Template:Kyoto/Notebook/Sep 17

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Sep 17

Sep 18

Restriction Enzyme Digestion

No name

	9/18Plux	SpeI	PstI	Buffer	BSA	MilliQ	total
2cuts	3.2µL	1µL	1µL	3µL	3µL	18.8µL	30µL
NC	1.7µL	0µL	0µL	1µL	1µL	6.3µL	10µL

	9/18Pcon-pT181antisense-spinach-DT②	XbaI	PstI	Buffer	BSA	MilliQ	total
2cuts	7.5µL	1µL	1µL	3µL	3µL	14.5µL	30µL
NC	1.4icro;L	0µL	0µL	1µL	1µL	6.6µL	10µL

	9/16 Plac②	SpeI	PstI	Buffer	BSA	MilliQ	total
2cuts	13.8µL	1µL	1µL	3µL	3µL	14.5µL	30µL
NC	0.7µL	0µL	0µL	1µL	1µL	7.3µL	10µL

	9/16 Spinach-DT	XbaI	PstI	Buffer	BSA	MilliQ	total
2cuts	17.6µL	1µL	1µL	3µL	3µL	4.4µL	30µL
NC	0.9µL	0µL	0µL	1µL	1µL	7.1µL	10µL

	9/8 Pcon J23100	SpeI	PstI	Buffer	BSA	MilliQ	total
2cuts	5.7µL	1µL	1µL	3µL	3µL	16.3µL	30µL
NC	0.3µL	0µL	0µL	1µL	1µL	7.7µL	10µL

	9/16 RBS-lacZα-DT	XbaI	PstI	Buffer	BSA	MilliQ	total
2cuts	8.4µL	1µL	1µL	3µL	3µL	13.6µL	30µL
NC	0.4µL	0µL	0µL	1µL	1µL	7.6µL	10µL

	9/11 Pcon-pT181antisense-spinach-DT&#9312:	XbaI	PstI	Buffer	BSA	MilliQ	total
2cuts	7.2µL	1µL	1µL	3µL	3µL	14.8µL	30µL
NC	0.4µL	0µL	0µL	1µL	1µL	7.6µL	10µL

	9/6 apt12-1R-DT	XbaI	PstI	Buffer	BSA	MilliQ	total
2cuts	7.9µL	1µL	1µL	3µL	3µL	14.1µL	30µL
NC	0.4µL	0µL	0µL	1µL	1µL	7.6µL	10µL

	9/3 pT181antisense	XbaI	PstI	Buffer	BSA	MilliQ	total
2cuts	6.3µL	1µL	1µL	3µL	3µL	15.7µL	30µL
NC	0.3µL	0µL	0µL	1µL	1µL	7.7µL	10µL

	9/14 pSB4K5	EcoRI	SpeI	Buffer	BSA	MilliQ	total
2cuts	7.3µL	1µL	1µL	3µL	3µL	14.7µL	30µL
NC	0.4µL	0µL	0µL	1µL	1µL	7.6µL	10µL

	8/31 Plux-RBS-GFP-DT	EcoRI	SpeI	Buffer	BSA	MilliQ	total
2cuts	9.2µL	1µL	1µL	3µL	3µL	12.8µL	30µL
NC	0.5µL	0µL	0µL	1µL	1µL	7.5µL	10µL

	9/12 RBS-lysis3-DT	XbaI	PstI	Buffer	BSA	MilliQ	total
2cuts	5.2µL	1µL	1µL	3µL	3µL	16.8µL	30µL
NC	0.3µL	0µL	0µL	1µL	1µL	7.6µL	10µL

	9/18 Pcon-pT181attenuator	EcoRI	SpeI	Buffer	BSA	MilliQ	total
2cuts	14.3µL	1µL	1µL	3µL	3µL	7.7µL	30µL
NC	0.7µL	0µL	0µL	1µL	1µL	7.3µL	10µL

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	Plae②	SpeI	PstI
2	RBS-lysis3-DT	XbaI	PstI
3	Plux-RBS-GFP-DT	EcoRI	SpeI
4	PSB4K5	EcoRI	SpeI
5	Pcon	SpeI	PstI
6	Plux	SpeI	PstI
7	1kbp ladder	--	--
8	Plae②	--	--
9	RBS-lysis3-DT	--	--
10	Plux-RBS-GFP-DT	--	--
11	PSB4K5	--	--
12	Pcon	--	--
13	Plux	--	--

Electrophoresis

Okazaki

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	Pcon pT181antisense spinach-DT②	XbaI	PstI
3	Spinach-DT	XbaI	PstI
4	RBS-lacZα-DT	XbaI	PstI
5	Pcon pT181antisense-Spinach-DT①	XbaI	PstI
6	aptamer12-1R-DT	XbaI	PstI
7	pT181attenuator	XbaI	PstI
8	Pcon attenuator	EcoRI	SpeI

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	Pcon pT181antisense spinach-DT②	XbaI	PstI
3	Spinach-DT	XbaI	PstI
4	RBS-lacZα-DT	XbaI	PstI
5	Pcon pT181antisense-Spinach-DT①	XbaI	PstI
6	apt12-1R-DT	XbaI	PstI
7	pT181attenuator	XbaI	PstI
8	Pcon attenuator	EcoRI	SpeI

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	Pcon pT181antisense spinach-DT②	--	--
3	Spinach-DT	--	--
4	RBS-lacZα-DT	--	--
5	Pcon pT181antisense-Spinach-DT①	--	--
6	apt12-1R-DT	--	--
7	pT181attenuator	--	--
8	Pcon attenuator	--	--

Gel Extraction

No name

Lane	DNA	Enzyme
1	1kbp ldder	--
2	Plac②	SpeI+PstI
3	Plac②	SpeI+PstI
4	--	--
5	RBS-lysis3-DT	XbaI+PstI
6	RBS-lysis3-DT	XbaI+PstI
7	--	--
8	Plux-RBS-GFP-DT	EcoRI+SpeI
9	Plux-RBS-GFP-DT	EcoRi+SpeI
10	--	--
11	PSB4KS	EcoRI+SpeI
12	PSB4KS	EcoRI+SpeI

Lane	DNA	Enzyme
1	1kbp ldder	--
2	Pcon	SpeI+PstI
3	Pcon	SpeI+PstI
4	--	--
5	Plux	SpeI+PStI
6	Plux	SpeI+PStI

Lane	DNA	Enzyme
1	Pcon PT181 antisense spinach-DT	XbaI+PstI
2	Pcon PT181 antisense Spinach-DT	XbaI+PstI
3	100bp ladder	--
4	Pcon PT181 antisense Spinach-DT	XbaI+PstI
5	Pcon PT181 antisense Spinach-DT	XbaI+PstI
6	1kbp ladder	--

Lane	DNA	Enzyme
1	100bp ladder	--
2	apt12-1R-DT	XbaI+PstI
3	apt12-1R-DT	XbaI+PstI
4	--	--
5	PT181 attenuator	XbaI+PstI
6	PT181 attenuator	XbaI+PstI

Lane	DNA	Enzyme
1	100bp ladder	--
2	Pcoa attenuator	EcoRI+PstI
3	Pcoa attenuator	EcoRI+PstI
4	--	--
5	RBS-lacZα-DT	XbaI+PstI
6	RBS-lacZα-DT	XbaI+PstI

Sep 19

Sep 20

Ligation

Okazaki, Kanaoka, Tatsui and Hirano

state	Vector		Inserter		Ligation High ver.2
experiment	9/18 Plac (SpeI & PstI)	3.0 µL	9/18 RBS-lysis3-DT (XbaI & PstI)	12.4 µL	3.5 µL
experiment	9/12 Ptet-pT181 antisense	1.0 µL	9/18 spinach-DT	11.9 µL	3.5 µL
experiment	9/13 Pcon-RBS-luxR-DT	2.1 µL	9/10 Plux-RBS-GFP-DT	5.6 µL	3.5 µL
experiment	9/13 Pcon-pT181 attenuator (SpeI & PstI)	2.0 µL	9/18 aptamer 12-1R-DT (XbaI & PstI)	6.3 µL	3.5 µL
experiment	9/18 Plac ②(SpeI & PstI)	3.0 µL	9/18 aptamer 12-1R-DT(XbaI & PstI)	4.5µL	x3.5 µL

Transformation

Tatsui

Name	Sample	Competent Cells	Plate
9/17 pSB1C3-Pcon-pT181 attenuator-DT	2 µL	20 µL	CP
9/17 pSB1C3-Pcon-aptamer 12_1R-DT	2 µL	20 µL	CP
9/17 pSB1C3-Pcon-antisense-spinach-DT	2 µL	20 µL	CP
9/17 pSB1C3-Pcon-RBS-tetR-DT x2	2 µL	20 µL	CP
9/14 pSB1C3-PkaiBC	2 µL	20 µL	CP
9/14 pSB1C3-RpaB	2 µL	20 µL	CP
9/16 pSB1C3-pT181 attenuator(EcoRI&SpeI)	2 µL	20 µL	CP
9/17 pSB1C3-pT181 attenuator(XbaI&PstI)	2 µL	20 µL	CP
9/17 pSB1C3-Pcon-spinach-DT	2 µL	20 µL	CP
9/14 RBS-GFP-DT-PkaiBC	2 µL	20 µL	CP
Plux-RBS-lysis3-DT	2 µL	20 µL	CP
9/20 Ptet-pT181 antisense-spinach-DT	2 µL	20 µL	CP
9/20 Pcon-spinach-DT(pSB4K5)	2 µL	20 µL	Kan
9/20 Pcon-pT181 antisense-spinach-DT(pSB4K5)	2 µL	20 µL	Kan
9/20 KaiABC	2 µL	20 µL	Kan
9/20 KaiABC	2 µL	20 µL	Kan
9/20 Plac-RBS-lysis3-DT	2 µL	20 µL	Amp
9/20 Plac-aptamer 12_1R	2 µL	20 µL	Amp
9/20 Plux-RBS-GFP-DT-Pcon-RBS-luxR-DT	2 µL	20 µL	Amp
Plac-aptamer12_1R-DT	2 µL	20 µL	Amp
9/20 Plac-aptamer12_1R-DT	2 µL	20 µL	Amp
9/20Pcon-pT181attenuator-aptamer-DT	2 µL	20 µL	Amp
9/17 Plac-pT181-attenuatorx2	2 µL	20 µL	Amp
9/15 Pcon-RBS-lacZα-DT	2 µL	20 µL	Amp
9/14 Pcon-pT181attenuator-RBS-lacZα-DT	2 µL	20 µL	Amp
PUC19	2 µL	20 µL	Amp

PCR

Tatsui

Sample	base pair
pT181 attenuator(pSB1C3)	--
Pcon-RBS-lacZα-DT	--

PreDenature	Denature	Annealing	Extension	cycle
94 °C	94 °C	55 °C	68 °C	--
5 min	30 s	30 s	3 min	30 cycles

Electrophoresis

No name

Lane	Sample	Enzyme
1	Pcon-RBS-lacZα-DT	--
2	1kb ladder	--
3	pSB1C3-pT181attenuator	--

