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Secretion Notebook

February 7

Preculture
We started preculture at 12:10.

February 8

Culture
We start culturing with 300[mL] of LB medium.

time	OD600
12:00	start
14:10	0.019
14:45	0.154
15:05	0.267
15:21	0.64

Making Competent Cell
We made competent cells.

Transformation
pGEM_TAP
Lacp (BBa_R0011)
DT (BBa_B0015)

Making Culture Medium Plates
We made 200mL of ampicillin culture, kanamycin culture, and chloramphenicol culture.

Transformation
GFP(BBa_E0040) in pSB1A2
DT(BBa_B0015) in pSB1AK3
ara(BBa_I0500) in pSB2K3
Lacp(BBa_R0011) in pSB1A2

February 9

transformation
BBa-E0040(GFP)(Mr.Fujita)

Liquid culture
DT.leap colony transformed on February 8
colony of competent cell made on February 8

February 10

Miniprep
DT2 43.9μg/ml(1.34 260/230 1.74 260/280)
lacp1 17.1μg/ml(1.54 260/280 0.83 260/230)
lacp2 18.0μg/ml(1.58 260/280 0.87 260/230)

transformation
B0040 1.4k PsB1A2 B0034 1.2M pSB1A2(from iGEM parts plate)

Competent cell
We did preculture for overnight. We put 1.5mL of preculture on 150mL of LB culture.

time	OD600
11:45	start
13:30	0.048
14:30	0.168
15:03	0.256
15:20	0.405
15:35	0.459
at last	0.576

February 11

Checking Transformation efficiency
Conpetent cell's transformation efficiency is 1.3x10^4colonys/μg

February 13

Transformation
Const promoter J23110,J23109,J23100

DNA	Competent cell	total
1μL	20	21

No colony was there on February 14

Liquid culture
lacP,DT,RBS(BBa_B0034),GFP
start at 20:00
in Plus grow with Ampicilin 3mL

February 14

Miniprep
concentration[μg/μL]

lacP3	39.6
lacP4	40.8
lacP5	28.9
RBS1	28.2
RBS2	57.4
RBS3	13.2
DT3	69.7
DT4	64.4
DT5	61.5
GFP1	64.0
GFP2	50.5
GFP3	66.0

Restriction
Const promoterJ23100

DNA	Spe1	Pst1	Buffer2	BSA	MilliQ	total
20	0.5	0.5	3	0.5	5.5	30

at 37℃ for overnight

February 15

Making gel
1% Agarose gel

Agarose	TAE
1.6g	160mL

Electrophoresis

No.1

Gel No.1

Restriction product	loading dye
5μL	1

The marker was 1kb ladder
It seemed that this restriction product was not cut.

No.2

Gel No.2
Lane1 : 1kb ladder
Lane2 : J23100 2μL + 6*Loading dye 1μL
Lane3 : J23100(Spe1,Pst1) 5μL
Lane4 : J23100(Spe1,Pst1) 2μL

There were bands on lane_2 and we cannot identify these bands because the sample of lane_2 was not cut with any restriction enzyme.
There must have been bands at 2100bp and 883bp on lane_3 and lane_4.

Testing whether restriction enzyme were deactivated or not

DNA(DT)	restriction enzyme	Buffer	BSA	MilliQ	total
10	0.5	3	0.5	16	30

at 37℃ for Oveernight
Restriction enzyme means Spe1(1,2) Pst1(1,2,3) in this time.

February 16

Electrophoresis

1. 1kb ladder
2. DT2
3. DT3
4. DT2 (Spe1-1)
5. DT2 (Spe1-2)
6. DT2 (Pst1-1)
7. DT2 (Pst1-2)
8. DT2 (Pst1-3)
9. DT3 (Pst1-4)
10. 1kb ladder

Pst1-1, Pst1-2, and Pst1-3 did not cut DNAs. They seemed to be deactivated.

