Template:Kyoto/Notebook/Sep 17

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Contents

Sep 17

Sep 18

Restriction Enzyme Digestion

No name

9/18Plux SpeIPstIBufferBSAMilliQtotal
2cuts3.2µL1µL1µL3µL3µL18.8µL 30µL
NC1.7µL0µL0µL1µL1µL6.3µL 10µL
9/18Pcon-pT181antisense-spinach-DT②XbaIPstIBufferBSAMilliQtotal
2cuts7.5µL1µL1µL3µL3µL14.5µL 30µL
NC1.4icro;L0µL0µL1µL1µL6.6µL 10µL
9/16 Plac②SpeIPstIBufferBSAMilliQtotal
2cuts13.8µL1µL1µL3µL3µL14.5µL 30µL
NC0.7µL0µL0µL1µL1µL7.3µL 10µL
9/16 Spinach-DTXbaIPstIBufferBSAMilliQtotal
2cuts17.6µL1µL1µL3µL3µL4.4µL 30µL
NC0.9µL0µL0µL1µL1µL7.1µL 10µL
9/8 Pcon J23100SpeIPstIBufferBSAMilliQtotal
2cuts5.7µL1µL1µL3µL3µL16.3µL 30µL
NC0.3µL0µL0µL1µL1µL7.7µL 10µL
9/16 RBS-lacZα-DTXbaIPstIBufferBSAMilliQtotal
2cuts8.4µL1µL1µL3µL3µL13.6µL 30µL
NC0.4µL0µL0µL1µL1µL7.6µL 10µL
9/11 Pcon-pT181antisense-spinach-DT&#9312:XbaIPstIBufferBSAMilliQtotal
2cuts7.2µL1µL1µL3µL3µL14.8µL 30µL
NC0.4µL0µL0µL1µL1µL7.6µL 10µL
9/6 apt12-1R-DTXbaIPstIBufferBSAMilliQtotal
2cuts7.9µL1µL1µL3µL3µL14.1µL 30µL
NC0.4µL0µL0µL1µL1µL7.6µL 10µL
9/3 pT181antisenseXbaIPstIBufferBSAMilliQtotal
2cuts6.3µL1µL1µL3µL3µL15.7µL 30µL
NC0.3µL0µL0µL1µL1µL7.7µL 10µL
9/14 pSB4K5EcoRISpeIBufferBSAMilliQtotal
2cuts7.3µL1µL1µL3µL3µL14.7µL 30µL
NC0.4µL0µL0µL1µL1µL7.6µL 10µL
8/31 Plux-RBS-GFP-DT EcoRISpeIBufferBSAMilliQtotal
2cuts9.2µL1µL1µL3µL3µL12.8µL 30µL
NC0.5µL0µL0µL1µL1µL7.5µL 10µL
9/12 RBS-lysis3-DT XbaIPstIBufferBSAMilliQtotal
2cuts5.2µL1µL1µL3µL3µL16.8µL 30µL
NC0.3µL0µL0µL1µL1µL7.6µL 10µL
9/18 Pcon-pT181attenuatorEcoRISpeIBufferBSAMilliQtotal
2cuts14.3µL1µL1µL3µL3µL7.7µL 30µL
NC0.7µL0µL0µL1µL1µL7.3µL 10µL

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1Plae② SpeIPstI
2RBS-lysis3-DTXbaIPstI
3Plux-RBS-GFP-DTEcoRISpeI
4PSB4K5EcoRISpeI
5PconSpeIPstI
6PluxSpeIPstI
71kbp ladder----
8Plae② ----
9RBS-lysis3-DT----
10Plux-RBS-GFP-DT----
11PSB4K5----
12Pcon----
13Plux----

Igku 9183.jpg

Electrophoresis

Okazaki

LaneSampleEnzyme1Enzyme2
1100bp ladder -- --
2Pcon pT181antisense spinach-DT②XbaIPstI
3Spinach-DTXbaIPstI
4RBS-lacZα-DT XbaIPstI
5Pcon pT181antisense-Spinach-DT① XbaIPstI
6aptamer12-1R-DTXbaIPstI
7pT181attenuatorXbaIPstI
8Pcon attenuatorEcoRISpeI

Igku 9181.jpg

LaneSampleEnzyme1Enzyme2
1100bp ladder -- --
2Pcon pT181antisense spinach-DT②XbaIPstI
3Spinach-DTXbaIPstI
4RBS-lacZα-DTXbaIPstI
5Pcon pT181antisense-Spinach-DT①XbaIPstI
6apt12-1R-DTXbaIPstI
7pT181attenuatorXbaIPstI
8Pcon attenuatorEcoRISpeI
LaneSampleEnzyme1Enzyme2
1100bp ladder -- --
2Pcon pT181antisense spinach-DT②----
3Spinach-DT----
4RBS-lacZα-DT----
5Pcon pT181antisense-Spinach-DT①----
6apt12-1R-DT----
7pT181attenuator----
8Pcon attenuator----

Igku 9182.jpg

Gel Extraction

No name

LaneDNAEnzyme
11kbp ldder--
2Plac②SpeI+PstI
3Plac②SpeI+PstI
4----
5RBS-lysis3-DTXbaI+PstI
6RBS-lysis3-DTXbaI+PstI
7----
8Plux-RBS-GFP-DTEcoRI+SpeI
9Plux-RBS-GFP-DTEcoRi+SpeI
10----
11PSB4KSEcoRI+SpeI
12PSB4KSEcoRI+SpeI

Igku 9188.jpg
Igku 9189.jpg

LaneDNAEnzyme
11kbp ldder--
2PconSpeI+PstI
3 PconSpeI+PstI
4----
5PluxSpeI+PStI
6 PluxSpeI+PStI

Igku 9184.jpg
Igku 9185.jpg

LaneDNAEnzyme
1Pcon PT181 antisense spinach-DTXbaI+PstI
2 Pcon PT181 antisense Spinach-DTXbaI+PstI
3100bp ladder --
4Pcon PT181 antisense Spinach-DTXbaI+PstI
5 Pcon PT181 antisense Spinach-DTXbaI+PstI
6 1kbp ladder--

Igku 9186.jpg
Igku 9187.jpg

LaneDNAEnzyme
1100bp ladder --
2apt12-1R-DTXbaI+PstI
3apt12-1R-DTXbaI+PstI
4----
5PT181 attenuatorXbaI+PstI
6PT181 attenuatorXbaI+PstI

Igku 91810.jpg
Igku 91811.jpg

LaneDNAEnzyme
1100bp ladder --
2Pcoa attenuatorEcoRI+PstI
3Pcoa attenuatorEcoRI+PstI
4----
5RBS-lacZα-DTXbaI+PstI
6RBS-lacZα-DT XbaI+PstI

Igku 91812.jpg
Igku 91813.jpg

Sep 19

Sep 20

Ligation

Okazaki, Kanaoka, Tatsui and Hirano

stateVectorInserterLigation High ver.2
experiment9/18 Plac (SpeI & PstI)3.0 µL9/18 RBS-lysis3-DT (XbaI & PstI)12.4 µL3.5 µL
experiment9/12 Ptet-pT181 antisense1.0 µL9/18 spinach-DT11.9 µL3.5 µL
experiment9/13 Pcon-RBS-luxR-DT2.1 µL9/10 Plux-RBS-GFP-DT5.6 µL3.5 µL
experiment9/13 Pcon-pT181 attenuator (SpeI & PstI)2.0 µL9/18 aptamer 12-1R-DT (XbaI & PstI)6.3 µL3.5 µL
experiment9/18 Plac ②(SpeI & PstI)3.0 µL9/18 aptamer 12-1R-DT(XbaI & PstI)4.5µLx3.5 µL

Transformation

Tatsui

NameSampleCompetent CellsPlate
9/17 pSB1C3-Pcon-pT181 attenuator-DT2 µL20 µLCP
9/17 pSB1C3-Pcon-aptamer 12_1R-DT2 µL20 µLCP
9/17 pSB1C3-Pcon-antisense-spinach-DT2 µL20 µLCP
9/17 pSB1C3-Pcon-RBS-tetR-DT x22 µL20 µLCP
9/14 pSB1C3-PkaiBC2 µL20 µLCP
9/14 pSB1C3-RpaB2 µL20 µLCP
9/16 pSB1C3-pT181 attenuator(EcoRI&SpeI) 2 µL20 µLCP
9/17 pSB1C3-pT181 attenuator(XbaI&PstI) 2 µL20 µLCP
9/17 pSB1C3-Pcon-spinach-DT2 µL20 µLCP
9/14 RBS-GFP-DT-PkaiBC2 µL20 µLCP
Plux-RBS-lysis3-DT2 µL20 µLCP
9/20 Ptet-pT181 antisense-spinach-DT2 µL20 µLCP
9/20 Pcon-spinach-DT(pSB4K5) 2 µL20 µLKan
9/20 Pcon-pT181 antisense-spinach-DT(pSB4K5) 2 µL20 µLKan
9/20 KaiABC2 µL20 µLKan
9/20 KaiABC2 µL20 µLKan
9/20 Plac-RBS-lysis3-DT2 µL20 µLAmp
9/20 Plac-aptamer 12_1R2 µL20 µLAmp
9/20 Plux-RBS-GFP-DT-Pcon-RBS-luxR-DT2 µL20 µLAmp
Plac-aptamer12_1R-DT2 µL20 µLAmp
9/20 Plac-aptamer12_1R-DT2 µL20 µLAmp
9/20Pcon-pT181attenuator-aptamer-DT2 µL20 µLAmp
9/17 Plac-pT181-attenuatorx22 µL20 µLAmp
9/15 Pcon-RBS-lacZα-DT2 µL20 µLAmp
9/14 Pcon-pT181attenuator-RBS-lacZα-DT2 µL20 µLAmp
PUC192 µL20 µLAmp

PCR

Tatsui

Samplebase pair
pT181 attenuator(pSB1C3)--
Pcon-RBS-lacZα-DT--
PreDenatureDenatureAnnealingExtensioncycle
94 °C94 °C55 °C68 °C--
5 min30 s30 s3 min30 cycles

Electrophoresis

No name

LaneSampleEnzyme
1Pcon-RBS-lacZα-DT--
21kb ladder--
3pSB1C3-pT181attenuator --

Sep 21

Electrophoresis

no name

LaneSampleEnzyme1Enzyme2
1Pcon-RBS-lacZα-DT----
21kbp ladder----
3pSB1C3+pT181 attenuator----

Igku 9211.jpg

Liquid Culture

No name

Samplemedium
9/2 spinach-DT-2--
8/16 Plux-4--
8/27 Plux-RBS-GFP-DT-1--
9/5 Pcon-pT181 attenuator-1--

