0.636
7494: 0.943

Notes

200μl sampling & OD measurement of CV+- of ΔRE+re
Sampling & OD measurement of pRK(DH5α),pRK(BL21),pRK+7494,7494
∙ 14:30 Start of main culture of ΔREpRK+re for fluorescence microscopy from 9/11
Started pre-culture with 5ml of LB from 9/12 for fluorescence microscopy in case OD had overgrown.
Photograph streaks of pRK, pRK+709, and pRK+pETCK again on plates with appropriate antibiotics
Glycerol stock preparation (ΔREpRK + re(cv),ΔREpRK + 7494,ΔREpRK + 9474,ΔREpRK + 709
200 µl sampling & OD measurement of CV+- of ΔREpRK+re
Sampling & OD measurement of pRK(DH5α),pRK(BL21),pRK+7494,7494

2023/09/11 (JST)

10:00 (JST) - 23:00 (JST)

Participants

Yuta Yamagishi
Kaisei Otake
Keisuke Nishimoto
Jun Sakai

Methods

Results

none

Notes

Diluted to an OD of 2, incubated
Sampling CV+, CV- of ΔREpRK + re, OD measurement
Sampling and OD measurement of pRK(DH5α), pRK(BL21), pRK + 7494, 7494
Fluorescence microscopy

2023/09/12 (JST)

14:30 (JST) - 20:00 (JST)

Participants

Yuta Yamagishi
Jun Sakai

Methods

Results

none

Notes

CV+, -, pRK(DH5α), pRK(BL21), pRK+7494, 200 μl sampling of ΔREpRK+re & OD measurement of 7494 (14:45)
(24h) OD measurement & fluorescence microscopy of CV+- of ΔREpRK of 4103
200 μl sampling & OD measurement of pRK(DH5α), pRK(BL21), pRK+7494, 7494 (18:10)
CV+ and - of ΔREpRK+r 200 µl sampling & OD measurement (19:30)
Preparation of agar medium containing tet12.5+cb

2023/09/13 (JST)

12:00 (JST) - 21:15 (JST)

Participants

Yuta Yamagishi
Arisa Imazu
Jun Sakai

Methods

200μl sampling & OD measurement of pRK(DH5α),pRK(BL21),pRK+7494,7494
OD measurement
PCR & electrophoresis for Gibson assembly (ΔREpRK fr1,fr2,74for ΔREpRK )
Autoclave
Culture of ΔREpRK + re in 5ml of LB
200μl sampling & OD measurement of ΔREpRK+re CV+, -,pRK(DH5α),pRK(BL21),pRK+7494,7494
Excess culture was put in Falcon tube and stored at -80°C with contents identified
PCR & electrophoresis of 709

Results

pRK(DH5α):4.835
pRK(BL21):7.940
pRK+7494:8.755
7494:9.785
Correct amplification was achieved.
Amplified

Notes

2023/09/14 (JST)

12:00 (JST) - 17:00 (JST)

Participants

Arisa Imazu
Kaisei Otake

Methods

Fragment purification, nanodrop
Fragment purification
Gibson assembly of 709,74 into ΔREpRK, transformation into DH5α
Transformation to DH5α
Streak of ΔREpRK+709, ΔREpRK+pETCK, and ΔREpRK
Preparation of plate with tet12.5
Agar medium preparation
OD measurement of ΔREpRK +RE pre-culture medium
OD measurement
Dilution with TB to 0.007 (OD8 expected at 17:00)

Results

fr1:175.0(ng/μl)
fr2:117.7
74:183.4
709:143.8
DNA concentration was changed to 0.09 because it exceeded 10 μl when performed at 0.1. incubation started at 15:30.
OD value 1.361
0.5 ml of pre-culture solution and 99.5 ml of TB

Notes

2023/09/15 (JST)

12:00 (JST) - 17:00 (JST)

Participants

Yuta Yamagishi
Keisuke Nishimoto
Jun Sakai

Methods

Results

none

Notes

OD measurement of ΔREpRK+re
Check if ΔREpRK+709 (DH5α) and ΔREpRK+74 (DH5α) incubated at 37°C are growing & shaken in 5ml of LB
Fluorescence microscopy
Start of pre-culture of ΔREpRK+re

