Template:Kyoto/Notebook/Sep 12

From 2013.igem.org

Sep 12

Miniprep

Nakamoto

DNA	concentration[µg/mL]	260/280	260/230
Pcon-pT181attenuator-DT	293.6	1.88	1.84
Pcon-apt12-1R-DT	303.8	1.75	1.64
Fusion1attenuator(pSB1c3)	172.0	1.96	2.31
RBS-lisis1-DT	235.1	1.59	1.60
RBS-lisis2-DT	387.7	1.91	2.01
RBS-lisis3-DT	387.9	1.91	2.09
Plux	166.7	1.98	1.63

Electrophoresis

Nakamoto

Lane	Sample
1	100bp ladder
2	9/11 P-RBS-lisis1-DT(Colony PCR prodution)

Liquid Culture

Nakamoto

Sample	medium
9/10 Pcon-apt12-1R-DT1	Plusgrow medium(+Amp)
9/10 DT-Pcon-pT181attenuator1	Plusgrow medium(+CP)
9/10 Fusion1-attenuator(pSB1c3)	Plusgrow medium(+CP)

Electrophoresis

Tatsui

Lane	Sample
1	1kb ladder
2	pSB4K5(EcoRI&SpeI)
3	pSB4K5 NC(EcoRI&SpeI)
4	RBS-lacz-DT(EcoRI&SpeI)
5	EcoRI&SpeI NC( EcoRI&SpeI)
6	Pcon-RBS-lacz-DT( EcoRI&SpeI)
7	Pcon-RBS-lacz-DT NC( EcoRI&SpeI)
8	1kb ladder

2

Lane	Sample
1	1kb ladder
2	Ptet-pT181antisense(SpeI&PstI)
3	Ptet-pT181antisense NC(SpeI&PstI)
4	RBS-lysis2-DT(XbaI&PstI)
5	EcoRI&SpeI NC( EcoRI&SpeI)
6	--
7	--
8	1kb ladder

Gel Extraction

Tatsui

Lane	DNA	Enzyme
1	1kb ladder	--
2	RBS-lacZα-DT	EcoRI&SpeI
3	RBS-lacZα-DT	EcoRI&SpeI
4	RBS-lacZα-DT	EcoRI&SpeI
5	--	--
6	Pcon-RBS-lacZα-DT	EcoRI&SpeI
7	Pcon-RBS-lacZα-DT	EcoRI&SpeI
8	Pcon-RBS-lacZα-DT	EcoRI&SpeI
9	1kb ladder	--

Name	concentration[µg/mL]	260/280	260/230
RBS-lacZα-DT(EcoRI&SpeI)	9.8	1.50	0.39
Pcon-RBS-lacZα-DT(EcoRI&SpeI)	11.2	1.47	0.08

Gel Extraction

Tatsui

Lane	DNA	Enzyme
1	1kb ladder	--
2	RBS-lysis2-DT	XbaI&PstI
3	RBS-lysis2-DT	XbaI&PstI
4	RBS-lysis2-DT	XbaI&PstI
5	--	--
6	1kb ladder	--

Name	concentration[µg/mL]	260/280	260/230
RBS-lysis2-DT(XbaI&PstI)	5.4	1.43	0.77

Electrophoresis

No name

Lane	Sample
1	1kb ladder
2	Pλ-RBS-luxI-DT(PCR product)
3	Pbad/araC-RBS-RFP(PCR product)

Colony PCR

Honda

Sample	base pair
9/11 apt12-1M(pSB1C3) 1	513
9/11 apt12-1M(pSB1C3) 2	513
9/11 Fusion3m2a09ttenuator(pSB1C3) 1	609
9/11 Fusion6 antisense(pSB1C3)1	431
9/11 aptamer 12-P(pSB1C3)1	515
9/11 Fusion1 antisense(pSB1C3) 1	420
9/11 Fusion1 antisense(pSB1C3) 2	420
9/11 Plac(BBa-R0011) 1	293
9/11 Plac(BBa-R0011) 2	293
9/11 Plac(BBa-R0011) 3	293
9/11 Plac(BBa-R0011) 4	293
9/11 Pcon-luxR-Plux-GFP 1	2138
9/11 Pcon-RBS-lacZα-DT(pSB4K5) 1	712