Genomic PCR

10*Buffer for KOD Plus	2mM dNTPs	25mM MgSO4	10μM primer-f	10μM primer-r	158ng/μL Genomic DNA	KOD plus	MilliQ	total
5	5	3	1.5	1.5	1	1	32	50

Electrophoresis

1. 1kb ladder
2. tatABCD (2.5kb)
3. TAMO reductase (2.7kb)
4. Negative control
We got bands of tatABCD but there were nonspecific amplification products.
We failed amplification of TAMO reductase.

Transformation
Constitutive promoter (BBa_J23107 , BBA_J23117)
High copy plasmid (pSB1AT3)

DNA	competent cell
1μL	10μL

February 17

PCR
We did PCR to amplify products of PCR that we had done yesterday but we could not amplify tatABCD.

Genomic PCR

Buffer	dNTPs	MgSO4	primer-f	primer-r	genomic DNA	KOD plus	MilliQ	total
2.5	2.5	5	0.75	0.75	0.5	0.5	16	50

Predenature 94℃, 2min
Denature 98℃, 10sec
Annealing 57℃, 30sec
Extension 68℃, 2.5min
(30cycles)

Electrophoresis

1. 1kb ladder
2. TAMO reductase
3. Negative control

Restriction

J23100	Spe1	Pst1	Buffer2	BSA	MilliQ	total
10	0.5	0.5	3	0.5	15.5	30

Genomic PCR

	Buffer	dNTPs	MgSO4	primer-f	primer-r	genomic DNA	KOD plus	MilliQ	total
TMAO reductase	2.5	2.5	3	0.75	0.75	0.5	0.5	15.5	25
tatABCD	2.5	2.5	2	0.75	0.75	0.5	0.5	15.5	25
tatABCD	2.5	2.5	2	0.75	0.75	5	0.5	10.5	25

Electrophoresis

1. 1kb ladder
2.3. TAMO reductase
4. tatABCD
5. tatABCD(10 times amount of genome)

Checking of restriction enzyme

DT	enzyme	Buffer	BSA	MilliQ	total
2	0.5	3	0.5	24	30

at 37℃ for overnight
We checked EcoR1 and Xba1.

February 18

Miniprep

	μg/mL	260/280	230/260
JS3117-1	135	1.5	2.06
JS3117-2	75	1.6	1.63
JS3109-1	115	1.5	1.88
JS3109-2	75	1.65	1.71
pSB1AT3-1	70	1.66	1.83
pSB1AT3-2	100	1.52	1.54

diluted to 25 times

Competent cell
We put 3mL of preculture product on yesterday onto 300mL of LB medium

time	OD600
10:30	start
12:10	0.118
13:00	0.270
13:30	0.502

Transformation

pSB1AT3-2	competent cell	MilliQ	total
0	20	10	30
2	20	8	30
10	20	0	30

Results(on Feb. 19)

pSB1AT3-2	number of colony
0	0
2	177
10	590

Transformation efficiency 7.4x10^4 colonys/μg

February 20

Restriction
sample 1

DT plasmid	EcoR1	Xba1	Buffer	BSA	MilliQ	total
7.5	0.5	0.5	3	0.5	18	30

sample2

GFP plasmid	EcoR1	Spe1	Buffer	BSA	MilliQ	total
10	0.5	0.5	3	0.5	15.5	30

Electrophoresis

sample1

a. sample1 2μL + MilliQ 3μL + 6×Loading Dye 1μL
b. sample1 5μL + 6×Loading Dye 1μL
lane_1 1kb ladder
lane_2 a
lane_3 b
lane_4 a
lane_5 b
lane_6 a
lane_7 b
lane_8 1kb ladder

sample2

c. sample2 2μL + MilliQ 3μL + 6×Loading Dye 1μL
d. sample2 5μL + 6×Loading Dye 1μL
lane_1 1kb ladder
lane_2 c
lane_3 d
lane_4 1kb ladder