Sep 21

Electrophoresis

no name

Lane	Sample	Enzyme1	Enzyme2
1	Pcon-RBS-lacZα-DT	--	--
2	1kbp ladder	--	--
3	pSB1C3+pT181 attenuator	--	--

Liquid Culture

No name

Sample	medium
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

Number	Use LB plate
1	8/27 Pλ-luxI-1(+Amp)
2	9/10 Plac(BBa-R0011)-3(+Amp)
3	9/5 Pcon-RBS-tetR-DT-1(+Amp)
4	9/5 Pcon-pT181 antisense-1(+Amp)
5	9/15 Pcon-pT181 attenuator(+Amp)
6	9/- Pcon-apt12_1R-DT-1(+Amp)
7	9/5 Pcon-spinach-DT-3(+Amp)
8	8/27 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT(+Amp)

Number	Use LB plate
1	9/4 pT181 antisense(pSB1C3)-1(+CP)
2	9/11 Fusion1 antisense(pSB1C3)-4(+CP)
3	9/11 Fusion6 antisense(pSB1C3)-1(+CP)
4	9/4 pT181 attenuator(pSB1C3)-1(+CP)
5	9/10 Fusion1 attenuator(pSB1C3)-1(+CP)
6	9/11 Fusion3n2 attenuator(pSB1C3)-1(+CP)
7	9/4 aptamer12_1R(pSB1C3)-1(+CP)
8	9/11 aptamer12_P(pSB1C3)-3(+CP)
9	9/11 aptamer12_1M(pSB1C3)-1(+CP)

</div>

Number	Use LB plate
1	8/29 Pbad/araC-RBS-RFP-DT-1(+CP)
2	9/4 RBS-lysis3-DT-3(+CP)
3	9/10 Pcon-pT181 attenuator-DT-1(+CP)
4	9/5 Ptet-pT181 antisense-1(+CP)
5	9/4aptamer 12_1R-DT-1(+CP)
6	9/5 Ptet-RBS-lacZα-DT-4(+CP)
7	8/10 RBS-lacI-DT-2(+CP)
8	8/29 Plux-RBS-GFP-DT-1(+CP)

</div>

Restriction Enzyme Digestion

Noname

	8/17 RBS-DT	EcoRI	SpeI	XbaI	PstI	Buffer	BSA	MilliQ	total
2cuts(XbaI&PstI)	8.1	0µL	0µL	1µL	1µL	3µL	3µL	13.9µL	30µL
NC	0.4	0µL	0µL	0µL	0µL	1µL	1µL	7.6µL	10µL

	8/17 RBS-DT	EcoRI	SpeI	XbaI	PstI	Buffer	BSA	MilliQ	total
2cut(EcoRI&XbaI)	16.2	1µL	1µL	0µL	0µL	3µL	3µL	5.8µL	30µL

	9/15 Pcon-pT181attenuator	EcoRI	SpeI	XbaI	PstI	Buffer	BSA	MilliQ	total
2cuts(EcoRI&PstI)	12	1µL	0µL	0µL	1µL	3µL	3µL	10µL	30µL
NC	0.6	0µL	0µL	0µL	0µL	1µL	1µL	7.4µL	10µL

	8/20 Pcon=RBS-GFP-DT-1	EcoRI	SpeI	XbaI	PstI	Buffer	BSA	MilliQ	total
2cut(EcoRI&XbaI)	6	1µL	0µL	1µL	0µL	3µL	3µL	0µL	14µL
NC	0.3	0µL	0µL	0µL	0µL	1µL	1µL	7.3µL	10µL

incubate 37°C 1hour

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	RBS-GFP-DT	XbaI	PstI
2	RBS-GFP-DT	--	--
3	RBS-GFP-DT	EcoRI	XbaI
4	1kbp ladder	--	--
5	Pcon-pT181attenuator	SpeI	PstI
6	Pcon-pT181attenuator	--	--
7	Pcon-RBs-GFP-DT	EcoRI	XbaI
8	Pcon-RBs-GFP-DT	--	--

PCR

Nakamoto

Sample	base 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

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	57°C	68°C	--
2min	10s	30s	35s	30cycles

Gel Extraction

No name

Lane	DNA	Enzyme
1	1kb ladder	--
3	RBS-GFP-DT	XbaI&PstI
4	RBS-GFP-DT	XbaI&PstI
5	RBS-GFP-DT	XbaI&PstI

Name	concentration[µg/mL]	260/280	260/230
RBS-GFP-DT(XbaI&PstI)	14.8	1.74	0.47
RBS-GFP-DT(EcoRI&SpeI)	88.4	1.85	1.49
Pcon-RBS-GFP-DT(EcoRI&XbaI)	47.5	1.82	1.12

Sep 22

Colony PCR

Tatsui

Sample	base 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

Electrophoresis

Nakamoto

Lane	Sample
1	Pcon-apt12_1R-DT(E-1A)
2	Pcon-spinach-DT(E-1A)
3	Pcon-pT181 attenuator(E-1A)
4	DT(1-3)
5	tetR aptamer 12_1R-DT(1-3)
6	100bp ladder
7	Pcon-pT181 attenuator(1-SA)
8	spinach-DT(2-1)
9	Pcon-pT181 attenuator(E-0)
10	Pcon-pT181 attenuator(2-SA)
11	spinach-DT(3-2)
12	Ptet-PT181 anisense

Miniprep

Honda

DNA	concentration[µg/mL]	260/280	260/230
9/21 spinach-DT	197.9	1.67	1.54
9/21 Plux	125.9	1.71	1.27
9/21 Plux-RBS-GFP-DT-(1)	168.0	1.62	1.73
9/21 Pcon-PT181 attenuator-(1)	368.3	1.57	1.62
9/21 RBS-GFP-DT	532.8	1.75	1.78
9/21 Pcon-RBS-GFP-DT	845.2	2.02	1.70

Colony PCR

Honda

Sample	base pair
9/20 Plux-RBS-GFP-DT-Pcon-RBS-luxR-DT-(1~2)	283

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	2min10s	30cycles

Gel Extraction

No name

Lane	DNA	Enzyme
1	100bp ladder	--
2~4	Pcon-aptamer12-1R-DT(E-1A)	--
6~8	Pcon-spinach-DT(E-1A)	--
10~12	Pcon-attenuator PT181(E-1A)	--

Lane	DNA	Enzyme
1	100bp ladder	--
2~4	DT(1-3)	--
6~8	aptamer12-1RDT(1-3)	--
10~12	Pcon-PT181 attenuator(1-5A)	--

Name	concentration[µg/mL]	260/280	260/230
Pcon-aptamer12-1R-DT(E-1A)	31.2	1.87	0.30
Pcon-spinach-DT(E-1A)	25.6	1.88	0.99
Pcon-attenuator PT181(E-1A)	25.3	1.99	0.86
DT(1-3)	22.1	1.82	0.20
aptamer12-1RDT(1-3)	33.5	1.99	0.05
Pcon-PT181 attenuator(1-5A)	36.9	1.12	0.96

Restriction Enzyme Digestion

No name

	9/16 Plac	XbaI	SpeI	PstI	BufferB	BSA	MilliQ	total
2cuts	12.5µL	0µL	1µL	1µL	3µL	3µL	9.5µL	30µL
NC	0.8µL	0µL	0µL	0µL	1µL	1µL	7.4µL	10µL

	9/22 Plux	XbaI	SpeI	PstI	BufferB	BSA	MilliQ	total
2cuts	16µL	0µL	1µL	1µL	3µL	3µL	14µL	30µL
NC	0.8µL	0µL	0µL	0µL	1µL	1µL	7.2µL	10µL

	9/20 RBS-tetR-2R-DT	XbaI	SpeI	PstI	BufferD	BSA	MilliQ	total
2cuts	8.5µL	1µL	0µL	1µL	3µL	3µL	13.5µL	30µL
NC	0.4µL	0µL	0µL	0µL	1µL	1µL	7.6µL	10µL

Gel Extraction

No name

Lane	DNA	Enzyme
1	100bp ladder	--
2~4	Pcon-attenuator PT181(E-0)	--

Lane	DNA	Enzyme
1	100bp ladder	--
2~4	Pcon-attenuator PT181(E-0)	--

Lane	DNA	Enzyme
2	100bp ladder	--
4~6	Pcon-attenuator PT181(2-SA)	--

Lane	DNA	Enzyme
1	100bp ladder	--
3~5	spinach-DT(3-2)	--
8~10	Ptet-antisense PT181(1-3)	--

Name	concentration[µg/mL]	260/280	260/230
Pcon-attenuator PT181(E-0)	2.4	1.80	0.17
Pcon-attenuator PT181(2-SA)	10.8	1.85	0.54
spinach-DT(3-2)	36.7	1.80	0.75
Ptet-antisense PT181(1-3)	16.8	1.74	0.78

Colony PCR

No name

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

Colony PCR

Tatsui

Sample	base pair
PconPT181antisense-spinach-DT(9~15)	723

Ligation

No name

state	Vector		Inserter		Ligation High ver.2
experiment	9/13 Pcon-PT181 attenuator (SpeI & PstI)	1.7 µL	9/18 aptamer-1R-DT (XbaI & PstI)	5.4µL	3.5µL
experiment	9/12 Plac(SpeI & PstI)	2.4 µL	9/18 aptamer-1R-DT (XbaI & PstI)	6.1µL	3.5 µL
experiment	9/13 Ptet(SpeI & PstI)	1.7 µL	9/10 Plux-RBS-GFP-DT	7.0 µL	4.4 µL
experiment	9/21 RBS-GFP-DT (EcoRI & XbaI)	0.6 µL	9/18 aptamer 12-1R-DT (XbaI & PstI)	5.6µL	3.1 µL
experiment	9/13 PSB1C3(XbaI & PstI)	1.9 µL	9/16 PT181 attenuator(XbaI & PstI)	4.6µL	3.3 µL

Liquid Culture

No name

Sample	medium
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 antisense	EcoRI	SpeI	BufferM	MilliQ	total
2cuts	8.3µL	1µL	1µL	3µL	16.7µL	30µL
NC	0.4µL	0µL	0µL	1µL	8.3µL	10µL

	9/7 Pcon-RBS-tetR-DT	EcoRI	XbaI	SpeI	PstI	BufferD	BSA	MilliQ	total
2cut	14µL	1µL	1µL	0µL	0µL	3µL	3µL	18.2µL	30µL
NC	0.7µL	0µL	0µL	0µL	0µL	1µL	1µL	7.3µL	10µL

	9/22 RBS-GFP-DT	EcoRI	XbaI	SpeI	PstI	BufferD	BSA	MilliQ	total
2cuts	3.8µL	1µL	1µL	0µL	0µL	3µL	3µL	18.2µL	30µL
NC	0.2µL	0µL	0µL	0µL	0µL	1µL	1µL	7.8µL	10µL