Master Plate

No name

NumberUse LB plate
18/27 Pλ-luxI-1(+Amp)
29/10 Plac(BBa-R0011)-3(+Amp)
39/5 Pcon-RBS-tetR-DT-1(+Amp)
49/5 Pcon-pT181 antisense-1(+Amp)
59/15 Pcon-pT181 attenuator(+Amp)
69/- Pcon-apt12_1R-DT-1(+Amp)
79/5 Pcon-spinach-DT-3(+Amp)
88/27 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT(+Amp)
NumberUse LB plate
19/4 pT181 antisense(pSB1C3)-1(+CP)
29/11 Fusion1 antisense(pSB1C3)-4(+CP)
39/11 Fusion6 antisense(pSB1C3)-1(+CP)
49/4 pT181 attenuator(pSB1C3)-1(+CP)
59/10 Fusion1 attenuator(pSB1C3)-1(+CP)
69/11 Fusion3n2 attenuator(pSB1C3)-1(+CP)
79/4 aptamer12_1R(pSB1C3)-1(+CP)
89/11 aptamer12_P(pSB1C3)-3(+CP)
99/11 aptamer12_1M(pSB1C3)-1(+CP)

</div>

NumberUse LB plate
18/29 Pbad/araC-RBS-RFP-DT-1(+CP)
29/4 RBS-lysis3-DT-3(+CP)
39/10 Pcon-pT181 attenuator-DT-1(+CP)
49/5 Ptet-pT181 antisense-1(+CP)
59/4aptamer 12_1R-DT-1(+CP)
69/5 Ptet-RBS-lacZα-DT-4(+CP)
78/10 RBS-lacI-DT-2(+CP)
88/29 Plux-RBS-GFP-DT-1(+CP)

</div>

Restriction Enzyme Digestion

Noname

8/17 RBS-DTEcoRISpeIXbaIPstI BufferBSAMilliQtotal
2cuts(XbaI&PstI)8.10µL0µL1µL1µL3µL3µL13.9µL30µL
NC0.40µL0µL0µL0µL1µL1µL7.6µL10µL
8/17 RBS-DTEcoRISpeIXbaIPstIBufferBSAMilliQtotal
2cut(EcoRI&XbaI)16.21µL1µL0µL0µL3µL3µL5.8µL30µL
9/15 Pcon-pT181attenuatorEcoRISpeIXbaIPstI BufferBSAMilliQtotal
2cuts(EcoRI&PstI)121µL0µL0µL1µL3µL3µL10µL30µL
NC0.60µL0µL0µL0µL1µL1µL7.4µL10µL
8/20 Pcon=RBS-GFP-DT-1EcoRISpeIXbaIPstIBufferBSAMilliQtotal
2cut(EcoRI&XbaI)61µL0µL1µL0µL3µL3µL0µL14µL
NC0.30µL0µL0µL0µL1µL1µL7.3µL10µL
incubate 37°C 1hour

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1RBS-GFP-DTXbaIPstI
2RBS-GFP-DT----
3RBS-GFP-DTEcoRIXbaI
41kbp ladder----
5Pcon-pT181attenuatorSpeIPstI
6Pcon-pT181attenuator----
7Pcon-RBs-GFP-DTEcoRIXbaI
8Pcon-RBs-GFP-DT----

Igku 9212.jpg

PCR

Nakamoto

Samplebase pair
Pcon-apt12_1R-DT(E-1A)50
Pcon-spinach-DT(E-1A)402
Pcon-pT181 attenuator(E-1A)400
DT(1-3)65
tetR aptamer 12_1R-DT(1-3)283
Pcon-pT181 attenuator(1-SA)303
spinach-DT(2-1)338
Pcon-pT181 attenuator(E-0)403
Pcon-pT181 attenuator(2-SA)403
spinach-DT(3-2)395
Ptet-PT181 anisense 400
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C57°C68°C--
2min10s30s35s30cycles

Gel Extraction

No name

LaneDNAEnzyme
11kb ladder--
3RBS-GFP-DTXbaI&PstI
4RBS-GFP-DTXbaI&PstI
5RBS-GFP-DTXbaI&PstI

Igku 9213.jpg
Igku 9214.jpg

Nameconcentration[µg/mL]260/280260/230
RBS-GFP-DT(XbaI&PstI)14.81.740.47
RBS-GFP-DT(EcoRI&SpeI)88.41.851.49
Pcon-RBS-GFP-DT(EcoRI&XbaI)47.51.821.12

Sep 22

Colony PCR

Tatsui

Samplebase pair
9/20 Plac-PT181 attenuator(1~4)664
9/20 Plac-PT181 antisense(1~6)369
9/20 Pcon-PT181 attenuator-aptamer-DT(1~6)859
9/20 Pcon-PT181 antisense-spinach-DT(1~8)723

Igku 0903 E1.jpg
Igku 0922 E2.jpg

Electrophoresis

Nakamoto

LaneSample
1Pcon-apt12_1R-DT(E-1A)
2Pcon-spinach-DT(E-1A)
3Pcon-pT181 attenuator(E-1A)
4DT(1-3)
5tetR aptamer 12_1R-DT(1-3)
6100bp ladder
7Pcon-pT181 attenuator(1-SA)
8spinach-DT(2-1)
9Pcon-pT181 attenuator(E-0)
10Pcon-pT181 attenuator(2-SA)
11spinach-DT(3-2)
12Ptet-PT181 anisense

Igku 0922 E3.jpg
Igku 0922 E4.jpg

Miniprep

Honda

DNAconcentration[µg/mL]260/280260/230
9/21 spinach-DT197.91.671.54
9/21 Plux125.91.711.27
9/21 Plux-RBS-GFP-DT-(1)168.01.621.73
9/21 Pcon-PT181 attenuator-(1)368.31.571.62
9/21 RBS-GFP-DT532.81.751.78
9/21 Pcon-RBS-GFP-DT845.22.021.70

Colony PCR

Honda

Samplebase pair
9/20 Plux-RBS-GFP-DT-Pcon-RBS-luxR-DT-(1~2)283
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s2min10s30cycles

Igku 0922 E5.jpg
Igku 0922 E6.jpg

Gel Extraction

No name

LaneDNAEnzyme
1100bp ladder--
2~4Pcon-aptamer12-1R-DT(E-1A)--
6~8Pcon-spinach-DT(E-1A)--
10~12Pcon-attenuator PT181(E-1A)--
LaneDNAEnzyme
1100bp ladder--
2~4DT(1-3)--
6~8aptamer12-1RDT(1-3)--
10~12Pcon-PT181 attenuator(1-5A)--
Nameconcentration[µg/mL]260/280260/230
Pcon-aptamer12-1R-DT(E-1A)31.21.870.30
Pcon-spinach-DT(E-1A)25.61.880.99
Pcon-attenuator PT181(E-1A)25.31.990.86
DT(1-3)22.11.820.20
aptamer12-1RDT(1-3)33.51.990.05
Pcon-PT181 attenuator(1-5A)36.91.120.96

Igku 0922 G1.jpg
Igku 0922 G2.jpg
Igku 0922 G3.jpg
Igku 0922 G4.jpg

Restriction Enzyme Digestion

No name

9/16 PlacXbaISpeIPstIBufferBBSAMilliQtotal
2cuts12.5µL0µL1µL1µL3µL3µL9.5µL30µL
NC0.8µL0µL0µL0µL1µL1µL7.4µL10µL
9/22 PluxXbaISpeIPstIBufferBBSAMilliQtotal
2cuts16µL0µL1µL1µL3µL3µL14µL30µL
NC0.8µL0µL0µL0µL1µL1µL7.2µL10µL
9/20 RBS-tetR-2R-DT XbaISpeIPstIBufferDBSAMilliQtotal
2cuts8.5µL1µL0µL1µL3µL3µL13.5µL30µL
NC0.4µL0µL0µL0µL1µL1µL7.6µL10µL

Igku 0922 E7.jpg

Gel Extraction

No name

LaneDNAEnzyme
1100bp ladder--
2~4Pcon-attenuator PT181(E-0)--
LaneDNAEnzyme
1100bp ladder--
2~4Pcon-attenuator PT181(E-0)--
LaneDNAEnzyme
2100bp ladder--
4~6Pcon-attenuator PT181(2-SA)--
LaneDNAEnzyme
1100bp ladder--
3~5spinach-DT(3-2)--
8~10Ptet-antisense PT181(1-3)--
Nameconcentration[µg/mL]260/280260/230
Pcon-attenuator PT181(E-0)2.41.800.17
Pcon-attenuator PT181(2-SA)10.81.850.54
spinach-DT(3-2)36.71.800.75
Ptet-antisense PT181(1-3)16.81.740.78

Igku 0922 G5.jpg
Igku 0922 G6.jpg
Igku 0922 G7.jpg
Igku 0922 G8.jpg
Igku 0922 G9.jpg
Igku 0922 G10.jpg

Colony PCR

No name

Samplebase pair
9/20 Pcon-PT181 attenuator-aptamer12-1R-DT-(7~14)859

Colony PCR

Tatsui

Samplebase pair
PconPT181antisense-spinach-DT(9~15)723

Igku 0922 E8.jpg

Ligation

No name

stateVectorInserterLigation High ver.2
experiment9/13 Pcon-PT181 attenuator (SpeI & PstI)1.7 µL9/18 aptamer-1R-DT (XbaI & PstI)5.4µL 3.5µL
experiment9/12 Plac(SpeI & PstI)2.4 µL9/18 aptamer-1R-DT (XbaI & PstI)6.1µL3.5 µL
experiment9/13 Ptet(SpeI & PstI)1.7 µL9/10 Plux-RBS-GFP-DT7.0 µL4.4 µL
experiment9/21 RBS-GFP-DT (EcoRI & XbaI)0.6 µL9/18 aptamer 12-1R-DT (XbaI & PstI)5.6µL3.1 µL
experiment9/13 PSB1C3(XbaI & PstI)1.9 µL9/16 PT181 attenuator(XbaI & PstI)4.6µL3.3 µL

Liquid Culture

No name

Samplemedium
9/20 Plac-antisense-1--
9/20 Plac-attenuator-1--
9/20 Plac-antisense-1--
9/20 Plac-attenuator-1--

digestion

No name

9/8 Pcon-PT181 antisenseEcoRISpeI BufferMMilliQtotal
2cuts8.3µL1µL1µL3µL16.7µL30µL
NC0.4µL0µL0µL1µL8.3µL10µL
9/7 Pcon-RBS-tetR-DTEcoRIXbaISpeIPstIBufferDBSAMilliQtotal
2cut14µL1µL1µL0µL0µL3µL3µL18.2µL30µL
NC0.7µL0µL0µL0µL0µL1µL1µL7.3µL10µL
9/22 RBS-GFP-DTEcoRIXbaISpeIPstI BufferDBSAMilliQtotal
2cuts3.8µL1µL1µL0µL0µL3µL3µL18.2µL30µL
NC0.2µL0µL0µL0µL0µL1µL1µL7.8µL10µL
9/6 aptamer12-1R-DTEcoRIXbaISpeIPstIBufferDBSAMilliQ
2cut8µL1µL1µL0µL0µL3µL3µL16µL
NC0.4µL0µL0µL0µL0µL1µL1µL7.6µL
incubate 37°C 1hour