2023/09/16 (JST)

13:30 (JST) - 23:30 (JST)

Participants

Yuta Yamagishi
Kaisei Otake

Methods

Results

Gotaq confirmed that the assembly of the miniprep plasmid was well accomplished for pRK(ΔRE)74.
1kb ladder,2 lanes are 74(template)(posicon), 3~5 lanes are pRK(ΔRE)74 yellow colonies, 6~8 lanes are pRK(ΔRE)74 white colonies, 9 lanes are 709(template)(posicon), 10~12 lanes are pRK(ΔRE)709 yellow colonies, 13,14,17 are pRK(ΔRE)709 white colonies

Notes

Transformation of pRK(ΔRE)-74 and pRK(ΔRE)709 into BL21(DE3)
Transformation of BL21(DE3) on
colony PCR of pRK(ΔRE)pRK-74 and pRK(ΔRE)pRK-709
Start of pre-culture of pRK(ΔRE)+RE

2023/09/17 (JST)

13:45 (JST) - 19:30 (JST)

Participants

Yuta Yamagishi
Jun Sakai

Methods

Results

electrophoresis
Bands 709 and 709 1 were in the same location.

Notes

Electrophoresis of 709 ポ,709 1-6,.
Streak of ΔREpRK709 into LB5mL, plate
Streak of ΔREpRK74 into LB5mL, plate
Culture for miniprep of ΔREpRK74
Start of main culture of ΔREpRK+re
TB media preparation

2023/09/18 (JST)

14:00 (JST) - 22:00 (JST)

Participants

Yuta Yamagishi
Jun Sakai
Kaisei Otake

Methods

Results

none

Notes

Fluorescence microscopy of ΔREpRK+re
ΔREpRK74 miniprep& ethanol precipitation, transformation into BL21
Start of main culture of ΔREpRK709 & grist production from single colony
TB media preparation
Photographs of plates used to test hypothesis of inhibition of pTaq downstream enzyme expression by overexpression of enzyme downstream of T7 promoter

2023/09/19 (JST)

12:00 (JST) - 22:40 (JST)

Participants

Yuta Yamagishi
Arisa Imazu
Kaisei Otake

Methods

OD measurement & fluorescence microscopy of CV+- of ΔREpRK+RE
OD measurement
Fluorescence Microscopy
200μl sampling & OD measurement from ΔREpRK709
OD measurement
Transformation of ΔREpRK74 into BL21(DE3)
Transformation of BL21
MS
HP-LC-MS_MS analysis

Results

Conducted in 5 µl because of low transformation efficiency
Considering the toxicity of the plasmid, transformation to DH5α was also performed at the same time.
Yesterday's transformation was centrifuged (13000 rpm, 5 min), 900 µl was removed, suspended, and 50 µl spread.
https://docs.google.com/presentation/d/1cGNZh9oJ2LBNzBOe2YfezLG0MPes7K92gIQRzJ9qLo4/edit?usp=sharing

Notes

2023/09/20 (JST)

12:00 (JST) - 22:40 (JST)

Participants

Yuta Yamagishi
Arisa Imazu
Kaisei Otake

Methods

OD measurement & fluorescence microscopy of CV+- of ΔREpRK+re
OD measurement
Fluorescence Microscopy
200 μl sampling & OD measurement from ΔREpRK709
OD measurement
ΔREpRK74 incubated at 37°C, check if pRK is growing. If they are growing, poke the colony and start shaking incubation at 5 ml of LB and 37°C.
At the same time, poke the ΔREpRK on the tabletop and start shaking culture with 5ml of LB.
Culture for fluorescence microscopy
Streak from a single colony of ΔREpRK74 to tet12.5LB agar medium

Results

Observation at 22:11 was not possible due to loss of POLY-L-LYSINE. Samples were stored at -20°C.
Incubation started 13:23
Incubation started 13:27

Notes

2023/09/21 (JST)

12:00 (JST) -

Participants

Yuta Yamagishi
Arisa Imazu
Kaisei Otake

Methods

OD measurement & fluorescence microscopic observation of CV+- of ΔREpRK+re
OD measurement
Fluorescence Microscopy
200μl sampling from ΔREpRK709 & OD measurement
OD measurement
ΔREpRK74, pRK(BL21(DE3)) main culture started , OD measurement every 1h
Culture for fluorescence microscopy
OD measurement