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	2min12s	30cycles

Restriction Enzyme Digestion

Tatsui

	9/5 Pcon	EcoRI	SpeI	XbaI	PstI	Buffer	BSA	MilliQ	total
1cuts(PstI)	5.7	0µL	0µL	0µL	1µL	3µL	3µL	17.3µL	30µL
NC(PstI)	1.9	0µL	0µL	0µL	0µL	1µL	1µL	6.1µL	10µL

	8/17 DT	EcoRI	Buffer	BSA	MilliQ	total
1cut(EcoRI)	10.6	1µL	3µL	3µL	12.4µL	30µL
NC(EcoRI)	3.5	0µL	1µL	1µL	4.5µL	10µL

Colony PCR

Nakamoto and Tatsui

Sample	base pair
9/11 Pcon-pT181attenuator-RBS-lacZα-DT	965
9/11 Plux-PBS-lysis1-DT 1	613
9/11 Pcon-pT181attenuator-aptamer12-1R-DT 1	859
9/11 Pcon-pT181attenuator-aptamer12-1R-DT 2	859
9/11 Plux-RBS-lysis3-DT 1	1210
9/11 Plux-RBS-lysis3-DT 2	1210
Pcon-Spinach-DT(pSB4K5) 1	605

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	1min12s	30cycles

Transformation

Tatsui

Name	Sample	Competent Cells	Plate
9/7 pSB4K5	2µL	20µL	Amp

Electrophoresis

No name

1

Lane	Sample
1	Fusion6 antisense
2	Fusion1 antisense-1
3	Fusion1 antisense-2
4	100bp ladder
5	Plac(BBa-R0011) 1
6	Plac(BBa-R0011) 2
7	Plac(BBa-R0011) 3
8	Plac(BBa-R0011) 4

2

Lane	Sample
1	1kb ladder
2	aptamer12-1M 1
3	aptamer12-1M 2
4	Fusion3m2 attenuator
5	aptamer12-P1
6	Plux-RBS-GFP-DT-Pcon-RBS-luxR-DT
7	Pcon-RBS-lacZα=DT
8	1kb ladder

Electrophoresis

No name

Lane	Sample	Enzyme
1	Pcon-pT181attenuator-RBS-lacZα-DT 1	--
2	Pcon-pT181attenuator-RBS-lacZα-DT 2	--
3	Plux-RBS-lysis1-DT 1	--
4	Pcon-pT181attenuator-aptamer12-1R-DT 1	--
5	Pcon-pT181attenuator-aptamer12-1R-DT 2	--
6	Plux-RBS-lysis3-DT 1	--
7	1kbp ladder	--
8	Plux-RBS-lysis3-DT 2	--
9	Pcon-spinach-DT(pSB4K5) 1	--
10	9/5 Pcon	PstI
11	9/5 Pcon NC	--
12	8/17 DT	EcoRI
13	8/17 DT	--

Liquid Culture

Nakamoto

Sample	medium
9/11Fusion6 antisense-1	Plusgrow medium(+CP)
Plac(BBa-R0011)-3	Plusgrow medium(+Amp)
Plac(BBa-R0011)-4	Plusgrow medium(+Amp)
Fusion3m2 attenuator -1	Plusgrow medium(+CP)
Pλ-luxI-2	Plusgrow medium(+Amp)
9/11 aptamer12-1M-2	Plusgrow medium(+CP)

Colony PCR

Nakamoto

Sample	base pair
9/11 Fusion1 antisense 3	394
9/11 Fusion1 antisense 4	394
9/11 aptamer12-P 2	378
9/11 aptamer12-P 3	378

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	36s	30cycles

Electrophoresis

Nakamoto

Lane	Sample	Enzyme
1	Fusion1 antisense 2	--
2	Fusion1 antisense 3	--
3	1kb ladder	--
4	apt12-P 2	--
5	apt12-P 3	--

Restriction Enzyme Digestion

Tatsui

	9/12 RBS-lysis2-DT	XbaI	PstI	Buffer	BSA	MilliQ	total
2 cuts	5.2	1.0µL	1.0µL	3.0µL	3.0µL	16.8µL	30µL
NC	0.3µL	0µL	0µL	1.0µL	1.0µL	7.7µ	10µL