Igku 0922 E9.jpg

Gel Extraction

No name

LaneDNAEnzyme
11kbp ladder--
3~5Pcon-PT181 antisense (EcoRI&SpaI)--

Igku 0922 G11.jpg
Igku 0922 G12.jpg

Transformation

No name

NameSampleCompetent CellsPlate
9/20 Plac-aptamer12-1R-DT(XbaI&PstI)2 µL20 µLAmp
9/20 pT181 attenuator(1C3)(EcoRI&SpeI)2 µL20 µLCP
9/17 pT181 attenuator(1C3)(XbaI&PstI)2 µL20 µLCP
9/20 Pcon-pT181 attenuator(SpaI&PstI)+aptamer12-1R-DT(XbaI&PstI)2 µL20 µLAmp
kaiABC2 µL20 µLAmp
9/22 pSB1C3-RpaB(XbaI&PstI)2 µL20 µLAmp
9/22 pSB1C3-pT181 attenuator(XbaI&PstI) 2 µL20 µLAmp
9/22 Ptet(SpaI&PstI)+RBS-GFP-DT(XbaI&PstI)2 µL20 µLCP
9/22 RBS-GFP-DT+Pcon-attenuator pT181 (EcoRI&SpeI)2 µL20 µLCP
9/22 pT181 attenuator(XbaI&PstI)(1C3)2 µL20 µLCP

PCR

No name

Samplebase pair
Pcon-RBS-tetR-DT(1-2(A))--
Pcon-RBS-tetR-DT(3-2(A))--
Pcon-RBS-GFP-DT(1-S(A))--
Pcon-RBS-GFP-DT(2-S(A))--
Pcon-tetR-DT(E-2(A))--
PreDenatureDenatureAnnealingExtensioncycle
94 °C94 °C57 °C68 °C--
2 min10 s30 s1min5s30 cycles

Igku 0922 E10.jpg

Electrophoresis

No name

LaneSample
11kbp ladder
2Pcon-RBS-tetR-DT(1-2Ⓐ)
3Pcon-RBS-tetR-DT(3-2Ⓐ)
4Pcon-RBS-GFP-DT(1-SⒶ)
5Pcon-RBS-GFP-DT(2-SⒶ)
6Pcon-tetR-DT(E-2Ⓐ)

Igku 0922 E10.jpg

Sep 23

Transformation

No name

NameSampleCompetent CellsPlate
9/14 pSB1C3+PkaiBC2 µL20 µL--
9/14 pSB1C3+RpaB2 µL20 µL--
9/14 pkaiBC+Rbs-GFP-DT2 µL20 µL--

Gel Extraction

No Name

LaneDNAEnzyme
1 1kbp ladder
3-4 Pcon-RBS-tetR-PT

Igku Sep23 Gel Extraction1.jpg
Igku Sep23 Gel Extraction2.jpg

Nameconcentration[µg/mL]260/280260/230
Pcon-PT181 anti EcoRI+SpeI46.7 1.06 0.93
Pcon-RBS-tetR-DT EcoRI+XbaI119.3 0.99
RBS-8FP-DT EcoRI+XbaI87.7 1.00 0.98
ap42_1R-DT EcoRI+XbaI68.8 1.32 1.03
Pcon-RBS-tetR-DT 1-2 A203.0 1.66 1.79
Pcon-RBS-tetR-DT 3-2 A67.7 2.64 1.28
Pcon-RBS-GFP-DT 1-S A185.6 1.88 2.04
Pcon-RBS-GFP-DT 2-S A298.6 1.68 1.55
Pcon-tetR-DT E-2 A202.4 1.57 1.66


PCR

No name

genome DNAKOD plus10x bufferdNTPMgSO4SasA_fwd primerSasA_rev primerMilliQtotal
9/8 Pcon-pT181 anti 0.52.52.51.50.750.7515.5 25
8/20 Ptet(1) 0.52.52.51.50.750.7515.5 25
9/16 Plac 2 0.52.52.51.50.750.7515.5 25
PreDenatureDenatureAnnealingExtensioncycle
94 °C98 °C57 °C68 °C--
2min 10sec 30sec 20sec 30cycles

Ligation

Yoshida

stateVectorInserterLigation High ver.2
experiment9/21 Pcon-RBS-GFP-DT EcoRI+XbaI47.5 µg/mL9/15 Pcon-RBS-tetR-DT EcoRI+SpeI59 µg/mL2.3 µg/mL
experiment9/21 Pcon-RBS-GFP-DT EcoRI+XbaI47.5 µg/mL9/15 Pcon-apt12_1R-DT EcoRI+SpeI17 µg/mL2.4 µg/mL
experiment9/21 Pcon-RBS-GFRDT EcoRI+XbaI2.1 µg/mL9/15 Pcon-DT181 attenuator-DT21 µg/mL3.0 µg/mL
experiment9/22 apt 12_1R-DT EcoRI+XbaI67.8 µg/mL9/18 Pcon-DT181 attenuator EcoRI+SpeI5.7 µg/mL3.5 µg/mL
experiment9/22RBS-GFP-DT EcoRI+XbaI87.7 µg/mL9/18 Pcon-DT18 attenuator EcoRI+SpeI6.7 µg/mL3.5 µg/mL
experiment9/13 DT EcoRI+XbaI25.4 µg/mL9/22 Pcon-DT181 antisense EcoRI+SpeI46.8 µg/mL2.3 µg/mL


PCR

No name

genome DNAKOD plus10x bufferdNTPMgSO4SasA_fwd primerSasA_rev primerMilliQtotal
Pcon-attenuator-DT (E-1A) 0.52.52.51.50.750.7577.5 25
Pcon-attenuator-DT (2-1A) 0.52.52.51.50.750.7577.5 25
Pcon-antisense-spinach-DT (1-2A) 0.52.52.51.50.750.7577.5 25
Pcon-antisense-spinach-DT (E-1A) 0.52.52.51.50.750.7577.5 25
PreDenatureDenatureAnnealingExtensioncycle
94 °C98 °C57 °C68 °C--
2min 10s 30s 45s 30cycle


Miniprep

No name

DNAconcentration[µg/mL]260/280260/230
9/22 Plac-DT181 attenuator-1204.0 2.00 1.95
9/22 Plac-DT181 attenuator-2195.3 1.84 1.62
9/22 Plac-DT181 antisense-1188.2 1.87 2.15
9/22 Plac-DT181 antisense-2165.0 1.95 2.66

Electrophoresis

Hirano

LaneSampleEnzyme1Enzyme2
1,6100bp ladder(Promega)----
2Pcon-attenuator-DT (E-1A)----
3Pcon-attenuator-DT (2-1A)----
4Pcon-antisense-spinach-DT (1-2A)----
5Pcon-antisense-spinach-DT (E-1A)----

Igku 9232.png

Gel Extraction

Hirano

Nameconcentration[µg/mL]260/280260/230
Pcon-pT181 attenuator-DT(E-1A)166.7 1.51 0.99
Pcon-pT181 attenuator-DT(2-1A)122.1 1.75 1.20
Pcon-pT181 antisense-spinach-DT(1-2A)99.1 1.86 1.81

Electrophoresis

Hirano

LaneSampleEnzyme1Enzyme2
1100bp ladder----
29/8 Pcon-pT181 antisense(E-1A)----
38/20 Ptet(1)(2-S)----
49/16 Plac2(E-1A)----

Igku 9231.png

Transformation

No Name

NameSampleCompetent CellsTotalPlate
pSB1C32µL20µL22µL


Ligation

No Name

stateVectorInserterLigation High ver.2
experiment9/19 Plac SpeI+PstI24.6 µg/mL9/18 ape12_1R-DT XbaI+PstI4.5 µg/mL2 µg/mL
experiment9/21 RBS-GFP-DT EcoRI+XbaI88.4 µg/mL9/15 Pcon-pT181 attenuator EcoRI+SpeI21 µg/mL2 µg/mL
experiment9/13 PSB1C3 XbaI+PstI26.0 µg/mL9/16 pT181 attenuator XbaI+PstI8.2 µg/mL2 µg/mL


Colony PCR

Tatsui

Samplebase pair
9/22 Pcon-PT181 attenuator12-1R-DT(1~8)--
9/22 Pcon-PT181 attenuator(1~4)--
9/20 Plac+aptamer12-1R-DT(1~4)--
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s54s30cycle

Sep 24

Electrophoresis

No name

LaneSample
19/20 Pcon-pT181aptamer12-1R-DT 1
29/20 Pcon-pT181aptamer12-1R-DT 2
39/20 Pcon-pT181aptamer12-1R-DT 3
49/20 Pcon-pT181aptamer12-1R-DT 4
59/20 Pcon-pT181aptamer12-1R-DT 5
69/20 Pcon-pT181aptamer12-1R-DT 6
79/20 Pcon-pT181aptamer12-1R-DT 7
89/20 Pcon-pT181aptamer12-1R-DT 8
9100bp ladder
109/22 Pcon-pT181attenuator-aptamer12-1R-DT 1
119/22 Pcon-pT181attenuator-aptamer12-1R-DT 2
129/22 Pcon-pT181attenuator-aptamer12-1R-DT 3
139/22 Pcon-pT181attenuator-aptamer12-1R-DT 4
149/20 Plac-aptamer12-1R-DT 1
159/20 Plac-aptamer12-1R-DT 2
169/20 Plac-aptamer12-1R-DT 3
179/20 Plac-aptamer12-1R-DT 4

Igku 0924 E1.jpg

Transformation

No name

NameSampleCompetent CellsTotalPlate
9/23 pSB1C3-pT181attenuator 2 µL20 µL 22 µLLB (+CP)
RBS-GFP-DT+Pcon-pT181attenuator 2 µL20 µL 22 µLLB (+CP)
9/23 Plac-+spinach-DT2 µL20 µL 22 µLLB (+Amp)
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 2 µL20 µL 22 µLLB (+Amp)
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 2 µL20 µL 22 µLLB (+Amp)
9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 2 µL20 µL 22 µLLB (+Amp)
9/23 Pcon-pT181antisense-DT 2 µL20 µL 22 µLLB (+CP)
9/23 Pcon-pT181attenuator-RBS-GFP-DT 2 µL20 µL 22 µLLB (+CP)
9/23 Pcon-pT181attenuator-aptamer12-1R-DT 2 µL20 µL 22 µLLB (+CP)
PUC192 µL20 µL 22 µLLB (+Amp)
Ptet-RBS-lacZα-DT 2 µL20 µL 22 µLLB (+CP)
pSB1C32 µL20 µL 22 µLLB (+CP)

Liquid Culture

No name

Samplemedium
aptamer12-1R-DT(Master plate) LB(+CP)

Restriction Enzyme Digestion(PCR product)