Results

When the protocol was followed without dilution, the CV+ was difficult to see because the bacteria overlapped, so it was re-taken with a 1.5x dilution.
In CV-, the bacteria hardly shone during fluorescence, so 2.5 µl of DAPI was added. Since there was still little change in the appearance of the bacteria, two patterns were taken: one with the exposure time set to CV+ and the other with the exposure time set to 1/2 second (0.5 second) during fluorescence.

Notes

2023/09/22 (JST)

15:00 (JST) - 23:30 (JST)

Participants

Yuta Yamagishi
Kaisei Otake
Keisuke Nishimoto

Methods

Results

None

Notes

OD measurement of pRK(ΔRE)+RE CV+,-, pRK74(ΔRE), pRK(BL21(DE3))
Sampling and OD measurement of pRK709(ΔRE)
Microscopic observation of samples
Start of pre-culture of pRK709(ΔRE), pRK(ΔRE)+709, pRK(ΔRE)+RE , pUPGFP, pRK74(ΔRE)
MS

2023/09/23 (JST)

11:30 (JST) - 17:30 (JST)

Participants

Yuta Yamagishi
Keisuke Taira
Jun Sakai
Hinata Yokokawa

Methods

Results

No preculture turbidity was observed for ΔREpRK74

Notes

Start of main culture of ΔREpRK709 and ΔREpRK+709
pUPGFPのminiprep
Pre-culture of ΔREpRK74 begins (14:40)
OD measurement of ΔREpRK709 and ΔREpRK+709
Education②Preparation with Pedi
Dilution of ΔREpRK709 and ΔREpRK+709

2023/09/24 (JST)

13:00 (JST) - 20:00 (JST)

Participants

Jun Sakai
Kaisei Otake

Methods

Results

OD at the start of this culture
2.91

Notes

Start of main culture of ΔREpRK74
ΔREpRK709,ΔREpRK+709,ΔREIpRK,pRK pre-culture start
Preparation of TB media
OD measurement of ΔREpRK74 at 19:30

2023/09/25 (JST)

12:00 (JST) - 20:00 (JST)

Participants

Arisa Imazu
Kaisei Otake

Methods

ΔREpRK74, ΔREpRK709, ΔREpRK+709Confirmation of turbidity
Autoclave flasks
Start of pre-culture of ΔREpRK, ΔREpRK74, ΔREpRK709, ΔREpRK+709, and pRK
Culture for fluorescence microscopy
Preparation of tet12.5 plate
Agar medium preparation
Streak of ΔREpRK, ΔREpRK74, ΔREpRK709, ΔREpRK+709, pRK to plate
Inoculation of E.coli Nissle 1917

Results

709 was not turbid, and for +74 and +709, the OD was 0.265 and 0.148, respectively, although slightly turbid, so three bottles were autoclaved.
Start of incubation 17:30

Notes

2023/09/26 (JST)

17:30 (JST) - 19:30 (JST)

Participants

Jun Sakai

Methods

Culture for substance production

Results

Check if E.coli Nissle 1917 strain is growing
It was growing.
Confirmation of successful preculture of ΔREpRK74,ΔREpRK709,ΔREpRK+709,ΔREpRK,pRK
It didn't grow.

Notes

Check if E.coli Nissle 1917 strain is growing
Confirmation of successful preculture of ΔREpRK74,ΔREpRK709,ΔREpRK+709,ΔREpRK,pRK
Confirmation of ΔREpRK74,ΔREpRK709,ΔREpRK+709,ΔREpRK,pRK plates
ΔREpRK709,ΔREpRK+709,ΔREpRK,pRK pre-culture
ΔREpRK74,ΔREpRK+7494 streak
Pre-culture of Nissle strain

2023/09/27 (JST)

12:00 (JST) - 20:30 (JST)