	9/12 RBS-lysis3-DT	XbaI	PstI	Buffer	BSA	MilliQ	total
2 cuts	5.2	1.0µL	1.0µL	3.0µL	3.0µL	16.8µL	30µL
NC	0.3µL	0µL	0µL	1.0µL	1.0µL	7.7µ	10µL

	9/11 RBS-lysis1-DT	XbaI	PstI	Buffer	BSA	MilliQ	total
2 cuts	8.5	1.0µL	1.0µL	3.0µL	3.0µL	13.5µL	30µL
NC	0.4µL	0µL	0µL	1.0µL	1.0µL	7.6µ	10µL

	8/20 J23100-RBS-luxR -DT	EcoRI	Buffer	BSA	MilliQ	total
1cut	5.8	1.0µL	3.0µL	3.0µL	17.2µL	30µL
NC	0.3µL	0µL	1.0µL	1.0µL	7.7µ	10µL

	9/10 pSB1C3	EcoRI	SpeI	Buffer	BSA	MilliQ	total
2cuts	8.1	1.0µL	1.0µL	3.0µL	3.0µL	13.9µL	30µL
NC	0.4µL	0µL	0µL	1.0µL	1.0µL	7.6µ	10µL

	9/12 Plux	PstI	Buffer	BSA	MilliQ	total
1cut	12.0	1.0µL	3.0µL	3.0µL	11.0micro;L	30µL
NC	0.6µL	0µL	1.0µL	1.0µL	7.4µ	10µL

	9/10 pSB1C3	XbaI	PstI	Buffer	BSA	MilliQ	total
2cuts	8.1	1.0µL	1.0µL	3.0µL	3.0µL	13.9µL	30µL

Electrophoresis

Tatsui

Lane	Sample	Enzyme1	Enzymel2
1	9/12 RBS-lysis2-DT	XbaI	PstI
2	9/12 RBS-lysis2-DT NC	--	--
3	9/11 RBS-lysis1-DT	XbaI	PstI
4	9/11 RBS-lysis1-DT NC	--	--
5	9/11 RBS-lysis3-DT	XbaI	PstI
6	9/11 RBS-lysis3-DT NC1	--	--
7	1kb ladder	--	--
8	100bp ladder	--	--
9	J23100-RBS-luxR-DT	EcoRI	--
10	J23100-RBS-luxR-DT	--	--
11	9/10 pSB1C3	EcoRI	SpeI
12	9/10 pSB1C3 NC	--	---
13	9/10 pSB1C3	XbaI	PstI
14	9/12 Plux	PstI	--
15	9/12 Plux NC	--	--

Observation through a confocal microscope

We used the liquid culture media with 200μL 9/10Pcon-pT181antisense-spinach-DT& 200μL 9/10 Pcon-spinach-DT& 200μL 9/10 JM109(overnight culture).
We translated them into each 1.5mL tube.

We elminated each supernatant using a 5000xg centrifuge for 1minute,.
Then,we resuspended with 100µL M9(distilled water) and eliminated each supernatant using a 5000xg centrifuge for 1 minute.x2

Adding 100µL M9(in 20µM DFHBI), we resuspended the pret.

15min after incubating in shield light, we eliminated each supernatant using a 5000xg centrifuge for 1 minute.

We looked at the fluorescence of the pret.
We failed to observe it.

Liquid Culture

Hirano

Sample	medium
9/10 Pcon-pT181antisense-spinach-DT	Plusgrow medium
9/10 Pcon-spinach-DT	Plusgrow medium
9/1 spinach-DT(RNA master plate)	Plusgrow medium

Template:Kyoto/Notebook/Sep 12

From 2013.igem.org

Contents

Sep 12

Miniprep

Electrophoresis

Liquid Culture

Electrophoresis

Gel Extraction

Gel Extraction

Electrophoresis

Colony PCR

Restriction Enzyme Digestion

Colony PCR

Transformation

Electrophoresis

Electrophoresis

Liquid Culture

Colony PCR

Electrophoresis

Restriction Enzyme Digestion

Electrophoresis

Observation through a confocal microscope

Liquid Culture