Hirano

Concentrate PCR product by evaporator until volume become less than 1µL, add all sulution and pipetting.
10*cut smart bufferBsaI-HFMilliQtotal
9/22 Pcon-pT181 attenuator (E-0) 2.0 µL 0.3 µL 17 µL 19.3µL
9/22 Pcon-pT181 attenuator (2-3A) 2.0 µL 0.3 µL 17 µL 19.3µL
9/22 spinach-DT(3-2) 2.0 µL 0.3 µL 17 µL 19.3µL
9/22 Ptet-pT181 antisense (1-3) 2.0 µL 0.3 µL 17 µL 19.3µL
9/22 Pcon-tet aptamer-DT (E-1A) 2.0 µL 0.3 µL 17 µL 19.3µL
9/22 Pcon-spinach-DT (E-1A) 2.0 µL 0.3 µL 17 µL 19.3µL
9/22 Pcon-pT181 attenuator (E-1A) 2.0 µL 0.3 µL 17 µL 19.3µL
9/22 DT (1-3) 2.0 µL 0.3 µL 17 µL 19.3µL
9/22 tet aptamer-DT (1-3) 2.0 µL 0.3 µL 17 µL 19.3µL
9/22 Pcon-pT181 attenuator (1-SA) 2.0 µL 0.3 µL 17 µL 19.3µL
9/22 Pcon-RBS-tetR-DT (1-2A) 2.0 µL 0.3 µL 17 µL 19.3µL
9/22 Pcon-RBS-tetR-DT (3-2A) 2.0 µL 0.3 µL 17 µL 19.3µL
9/22 Pcon-RBS-tetR-DT (E-2A) 2.0 µL 0.3 µL 17 µL 19.3µL
9/22 Pcon-RBS-GFP-DT (1-2A) 2.0 µL 0.3 µL 17 µL 19.3µL
9/22 Pcon-RBS-GFP-DT (2-SA) 2.0 µL 0.3 µL 17 µL 19.3µL
9/23 Pcon-pT181 attenuator-DT (E-1A) 2.0 µL 0.3 µL 17 µL 19.3µL
9/23 Pcon-pT181 attenuator-DT (0-1A) 2.0 µL 0.3 µL 17 µL 19.3µL
9/23 Pcon-pT181 antisense-spinach-DT (1-2A) 2.0 µL 0.3 µL 17 µL 19.3µL
9/23 Pcon-pT181 antisense (1-2A) 2.0 µL 0.3 µL 17 µL 19.3µL
9/23 Ptet-1 (2-S) 2.0 µL 0.3 µL 17 µL 19.3µL
9/23 Plac-2 (E-1A) 2.0 µL 0.3 µL 17 µL 19.3µL
Incubate 37°C 12hour

Column Refining

Kojima

Nameconcentration[µg/mL]260/280260/230
8/22 Pcon-pT181 attenuator(E-0)7.81.700.03
8/22 Pcon-pT181 attenuator(2-SA)17.31.930.03
8/22 spinach-DT (3-2)22.41.930.09
8/22 Ptet-PT181 anisense (1-3)18.31.580.14
8/22 Pcon-aptamer12_1R-DT (E-1A)8.22.100.05
8/22 Pcon-spinach-DT(E-1A)5.60.910.05
8/22 Pcon-pT181 attenuator(E-1A)18.51.800.12
8/22 DT(1-3)7.31.130.04
8/22 tetR aptamer 12_1R-DT(1-3)14.61.740.06
8/22 Pcon-pT181 attenuator(1-SA)7.91.570.04
8/22 Pcon-RBS-tetR-DT(1-2Ⓐ)41.61.810.25
8/22 Pcon-RBS-tetR-DT(3-2Ⓐ)30.21.750.15
8/22 Pcon-RBS-tetR-DT(E-2Ⓐ)36.91.750.56
8/22 Pcon-RBS-GFP-DT(1-SⒶ)43.51.830.33
8/22 Pcon-RBS-GFP-DT(2-SⒶ)64.61.820.30
8/23 Pcon-pT181 attenuator-DT (E-1A)31.21.830.09
8/23 Pcon-pT181 attenuator-DT (0-1A)64.51.800.43
8/23 Pcon-pT181 antisense-spinach-DT (1-2A)36.41.880.21
8/23 Pcon-pT181 antisense-spinach-DT (E-1A)27.91.920.16
8/23 Ptet(1)(2-S)37.01.910.16
9/23 Plac-2(E-1A) 21.21.820.13

Transformation

No name

NameSampleCompetent CellsTotalPlate
9/23 Pcon-attenuator-aptamer12-1R-DT 2 µL20 µL 22 µLLB (+CP)
9/23 pT181attenuator(1C3) 2 µL20 µL 22 µLLB (+CP)
9/23 Pcon-pT181antisense-DT2 µL20 µL 22 µLLB (+CP)
9/23 Pcon-pT181attenuator-RBS--GFP-DT 2 µL20 µL 22 µLLB (+CP)

Liquid Culture

No name

Samplemedium
pSB1C3 (Master plate-1) LB(+CP)

Restriction Enzyme Digestion

Kojima

8/21 pT181attenuator (330 µg/mL) XbaIPstIbuffer BSAMilliQtotal
2 cuts6.1113315.930
NC0.300117.710
9/22 Pcon-pT181attenuator (368.3 µg/mL) EcoRISpeIbuffer BSAMilliQtotal
2 cuts5.4113019.630
NC0.300108.710
9/16 pSB1C3EcoRISpeIbuffer BSAMilliQtotal
2 cuts1811307.030
NC0.900108.110

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
11kb ladder--
2pT181attenuator XbaIPstI
3 pT181attenuator ----
4Pcon-pT181attenuator EcoRISpeI
5 Pcon-pT181attenuator ----
6pSB1C3 EcoRISpeI
7 pSB1C3 ----

Igku 0924 E2.jpg

Miniprep

No name

DNAconcentration[µg/mL]260/280260/230
9/24 aptamer12-1R-DT144.71.031.21

Gel Extraction

Kojima

LaneDNAEnzyme1Enzyme2
1100kb ladder----
2pT181attenuatorXbaIPstI
3 pT181attenuatorXbaIPstI
4 pT181attenuatorXbaIPstI
5------
6Pcon-pT181attenuatorEcoRISpeI
7 Pcon-pT181attenuatorEcoRISpeI
8 Pcon-pT181attenuatorEcoRISpeI
9------
10pSB1C3EcoRISpeI
11 pSB1C3EcoRISpeI
12 pSB1C3EcoRISpeI

Igku 0924 G1.jpg
Igku 0924 G2.jpg

Nameconcentration[µg/mL]260/280260/230
pT181attenuator(XbaI&PstI)10.1 2.02 0.43
Pcon-pT181attenuator(EcoRI&SpeI)10.3 1.81 0.32
pSB1C3(EcoRI&SpeI)10.02.810.02

Colony PCR

Nakamoto

Samplebase pair
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 11414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 21414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 31414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 41414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 51414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 61414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 71414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 81414
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 11631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 21631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 31631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 41631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 51631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 61631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 71631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 81631
9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 12042
9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 22042
9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 32042
9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 42042
9/23 Pcon-pT181antisense-DT 1508
9/23 Pcon-pT181antisense-DT 2508
9/23 Pcon-pT181antisense-DT 3508
9/23 Pcon-pT181antisense-DT 4508
9/23 Pcon-pT181antisense-DT 5508
9/23 Pcon-pT181antisense-DT 6508
9/23 Pcon-pT181antisense-DT 7508
9/23 Pcon-pT181antisense-DT 8508
PreDenatureDenatureAnnealingExtensioncycle
94 °C94 °C55 °C68 °C--
5 min30 sec30 sec2min30 cycles
LaneSample
19/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 1
29/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 2
39/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 3
49/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 4
59/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 5
69/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 6
79/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 7
89/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 8
91kb ladder
109/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 1
119/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 2
129/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 3
139/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 4
149/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 5
159/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 6
169/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 7
179/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 8

Igku 0924 E3.jpg

LaneSample
19/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 1
29/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 2
39/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 3
49/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 4
59/23 Pcon-pT181antisense-DT 1
69/23 Pcon-pT181antisense-DT 2
71kb ladder
89/23 Pcon-pT181antisense-DT 3
99/23 Pcon-pT181antisense-DT 4
109/23 Pcon-pT181antisense-DT 5
119/23 Pcon-pT181antisense-DT 6
129/23 Pcon-pT181antisense-DT 7
139/23 Pcon-pT181antisense-DT 8
14--

Igku 0924 E4.jpg

Restriction Enzyme Digestion

Nakamoto

9/24 aptamer12-1R-DT EcoRIXbaI bufferBSAMilliQtotal
2cuts17.5µL1.0µL 1.0µL 3.0µL 3.0µL 4.5µL 30µL
NC0.9µL 0µL 0µL 1.0µL 1.0µL 7.1µL 10µL
9/20 PtetSpeIPstI bufferBSAMilliQtotal
2cuts9.5µL1.0µL 1.0µL 3.0µL 0µL 15.5µL 30µL
NC0.5µL 0µL 0µL 1.0µL 0µL 8.5µL 10µL
9/22 RBS-GFP-DTEcoRI SpeI bufferBSAMilliQtotal
2cuts3.8µL1.0µL 1.0µL 3.0µL 0µL 21.2µL 30µL
NC0.2µL 0µL 0µL 1.0µL 0µL 8.8µL 10µL
9/22 Pcon-RBS-GFP-DTEcoRI XbaI bufferBSAMilliQtotal
2cuts2.4µL1.0µL 1.0µL 3.0µL 3.0µL 19.6µL 30µL
NC0.1µL 0µL 0µL 1.0µL 1.0µL 7.9µL 10µL
9/22 Pcon-pT181attenuator 1 EcoRI XbaI bufferBSAMilliQtotal
2cuts5.4µL1.0µL 1.0µL 3.0µL 3.0µL 16.6µL 30µL
NC0.3µL 0µL 0µL 1.0µL 1.0µL 7.7µL 10µL
9/22 RBS-GFP-DTXbaI PstI bufferBSAMilliQtotal
2cuts3.8µL1.0µL 1.0µL 3.0µL 3.0µL 18.2µL 30µL
NC0.2µL 0µL 0µL 1.0µL 1.0µL 7.8µL 10µL

Ligation

Nakamoto

stateVectorInserterLigation High ver.2
experiment9/22 aptamer12-1R-DT(EcoRI&XbaI) 1.4 µL9/24 Pcon-pT181attenuator(EcoRI&SpeI)26 µL3.5 µL
experiment9/4 Ptet(SpeI&PstI)2.0 µL9/21 RBS-GFP-DT(XbaI&PstI)9.0 µL3.5 µL
  • incubated at 37 °C for 1 hour.

colony PCR

Kojima

Samplebase pair
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 52042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 62042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 72042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 82042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 92042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 102042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 112042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 122042bp
9/23 Pcon-pT181antisense-DT 9508bp
9/23 Pcon-pT181antisense-DT 10508bp
9/23 Pcon-pT181antisense-DT 11508bp
9/23 Pcon-pT181antisense-DT 12508bp
9/23 Pcon-pT181antisense-DT 13508bp
9/24 Pcon-pT181attenuator-RBS-GFP-DT 11527bp
9/24 Pcon-pT181attenuator-RBS-GFP-DT 21527bp
9/24 Pcon-pT181attenuator-RBS-GFP-DT 31527bp
9/24 Pcon-pT181attenuator-RBS-GFP-DT 41527bp
9/24 Pcon-pT181attenuator-aptamer12-1R-DT 1859bp
9/24 Pcon-pT181attenuator-aptamer12-1R-DT 2859bp
9/24 Pcon-pT181attenuator-aptamer12-1R-DT 3859bp
9/24 Pcon-pT181attenuator-aptamer12-1R-DT 4859bp
9/24 pT181attenuator(pSB1C3) 1390bp
9/24 pT181attenuator(pSB1C3) 2390bp
9/24 pT181attenuator(pSB1C3) 3390bp
9/24 pT181attenuator(pSB1C3) 4390bp

Tatsui

PreDenatureDenatureAnnealingExtensioncycle
 94°C 94°C 55°C 68°C--
5min30s 30s 2min30cycle