Participants

Yuta Yamagishi
Arisa Imazu
Jun Sakai

Methods

Autoclave flasksCheck if ΔREpRK74 and ΔREpRK+7494 are growing during incubation at 37°C, and start pre-culture
ΔREpRK709,ΔREpRK+709,ΔREpRK, pRK main culture started, re-start pre-culture
Culture for fluorescence microscopy
pRK(nissle),ΔREpRK(nissle),ΔREpRK + REI(CV)(nissle) elepo
electroporation
Plating of pRK(nissle),ΔREpRK(nissle),ΔREIpRK+RE(CV)(nissle)

Results

ΔREpRK74 was not growing, so only ΔREpRK+7494 started at 12:30.
Re-incubated pre-culture since the shaken culture was not turbid.
The main culture was also started by adding appropriate antibiotics to 48 ml of TB and 2 ml of the pre-culture solution. Both started at 17:15.

Notes

2023/09/28 (JST)

12:00 (JST) - 0:00 (JST)

Participants

Yuta Yamagishi
Arisa Imazu
Kaisei Otake

Methods

ΔREpRK709, ΔREpRK+709, ΔREpRK, pRK during the main culture, check for pRK turbidity
Confirmation of turbidity of ΔREpRK709, ΔREpRK+709, ΔREpRK, and pRK during pre-culture
Confirmation of the growth of nissle strain
Dissolve all the greaseㇳ of ΔREpRK74 and start incubation with 5ml of LB.
Plating of pRK(nissle),ΔREpRK(nissle),ΔREpRK+re(CV)(nissle)
Started pre-culture of ΔREpRK+709
https://docs.google.com/document/d/1lNBXAWCkBIBCbDjVZeQTuMB2lren4PAkArNoL7LgynY/edit
PCDFduet7494 pre-culture and streaking
https://docs.google.com/document/d/1lNBXAWCkBIBCbDjVZeQTuMB2lren4PAkArNoL7LgynY/edit
pRK, ΔREpRK main culture started, ΔREpRK709 pre-culture started
https://docs.google.com/document/d/1lNBXAWCkBIBCbDjVZeQTuMB2lren4PAkArNoL7LgynY/edit
Start of pre-culture for preparation of nissle elepo-Compi
https://docs.google.com/document/d/1lNBXAWCkBIBCbDjVZeQTuMB2lren4PAkArNoL7LgynY/edit
Crocetin extraction
https://static.igem.wiki/teams/4955/wiki/protocols/extraction-of-crocetin-crocin.pdfhttps://docs.google.com/document/d/1 lNBXAWCkBIBCbDjVZeQTuMB2lren4PAkArNoL7LgynY/edit

Results

All were 0,000 and were autoclaved.
ΔREpRK709 and pRK were turbid; ΔREpRK was turbid but low in turbidity; ΔREpRK+709 was clear; ΔREpRK+709 was clear; ΔREpRK+rel was clear.
Only 50 µl of ΔREpRK + re was sprinkled with minute white material was seen on the plate surface, but otherwise it did not grow.
Start at 13:50
Incubation started at 14:48 with 50 µl of yesterday's transformed and concentrated res.
Started at 16:24
Started at 16:40
Main culture started at 17:03 with 48 ml of TB and 2 ml of pre-culture solution. Pre-culture started at 17:10.

Notes

2023/09/29 (JST)

10:00 (JST) - 11:50 (JST)

Participants

Kaisei Otake
Jun Sakai

Methods

Results

none

Notes

Crocetin extraction
ΔREpRK(Nissle) incubated in LB 5ml antibiotic tet (17:30)
OD measurement of pRK,ΔREpRK (17:45)
IPTG induction of pRK,ΔpRK (20:00)
Start of pre-culture of ΔREpRK+709
Start of main culture of ΔREpRK709 (20:30)
Start of pre-culture of pRK+7494
Primer dilution
pCDFDuet7494PCR
Pre-culture of ΔREpRK (BL21)

2023/09/30 (JST)

12:00 (JST) - 17:00 (JST)

Participants

Yuta Yamagishi
Jun Sakai

Methods

Results

Electrophoresis of yesterday's PCR products
A band occurs between 5kb and 6kb
The blue seal, 7494, was shorter than its original length.
The DNA concentration in tube "1" was 22.8 ng/μl.