PCR

	Buffer	dNTPs	MgSO4	primer-f	primer-r	genomic DNA	KOD plus	MilliQ	total
tatABCD1	2.5	2.5	1.5	0.75	0.75	0.5	0.5	16	25
tatABCD2	2.5	2.5	1.5	0.75	0.75	0.5	0.5	16	25
TMAO reductase1	2.5	2.5	2	0.75	0.75	0.5	0.5	15.5	25
TMAO reductase2	2.5	2.5	2	0.75	0.75	5	0.5	10.5	25

Predenature 94℃ 2min
Denature 98℃ 10sec
Annealing 59℃ 30sec
Extension 68℃ 2.5min
→30cycles

	Buffer	dNTPs	MgSO4	primer-f	primer-r	PCR products	genomic DNA	KOD plus	MilliQ	total
tatABCD1	2.5	2.5	1.5	0.75	0.75	0	0.5	0.5	16	25
tatABCD2	2.5	2.5	1.5	0.75	0.75	1	0	0.5	15.5	25
TMAO reductase1	2.5	2.5	2	0.75	0.75	0	0	0.5	16	25
TMAO reductase2	2.5	2.5	2	0.75	0.75	0	0	0.5	16	25

Predenature 94℃ 2min
Denature 98℃ 10sec
Annealing 59℃ 30sec
Extension 68℃ 2.5min
→35cycles

Electrophoresis

lane_1.1kb ladder
lane_2.tatABCD
lane_3.tatABCD
lane_4.TAMO1
lane_5.TAMO2
lane_6.1kb ladder

lane_1.1kb ladder
lane_2.tatABCD1
lane_3.tatABCD2
lane_4.TAMO
lane_5.TAMO
lane_6.1kb ladder

February 21

PCR (Advantage HF protocol)

	buffer	dNTPs	primer-f	primer-r	gDNA	PCR products	DW	polymerase	total
tatABCD	2.5	2.5	0.75	0.75	1	0	17	0.5	25
TMAO	2.5	2.5	0.75	0.75	0	1	17	0.5	25

Predenature 94℃ 1min
Denature 94℃ 30sec
Annealing 58℃ 30sec
Extension 68℃ 3min
→25cycles

Electrophoresis

1. 1kb ladder 2μL
2. tatABCD 5μL + 6×Loading Buffer 1μL
3. TAMO 5μL + 6×Loading Buffer 1μL
4. 1kb ladder 2μL

Liquid culture
pSB3C5-1,2
pSB4K5-1,2

February 22

Gel extraction
lane 1 of the gel 45.0μg/mL

PCR purification
product 38.2μg/mL

PCR
TMAO reductase

	Buffer	dNTPs	MgSO4	prefix primer-f	suffix primer-r	product of gel extract	product of PCR purification(1ng/μL)	KOD plus	MilliQ	total
1	5	5	4	1.5	1.5	0.5	0	1	32.5	50
2	5	5	4	1.5	1.5	1	0	1	32.5	50
3	5	5	4	1.5	1.5	0	0.5	1	32.5	50
4	5	5	4	1.5	1.5	0	1	1	32.5	50

94℃, 2min
98℃, 10sec
59℃, 30sec
68℃, 3min
→25cycles

Electrophoresis

1. 1kb ladder
2. TAMO1
3. TAMO2
4. TAMO3
5. TAMO4
6. constructive promoter 1-18C
7. constructive promoter Spe1
8. constructive promoter Pst1
9. 1kb ladder
PCR

	Buffer	dNTPs	MgSO4	prefix primer-f	suffix primer-r	product of PCR purification(1ng/μL)	KOD plus	MilliQ	total
1	5	5	3	1.5	1.5	0.5	1	32.5	50
2	5	5	3	1.5	1.5	1	1	32	50
3	5	5	3	1.5	1.5	2	1	31	50
4	5	5	3	1.5	1.5	3	1	30	50
5	5	5	3	1.5	1.5	10	1	29	50
6	5	5	3	1.5	1.5	0	1	33	50