Miniprep

No name

DNAconcentration[µg/mL]260/280260/230
pSB1C3183.9 1.91 1.59
Pcon-pT181attenuator-aptamer12-1R-DT231.71.822.35
Plac-aptamer12-1R-DT208.91.981.79

Electrophoresis

Nakamoto

LaneSampleEnzyme1Enzyme2
11kb ladder----
29/24 aptamer12-1R-DTEcoRIXbaI
3 9/24 aptamer12-1R-DT -- --
49/24 Ptet SpeI PstI
59/24 Ptet ----
69/24 RBS-GFP-DT EcoRISpeI
79/24 RBS-GFP-DT -- --
89/24 Pcon-RBS-GFP-DT EcoRIXbaI
89/24 Pcon-RBS-GFP-DT ----
99/24 Pcon-RBS-GFP-DT ----
109/24 Pcon-pT181attenuator 1 EcoRIXbaI
119/24 Pcon-pT181attenuator 1 ----
129/24 RBS-GFP-DT XbaIPstI
139/24 RBS-GFP-DT ----
141kb ladder----

Igku 0924 E5.jpg

Gel Extraction

No name 1

LaneDNAEnzyme1Enzyme2
11kb ladder----
2aptamer12-1R-DTEcoRIXbaI
3aptamer12-1R-DTEcoRIXbaI
4aptamer12-1R-DTEcoRIXbaI
5-- -- --
6PtetSpeIPstI
7PtetSpeIPstI
8PtetSpeIPstI
9------
10RBS-GFP-DTEcoRISpeI
11RBS-GFP-DTEcoRISpeI
12RBS-GFP-DTEcoRISpeI

Igku 0924 G3.jpg
Igku 0924 G4.jpg

LaneDNAEnzyme1Enzyme2
11kb ladder----
2Pcon-RBS-GFP-DTEcoRIXbaI
3Pcon-RBS-GFP-DTEcoRIXbaI
4Pcon-RBS-GFP-DTEcoRIXbaI
5------
6Pcon-pT181attenuatorEcoRIXbaI
7Pcon-pT181attenuatorEcoRIXbaI
8Pcon-pT181attenuatorEcoRIXbaI
9------
10RBS-GFP-DTXbaIPstI
11RBS-GFP-DTXbaIPstI
12RBS-GFP-DTXbaIPstI

Igku 0924 G5.jpg
Igku 0924 G6.jpg

Electrophoresis

No name
1

LaneSample
11kb ladder
2Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 5
3Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 6
4Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 7
5Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 8
6Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 9
7Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 10
81kb ladder


2

LaneSample
11kb ladder
2Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 11
3Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 12
4Pcon-pT181attenuator-RBS-GFP-DT 1
5Pcon-pT181attenuator-RBS-GFP-DT 2
6Pcon-pT181attenuator-RBS-GFP-DT 3
7Pcon-pT181attenuator-RBS-GFP-DT 4
81kb ladder

Igku 0924 E6.jpg


3

LaneSample
1Pcon-pT181antisense-DT 9
2 Pcon-pT181antisense-DT 10
3 Pcon-pT181antisense-DT 11
4 Pcon-pT181antisense-DT 12
5 Pcon-pT181antisense-DT 13
6100bp ladder
7Pcon-pT181attenuator-aptamer12-1R-DT 1
8 Pcon-pT181attenuator-aptamer12-1R-DT 2


4

LaneSample
1Pcon-pT181attenuator-aptamer12-1R-DT 3
2 Pcon-pT181attenuator-aptamer12-1R-DT 4
3 100bp ladder
4 pT181attenuator(pSB1C3) 1
5 pT181attenuator(pSB1C3) 2
6 pT181attenuator(pSB1C3) 3
7 pT181attenuator(pSB1C3) 4

Igku 0924 E7.jpg

Ligation

Hirano

stateVectorInserter1Inserter2Inserter3Inserter4(NEB)T4ligase(NEB)BuffermilliQtotal
experimentRBS-GFP-DT(pSB1C3)1µLtetR-aptamer12-1R-DT(1-3)1 µLPtet(2-S)0.3µLPcon-tetR-aptamer12-1R-DT(3-2A)1.1µLPlac(E-1A)0.5µL0.5µL0.5µL0.2 µL5µL
experimentRBS-GFP-DT(pSB1C3)1µLPcon-pT181attenuator(E-1A)1 µLPcon-tetR-aptamer12-1R-DT(1-2A)1µLPtet(2-S)0.3µL-- -- 0.5µL0.5µL0.7 µL5µL
experimentRBS-GFP-DT(pSB1C3)1µLPcon-tetRaptamer12-1R-DT(E-1A)2µLPcon-tetRaptamer12-1R-DT(1-2A)1µLPtet(2-S)0.3µL----0.5µL0.5µL0 µL5.3µL
experimentRBS-GFP-DT(pSB1C3)1µLPcon-spinach-DT(E-1A)3µLPcon-tetRaptamer12-1R-DT(1-2A)1µLPtet(2-S)0.3µL----0.5µL0.5µL0 µL6.3µL
experimentRBS-GFP-DT(pSB1C3)1µLPcon-pT181antisense-DT(E-1A)0.5µLDT(1-3)2µLPtet(2-S)0.3µLPcon-tetRaptamer12-1R-DT(3-2A)1.1µL0.5µL0.5µL0.1 µL5µL
experimentRBS-GFP-DT(pSB1C3)1µLPcon-pT181attenuator(E-1A)1µLtetRaptamer12-1R-DT(1-3)1µLPtet(2-S)0.3µLPcon-tetRaptamer12-1R-DT(3-2A)1.1µL0.5µL0.5µL0µL5.4µL
experimentRBS-GFP-DT(pSB1C3)1µLPtet(2-S)0.3µLPcon-tetRaptamer12-1R-DT(E-2A)1µL--------0.5µL0.5µL1.7µL5µL
experimentRBS-GFP-DT(pSB1C3)1µLPcon-GFP-DT(2-SA)0.5µL1µL----------0.5µL0.5µL1.5µL5µL
experimentRBS-GFP-DT(pSB1C3)1µLPcon-tetRaptamer12-1R-DT(E-1A)2µLPcon-pT181attenuator(1-SA)2.1µL--------0.5µL0.5µL0µL6.1µL
experimentRBS-GFP-DT(pSB1C3)1µLPcon-pT181attenuator-DT(E-1A)1µLPcon-pT181attenuator(1-SA)2.1µL--------0.5µL0.5µL0µL5.1µL
experimentRBS-GFP-DT(pSB1C3)1µLPcon-tetR12-1R-DT(E-1A)2µLPcon-GFP-DT(2-SA)0.5µLspinach-DT(3-2)1µLPtet-pT181antisense(1-3)1µL0.5µL0.5µL0µL6.5µL
experimentRBS-GFP-DT(pSB1C3)1µLPcon-tetRaptamer12-1R-DT(E-1A)2µLPcon-pT181antisense(1-2A)0.7µLPcon-pT181attenuator(2-SA)2.1µL----0.5µL0.5µL0µL6.8µL
experimentRBS-GFP-DT(pSB1C3)1µLPcon-tetRaptamer12-1R-DT(E-1A)2µLPcon-pT181attenuator(2-SA)2.1µLSpinach-DT(3-2)1µLPtet-pT181antisense(1-3)1µL0.5µL0.5µL0µL8.1µL


Concentrate total (more than 5µL) by evaporater until volume become less than 5 µL

PCR

No name

genome DNAKOD plus10x bufferdNTPMgSO4F primerR primerKOD-plusMilliQtotal
Pcon antisense(1C3-GGE-fwd,1A2-GG2-rev)1µL2.5µL2.5µL1.5µL0.75µL0.75µL0.5µL15.525µL
DT-(1C3-GG1-fwd,1C3-GG2-rev)1µL2.5µL2.5µL1.5µL0.75µL0.75µL0.5µL15.525µL
Spinach-DT-(1C3-GG1-fwd,1C3-GG2-rev)1µL2.5µL2.5µL1.5µL0.75µL0.75µL0.5µL15.525µL
Pcon-antisense-spinach-DT(1C3-GGE-fwd,1A2-GG1-rev) 1µL2.5µL2.5µL1.5µL0.75µL0.75µL0.5µL15.525µL
Ptet(1C3-GGE-fwd,1C3-GG0-rev) 1µL2.5µL2.5µL1.5µL0.75µL0.75µL0.5µL15.525µL
Pcon attenuator(1C3-GGE-fwd,1C3-GG0-rev) 1µL2.5µL2.5µL1.5µL0.75µL0.75µL0.5µL15.525µL
DT(1C3-GG2-fwd,1C3-GG1-rev)1µL2.5µL2.5µL1.5µL0.75µL0.75µL0.5µL15.525µL
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C57°C68°C--
2min10sec30sec36sec30

Sep 25

Electrophoresis

nakamoto

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2Pcon-antisense (1C3-66E-fwd A2-662 rev)----
3DT (1C3-661-fwd 1C3 662 rev)----
4spinach-DT(1C3-661-fwd 1C3 662 rev)----
5Pcon-antisense-spinach-DT(1C3-66E fwd A2-661 rev)----
6100bp ladder----

Igku 9251a.jpg

LaneSampleEnzyme1Enzyme2
2100bp ladder----
3Ptet (1C3 66E fwd-1C3 660 rev)----
4Pcon-attenuator(1C3-660 fwd A2-661 rev)----
5DT(1C3 662 fwd-1C3 661 rev)----
6Pcon-attenuator-aptamer-DT(1C3 66E fwd-A2-661-rev)----
7100bp ladder----

Igku 9251b.jpg

Colony PCR

Nakamoto

Samplebase pair
9/23 Pcon-PT181 attenuator-aptamer12-1R-DT-(5~12)859
9/23 Pcon-PT181 attenuator-RBS-GFP-DT-(5~8)1527
9/23 Pcon-PT181 attenuator-pSB1C3-(5~8)601
PreDenatureDenatureAnnealingExtensioncycle
94 °C94 °C55 °C68 °C--
2 min30 s30 s1min5s30 cycles


Electrophoresis

no name

LaneSampleEnzyme1Enzyme2
1Pcon-PT181 attenuator-aptamer12-1R-DT-5----
2Pcon-PT181 attenuator-aptamer12-1R-DT-6----
3Pcon-PT181 attenuator-aptamer12-1R-DT-7----
4Pcon-PT181 attenuator-aptamer12-1R-DT-8----
5Pcon-PT181 attenuator-aptamer12-1R-DT-9----
6Pcon-PT181 attenuator-aptamer12-1R-DT-10----
7Pcon-PT181 attenuator-aptamer12-1R-DT-11----
8Pcon-PT181 attenuator-aptamer12-1R-DT-12----
91kbp ladder----
10Pcon-PT181 attenuator-RBS-GFP-DT-5----
11Pcon-PT181 attenuator-RBS-GFP-DT-6----
12Pcon-PT181 attenuator-RBS-GFP-DT-7----
13Pcon-PT181 attenuator-RBS-GFP-DT-8----
14Pcon-PT181 attenuator-pSB1C3-5----
15Pcon-PT181 attenuator-pSB1C3-6----
16Pcon-PT181 attenuator-pSB1C3-7----
17Pcon-PT181 attenuator-pSB1C3-8----