Notes

ΔREpRK(Nissle),REpRK+709,pRK,ΔREpRK,ΔREpRK709 pre-culture
Electrophoresis of yesterday's PCR products
Purification of 7494 PCR fragments marked "1" on the tube.
in fusion
Started culture of ΔREpRK(Nissle) for TSS
TSS

2023/10/01 (JST)

12:45 (JST) - 22:40 (JST)

Participants

Jun Sakai
Kaisei Otake
Yuta Yamagishi

Methods

Results

OD(13:00)
pRK 13.93
ΔREpRK 16.15
OD(22:30)
pRK
ΔREpRK　11.10

Notes

OD measurement & 200 µl sampling of pRK and ΔREpRK
Start of main culture & OD measurement
Transformation of infused 7494 into DH5α
Extraction of appropriate samples
Started 30c culture of single colonies of pCDFDuet7494 in 5 ml magic medium.

2023/10/02 (JST)

13:00 (JST) - 20:45 (JST)

Participants

Yuta Yamagishi
Jun Sakai
Keisuke Nishimoto

Methods

Results

none

Notes

Sampling of pRK and ΔREpRK, OD measurement
OD measurement of ΔREpRK(Nissle), pRK, ΔREpRK, ΔREpRK709,pRK7494, IPTG induction
OD measurement of ΔREpRK+re(Nissle), dilution
Confirmation of ΔREpRK+re(Nissle), 7494(DH5α) plates
5 ml magic medium collected, suspended, sonicated, centrifuged sup in 5 aliquots, liquid nitrogen, -80 storage

2023/10/03 (JST)

12:45 (JST) - 20:10 (JST)

Participants

Yuta Yamagishi
Keisuke Nishimoto

Methods

Results

None

Notes

Sampling of pRK, pRK(ΔRE), OD measurement, and storage at -80°C
OD measurement and sampling of pRK(Nissle)(ΔRE), pRK, pRK(ΔRE), pRK709(ΔRE),pRK7494
OD measurement, CV induction, IPTG induction, and fluorescence microscopy of pRK(ΔRE)+RE(Nissle)
Pre-culture of pRK(ΔRE)+REI(Nissle)

2023/10/04 (JST)

15:20 (JST) -

Participants

Yuta Yamagishi
Kaisei Otake

Methods

Results

None

Notes

Started shaking culture of pRK(ΔRE)+RE(Nissle)①~④ in 5ml test tube, TB, final OD0.1.
Sampling of pRK(ΔRE)(Nissle), pRK, pRK(ΔRE), pRK(ΔRE)709, pRK(ΔRE)+7494, OD measurement
Extraction of Crocetin from Nissle and BL21 strains

2023/10/05 (JST)

17:45 (JST) - 21:30 (JST)

Participants

Yuta Yamagishi
Keisuke Nishimoto

Methods

Results

None

Notes

OD measurement of pRK(Nissle)(ΔRE), preparation of competent cells for electropolation, electropolation
Miniprep of 7494
OD measurement and sampling of pRK(Nissle)(ΔRE), pRK, pRK(ΔRE), pRK709(ΔRE), pRK7494

2023/10/06 (JST)

10:30 (JST) - 11:00 (JST)

Participants

Kaisei Otake

Methods

None

Results

None

Notes

Streak of pRK(ΔRE)+RE(Nissle) on CV+- plate
Inoculation of 4 colonies of pRK(ΔRE)+RE(Nissle) and pRK(ΔRE)709 into 5ml of LB

2023/10/07 (JST)

14:00 (JST) -

Participants

Jun Sakai
Kaisei Otake
Keisuke Taira

Methods

Results

Fluorescence microscopy
30min(18:00)
1
CV+ 0.720
CV－0.806
4
CV+ 0.700
CV－0.736
1h(18:30)
1
CV+ 0.853
CV－1.212
4
CV+ 0.780
CV－1.069
2h(19:30)
1
CV+ 0.345
CV－ 2.805
4
CV+ 0.225
CV－ 2.98
4h(21:30)
1
CV+0.693
CV－5.268
4
CV+0.781
CV－5.538

Notes

ΔREpRK+re(Nissle) in 5 ml of TB, starting at OD 0.05 and 0.1, respectively
Fluorescence microscopy