	9/6 aptamer12-1R-DT	EcoRI	XbaI	SpeI	PstI	BufferD	BSA	MilliQ
2cut	8µL	1µL	1µL	0µL	0µL	3µL	3µL	16µL
NC	0.4µL	0µL	0µL	0µL	0µL	1µL	1µL	7.6µL

incubate 37°C 1hour

Gel Extraction

No name

Lane	DNA	Enzyme
1	1kbp ladder	--
3~5	Pcon-PT181 antisense (EcoRI&SpaI)	--

Transformation

No name

Name	Sample	Competent Cells	Plate
9/20 Plac-aptamer12-1R-DT(XbaI&PstI)	2 µL	20 µL	Amp
9/20 pT181 attenuator(1C3)(EcoRI&SpeI)	2 µL	20 µL	CP
9/17 pT181 attenuator(1C3)(XbaI&PstI)	2 µL	20 µL	CP
9/20 Pcon-pT181 attenuator(SpaI&PstI)＋aptamer12-1R-DT(XbaI&PstI)	2 µL	20 µL	Amp
kaiABC	2 µL	20 µL	Amp
9/22 pSB1C3-RpaB(XbaI&PstI)	2 µL	20 µL	Amp
9/22 pSB1C3-pT181 attenuator(XbaI&PstI)	2 µL	20 µL	Amp
9/22 Ptet(SpaI&PstI)＋RBS-GFP-DT(XbaI&PstI)	2 µL	20 µL	CP
9/22 RBS-GFP-DT＋Pcon-attenuator pT181 (EcoRI&SpeI)	2 µL	20 µL	CP
9/22 pT181 attenuator(XbaI&PstI)(1C3)	2 µL	20 µL	CP

PCR

No name

Sample	base 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))	--

PreDenature	Denature	Annealing	Extension	cycle
94 °C	94 °C	57 °C	68 °C	--
2 min	10 s	30 s	1min5s	30 cycles

Electrophoresis

No name

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

Sep 23

Transformation

No name

Name	Sample	Competent Cells	Plate
9/14 pSB1C3＋PkaiBC	2 µL	20 µL	--
9/14 pSB1C3＋RpaB	2 µL	20 µL	--
9/14 pkaiBC＋Rbs-GFP-DT	2 µL	20 µL	--

Gel Extraction

No Name

Lane	DNA	Enzyme
1	1kbp ladder
3-4	Pcon-RBS-tetR-PT

Name	concentration[µg/mL]	260/280	260/230
Pcon-PT181 anti EcoRI+SpeI	46.7	1.06	0.93
Pcon-RBS-tetR-DT EcoRI+XbaI	119.3	0.99
RBS-8FP-DT EcoRI+XbaI	87.7	1.00	0.98
ap42_1R-DT EcoRI+XbaI	68.8	1.32	1.03
Pcon-RBS-tetR-DT 1-2 A	203.0	1.66	1.79
Pcon-RBS-tetR-DT 3-2 A	67.7	2.64	1.28
Pcon-RBS-GFP-DT 1-S A	185.6	1.88	2.04
Pcon-RBS-GFP-DT 2-S A	298.6	1.68	1.55
Pcon-tetR-DT E-2 A	202.4	1.57	1.66

PCR

No name

genome DNA	KOD plus	10x buffer	dNTP	MgSO4	SasA_fwd primer	SasA_rev primer	MilliQ	total
9/8 Pcon-pT181 anti	0.5	2.5	2.5	1.5	0.75	0.75	15.5	25
8/20 Ptet(1)	0.5	2.5	2.5	1.5	0.75	0.75	15.5	25
9/16 Plac 2	0.5	2.5	2.5	1.5	0.75	0.75	15.5	25

PreDenature	Denature	Annealing	Extension	cycle
94 °C	98 °C	57 °C	68 °C	--
2min	10sec	30sec	20sec	30cycles

Ligation

Yoshida

state	Vector		Inserter		Ligation High ver.2
experiment	9/21 Pcon-RBS-GFP-DT EcoRI+XbaI	47.5 µg/mL	9/15 Pcon-RBS-tetR-DT EcoRI+SpeI	59 µg/mL	2.3 µg/mL
experiment	9/21 Pcon-RBS-GFP-DT EcoRI+XbaI	47.5 µg/mL	9/15 Pcon-apt12_1R-DT EcoRI+SpeI	17 µg/mL	2.4 µg/mL
experiment	9/21 Pcon-RBS-GFRDT EcoRI+XbaI	2.1 µg/mL	9/15 Pcon-DT181 attenuator-DT	21 µg/mL	3.0 µg/mL
experiment	9/22 apt 12_1R-DT EcoRI+XbaI	67.8 µg/mL	9/18 Pcon-DT181 attenuator EcoRI+SpeI	5.7 µg/mL	3.5 µg/mL
experiment	9/22RBS-GFP-DT EcoRI+XbaI	87.7 µg/mL	9/18 Pcon-DT18 attenuator EcoRI+SpeI	6.7 µg/mL	3.5 µg/mL
experiment	9/13 DT EcoRI+XbaI	25.4 µg/mL	9/22 Pcon-DT181 antisense EcoRI+SpeI	46.8 µg/mL	2.3 µg/mL

PCR

No name

genome DNA	KOD plus	10x buffer	dNTP	MgSO4	SasA_fwd primer	SasA_rev primer	MilliQ	total
Pcon-attenuator-DT (E-1A)	0.5	2.5	2.5	1.5	0.75	0.75	77.5	25
Pcon-attenuator-DT (2-1A)	0.5	2.5	2.5	1.5	0.75	0.75	77.5	25
Pcon-antisense-spinach-DT (1-2A)	0.5	2.5	2.5	1.5	0.75	0.75	77.5	25
Pcon-antisense-spinach-DT (E-1A)	0.5	2.5	2.5	1.5	0.75	0.75	77.5	25

PreDenature	Denature	Annealing	Extension	cycle
94 °C	98 °C	57 °C	68 °C	--
2min	10s	30s	45s	30cycle

Miniprep

No name

DNA	concentration[µg/mL]	260/280	260/230
9/22 Plac-DT181 attenuator-1	204.0	2.00	1.95
9/22 Plac-DT181 attenuator-2	195.3	1.84	1.62
9/22 Plac-DT181 antisense-1	188.2	1.87	2.15
9/22 Plac-DT181 antisense-2	165.0	1.95	2.66

Electrophoresis

Hirano

Lane	Sample	Enzyme1	Enzyme2
1,6	100bp ladder(Promega)	--	--
2	Pcon-attenuator-DT (E-1A)	--	--
3	Pcon-attenuator-DT (2-1A)	--	--
4	Pcon-antisense-spinach-DT (1-2A)	--	--
5	Pcon-antisense-spinach-DT (E-1A)	--	--

Gel Extraction

Hirano

Name	concentration[µg/mL]	260/280	260/230
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

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	9/8 Pcon-pT181 antisense(E-1A)	--	--
3	8/20 Ptet(1)(2-S)	--	--
4	9/16 Plac2(E-1A)	--	--

Transformation

No Name

Name	Sample	Competent Cells	Total	Plate
pSB1C3	2µL	20µL	22µL

Ligation

No Name

state	Vector		Inserter		Ligation High ver.2
experiment	9/19 Plac SpeI+PstI	24.6 µg/mL	9/18 ape12_1R-DT XbaI+PstI	4.5 µg/mL	2 µg/mL
experiment	9/21 RBS-GFP-DT EcoRI+XbaI	88.4 µg/mL	9/15 Pcon-pT181 attenuator EcoRI+SpeI	21 µg/mL	2 µg/mL
experiment	9/13 PSB1C3 XbaI+PstI	26.0 µg/mL	9/16 pT181 attenuator XbaI+PstI	8.2 µg/mL	2 µg/mL

Colony PCR

Tatsui

Sample	base 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)	--

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	54s	30cycle

Sep 24

Electrophoresis

No name

Lane	Sample
1	9/20 Pcon-pT181aptamer12-1R-DT 1
2	9/20 Pcon-pT181aptamer12-1R-DT 2
3	9/20 Pcon-pT181aptamer12-1R-DT 3
4	9/20 Pcon-pT181aptamer12-1R-DT 4
5	9/20 Pcon-pT181aptamer12-1R-DT 5
6	9/20 Pcon-pT181aptamer12-1R-DT 6
7	9/20 Pcon-pT181aptamer12-1R-DT 7
8	9/20 Pcon-pT181aptamer12-1R-DT 8
9	100bp ladder
10	9/22 Pcon-pT181attenuator-aptamer12-1R-DT 1
11	9/22 Pcon-pT181attenuator-aptamer12-1R-DT 2
12	9/22 Pcon-pT181attenuator-aptamer12-1R-DT 3
13	9/22 Pcon-pT181attenuator-aptamer12-1R-DT 4
14	9/20 Plac-aptamer12-1R-DT 1
15	9/20 Plac-aptamer12-1R-DT 2
16	9/20 Plac-aptamer12-1R-DT 3
17	9/20 Plac-aptamer12-1R-DT 4

Transformation

No name

Name	Sample	Competent Cells	Total	Plate
9/23 pSB1C3-pT181attenuator	2 µL	20 µL	22 µL	LB (+CP)
RBS-GFP-DT+Pcon-pT181attenuator	2 µL	20 µL	22 µL	LB (+CP)
9/23 Plac-+spinach-DT	2 µL	20 µL	22 µL	LB (+Amp)
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT	2 µL	20 µL	22 µL	LB (+Amp)
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT	2 µL	20 µL	22 µL	LB (+Amp)
9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT	2 µL	20 µL	22 µL	LB (+Amp)
9/23 Pcon-pT181antisense-DT	2 µL	20 µL	22 µL	LB (+CP)
9/23 Pcon-pT181attenuator-RBS-GFP-DT	2 µL	20 µL	22 µL	LB (+CP)
9/23 Pcon-pT181attenuator-aptamer12-1R-DT	2 µL	20 µL	22 µL	LB (+CP)
PUC19	2 µL	20 µL	22 µL	LB (+Amp)
Ptet-RBS-lacZα-DT	2 µL	20 µL	22 µL	LB (+CP)
pSB1C3	2 µL	20 µL	22 µL	LB (+CP)

Liquid Culture

No name

Sample	medium
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 buffer	BsaI-HF	MilliQ	total
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

Name	concentration[µg/mL]	260/280	260/230
8/22 Pcon-pT181 attenuator(E-0)	7.8	1.70	0.03
8/22 Pcon-pT181 attenuator(2-SA)	17.3	1.93	0.03
8/22 spinach-DT (3-2)	22.4	1.93	0.09
8/22 Ptet-PT181 anisense (1-3)	18.3	1.58	0.14
8/22 Pcon-aptamer12_1R-DT (E-1A)	8.2	2.10	0.05
8/22 Pcon-spinach-DT(E-1A)	5.6	0.91	0.05
8/22 Pcon-pT181 attenuator(E-1A)	18.5	1.80	0.12
8/22 DT(1-3)	7.3	1.13	0.04
8/22 tetR aptamer 12_1R-DT(1-3)	14.6	1.74	0.06
8/22 Pcon-pT181 attenuator(1-SA)	7.9	1.57	0.04
8/22 Pcon-RBS-tetR-DT(1-2Ⓐ)	41.6	1.81	0.25
8/22 Pcon-RBS-tetR-DT(3-2Ⓐ)	30.2	1.75	0.15
8/22 Pcon-RBS-tetR-DT(E-2Ⓐ)	36.9	1.75	0.56
8/22 Pcon-RBS-GFP-DT(1-SⒶ)	43.5	1.83	0.33
8/22 Pcon-RBS-GFP-DT(2-SⒶ)	64.6	1.82	0.30
8/23 Pcon-pT181 attenuator-DT (E-1A)	31.2	1.83	0.09
8/23 Pcon-pT181 attenuator-DT (0-1A)	64.5	1.80	0.43
8/23 Pcon-pT181 antisense-spinach-DT (1-2A)	36.4	1.88	0.21
8/23 Pcon-pT181 antisense-spinach-DT (E-1A)	27.9	1.92	0.16
8/23 Ptet(1)(2-S)	37.0	1.91	0.16
9/23 Plac-2(E-1A)	21.2	1.82	0.13