Igku 9252.jpg

Transformation

kojima and nakamoto

NameSampleCompetent CellsTotalPlate
Plac+tet aptamer12+Pcon tetR-DT+Ptet+RBS-GFP-DT1µL10µL11µL--
Pcon-pT181attenuator-DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT1µL10µL11µL--
Pcon-tet aptamer12-DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT1µL10µL11µL--
Pcon-Spinach-DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT1µL10µL11µL--
Pcon-pT181antisense+DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT1µL10µL11µL--
Pcon-pT181attenuator+tet aptamer12-DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT1µL10µL11µL--
Pcon tetR-DT+Ptet+RBS-GFP-DT1µL10µL11µL--
Pcon tetR-DT+Pcon-GFP-DT+RBS-GFP-DT1µL10µL11µL--
Pcon-tet aptamer12-DT+Pcon-pT181attenuator+RBS-GFP-DT1µL10µL11µL--
Pcon-pT181attenuator-DT+Pcon-pT181attenuator+RBS-GFP-DT1µL10µL11µL--
Pcon-tet aptamer12-DT+Ptet-pT181antisense+Spinach-DT+Pcon-RBS-GFP-DT+RBS-GFP-DT1µL10µL11µL--
Pcon-tet aptamer12-DT+Pcon-pT181antisense+Spinach-DT+Pcon-pT181attenuator+RBS-GFP-DT1µL10µL11µL--
Pcon-tet aptamer12-DT+Ptet-pT181antisense+Spinach-DT+Pcon-pT181attenuator+RBS-GFP-DT1µL10µL11µL--

NameSampleCompetent CellsTotalPlate
aptamer12-DT(EcoRI+XbaI)+Pcon-pT181attenuator(EcoRI+SpeI)2µL20µL22µL--
Ptet(SpeI+PstI)+RBS-GFP-DT(XbaI+PstI))2µL20µL22µL--

Ligation

nakamoto

stateVectorInserterLigation High ver.2
experiment9/25 aptamer12_1R-DT(EcoRI&XbaI)4.5 µLPcon-pT181 attenuator(EcoRI&SpeI)7.7 µL6.1 µL
experiment9/17 pSB1C3(EcoRI&SpeI)2.0 µL9/3 pT181 attenuator(EcoRI&SpeI)14 µL8 µL
experiment9/25 Pcon-pT181 attenuator(EcoRI&XbaI)4.5 µL9/25 RBS-GFP-DT(EcoRI&SpeI)19.3 µL11.9 µL
experiment9/25 Pcon-RBS-GFP-DT(EcoRI&XbaI)4.2 µLPcon-RBS-tetR-DT(EcoRI&SpeI)20 µL12.1 µL

Restriction Enzyme Digestion

nakamoto>

9/24 Pcon-attenuator-aptamer-DT(µL)EcoRI(µL)SpeI(µL)Buffer(µL)MilliQ(µL)total(µL)
2 cuts8.611316.430
NC0.40018.610
9/11 DT(µL)EcoRI(µL)XbaI(µL)Buffer(µL)BSA(µL)MilliQ(µL)total(µL)
2 cuts10.4113311.630
NC0.500117.510

Liquid Culture

Kojima

Samplemedium
DT --
pT181attenuator --
Pcon-RBS-tetR-DT --

Gel Extraction

Kojima

LaneDNAEnzyme
1100bp ladder--
2Pcon-pT181antisense--
3
4
5----
6DT--
7
8
9----
10Spinach-DT--
11
12

Igku 9253.jpg
Igku 9254.jpg

LaneDNAEnzyme
1100bp ladder--
3Pcon-antisense-spinach-DT(E-1A)--
4Pcon-antisense-spinach-DT(E-1A)--
5Pcon-antisense-spinach-DT(E-1A)--

Igku 9255.jpg
Igku 9256.jpg

LaneDNAEnzyme
7100bp ladder--
9Ptet(E-0)--
10Ptet(E-0)--
11Ptet(E-0)--

Igku 9257.jpg
Igku 9258.jpg

LaneDNAEnzyme
13100bp ladder--
15Pcon-attenuator(0-1A)--
16Pcon-attenuator(0-1A)--
17Pcon-attenuator(0-1A)--

Igku 9259.jpg
||-- Igku 92510.jpg

LaneDNAEnzyme
19100bp ladder--
21Pcon-attenuator-aptamer-DT(E-1A)--
22Pcon-attenuator-aptamer-DT(E-1A)--
23Pcon-attenuator-aptamer-DT(E-1A)--

Igku 92511.jpg
Igku 92512.jpg

Nameconcentration[µg/mL]260/280260/230
DT(2-1)24.91.730.37
Ptet(E-0)12.81.590.90
Pcon-antisense(E-2A)16.51.721.07
Pcon-attenuator-aptamer-DT(E-1A)22.11.750.94
Pcon-antisense-spinach-DT(E-1A)31.01.781.15
spinach-DT(1-2)23.41.851.11
Pcon-attenuator(0-1A)22.51.760.46
DT(1-2)25.11.781.21

Electrophoresis

LaneSampleEnzyme1Enzyme2
11kbp ladder----
2Pcon-pT181 attenuator-aptamer 12_1R-DTEcoRISpeI
3Pcon-pT181 attenuator-aptamer 12_1R-DT----
4DTEcoRIXbaI
5DT----

Igku 92513.jpg

Gel Extraction

Nakamoto

LaneDNAEnzyme
1DTEcoRI+XbaI
2DTEcoRI+XbaI
3Pcon-pT181 attenuator-aptamer12_1R-DTEcoRI+SpeI
4Pcon-pT181 attenuator-aptamer12_1R-DTEcoRI+SpeI
5Pcon-pT181 attenuator-aptamer12_1R-DTEcoRI+SpeI

Igku 92514.jpg
Igku 92515.jpg

Nameconcentration[µg/mL]260/280260/230
Pcon-pT181 attenuator-aptamer12_1R-DT(EcoRI&SpeI)11.81.800.46
DT(EcoRI+XbaI)42.61.840.98

Liquid Culture

No name

Samplemedium
Pcon-pT181anntisense-spinach-DT(4k5)-

Ligation

Nakamoto and Tatsui

stateVectorInserterLigation High ver.2
experiment9/24 pSB1C3(EcoRI+Spel)10µL9/25 PconpT181attenuator-aptamer12-1R-DT(EcoRI+Spel)5.9µL8µL

PCR

Hirano

800pg/μL DTKOD plus10x bufferdNTPMgSO41C3_GG2_fwdiC3_GG1_revMilliQtotal
10.52.52.51.50.750.7515.525
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C57°C68°C--
2min10s30s24s30cycles

Transformation

Tatsui

NamePlate
aptamer12-1R-DTCP
pSB1C3(EcoRI+SpeI)+pT181attenuator(EcoRI+Spal)CP
Pcon-pT181attenuator(EcoRI+XbaI)+RBS-GFP-DT(EcoRI+SpeI)Amp
Pcon-RBS-GFP-DT(EcoRI+XbaI)+Pcon-RBS-tetR-DT(EcoRI+SpeI)Amp
pSB1C3(EcoRI+SpeI)+Pcon-pT181attenuator-aptamer12-1R-DT(EcoRI+SpeI)CP

Electrophoresis

Hirano

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2DT(2-1)----

Igku 92516.jpg

Column Refining

Nameconcentration[µg/mL]260/280260/230
8/25 DT(2-1)------

Restriction Enzyme Digestion

Concentrate PCR product by evaporator until volume become less than 3µL, add all sulution and pipetting.
9/25 DT(2-1)10*cut smart BufferBsaI-HFMilliQtotal
3.0 µL2.0 µL0.3 µL14.7 µL20.0 µL

Incubate 37°C 12hour

Liquid Culture

Hirano

Samplemedium
Pcon-GFP-DTLB(Amp)
RBS-GFP-DTLB(CP)
Pcon-spinach-DTLB(Amp)
Pcon-tetRaptamer-DTLB(Amp)
Pcon-attenuator-DTLB(Amp)
spinach-DTLB(CP)
Pcon-antisense-spinach-DTLB(Amp)

Plating

Hirano

Mix Liquid Culture 1mL and LB(+Amp), incubate 37°C 30min
OD600-1.445
SampleUse plateThe number of plate
Pcon-GFP-DTM9(+Amp)3
Pcon-GFP-DTLB(+Amp)3

</div> incubate 37°C

Colony PCR

No name

Samplebase pair
9/24 GGA-1(1~8)3177
9/24 GGA-2(1~8)3189
9/24 GGA-3(1~8)2989
9/24 GGA-4(1~8)2939
9/24 GGA-7(1)2489
GGA-16(1)2717
RBS-GFP-DT--
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s3min15s30cycles

RNA Extraction

Nakamoto
sumple 0.25mL
Add 1mLISOGEN-LS
Lysis or homogenization
Store for 5 min, at room temperature
Add 0.2mL Chloroform
Shake vigorously for 15sec.
Store for 2~3min.at room temperature
Centrifuge 12K*g for 15min. at 4°C
Extract aqueous phase
Add 0.5mL isopropanol
Store for 5~10min. at room temperature
Centrifuge 12K*g for 10min. at 4°C
Remove aqueous phase
Add at least 1mL 70% ethanol
Vortex
Centrifuge 7.5K*g for 5min. at 4°C
Remove aqueous phase
Dry briefly
Add ddH2O, TE(pH8.0) or 0.5% SDS
Dissolve
Total RNA solution

DNAconcentration[µg/mL]260/280260/230
Pcon-RBS-GFP-DT27831.951.48

Add 91.2µL, Store at freezer
Total 250µL</div>

cDNA Synthesis

Nakamoto

volume7 µL
7*cDNA Wipcout Buffer1 µL
Template RNA(9/25 Pcon-RBS-GFP-DT)3 µL
RNase-frec water4 µL

Incubate 42°C 2min, then on ice immediately
Add

5*Quantiscript Reverse Transcriptase0.5 µL
5*Quantiscript RT Buffer2 µL
RT Primer Mix0.5µL

Incubate 42°C 15min, then incubate 95°C 3min

Electrophoresis

Nakamoto

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2Pcon-RBS-GFP-DT(RNA)----

Igku 92517.jpg

Ligation

Tatsui

stateVectorInserterLigation High ver.2
experiment9/13 Pcon-pT181-attenuator(SpeI&PstI)3.5µL9/25 RBS-GFP-DT(XbaI&PstI)13.5µL8.5 µL

Sep 26

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1GGA1-1----
2GGA1-2----
3GGA1-3----
4GGA1-4----
5GGA1-5----
6GGA1-6----
7GGA1-7----
8GGA1-8----
91kbp ladder----
10GGA2-1----
11GGA2-2----
12GGA2-3----
13GGA2-4----
14GGA2-5----
15GGA2-6----
16GGA2-7----
17GGA2-8----

Igku 0926 E1.jpg

LaneSampleEnzyme1Enzyme2
1GGA3-1----
2GGA3-2----
3GGA3-3----
41kbp ladder----
5GGA3-4----
6GGA3-5----
7GGA3-6----
8GGA3-7----
LaneSampleEnzyme1Enzyme2
1GGA3-8----
2GGA4-1----
3GGA4-2----
4GGA4-3----
51kbp ladder----
6GGA4-4----
7GGA4-5----
8GGA4-6----

Igku 0926 E2.jpg


LaneSampleEnzyme1Enzyme2
1GGA4-7----
2GGA4-8----
31kbp ladder----
4GGA7-1----
5GGA16-1----
6NC(RBS-GFP-DT)----

Igku 0926 E3.jpg

Ligasion(Golden Gate Assenbly)

No name

stateVectorInserter1Inserter2Inserter3Inserter4Inserter5(NEB)T4 ligase(NEB)10*T4 ligaseMilliQ
experimentRBS-GFP-DT1.0 µLPcon-pT181 antisense(E-2A)0.6µLDT(2-1)0.5µLPcon-pT181 attenuator(1-SA)2.1 µL--------0.5 µL0.5 µLup to 5 µL
experimentpSB1C3(EcoRI&SpeI)1.8 µLPcon-pT181 antisense(E-2A)0.9 µLDT(2-1)0.5 µLPcon-GFP-DT(1-SA)1.5 µL--------0.5 µL0.5 µLup to 5 µL
experimentRBS-GFP-DT1.0 µLPtet(E-0)0.7µLPcon pT181 attenuator(0-1A)0.3µLtet aptamer(1-3)0.3 µLPcon-tetR-DT(3-2A)1.1 µLPtet(2-5)0.3 µL0.5 µL0.5 µLup to 5 µL
experimentRBS-GFP-DT1.0 µLPtet(E-0)0.7 µLPcon-pT181 attenuator-DT(0-1A)1.0 µLPcon-tetR-DT(1-2A)1.0 µLPtet(2-5)0.3µL----0.5 µL0.5 µLup to 5 µL
experimentRBS-GFP-DT1.0 µLPcon-pT181 attenuator-tetR-DT(E-1A)1.0 µLPcon antisense-spinach-DT(1-2A)0.7µLPcon-pT 181 attenuator(2-SA)2.1 µL--------0.5 µL0.5 µLup to 5 µL
experimentRBS-GFP-DT1.0 µLPcon antisense(E-1A)0.5 µLspinach-DT(1-2)1.0 µLPcon-GFP-DT(1-2)0.8 µL--------0.5 µL0.5 µLup to 5 µL
experimentpSB1C3(EcoRI&SpeI)1.8 µLPcon-pT181 antisense(E-1A)0.8µLspinach-DT(1-2)1.0µLPcon-GFP-DT(2-SA)0.8 µL--------0.5 µL0.5 µLup to 5 µL
experimentpSB1C3(EcoRI&SpeI)1.8 µLPcon-tet aptamer-DT(E-1A)3.0 µLPtet-pT181 antisense(1-3)1.5 µLspinach-DT(3-2)1.5 µLPcon-GFP-DT(2-5A)0.8 µL----0.5 µL0.5 µLup to 5 µL

Liquid Culture

No name

Samplemedium
Pcon-GFP-DTAmp
RBS-GFP-DTCP
Pcon-pT181 attenuator-DTAmp
Pcon-tet aptamer-DTAmp
Pcon-spinach-DTAmp
Pcon-pT181 antisense-spinach-DTAmp
spinach-DTCP

BSAI Digestion

No name

DNAMilliQBSAI-HF10*cut smarttotal
Pcon7 µL10.7 µL0.3 µL2 µL20 µL
RBS-luxR-DT10 µL7 µL0.3 µL2 µL20 µL

Igku 0926 E4.jpg

Colony PCR

Tatsui

Samplebase pair
GGA-1 (9~16)3177
GGA-2 (9~16)3189
GGA-3 (9~21)2989
GGA-4 (9~16)2939
GGA-6 (1)3359
GGA-16(2~4)2939
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s3min30s30cycles

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1GGA-1 (9)----
2GGA-1 (10)----
3GGA-1 (11)----
4GGA-1 (12)----
5GGA-1 (13)----
6GGA-1 (14)----
7GGA-1 (15)----
8GGA-1 (16)----
91kbp ladder----
10GGA-2 (9)----
11GGA-2 (10)----
12GGA-2 (11)----
13GGA-2 (12)----
14GGA-2 (13)----
15GGA-2 (14)----
16GGA-2 (15)----
17GGA-2 (16)----

Igku 0926 E6.jpg

LaneSampleEnzyme1Enzyme2
1GGA-4 (9)----
2GGA-4 (10)----
3GGA-4 (11)----
4GGA-4 (12)----
5GGA-4 (13)----
6GGA-4 (14)----
7GGA-4 (15)----
8GGA-4 (16)----
91kbp ladder----
10GGA-6 (1)----
11GGA-16 (2)----
12GGA-16 (3)----
13GGA-16 (4)----

Igku 0926 E7.jpg

LaneSampleEnzyme1Enzyme2
1GGA-3 (9)----
2GGA-3 (10)----
3GGA-3 (11)----
4GGA-3 (12)----
51kbp ladder----
6GGA-3 (13)----
7GGA-3 (15)----
8GGA-3 (14)----
LaneSampleEnzyme1Enzyme2
1GGA-3 (17)----
2GGA-3 (18)----
3GGA-3 (19)----
41kbp ladder----
5GGA-3 (20)----
6GGA-3 (21)----
7GGA-3 (16)----

Igku 0926 E8.jpg

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1RBS-GFP-DT(RNA)----
21kbp ladder----
3Pcon-RBS-GFP-DT(RNA)----

Igku 0926 E9.jpg

Liquid Culture

Hirano

Samplemedium
Pcon-GFP-DT--
RBS-GFP-DT--
Pcon-tet aptamer-DT--
Pcon-spinach-DT--
Pcon-pT181 antisense-spinach-DT--
spinach-DT--

Colony PCR

Hirano

Samplebase pair
9/25 Pcon-pH181 attenuator+RBS-GFP-DT-1--
9/25 Pcon-pH181 attenuator+RBS-GFP-DT-2--
9/25 Pcon-pH181 attenuator+RBS-GFP-DT-3--
9/25 Pcon-pH181 attenuator+RBS-GFP-DT-4--
9/24 Ptet-RBS-GFP-DT-1--
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s1min40s30cycles

Ligasion(Golden Gate Assenbly)

Hirano

stateVectorInserter1Inserter2Inserter3Inserter4Inserter5
experimentGGA2Pcon-attenuator(E-1A)Pcon-tetR-DT(1-2A)Ptet(2-S)RBS-GFP-DT(EcoRI&XbaI)
experimentGGA3Pcon-tetap-DT(E-1A)Pcon-tetR-DT(1-2A)Ptet(2-S)RBS-GFP-DT(EcoRI&XbaI)
experimentGGA4Pcon-spi-DT(E-1A)Pcon-tetR-DT(1-2A)Ptet(2-S)RBS-GFP-DT(EcoRI&XbaI)
experimentGGA5Pcon-antisense(E-1A)DT(1-3)Pcon-tetR-DT(3-2A)Ptet(2-S)RBS-GFP-DT(EcoRI&XbaI)
experimentGGA6Pcon-attenuator(E-1A)tetap-DT(1-3)Pcon-tetR-DT(3-2A)Ptet(2-S)RBS-GFP-DT(EcoRI&XbaI)
experimentGGA7Pcon-tetR-DT(E-2A)Ptet(2-S)RBS-GFP-DT(EcoRI&XbaI)
PreDenatureDenatureAnnealingExtensioncycle
37°C16°C50°C80°C--
3min4min5min5min25cycles

RNA Extraction

Nakamoto

Sample
Pcon-Spinach-DT
Pcon-tetRaptamer-DT
Pcon-attenuator-DT
Spinach-DT
Pcon-antisense-Spinach-DT

Liquid Culture

Kozima

Sample
Pcon-attemuater-DT
Pcon-aptamer-DT
Pcon-spinach-DT
Pcon-RBS-tetR-DT
Pcon-antisense-spinach-DT(Master 1)
Pcon-antisense

Colony PCR

Nakamoto Kozima

Samplebase pair
9/25 9/19pSB1C3&9/3PT181 attenuator(1~3)601bp
9/25 aptamer12_1R-DT&9/24Pcon-pT181 attenuator(1~4)859bp
Pcon-attenuator-aptamer-DT(1)859bp
PreDenatureDenatureAnnealingExtensioncycle
 94°C 94°C 55°C 65°C--
5min30sec 30sec 54sec 30

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
11kbp ladder----
29/25 9/23 1C3&9/3attenuator----
39/25 9/23 1C3&9/3attenuator----
49/25 9/23 1C3&9/3attenuator----
59/25aptamer-DT&9/24Pcon-attenuator----
69/25aptamer-DT&9/24Pcon-attenuator----
79/25aptamer-DT&9/24Pcon-attenuator----
89/24 Pcon attenuator-aptamer-DT(1)----
99/25aptamer-DT&9/24Pcon-attenuator----

Igku 0926 E10.jpg

Plating

Hirano

SampleUse plate
9/26 GGA2 LB(CP)
9/26 GGA3 LB(CP)
9/26 GGA4LB(CP)
9/26 GGA5 LB(CP)
9/26 GGA6 LB(CP)
9/26 GGA7 LB(CP)
9/26 GGA11 LB(CP)
9/26 GGA312LB(CP)
9/26 GGA13 LB(CP)
9/26 GGA14LB(CP)
9/26 GGA15 LB(CP)
9/26 GGA20LB(CP)
9/26 GGA23 LB(CP)
9/26 GGA24LB(CP)
9/26 Ptet+RBS-GFP-DT LB(CP)

incubate 37°C

Restriction Enzyme Digestion

Kojima

9/24 pSB1C3EcoRISpeIbufferMilliQtotal
2cuts11µL1µL1µL3µL14µL30µL
NC0.5µL0µL0µL1µL8.5µL10µL
9/24 Pcon-pT181attenuator-aptamer12-1R-DTEcoRISpeIbufferMilliQtotal
2cuts8.7µL1µL1µL3µL16.3µL30µL
NC0.4µL0µL0µL1µL8.6µL10µL
9/24 pT181attenuatorEcoRISpeIbufferMilliQtotal
2cuts3.1µL1µL1µL3µL21.9µL30µL
NC0.2µL0µL0µL1µL8.8µL10µL
8/17 RBS-GFP-DTEcoRIBSA bufferMilliQtotal
1cut16.4µL1µL3µL3µL6.6µL30µL
NC0.4µL0µL1µL1µL7.6µL10µL
9/17 pSB4K5EcoRIPstIBSA bufferMilliQtotal
2cuts8.5µL1µL1µL3µL3µL13.5µL30µL
NC0.4µL0µL0µL1µL1µL7.6µL10µL
9/14 pSB4K5EcoRIPstIBSA bufferMilliQtotal
2cuts7.3µL1µL1µL3µL3µL14.7µL30µL
NC0.4µL0µL0µL1µL1µL7.6µL10µL
9/22 Pcon-pT181attenuator-DTEcoRISpeI bufferMilliQtotal
2cuts5.4µL1µL1µL3µL19.6µL30µL
NC0.3µL0µL0µL1µL8.7µL10µL
8/7 RBS-GFP-DT 1XbaIPstIBSA bufferMilliQtotal
2cuts8.1µL1µL1µL3µL3µL13.9µL30µL
NC0.4µL0µL0µL1µL1µL7.6µL10µL
9/26 RBS-GFP-DT 1XbaI BSA bufferMilliQtotal
1cut8.1µL1µL3µL3µL14.9µL30µL
9/16 Pλ-luxIEcoRIPstIBSA bufferMilliQtotal
2cuts14.7µL1µL1µL3µL3µL7.3µL30µL
NC0.7µL0µL0µL1µL1µL7.3µL10µL
9/21 PtetEcoRISpeI bufferMilliQtotal
2cuts14.2µL1µL1µL3µL10.8µL30µL
NC0.7µL0µL0µL1µL8.3µL10µL
9/24 Pcon-pT181attenuator-aptamer12-1R-DTXbaIPstIBSA bufferMilliQtotal
2cuts8.7µL1µL1µL3µL3µL13.3µL30µL
NC0.4µL0µL0µL1µL1µL7.6µL10µL