Transformation

No name

Name	Sample	Competent Cells	Total	Plate
9/23 Pcon-attenuator-aptamer12-1R-DT	2 µL	20 µL	22 µL	LB (+CP)
9/23 pT181attenuator(1C3)	2 µL	20 µL	22 µL	LB (+CP)
9/23 Pcon-pT181antisense-DT	2 µL	20 µL	22 µL	LB (+CP)
9/23 Pcon-pT181attenuator-RBS--GFP-DT	2 µL	20 µL	22 µL	LB (+CP)

Liquid Culture

No name

Sample	medium
pSB1C3 (Master plate-1)	LB(+CP)

Restriction Enzyme Digestion

Kojima

	8/21 pT181attenuator (330 µg/mL)	XbaI	PstI	buffer	BSA	MilliQ	total
2 cuts	6.1	1	1	3	3	15.9	30
NC	0.3	0	0	1	1	7.7	10

	9/22 Pcon-pT181attenuator (368.3 µg/mL)	EcoRI	SpeI	buffer	BSA	MilliQ	total
2 cuts	5.4	1	1	3	0	19.6	30
NC	0.3	0	0	1	0	8.7	10

	9/16 pSB1C3	EcoRI	SpeI	buffer	BSA	MilliQ	total
2 cuts	18	1	1	3	0	7.0	30
NC	0.9	0	0	1	0	8.1	10

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	1kb ladder	-	-
2	pT181attenuator	XbaI	PstI
3	pT181attenuator	--	--
4	Pcon-pT181attenuator	EcoRI	SpeI
5	Pcon-pT181attenuator	--	--
6	pSB1C3	EcoRI	SpeI
7	pSB1C3	--	--

Miniprep

No name

DNA	concentration[µg/mL]	260/280	260/230
9/24 aptamer12-1R-DT	144.7	1.03	1.21

Gel Extraction

Kojima

Lane	DNA	Enzyme1	Enzyme2
1	100kb ladder	--	--
2	pT181attenuator	XbaI	PstI
3	pT181attenuator	XbaI	PstI
4	pT181attenuator	XbaI	PstI
5	--	--	--
6	Pcon-pT181attenuator	EcoRI	SpeI
7	Pcon-pT181attenuator	EcoRI	SpeI
8	Pcon-pT181attenuator	EcoRI	SpeI
9	--	--	--
10	pSB1C3	EcoRI	SpeI
11	pSB1C3	EcoRI	SpeI
12	pSB1C3	EcoRI	SpeI

Name	concentration[µg/mL]	260/280	260/230
pT181attenuator(XbaI&PstI)	10.1	2.02	0.43
Pcon-pT181attenuator(EcoRI&SpeI)	10.3	1.81	0.32
pSB1C3(EcoRI&SpeI)	10.0	2.81	0.02

Colony PCR

Nakamoto

Sample	base pair
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 1	1414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 2	1414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 3	1414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 4	1414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 5	1414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 6	1414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 7	1414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 8	1414
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 1	1631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 2	1631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 3	1631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 4	1631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 5	1631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 6	1631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 7	1631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 8	1631
9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 1	2042
9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 2	2042
9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 3	2042
9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 4	2042
9/23 Pcon-pT181antisense-DT 1	508
9/23 Pcon-pT181antisense-DT 2	508
9/23 Pcon-pT181antisense-DT 3	508
9/23 Pcon-pT181antisense-DT 4	508
9/23 Pcon-pT181antisense-DT 5	508
9/23 Pcon-pT181antisense-DT 6	508
9/23 Pcon-pT181antisense-DT 7	508
9/23 Pcon-pT181antisense-DT 8	508

PreDenature	Denature	Annealing	Extension	cycle
94 °C	94 °C	55 °C	68 °C	--
5 min	30 sec	30 sec	2min	30 cycles

Lane	Sample
1	9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 1
2	9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 2
3	9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 3
4	9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 4
5	9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 5
6	9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 6
7	9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 7
8	9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 8
9	1kb ladder
10	9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 1
11	9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 2
12	9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 3
13	9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 4
14	9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 5
15	9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 6
16	9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 7
17	9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 8

Lane	Sample
1	9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 1
2	9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 2
3	9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 3
4	9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 4
5	9/23 Pcon-pT181antisense-DT 1
6	9/23 Pcon-pT181antisense-DT 2
7	1kb ladder
8	9/23 Pcon-pT181antisense-DT 3
9	9/23 Pcon-pT181antisense-DT 4
10	9/23 Pcon-pT181antisense-DT 5
11	9/23 Pcon-pT181antisense-DT 6
12	9/23 Pcon-pT181antisense-DT 7
13	9/23 Pcon-pT181antisense-DT 8
14	--

Restriction Enzyme Digestion

Nakamoto

	9/24 aptamer12-1R-DT	EcoRI	XbaI	buffer	BSA	MilliQ	total
2cuts	17.5µL	1.0µL	1.0µL	3.0µL	3.0µL	4.5µL	30µL
NC	0.9µL	0µL	0µL	1.0µL	1.0µL	7.1µL	10µL

	9/20 Ptet	SpeI	PstI	buffer	BSA	MilliQ	total
2cuts	9.5µL	1.0µL	1.0µL	3.0µL	0µL	15.5µL	30µL
NC	0.5µL	0µL	0µL	1.0µL	0µL	8.5µL	10µL

	9/22 RBS-GFP-DT	EcoRI	SpeI	buffer	BSA	MilliQ	total
2cuts	3.8µL	1.0µL	1.0µL	3.0µL	0µL	21.2µL	30µL
NC	0.2µL	0µL	0µL	1.0µL	0µL	8.8µL	10µL

	9/22 Pcon-RBS-GFP-DT	EcoRI	XbaI	buffer	BSA	MilliQ	total
2cuts	2.4µL	1.0µL	1.0µL	3.0µL	3.0µL	19.6µL	30µL
NC	0.1µL	0µL	0µL	1.0µL	1.0µL	7.9µL	10µL

	9/22 Pcon-pT181attenuator 1	EcoRI	XbaI	buffer	BSA	MilliQ	total
2cuts	5.4µL	1.0µL	1.0µL	3.0µL	3.0µL	16.6µL	30µL
NC	0.3µL	0µL	0µL	1.0µL	1.0µL	7.7µL	10µL

	9/22 RBS-GFP-DT	XbaI	PstI	buffer	BSA	MilliQ	total
2cuts	3.8µL	1.0µL	1.0µL	3.0µL	3.0µL	18.2µL	30µL
NC	0.2µL	0µL	0µL	1.0µL	1.0µL	7.8µL	10µL

Ligation

Nakamoto

state	Vector		Inserter		Ligation High ver.2
experiment	9/22 aptamer12-1R-DT(EcoRI&XbaI)	1.4 µL	9/24 Pcon-pT181attenuator(EcoRI&SpeI)	26 µL	3.5 µL
experiment	9/4 Ptet(SpeI&PstI)	2.0 µL	9/21 RBS-GFP-DT(XbaI&PstI)	9.0 µL	3.5 µL

incubated at 37 °C for 1 hour.

colony PCR

Kojima

Sample	base pair
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 5	2042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 6	2042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 7	2042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 8	2042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 9	2042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 10	2042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 11	2042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 12	2042bp
9/23 Pcon-pT181antisense-DT 9	508bp
9/23 Pcon-pT181antisense-DT 10	508bp
9/23 Pcon-pT181antisense-DT 11	508bp
9/23 Pcon-pT181antisense-DT 12	508bp
9/23 Pcon-pT181antisense-DT 13	508bp
9/24 Pcon-pT181attenuator-RBS-GFP-DT 1	1527bp
9/24 Pcon-pT181attenuator-RBS-GFP-DT 2	1527bp
9/24 Pcon-pT181attenuator-RBS-GFP-DT 3	1527bp
9/24 Pcon-pT181attenuator-RBS-GFP-DT 4	1527bp
9/24 Pcon-pT181attenuator-aptamer12-1R-DT 1	859bp
9/24 Pcon-pT181attenuator-aptamer12-1R-DT 2	859bp
9/24 Pcon-pT181attenuator-aptamer12-1R-DT 3	859bp
9/24 Pcon-pT181attenuator-aptamer12-1R-DT 4	859bp
9/24 pT181attenuator(pSB1C3) 1	390bp
9/24 pT181attenuator(pSB1C3) 2	390bp
9/24 pT181attenuator(pSB1C3) 3	390bp
9/24 pT181attenuator(pSB1C3) 4	390bp

Tatsui

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	2min	30cycle

Miniprep

No name

DNA	concentration[µg/mL]	260/280	260/230
pSB1C3	183.9	1.91	1.59
Pcon-pT181attenuator-aptamer12-1R-DT	231.7	1.82	2.35
Plac-aptamer12-1R-DT	208.9	1.98	1.79

Electrophoresis

Nakamoto

Lane	Sample	Enzyme1	Enzyme2
1	1kb ladder	--	--
2	9/24 aptamer12-1R-DT	EcoRI	XbaI
3	9/24 aptamer12-1R-DT	--	--
4	9/24 Ptet	SpeI	PstI
5	9/24 Ptet	--	--
6	9/24 RBS-GFP-DT	EcoRI	SpeI
7	9/24 RBS-GFP-DT	--	--
8	9/24 Pcon-RBS-GFP-DT	EcoRI	XbaI
8	9/24 Pcon-RBS-GFP-DT	--	--
9	9/24 Pcon-RBS-GFP-DT	--	--
10	9/24 Pcon-pT181attenuator 1	EcoRI	XbaI
11	9/24 Pcon-pT181attenuator 1	--	--
12	9/24 RBS-GFP-DT	XbaI	PstI
13	9/24 RBS-GFP-DT	--	--
14	1kb ladder	--	--

Gel Extraction

No name 1

Lane	DNA	Enzyme1	Enzyme2
1	1kb ladder	--	--
2	aptamer12-1R-DT	EcoRI	XbaI
3	aptamer12-1R-DT	EcoRI	XbaI
4	aptamer12-1R-DT	EcoRI	XbaI
5	--	--	--
6	Ptet	SpeI	PstI
7	Ptet	SpeI	PstI
8	Ptet	SpeI	PstI
9	--	--	--
10	RBS-GFP-DT	EcoRI	SpeI
11	RBS-GFP-DT	EcoRI	SpeI
12	RBS-GFP-DT	EcoRI	SpeI

Lane	DNA	Enzyme1	Enzyme2
1	1kb ladder	--	--
2	Pcon-RBS-GFP-DT	EcoRI	XbaI
3	Pcon-RBS-GFP-DT	EcoRI	XbaI
4	Pcon-RBS-GFP-DT	EcoRI	XbaI
5	--	--	--
6	Pcon-pT181attenuator	EcoRI	XbaI
7	Pcon-pT181attenuator	EcoRI	XbaI
8	Pcon-pT181attenuator	EcoRI	XbaI
9	--	--	--
10	RBS-GFP-DT	XbaI	PstI
11	RBS-GFP-DT	XbaI	PstI
12	RBS-GFP-DT	XbaI	PstI

Electrophoresis

No name
1

Lane	Sample
1	1kb ladder
2	Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 5
3	Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 6
4	Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 7
5	Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 8
6	Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 9
7	Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 10
8	1kb ladder

2

Lane	Sample
1	1kb ladder
2	Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 11
3	Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 12
4	Pcon-pT181attenuator-RBS-GFP-DT 1
5	Pcon-pT181attenuator-RBS-GFP-DT 2
6	Pcon-pT181attenuator-RBS-GFP-DT 3
7	Pcon-pT181attenuator-RBS-GFP-DT 4
8	1kb ladder

3

Lane	Sample
1	Pcon-pT181antisense-DT 9
2	Pcon-pT181antisense-DT 10
3	Pcon-pT181antisense-DT 11
4	Pcon-pT181antisense-DT 12
5	Pcon-pT181antisense-DT 13
6	100bp ladder
7	Pcon-pT181attenuator-aptamer12-1R-DT 1
8	Pcon-pT181attenuator-aptamer12-1R-DT 2

4

Lane	Sample
1	Pcon-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

Ligation

Hirano

state	Vector		Inserter1		Inserter2		Inserter3		Inserter4		(NEB)T4ligase	(NEB)Buffer	milliQ	total
experiment	RBS-GFP-DT(pSB1C3)	1µL	tetR-aptamer12-1R-DT(1-3)	1 µL	Ptet(2-S)	0.3µL	Pcon-tetR-aptamer12-1R-DT(3-2A)	1.1µL	Plac(E-1A)	0.5µL	0.5µL	0.5µL	0.2 µL	5µL
experiment	RBS-GFP-DT(pSB1C3)	1µL	Pcon-pT181attenuator(E-1A)	1 µL	Pcon-tetR-aptamer12-1R-DT(1-2A)	1µL	Ptet(2-S)	0.3µL	--	--	0.5µL	0.5µL	0.7 µL	5µL
experiment	RBS-GFP-DT(pSB1C3)	1µL	Pcon-tetRaptamer12-1R-DT(E-1A)	2µL	Pcon-tetRaptamer12-1R-DT(1-2A)	1µL	Ptet(2-S)	0.3µL	--	--	0.5µL	0.5µL	0 µL	5.3µL
experiment	RBS-GFP-DT(pSB1C3)	1µL	Pcon-spinach-DT(E-1A)	3µL	Pcon-tetRaptamer12-1R-DT(1-2A)	1µL	Ptet(2-S)	0.3µL	--	--	0.5µL	0.5µL	0 µL	6.3µL
experiment	RBS-GFP-DT(pSB1C3)	1µL	Pcon-pT181antisense-DT(E-1A)	0.5µL	DT(1-3)	2µL	Ptet(2-S)	0.3µL	Pcon-tetRaptamer12-1R-DT(3-2A)	1.1µL	0.5µL	0.5µL	0.1 µL	5µL
experiment	RBS-GFP-DT(pSB1C3)	1µL	Pcon-pT181attenuator(E-1A)	1µL	tetRaptamer12-1R-DT(1-3)	1µL	Ptet(2-S)	0.3µL	Pcon-tetRaptamer12-1R-DT(3-2A)	1.1µL	0.5µL	0.5µL	0µL	5.4µL
experiment	RBS-GFP-DT(pSB1C3)	1µL	Ptet(2-S)	0.3µL	Pcon-tetRaptamer12-1R-DT(E-2A)	1µL	--	--	--	--	0.5µL	0.5µL	1.7µL	5µL
experiment	RBS-GFP-DT(pSB1C3)	1µL	Pcon-GFP-DT(2-SA)	0.5µL	1µL	--	--	--	--	--	0.5µL	0.5µL	1.5µL	5µL
experiment	RBS-GFP-DT(pSB1C3)	1µL	Pcon-tetRaptamer12-1R-DT(E-1A)	2µL	Pcon-pT181attenuator(1-SA)	2.1µL	--	--	--	--	0.5µL	0.5µL	0µL	6.1µL
experiment	RBS-GFP-DT(pSB1C3)	1µL	Pcon-pT181attenuator-DT(E-1A)	1µL	Pcon-pT181attenuator(1-SA)	2.1µL	--	--	--	--	0.5µL	0.5µL	0µL	5.1µL
experiment	RBS-GFP-DT(pSB1C3)	1µL	Pcon-tetR12-1R-DT(E-1A)	2µL	Pcon-GFP-DT(2-SA)	0.5µL	spinach-DT(3-2)	1µL	Ptet-pT181antisense(1-3)	1µL	0.5µL	0.5µL	0µL	6.5µL
experiment	RBS-GFP-DT(pSB1C3)	1µL	Pcon-tetRaptamer12-1R-DT(E-1A)	2µL	Pcon-pT181antisense(1-2A)	0.7µL	Pcon-pT181attenuator(2-SA)	2.1µL	--	--	0.5µL	0.5µL	0µL	6.8µL
experiment	RBS-GFP-DT(pSB1C3)	1µL	Pcon-tetRaptamer12-1R-DT(E-1A)	2µL	Pcon-pT181attenuator(2-SA)	2.1µL	Spinach-DT(3-2)	1µL	Ptet-pT181antisense(1-3)	1µL	0.5µL	0.5µL	0µL	8.1µL

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

PCR

No name

genome DNA	KOD plus	10x buffer	dNTP	MgSO4	F primer	R primer	KOD-plus	MilliQ	total
Pcon antisense(1C3-GGE-fwd,1A2-GG2-rev)	1µL	2.5µL	2.5µL	1.5µL	0.75µL	0.75µL	0.5µL	15.5	25µL
DT-(1C3-GG1-fwd,1C3-GG2-rev)	1µL	2.5µL	2.5µL	1.5µL	0.75µL	0.75µL	0.5µL	15.5	25µL
Spinach-DT-(1C3-GG1-fwd,1C3-GG2-rev)	1µL	2.5µL	2.5µL	1.5µL	0.75µL	0.75µL	0.5µL	15.5	25µL
Pcon-antisense-spinach-DT(1C3-GGE-fwd,1A2-GG1-rev)	1µL	2.5µL	2.5µL	1.5µL	0.75µL	0.75µL	0.5µL	15.5	25µL
Ptet(1C3-GGE-fwd,1C3-GG0-rev)	1µL	2.5µL	2.5µL	1.5µL	0.75µL	0.75µL	0.5µL	15.5	25µL
Pcon attenuator(1C3-GGE-fwd,1C3-GG0-rev)	1µL	2.5µL	2.5µL	1.5µL	0.75µL	0.75µL	0.5µL	15.5	25µL
DT(1C3-GG2-fwd,1C3-GG1-rev)	1µL	2.5µL	2.5µL	1.5µL	0.75µL	0.75µL	0.5µL	15.5	25µL

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	57°C	68°C	--
2min	10sec	30sec	36sec	30

Sep 25

Electrophoresis

nakamoto

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	Pcon-antisense (1C3-66E-fwd A2-662 rev)	--	--
3	DT (1C3-661-fwd 1C3 662 rev)	--	--
4	spinach-DT(1C3-661-fwd 1C3 662 rev)	--	--
5	Pcon-antisense-spinach-DT(1C3-66E fwd A2-661 rev)	--	--
6	100bp ladder	--	--

Lane	Sample	Enzyme1	Enzyme2
2	100bp ladder	--	--
3	Ptet (1C3 66E fwd-1C3 660 rev)	--	--
4	Pcon-attenuator(1C3-660 fwd A2-661 rev)	--	--
5	DT(1C3 662 fwd-1C3 661 rev)	--	--
6	Pcon-attenuator-aptamer-DT(1C3 66E fwd-A2-661-rev)	--	--
7	100bp ladder	--	--

Colony PCR

Nakamoto

Sample	base 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

PreDenature	Denature	Annealing	Extension	cycle
94 °C	94 °C	55 °C	68 °C	--
2 min	30 s	30 s	1min5s	30 cycles

Electrophoresis

no name

Lane	Sample	Enzyme1	Enzyme2
1	Pcon-PT181 attenuator-aptamer12-1R-DT-5	--	--
2	Pcon-PT181 attenuator-aptamer12-1R-DT-6	--	--
3	Pcon-PT181 attenuator-aptamer12-1R-DT-7	--	--
4	Pcon-PT181 attenuator-aptamer12-1R-DT-8	--	--
5	Pcon-PT181 attenuator-aptamer12-1R-DT-9	--	--
6	Pcon-PT181 attenuator-aptamer12-1R-DT-10	--	--
7	Pcon-PT181 attenuator-aptamer12-1R-DT-11	--	--
8	Pcon-PT181 attenuator-aptamer12-1R-DT-12	--	--
9	1kbp ladder	--	--
10	Pcon-PT181 attenuator-RBS-GFP-DT-5	--	--
11	Pcon-PT181 attenuator-RBS-GFP-DT-6	--	--
12	Pcon-PT181 attenuator-RBS-GFP-DT-7	--	--
13	Pcon-PT181 attenuator-RBS-GFP-DT-8	--	--
14	Pcon-PT181 attenuator-pSB1C3-5	--	--
15	Pcon-PT181 attenuator-pSB1C3-6	--	--
16	Pcon-PT181 attenuator-pSB1C3-7	--	--
17	Pcon-PT181 attenuator-pSB1C3-8	--	--

Transformation

kojima and nakamoto

Name	Sample	Competent Cells	Total	Plate
Plac+tet aptamer12+Pcon tetR-DT+Ptet+RBS-GFP-DT	1µL	10µL	11µL	--
Pcon-pT181attenuator-DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT	1µL	10µL	11µL	--
Pcon-tet aptamer12-DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT	1µL	10µL	11µL	--
Pcon-Spinach-DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT	1µL	10µL	11µL	--
Pcon-pT181antisense+DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT	1µL	10µL	11µL	--
Pcon-pT181attenuator+tet aptamer12-DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT	1µL	10µL	11µL	--
Pcon tetR-DT+Ptet+RBS-GFP-DT	1µL	10µL	11µL	--
Pcon tetR-DT+Pcon-GFP-DT+RBS-GFP-DT	1µL	10µL	11µL	--
Pcon-tet aptamer12-DT+Pcon-pT181attenuator+RBS-GFP-DT	1µL	10µL	11µL	--
Pcon-pT181attenuator-DT+Pcon-pT181attenuator+RBS-GFP-DT	1µL	10µL	11µL	--
Pcon-tet aptamer12-DT+Ptet-pT181antisense+Spinach-DT+Pcon-RBS-GFP-DT+RBS-GFP-DT	1µL	10µL	11µL	--
Pcon-tet aptamer12-DT+Pcon-pT181antisense+Spinach-DT+Pcon-pT181attenuator+RBS-GFP-DT	1µL	10µL	11µL	--
Pcon-tet aptamer12-DT+Ptet-pT181antisense+Spinach-DT+Pcon-pT181attenuator+RBS-GFP-DT	1µL	10µL	11µL	--