Electrophoresis

Kojima

LaneSample Enzyme1 Enzyme2
1pSB1C3EcoRISpeI
2pSB1C3----
3Pcon-pT181attenuator-aptamer12-1R-DTEcoRISpeI
4Pcon-pT181attenuator-aptamer12-1R-DT----
51kb ladder----
6pT181attenuatorEcoRISpeI
7pT181attenuator----
8RBS-GFP-DTEcoRI--
9RBS-GFP-DT----

Igku Sep26 Electrophoresis(E11) 1.jpg

Sep 27

Gel Extraction

Kojima

LaneDNAEnzyme
11kbp ladder--
2pSB1C3EcoRI+SpeI
3
4
6Pcon-attenuator-aptmer-DTEcoRI+SpeI
7
8
10Pcon-attenuator-aptmer-DTEcoRI+SpeI
11
12

Igku 9272.jpg
Igku 9273.jpg

Nameconcentration[µg/mL]260/280260/230
Pcon-attenuator-DT(EcoRI+SpeI)6.81.980.43
pT181-attenuator(EcoRI+SpeI)7.51.840.06
pSB1C3(EcoRI+SpeI)17.71.970.71

Gel Extraction

No name

LaneDNAEnzyme
11kbp ladder--
3pSB4K5EcoRI+PstI
4pSB4K5EcoRI+PstI
5pSB4K5EcoRI+PstI
7pSB6A1EcoRI+PstI
8pSB6A1EcoRI+PstI
9pSB6A1EcoRI+PstI

Igku 9274.jpg
Igku 9275.jpg

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1pSB4K5EcoRIPstI
2pSB4K5--
31kbp ladder--
4Pcon-attenuatorEcoRISpeI
5Pcon-attenuator--


LaneSampleEnzyme1Enzyme2
1RBS-GFP-DTXbaIPstI
2RBS-GFP-DT--
3RBS-GFP-DTEcoRIXbaI
41kbp ladder--
5PtetEcoRISpeI
6Ptet--

Igku 0927 G5.jpg

Liquid Culture

No name

Samplemedium
Pcon-GFPAmp
RBS-GFP-DTCP
Pcon-attenuatorAmp
Pcon-attenuator-aptamer-DTAmp
Pcon-tetR-DTAmp

Ligation

No name

stateVectorInserterLigation High ver.2
experiment9/17 pSB1C3 (EcoRI+SpeI) 10.0ng/µL10µL9/26 pT181 attenuator(EcoRI+SpeI) 7.5ng/µL1.3µL3.5 µL
experiment9/25 DT (EcoRI+XbaI) 42.8ng/µL2.3µL9/22 Pcon-pT181 antisense(EcoRI+SpeI) 46.7ng/µL1.5µL1.9 µL
experiment9/27 pSB4K5(EcoRI+PstI) 12.9ng/µL7.8µL9/10 Pcon-attenuator(EcoRI+SpeI) 13.8ng/µL8.0µL3.5 µL
experiment9/27 pSB4K5(EcoRI+PstI) 12.9ng/µL7.8µL9/25 RBS-GFP-DT(XbaI+PstI) 13.9ng/µL12µL3.5 µL
experiment9/8 pSB4K5 (EcoRI+SpeI) 17.6ng/µL5.7 µL8/21 Pcon-GFP-DT(EcoRI+SpeI) 11ng/µL12.6µL3.5 µL

Liquid Culture

Hirano

Samplemedium
Pcon-spinach-DTplusgrow Amp
Pcon-tetRaptamer-DTplusgrow Amp 
Pcon-antisense-spinach-DTplusgrow Amp
spinach-DTplusgrow CP

RNA Extraction

Nakamoto

Nameconcentration[µg/mL]260/280260/230
9/26 Pcon-GFP-DT423.01.831.46
9/26 Pcon-GFP-DT K 298.71.821.50

Electrophoresis

No name

LaneSample
19/27 Pcon-GFP-DT(RNA)
21kb ladder
39/27 Pcon-GFP-DT K(RNA)

Igku 9271.jpg

Miniprep

Tatsui

DNA
Pcon-aptamer-DT
Pcon-spinach-DT
Pcon-antisense
Pcon-tetR-DT
Pcon-attenuator-DT
Pcon-antisense-spinach-DT

Transformation

Hirano

Name1Name2Sample1(µL)Sample2(µL)Competent Cells(µL)Total(µL)
Pcon-attenuator+RBS-GFP-DT(4K5)Pcon-attenuator-DT(1A2)213033
Pcon-attenuator+RBS-GFP-DT(4K5)Pcon-antisense-spinach-DT(1A2)213033
Pcon-attenuator+RBS-GFP-DT(4K5)Pcon-tetR aptamer-DT(1A2)213033
Pcon-RBS-GFP-DT(4K5)Pcon-attenuator-DT(1A2)213033
Pcon-RBS-GFP-DT(4K5)Pcon-antisense-spinach-DT(1A2)213033
Pcon-RBS-GFP-DT(4K5)Pcon-tetR aptamer-DT(1A2)213033
Pcon-attenuator+RBS-GFP-DT(4K5)--2--2022
Pcon-RBS-GFP-DT(4K5)--2--2022
attenuator(1C3)--2--2022

Miniprep

No name

DNAconcentration[µg/mL]260/280260/230
Pcon-RBS-GFP-DT137.61.951.78
RBS-GFP-DT77.02.061.96
Pcon-PT181 attenuator231.01.891.89
Pcon-antisense-Spinach-DT(2)130.01.561.62
Pcon-aptamer-DT218.81.921.72
Pcon-attenuator-DT180.71.951.78
Pcon-Spinach-DT177.61.961.06
Pcon-antisense-Spinach-DT(1)181.11.961.92
Pcon-antisense152.92.021.96
Pcon-RBS+ezR-DT164.11.931.91

Restriction Enzyme Digestion

No name

9/27 RBS-GFP-DTXbaIPstIBufferBSAMilliQtotal
2 cuts12µL1µL1µL3µL3µL20µL40µL
NC1.3µL0µL0µL1µL1µL6.7µL10µL
9/27 Pcon-tetR-DTEcoRIPstIBufferMilliQtotal
2 cuts12.2µL1µL1µL3µL12.8µL 30µL
NC0.6µL0µL0µL1µL8.4µL10µL

RNA Extraction

Nakamoto

Nameconcentration[µg/mL]260/280260/230
9/27 Pcon-RBS-GFP-DT686.61.991.34
9/27 Pcon-RBS-GFP-DT (+High salt)127.61.760.27

DNA sythesis

No name

genome DNADNA volumegenome DNA wipeout bufferRNase freewaterRTRT bufferPrimer Mixtotal
9/27 Pcon-RBS-GFP-DT0.8µL1µL5.2µL 0.5µL 2µL0.5µL 10µL
9/27 pcon-RBS-GFP-DT(+high salt)2.5µL1µL3.5µL 0.5µL 2µL 0.5µL 10µL
9/27 Pcon-RBS-GFP-DT(revised concentration) 0.5µL1µL5.5µL0.5µL2µL0.5µL10µL

Electrophoresis

No name

LaneSample
11kb ladder
2Pcon-RBS-GFP-DT
3 Pcon-RBS-GFP-DT

Igku 0927 E2.jpg

Liquid Culture

No name

Samplemedium
Pcon spinach DT plusgrow 2µL(Amp)
Pcon aptamer DT plusgrow 2µL(Amp)
Pcon antisense Spinach DT plusgrow 2µL(Amp)
Spinach DTplusgrow 4µL(CP) 

RT PCR

No name

genome DNAKOD plus10x buffer for KOD-plus-ver.22nM dNTPs25mM MgSO4F primerR primerMilliQtotal
0.512.52.51.50.750.7515.525
PreDenatureDenatureAnnealingExtensioncycle
 94°C 98°C 49.8°C 68°C--
2min 10sec 30sec 10sec 30

Electrophoresis

No name

LaneSample
1Pcon-GFP-DT(GFP)
2Pcon-GFP-DT(internal)
3Pcon-GFP-DT(HS)(GFP)
4Pcon-GFP-DT(internal)
LaneSample
1Pcon-GFP-DT(GFP)
2Pcon-GFP-DT(internal)
3100bp ladder

Igku 927E4.jpg

Digesting cut check

No name

LaneSample
1RBS-GFP-DT(XbaI&PstI)
2RBS-GFP-DT NC(XbaI&PstI)
31kbp ladder
4Pcon-RBS-tetR-DT(EcoRI&SpeI)
5Pcon-RBS-tetR-DT NC(EcoRI&SpeI)

Igku 927 E3.jpg

Gel Extraction

No name

LaneDNAEnzyme
11kbp ladder--
3RBS-GFP-DTXbal+PstI
4RBS-GFP-DTXbal+PstI
5RBS-GFP-DTXbal+PstI
71kbp ladder--
9Pcon-tetR-DTEcoRI+SpeI
10Pcon-tetR-DTEcoRI+SpeI
11Pcon-tetR-DTEcoRI+SpeI

Igku 0927 G7.jpg
Igku 0927 G8.jpg
Igku 0927 G9.jpg
Igku 0927 G10.jpg

RNA Extraction

Nakamoto

05min15min20min23min28min33min36min
aptamer generator0.3480.4080.5500.865
Pcon-spinach-DT0.2500.2400.3050.4260.4710.5540.6680
GFP generator0.3890.4200.5160.884
antisense-spinach0.3710.4380.5370.733
Spinach-DT0.0840.1100.1340.4330.2690.3260.4090.742
Nameconcentration[µg/mL]260/280260/230
Spinach-DT83.91.630.17
Pcon-tetRaptamer-DT425.12.100.64
Pcon-RBS-GFP-DT262.42.010.84
Pcon-antisense-Spinach-DT489.12.050.68
Pcon-Spinach-DT266.62.010.66

cDNA Synthesis

Nakamoto

9/27 Spinach-DT9/23 Pcon-tetRaptamer-DT9/23 Pcon-RBS-GFP-DT9/23 Pcon-antisense-spinach-DT9/23 Pcon-Spinach-DT
RNA11111
gDNA wipeout61.21.91.01.9
RNase-free water04.84.15.04.1
RT0.50.50.50.50.5
RT Buffer22222
Primer mix0.50.50.50.50.5
total10µL10µL10µL10µL10µL