Name	Sample	Competent Cells	Total	Plate
aptamer12-DT(EcoRI+XbaI)+Pcon-pT181attenuator(EcoRI+SpeI)	2µL	20µL	22µL	--
Ptet(SpeI+PstI)+RBS-GFP-DT(XbaI+PstI))	2µL	20µL	22µL	--

Ligation

nakamoto

state	Vector		Inserter		Ligation High ver.2
experiment	9/25 aptamer12_1R-DT(EcoRI&XbaI)	4.5 µL	Pcon-pT181 attenuator(EcoRI&SpeI)	7.7 µL	6.1 µL
experiment	9/17 pSB1C3(EcoRI&SpeI)	2.0 µL	9/3 pT181 attenuator(EcoRI&SpeI)	14 µL	8 µL
experiment	9/25 Pcon-pT181 attenuator(EcoRI&XbaI)	4.5 µL	9/25 RBS-GFP-DT(EcoRI&SpeI)	19.3 µL	11.9 µL
experiment	9/25 Pcon-RBS-GFP-DT(EcoRI&XbaI)	4.2 µL	Pcon-RBS-tetR-DT(EcoRI&SpeI)	20 µL	12.1 µL

Restriction Enzyme Digestion

nakamoto>

	9/24 Pcon-attenuator-aptamer-DT(µL)	EcoRI(µL)	SpeI(µL)	Buffer(µL)	MilliQ(µL)	total(µL)
2 cuts	8.6	1	1	3	16.4	30
NC	0.4	0	0	1	8.6	10

	9/11 DT(µL)	EcoRI(µL)	XbaI(µL)	Buffer(µL)	BSA(µL)	MilliQ(µL)	total(µL)
2 cuts	10.4	1	1	3	3	11.6	30
NC	0.5	0	0	1	1	7.5	10

Liquid Culture

Kojima

Sample	medium
DT	--
pT181attenuator	--
Pcon-RBS-tetR-DT	--

Gel Extraction

Kojima

Lane	DNA	Enzyme
1	100bp ladder	--
2	Pcon-pT181antisense	--
3
4
5	--	--
6	DT	--
7
8
9	--	--
10	Spinach-DT	--
11
12

Lane	DNA	Enzyme
1	100bp ladder	--
3	Pcon-antisense-spinach-DT(E-1A)	--
4	Pcon-antisense-spinach-DT(E-1A)	--
5	Pcon-antisense-spinach-DT(E-1A)	--

Lane	DNA	Enzyme
7	100bp ladder	--
9	Ptet(E-0)	--
10	Ptet(E-0)	--
11	Ptet(E-0)	--

Lane	DNA	Enzyme
13	100bp ladder	--
15	Pcon-attenuator(0-1A)	--
16	Pcon-attenuator(0-1A)	--
17	Pcon-attenuator(0-1A)	--

||--

Lane	DNA	Enzyme
19	100bp ladder	--
21	Pcon-attenuator-aptamer-DT(E-1A)	--
22	Pcon-attenuator-aptamer-DT(E-1A)	--
23	Pcon-attenuator-aptamer-DT(E-1A)	--

Name	concentration[µg/mL]	260/280	260/230
DT(2-1)	24.9	1.73	0.37
Ptet(E-0)	12.8	1.59	0.90
Pcon-antisense(E-2A)	16.5	1.72	1.07
Pcon-attenuator-aptamer-DT(E-1A)	22.1	1.75	0.94
Pcon-antisense-spinach-DT(E-1A)	31.0	1.78	1.15
spinach-DT(1-2)	23.4	1.85	1.11
Pcon-attenuator(0-1A)	22.5	1.76	0.46
DT(1-2)	25.1	1.78	1.21

Electrophoresis

Lane	Sample	Enzyme1	Enzyme2
1	1kbp ladder	--	--
2	Pcon-pT181 attenuator-aptamer 12_1R-DT	EcoRI	SpeI
3	Pcon-pT181 attenuator-aptamer 12_1R-DT	--	--
4	DT	EcoRI	XbaI
5	DT	--	--

Gel Extraction

Nakamoto

Lane	DNA	Enzyme
1	DT	EcoRI+XbaI
2	DT	EcoRI+XbaI
3	Pcon-pT181 attenuator-aptamer12_1R-DT	EcoRI+SpeI
4	Pcon-pT181 attenuator-aptamer12_1R-DT	EcoRI+SpeI
5	Pcon-pT181 attenuator-aptamer12_1R-DT	EcoRI+SpeI

Name	concentration[µg/mL]	260/280	260/230
Pcon-pT181 attenuator-aptamer12_1R-DT(EcoRI&SpeI)	11.8	1.80	0.46
DT(EcoRI+XbaI)	42.6	1.84	0.98

Liquid Culture

No name

Sample	medium
Pcon-pT181anntisense-spinach-DT(4k5)	-

Ligation

Nakamoto and Tatsui

state	Vector		Inserter		Ligation High ver.2
experiment	9/24 pSB1C3(EcoRI+Spel)	10µL	9/25 PconpT181attenuator-aptamer12-1R-DT(EcoRI+Spel)	5.9µL	8µL

PCR

Hirano

800pg/μL DT	KOD plus	10x buffer	dNTP	MgSO4	1C3_GG2_fwd	iC3_GG1_rev	MilliQ	total
1	0.5	2.5	2.5	1.5	0.75	0.75	15.5	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	57°C	68°C	--
2min	10s	30s	24s	30cycles

Transformation

Tatsui

Name	Plate
aptamer12-1R-DT	CP
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

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	DT(2-1)	--	--

Column Refining

Name	concentration[µg/mL]	260/280	260/230
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 Buffer	BsaI-HF	MilliQ	total
3.0 µL	2.0 µL	0.3 µL	14.7 µL	20.0 µL

Incubate 37°C 12hour

Liquid Culture

Hirano

Sample	medium
Pcon-GFP-DT	LB(Amp)
RBS-GFP-DT	LB(CP)
Pcon-spinach-DT	LB(Amp)
Pcon-tetRaptamer-DT	LB(Amp)
Pcon-attenuator-DT	LB(Amp)
spinach-DT	LB(CP)
Pcon-antisense-spinach-DT	LB(Amp)

Plating

Hirano

Mix Liquid Culture 1mL and LB(+Amp), incubate 37°C 30min

OD600-1.445

Sample	Use plate	The number of plate
Pcon-GFP-DT	M9(+Amp)	3
Pcon-GFP-DT	LB(+Amp)	3

</div> incubate 37°C

Colony PCR

No name

Sample	base 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	--

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	3min15s	30cycles

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

DNA	concentration[µg/mL]	260/280	260/230
Pcon-RBS-GFP-DT	2783	1.95	1.48

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

cDNA　Synthesis

Nakamoto

volume	7 µL
7*cDNA Wipcout Buffer	1 µL
Template RNA(9/25 Pcon-RBS-GFP-DT)	3 µL
RNase-frec water	4 µL

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

5*Quantiscript Reverse Transcriptase	0.5 µL
5*Quantiscript RT Buffer	2 µL
RT Primer Mix	0.5µL

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

Electrophoresis

Nakamoto

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	Pcon-RBS-GFP-DT(RNA)	--	--

Ligation

Tatsui

state	Vector		Inserter		Ligation High ver.2
experiment	9/13 Pcon-pT181-attenuator(SpeI&PstI)	3.5µL	9/25 RBS-GFP-DT(XbaI&PstI)	13.5µL	8.5 µL

Sep 26

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	GGA1-1	--	--
2	GGA1-2	--	--
3	GGA1-3	--	--
4	GGA1-4	--	--
5	GGA1-5	--	--
6	GGA1-6	--	--
7	GGA1-7	--	--
8	GGA1-8	--	--
9	1kbp ladder	--	--
10	GGA2-1	--	--
11	GGA2-2	--	--
12	GGA2-3	--	--
13	GGA2-4	--	--
14	GGA2-5	--	--
15	GGA2-6	--	--
16	GGA2-7	--	--
17	GGA2-8	--	--

Lane	Sample	Enzyme1	Enzyme2
1	GGA3-1	--	--
2	GGA3-2	--	--
3	GGA3-3	--	--
4	1kbp ladder	--	--
5	GGA3-4	--	--
6	GGA3-5	--	--
7	GGA3-6	--	--
8	GGA3-7	--	--

Lane	Sample	Enzyme1	Enzyme2
1	GGA3-8	--	--
2	GGA4-1	--	--
3	GGA4-2	--	--
4	GGA4-3	--	--
5	1kbp ladder	--	--
6	GGA4-4	--	--
7	GGA4-5	--	--
8	GGA4-6	--	--

Lane	Sample	Enzyme1	Enzyme2
1	GGA4-7	--	--
2	GGA4-8	--	--
3	1kbp ladder	--	--
4	GGA7-1	--	--
5	GGA16-1	--	--
6	NC(RBS-GFP-DT)	--	--

Ligasion(Golden Gate Assenbly)

No name

state	Vector		Inserter1		Inserter2		Inserter3		Inserter4		Inserter5		(NEB)T4 ligase	(NEB)10*T4 ligase	MilliQ
experiment	RBS-GFP-DT	1.0 µL	Pcon-pT181 antisense(E-2A)	0.6µL	DT(2-1)	0.5µL	Pcon-pT181 attenuator(1-SA)	2.1 µL	--	--	--	--	0.5 µL	0.5 µL	up to 5 µL
experiment	pSB1C3(EcoRI&SpeI)	1.8 µL	Pcon-pT181 antisense(E-2A)	0.9 µL	DT(2-1)	0.5 µL	Pcon-GFP-DT(1-SA)	1.5 µL	--	--	--	--	0.5 µL	0.5 µL	up to 5 µL
experiment	RBS-GFP-DT	1.0 µL	Ptet(E-0)	0.7µL	Pcon pT181 attenuator(0-1A)	0.3µL	tet aptamer(1-3)	0.3 µL	Pcon-tetR-DT(3-2A)	1.1 µL	Ptet(2-5)	0.3 µL	0.5 µL	0.5 µL	up to 5 µL
experiment	RBS-GFP-DT	1.0 µL	Ptet(E-0)	0.7 µL	Pcon-pT181 attenuator-DT(0-1A)	1.0 µL	Pcon-tetR-DT(1-2A)	1.0 µL	Ptet(2-5)	0.3µL	--	--	0.5 µL	0.5 µL	up to 5 µL
experiment	RBS-GFP-DT	1.0 µL	Pcon-pT181 attenuator-tetR-DT(E-1A)	1.0 µL	Pcon antisense-spinach-DT(1-2A)	0.7µL	Pcon-pT 181 attenuator(2-SA)	2.1 µL	--	--	--	--	0.5 µL	0.5 µL	up to 5 µL
experiment	RBS-GFP-DT	1.0 µL	Pcon antisense(E-1A)	0.5 µL	spinach-DT(1-2)	1.0 µL	Pcon-GFP-DT(1-2)	0.8 µL	--	--	--	--	0.5 µL	0.5 µL	up to 5 µL
experiment	pSB1C3(EcoRI&SpeI)	1.8 µL	Pcon-pT181 antisense(E-1A)	0.8µL	spinach-DT(1-2)	1.0µL	Pcon-GFP-DT(2-SA)	0.8 µL	--	--	--	--	0.5 µL	0.5 µL	up to 5 µL
experiment	pSB1C3(EcoRI&SpeI)	1.8 µL	Pcon-tet aptamer-DT(E-1A)	3.0 µL	Ptet-pT181 antisense(1-3)	1.5 µL	spinach-DT(3-2)	1.5 µL	Pcon-GFP-DT(2-5A)	0.8 µL	--	--	0.5 µL	0.5 µL	up to 5 µL

Liquid Culture

No name

Sample	medium
Pcon-GFP-DT	Amp
RBS-GFP-DT	CP
Pcon-pT181 attenuator-DT	Amp
Pcon-tet aptamer-DT	Amp
Pcon-spinach-DT	Amp
Pcon-pT181 antisense-spinach-DT	Amp
spinach-DT	CP

BSAI Digestion

No name

	DNA	MilliQ	BSAI-HF	10*cut smart	total
Pcon	7 µL	10.7 µL	0.3 µL	2 µL	20 µL
RBS-luxR-DT	10 µL	7 µL	0.3 µL	2 µL	20 µL

Colony PCR

Tatsui

Sample	base 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

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	3min30s	30cycles

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	GGA-1 (9)	--	--
2	GGA-1 (10)	--	--
3	GGA-1 (11)	--	--
4	GGA-1 (12)	--	--
5	GGA-1 (13)	--	--
6	GGA-1 (14)	--	--
7	GGA-1 (15)	--	--
8	GGA-1 (16)	--	--
9	1kbp ladder	--	--
10	GGA-2 (9)	--	--
11	GGA-2 (10)	--	--
12	GGA-2 (11)	--	--
13	GGA-2 (12)	--	--
14	GGA-2 (13)	--	--
15	GGA-2 (14)	--	--
16	GGA-2 (15)	--	--
17	GGA-2 (16)	--	--

Lane	Sample	Enzyme1	Enzyme2
1	GGA-4 (9)	--	--
2	GGA-4 (10)	--	--
3	GGA-4 (11)	--	--
4	GGA-4 (12)	--	--
5	GGA-4 (13)	--	--
6	GGA-4 (14)	--	--
7	GGA-4 (15)	--	--
8	GGA-4 (16)	--	--
9	1kbp ladder	--	--
10	GGA-6 (1)	--	--
11	GGA-16 (2)	--	--
12	GGA-16 (3)	--	--
13	GGA-16 (4)	--	--

Lane	Sample	Enzyme1	Enzyme2
1	GGA-3 (9)	--	--
2	GGA-3 (10)	--	--
3	GGA-3 (11)	--	--
4	GGA-3 (12)	--	--
5	1kbp ladder	--	--
6	GGA-3 (13)	--	--
7	GGA-3 (15)	--	--
8	GGA-3 (14)	--	--

Lane	Sample	Enzyme1	Enzyme2
1	GGA-3 (17)	--	--
2	GGA-3 (18)	--	--
3	GGA-3 (19)	--	--
4	1kbp ladder	--	--
5	GGA-3 (20)	--	--
6	GGA-3 (21)	--	--
7	GGA-3 (16)	--	--

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	RBS-GFP-DT(RNA)	--	--
2	1kbp ladder	--	--
3	Pcon-RBS-GFP-DT(RNA)	--	--

Liquid Culture

Hirano

Sample	medium
Pcon-GFP-DT	--
RBS-GFP-DT	--
Pcon-tet aptamer-DT	--
Pcon-spinach-DT	--
Pcon-pT181 antisense-spinach-DT	--
spinach-DT	--

Colony PCR

Hirano

Sample	base 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	--

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	1min40s	30cycles

Ligasion(Golden Gate Assenbly)

Hirano

state	Vector	Inserter1	Inserter2	Inserter3	Inserter4	Inserter5
experiment	GGA2	Pcon-attenuator(E-1A)	Pcon-tetR-DT(1-2A)	Ptet(2-S)	RBS-GFP-DT(EcoRI&XbaI)
experiment	GGA3	Pcon-tetap-DT(E-1A)	Pcon-tetR-DT(1-2A)	Ptet(2-S)	RBS-GFP-DT(EcoRI&XbaI)
experiment	GGA4	Pcon-spi-DT(E-1A)	Pcon-tetR-DT(1-2A)	Ptet(2-S)	RBS-GFP-DT(EcoRI&XbaI)
experiment	GGA5	Pcon-antisense(E-1A)	DT(1-3)	Pcon-tetR-DT(3-2A)	Ptet(2-S)	RBS-GFP-DT(EcoRI&XbaI)
experiment	GGA6	Pcon-attenuator(E-1A)	tetap-DT(1-3)	Pcon-tetR-DT(3-2A)	Ptet(2-S)	RBS-GFP-DT(EcoRI&XbaI)
experiment	GGA7	Pcon-tetR-DT(E-2A)	Ptet(2-S)	RBS-GFP-DT(EcoRI&XbaI)

PreDenature	Denature	Annealing	Extension	cycle
37°C	16°C	50°C	80°C	--
3min	4min	5min	5min	25cycles

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

Sample	base 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

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	65°C	--
5min	30sec	30sec	54sec	30

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	1kbp ladder	--	--
2	9/25 9/23 1C3&9/3attenuator	--	--
3	9/25 9/23 1C3&9/3attenuator	--	--
4	9/25 9/23 1C3&9/3attenuator	--	--
5	9/25aptamer-DT&9/24Pcon-attenuator	--	--
6	9/25aptamer-DT&9/24Pcon-attenuator	--	--
7	9/25aptamer-DT&9/24Pcon-attenuator	--	--
8	9/24 Pcon attenuator-aptamer-DT(1)	--	--
9	9/25aptamer-DT&9/24Pcon-attenuator	--	--

Plating

Hirano

Sample	Use plate
9/26 GGA2	LB(CP)
9/26 GGA3	LB(CP)
9/26 GGA4	LB(CP)
9/26 GGA5	LB(CP)
9/26 GGA6	LB(CP)
9/26 GGA7	LB(CP)
9/26 GGA11	LB(CP)
9/26 GGA312	LB(CP)
9/26 GGA13	LB(CP)
9/26 GGA14	LB(CP)
9/26 GGA15	LB(CP)
9/26 GGA20	LB(CP)
9/26 GGA23	LB(CP)
9/26 GGA24	LB(CP)
9/26 Ptet+RBS-GFP-DT	LB(CP)

incubate 37°C

Restriction Enzyme Digestion

Kojima

	9/24 pSB1C3	EcoRI	SpeI	buffer	MilliQ	total
2cuts	11µL	1µL	1µL	3µL	14µL	30µL
NC	0.5µL	0µL	0µL	1µL	8.5µL	10µL

	9/24 Pcon-pT181attenuator-aptamer12-1R-DT	EcoRI	SpeI	buffer	MilliQ	total
2cuts	8.7µL	1µL	1µL	3µL	16.3µL	30µL
NC	0.4µL	0µL	0µL	1µL	8.6µL	10µL

	9/24 pT181attenuator	EcoRI	SpeI	buffer	MilliQ	total
2cuts	3.1µL	1µL	1µL	3µL	21.9µL	30µL
NC	0.2µL	0µL	0µL	1µL	8.8µL	10µL

	8/17 RBS-GFP-DT	EcoRI	BSA	buffer	MilliQ	total
1cut	16.4µL	1µL	3µL	3µL	6.6µL	30µL
NC	0.4µL	0µL	1µL	1µL	7.6µL	10µL

	9/17 pSB4K5	EcoRI	PstI	BSA	buffer	MilliQ	total
2cuts	8.5µL	1µL	1µL	3µL	3µL	13.5µL	30µL
NC	0.4µL	0µL	0µL	1µL	1µL	7.6µL	10µL

	9/14 pSB4K5	EcoRI	PstI	BSA	buffer	MilliQ	total
2cuts	7.3µL	1µL	1µL	3µL	3µL	14.7µL	30µL
NC	0.4µL	0µL	0µL	1µL	1µL	7.6µL	10µL

	9/22 Pcon-pT181attenuator-DT	EcoRI	SpeI	buffer	MilliQ	total
2cuts	5.4µL	1µL	1µL	3µL	19.6µL	30µL
NC	0.3µL	0µL	0µL	1µL	8.7µL	10µL

	8/7 RBS-GFP-DT 1	XbaI	PstI	BSA	buffer	MilliQ	total
2cuts	8.1µL	1µL	1µL	3µL	3µL	13.9µL	30µL
NC	0.4µL	0µL	0µL	1µL	1µL	7.6µL	10µL

	9/26 RBS-GFP-DT 1	XbaI	BSA	buffer	MilliQ	total
1cut	8.1µL	1µL	3µL	3µL	14.9µL	30µL

	9/16 Pλ-luxI	EcoRI	PstI	BSA	buffer	MilliQ	total
2cuts	14.7µL	1µL	1µL	3µL	3µL	7.3µL	30µL
NC	0.7µL	0µL	0µL	1µL	1µL	7.3µL	10µL

	9/21 Ptet	EcoRI	SpeI	buffer	MilliQ	total
2cuts	14.2µL	1µL	1µL	3µL	10.8µL	30µL
NC	0.7µL	0µL	0µL	1µL	8.3µL	10µL

	9/24 Pcon-pT181attenuator-aptamer12-1R-DT	XbaI	PstI	BSA	buffer	MilliQ	total
2cuts	8.7µL	1µL	1µL	3µL	3µL	13.3µL	30µL
NC	0.4µL	0µL	0µL	1µL	1µL	7.6µL	10µL

Electrophoresis

Kojima

Lane	Sample	Enzyme1	Enzyme2
1	pSB1C3	EcoRI	SpeI
2	pSB1C3	--	--
3	Pcon-pT181attenuator-aptamer12-1R-DT	EcoRI	SpeI
4	Pcon-pT181attenuator-aptamer12-1R-DT	--	--
5	1kb ladder	--	--
6	pT181attenuator	EcoRI	SpeI
7	pT181attenuator	--	--
8	RBS-GFP-DT	EcoRI	--
9	RBS-GFP-DT	--	--

Sep 27

Gel Extraction

Kojima

Lane	DNA	Enzyme
1	1kbp ladder	--
2	pSB1C3	EcoRI+SpeI
3
4
6	Pcon-attenuator-aptmer-DT	EcoRI+SpeI
7
8
10	Pcon-attenuator-aptmer-DT	EcoRI+SpeI
11
12

Name	concentration[µg/mL]	260/280	260/230
Pcon-attenuator-DT(EcoRI+SpeI)	6.8	1.98	0.43
pT181-attenuator(EcoRI+SpeI)	7.5	1.84	0.06
pSB1C3(EcoRI+SpeI)	17.7	1.97	0.71

Gel Extraction

No name

Lane	DNA	Enzyme
1	1kbp ladder	--
3	pSB4K5	EcoRI+PstI
4	pSB4K5	EcoRI+PstI
5	pSB4K5	EcoRI+PstI
7	pSB6A1	EcoRI+PstI
8	pSB6A1	EcoRI+PstI
9	pSB6A1	EcoRI+PstI

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	pSB4K5	EcoRI	PstI
2	pSB4K5	-	-
3	1kbp ladder	-	-
4	Pcon-attenuator	EcoRI	SpeI
5	Pcon-attenuator	-	-

Lane	Sample	Enzyme1	Enzyme2
1	RBS-GFP-DT	XbaI	PstI
2	RBS-GFP-DT	-	-
3	RBS-GFP-DT	EcoRI	XbaI
4	1kbp ladder	-	-
5	Ptet	EcoRI	SpeI
6	Ptet	-	-

Liquid Culture

No name

Sample	medium
Pcon-GFP	Amp
RBS-GFP-DT	CP
Pcon-attenuator	Amp
Pcon-attenuator-aptamer-DT	Amp
Pcon-tetR-DT	Amp

Ligation

No name

state	Vector		Inserter		Ligation High ver.2
experiment	9/17 pSB1C3 (EcoRI+SpeI) 10.0ng/µL	10µL	9/26 pT181 attenuator(EcoRI+SpeI) 7.5ng/µL	1.3µL	3.5 µL
experiment	9/25 DT (EcoRI+XbaI) 42.8ng/µL	2.3µL	9/22 Pcon-pT181 antisense(EcoRI+SpeI) 46.7ng/µL	1.5µL	1.9 µL
experiment	9/27 pSB4K5(EcoRI+PstI) 12.9ng/µL	7.8µL	9/10 Pcon-attenuator(EcoRI+SpeI) 13.8ng/µL	8.0µL	3.5 µL
experiment	9/27 pSB4K5(EcoRI+PstI) 12.9ng/µL	7.8µL	9/25 RBS-GFP-DT(XbaI+PstI) 13.9ng/µL	12µL	3.5 µL
experiment	9/8 pSB4K5 (EcoRI+SpeI) 17.6ng/µL	5.7 µL	8/21 Pcon-GFP-DT(EcoRI+SpeI) 11ng/µL	12.6µL	3.5 µL

Liquid Culture

Hirano

Sample	medium
Pcon-spinach-DT	plusgrow Amp
Pcon-tetRaptamer-DT	plusgrow Amp
Pcon-antisense-spinach-DT	plusgrow Amp
spinach-DT	plusgrow CP

RNA Extraction

Nakamoto

Name	concentration[µg/mL]	260/280	260/230
9/26 Pcon-GFP-DT	423.0	1.83	1.46
9/26 Pcon-GFP-DT K	298.7	1.82	1.50

Electrophoresis

No name

Lane	Sample
1	9/27 Pcon-GFP-DT(RNA)
2	1kb ladder
3	9/27 Pcon-GFP-DT K(RNA)

Miniprep

Tatsui

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

Transformation

Hirano

Name1	Name2	Sample1(µL)	Sample2(µL)	Competent Cells(µL)	Total(µL)
Pcon-attenuator+RBS-GFP-DT(4K5)	Pcon-attenuator-DT(1A2)	2	1	30	33
Pcon-attenuator+RBS-GFP-DT(4K5)	Pcon-antisense-spinach-DT(1A2)	2	1	30	33
Pcon-attenuator+RBS-GFP-DT(4K5)	Pcon-tetR aptamer-DT(1A2)	2	1	30	33
Pcon-RBS-GFP-DT(4K5)	Pcon-attenuator-DT(1A2)	2	1	30	33
Pcon-RBS-GFP-DT(4K5)	Pcon-antisense-spinach-DT(1A2)	2	1	30	33
Pcon-RBS-GFP-DT(4K5)	Pcon-tetR aptamer-DT(1A2)	2	1	30	33
Pcon-attenuator+RBS-GFP-DT(4K5)	--	2	--	20	22
Pcon-RBS-GFP-DT(4K5)	--	2	--	20	22
attenuator(1C3)	--	2	--	20	22

Miniprep

No name

DNA	concentration[µg/mL]	260/280	260/230
Pcon-RBS－GFP-DT	137.6	1.95	1.78
RBS-GFP-DT	77.0	2.06	1.96
Pcon-PT181 attenuator	231.0	1.89	1.89
Pcon-antisense-Spinach-DT(2)	130.0	1.56	1.62
Pcon-aptamer-DT	218.8	1.92	1.72
Pcon-attenuator-DT	180.7	1.95	1.78
Pcon-Spinach-DT	177.6	1.96	1.06
Pcon-antisense-Spinach-DT(1)	181.1	1.96	1.92
Pcon-antisense	152.9	2.02	1.96
Pcon-RBS+ezR-DT	164.1	1.93	1.91

Restriction Enzyme Digestion

No name

	9/27 RBS-GFP-DT	XbaI	PstI	Buffer	BSA	MilliQ	total
2 cuts	12µL	1µL	1µL	3µL	3µL	20µL	40µL
NC	1.3µL	0µL	0µL	1µL	1µL	6.7µL	10µL

	9/27 Pcon-tetR-DT	EcoRI	PstI	Buffer	MilliQ	total
2 cuts	12.2µL	1µL	1µL	3µL	12.8µL	30µL
NC	0.6µL	0µL	0µL	1µL	8.4µL	10µL

RNA Extraction

Nakamoto

Name	concentration[µg/mL]	260/280	260/230
9/27 Pcon-RBS-GFP-DT	686.6	1.99	1.34
9/27 Pcon-RBS-GFP-DT (+High salt)	127.6	1.76	0.27

DNA sythesis

No name

genome DNA	DNA volume	genome DNA wipeout buffer	RNase freewater	RT	RT buffer	Primer Mix	total
9/27 Pcon-RBS-GFP-DT	0.8µL	1µL	5.2µL	0.5µL	2µL	0.5µL	10µL
9/27 pcon-RBS-GFP-DT(+high salt)	2.5µL	1µL	3.5µL	0.5µL	2µL	0.5µL	10µL
9/27 Pcon-RBS-GFP-DT(revised concentration)	0.5µL	1µL	5.5µL	0.5µL	2µL	0.5µL	10µL

Electrophoresis

No name

Lane	Sample
1	1kb ladder
2	Pcon-RBS-GFP-DT
3	Pcon-RBS-GFP-DT

Liquid Culture

No name

Sample	medium
Pcon spinach DT	plusgrow 2µL(Amp)
Pcon aptamer DT	plusgrow 2µL(Amp)
Pcon antisense Spinach DT	plusgrow 2µL(Amp)
Spinach DT	plusgrow 4µL(CP)

RT PCR

No name

genome DNA	KOD plus	10x buffer for KOD-plus-ver.2	2nM dNTPs	25mM MgSO4	F primer	R primer	MilliQ	total
0.5	1	2.5	2.5	1.5	0.75	0.75	15.5	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	49.8°C	68°C	--
2min	10sec	30sec	10sec	30

Electrophoresis

No name

Lane	Sample
1	Pcon-GFP-DT(GFP)
2	Pcon-GFP-DT(internal)
3	Pcon-GFP-DT(HS)(GFP)
4	Pcon-GFP-DT(internal)

Lane	Sample
1	Pcon-GFP-DT(GFP)
2	Pcon-GFP-DT(internal)
3	100bp ladder

Digesting cut check

No name

Lane	Sample
1	RBS-GFP-DT(XbaI&PstI)
2	RBS-GFP-DT NC(XbaI&PstI)
3	1kbp ladder
4	Pcon-RBS-tetR-DT(EcoRI&SpeI)
5	Pcon-RBS-tetR-DT NC(EcoRI&SpeI)

Gel Extraction

No name

Lane	DNA	Enzyme
1	1kbp ladder	--
3	RBS-GFP-DT	Xbal+PstI
4	RBS-GFP-DT	Xbal+PstI
5	RBS-GFP-DT	Xbal+PstI
7	1kbp ladder	--
9	Pcon-tetR-DT	EcoRI+SpeI
10	Pcon-tetR-DT	EcoRI+SpeI
11	Pcon-tetR-DT	EcoRI+SpeI

RNA Extraction

Nakamoto

	0	5min	15min	20min	23min	28min	33min	36min
aptamer generator	0.348	0.408	0.550	0.865
Pcon-spinach-DT	0.250	0.240	0.305	0.426	0.471	0.554	0.668	0
GFP generator	0.389	0.420	0.516	0.884
antisense-spinach	0.371	0.438	0.537	0.733
Spinach-DT	0.084	0.110	0.134	0.433	0.269	0.326	0.409	0.742

Name	concentration[µg/mL]	260/280	260/230
Spinach-DT	83.9	1.63	0.17
Pcon-tetRaptamer-DT	425.1	2.10	0.64
Pcon-RBS-GFP-DT	262.4	2.01	0.84
Pcon-antisense-Spinach-DT	489.1	2.05	0.68
Pcon-Spinach-DT	266.6	2.01	0.66

cDNA Synthesis

Nakamoto

	9/27 Spinach-DT	9/23 Pcon-tetRaptamer-DT	9/23 Pcon-RBS-GFP-DT	9/23 Pcon-antisense-spinach-DT	9/23 Pcon-Spinach-DT
RNA	1	1	1	1	1
gDNA wipeout	6	1.2	1.9	1.0	1.9
RNase-free water	0	4.8	4.1	5.0	4.1
RT	0.5	0.5	0.5	0.5	0.5
RT Buffer	2	2	2	2	2
Primer mix	0.5	0.5	0.5	0.5	0.5
total	10µL	10µL	10µL	10µL	10µL

Template:Kyoto/Notebook/Sep 17

From 2013.igem.org

Contents

Sep 17

Sep 18

Restriction Enzyme Digestion

Electrophoresis

Electrophoresis

Gel Extraction

Sep 19

Sep 20

Ligation

Transformation

PCR

Electrophoresis

Sep 21

Electrophoresis

Liquid Culture

Master Plate

Restriction Enzyme Digestion

Electrophoresis

PCR

Gel Extraction

Sep 22

Colony PCR

Electrophoresis

Miniprep

Colony PCR

Gel Extraction

Restriction Enzyme Digestion

Gel Extraction

Colony PCR

Colony PCR

Ligation

Liquid Culture

digestion

Gel Extraction

Transformation

PCR

Electrophoresis

Sep 23

Transformation

Gel Extraction

PCR

Ligation

PCR

Miniprep

Electrophoresis

Gel Extraction

Electrophoresis

Transformation

Ligation

Colony PCR

Sep 24

Electrophoresis

Transformation

Liquid Culture

Restriction Enzyme Digestion(PCR product)

Column Refining

Transformation

Liquid Culture

Restriction Enzyme Digestion

Electrophoresis

Miniprep

Gel Extraction

Colony PCR

Restriction Enzyme Digestion

Ligation

colony PCR

Miniprep

Electrophoresis

Gel Extraction

Electrophoresis

Ligation

PCR

Sep 25

Electrophoresis

Colony PCR

Electrophoresis

Transformation

cDNA　Synthesis