94℃, 2min
98℃, 10sec
59℃, 30sec
68℃, 3min
→25cycles

Electrophoresis

Checking Dpn1

Buffer	lacp(28.7ng/μL)	Dpn1	MilliQ	total
2	10	0.5	7.5	20
2	10	0	5	17

February 23

Colony PCR
tatABCD(2 samples)

Buffer	dNTPs	MgSO4	primer-f	primer-r	KOD plus	MilliQ	total
5	5	3	1.5	1.5	1	33	50

Predenature 94℃ 1min
Denature 98℃ 10sec
Annealing 59℃ 30sec
Extension 68℃ 3min
→25cycles

Electrophoresis

1. 1kb ladder 2μL
2. tatABCD 1 5μL + 6×Loading Buffer 1μL
3. tatABCD 2 5μL + 6×Loading Buffer 1μL
4. 1kb ladder 2μL

Miniprep
pSB4K5 and pSB3C5
deluted it to 25 times and then measured it
pSB4K5 1 : 60.0 μg/ml 1.67(260/280) 1.98(260/230)
pSB4K5 2 : 55.0 μg/ml 1.49(260/280) 1.62(260/230)
pSB3C5-3 : 3.6 μg/ml 1.57(260/280) 3.00(260/230)
pSB3C5-4 : 1.3 μg/ml 1.44(260/280) 1.04(260/230)

Liquid culture
pSB3C5-3,4

Electrophoresis

1. 1kb ladder 2μL
2. pSB3C5-3 5μL, 6×loading dye 1μL
3. pSB3C5-4 5μL, 6×loading dye 1μL
4. 1kb ladder 2μL

February 27

Test of Dpn1

Buffer2	GFP2	BSA	MilliQ	Dpn1
3	3	0.3	23	1

Colony PCR

	buffer	dNTPs	MgSO4	Primer-f	Primer-r	MilliQ	KOD Plus	total
Colony PCR(2 samples)	5	5	3	1.5	1.5	33	1	50
Negative control	5	5	3	1.5	1.5	34	0	50

Predenature 94℃,2min
Denature 98℃,10sec
Annealing 59℃,30sec
Extension 68℃,3min
→25cycles

Electrophoresis

	sample	Loading Dye	MilliQ
1.1kb ladder	2	0	0
2.product of PCR1	5	1	0
3.product of PCR2	5	1	0
4.product of PCR(Negative control)	5	1	0
5.product of PCR(2/23)	5	1	0
6.GFP2(DPN1)	10	2	0
7.GFP2	3	2	7
8.1kb ladder	2	0	0

Results of liquid culture
We measure this after dilute it to 10 times.

pSB3C5-5	pSB3C5-6	pSB3C5-5(1% glucose)	pSB3C5-6(1% glucose)
8.5[µg/ml]	-1.8	-17.9	-18.2

PCR

	buffer	dNTPs	MgSO4	Primer-f(prefix)	Primer-r(suffix)	PCR purification product(1ng/µL)	MilliQ	KOD Plus	total
1	5	5	3	1.5	1.5	0.2	32.8	1	50
2	5	5	3	1.5	1.5	0.5	32.5	1	50

PCR purification product was that purification product(75ng/µL) of electrophoresis-3 deluted to 1ng/µL

Predenature 94℃,2min
Denature 98℃,10sec
Annealing 59℃,30sec
Extension 68℃,3min
→25cycles

February 28

Electrophoresis

1. 1kb ladder
2. PCR1 →Product of gel extraction : tatABCD with prefix and suffix 105[ng/µL]
3. PCR2

Restriction

Buffer2	plasmid(?)	enzyme	MilliQ	total
2	2	0.2	15.8	20

incubate 1 hour at 37℃

Electrophoresis

1. 1kb ladder
2. Control (without enzymes)
3. EcoR1
4. Xba1 (crystallized)
5. Xba1 (with seal)
6. Spe1
7. Pst1
8. 1kb ladder

PCR and Electrophoresis

Quick Taq Dye Mix	primer-f	primer-r	template	MilliQ	total
25	1.0	1.0	0.5	22.5	50

Predenature 94℃,2min
Denature 94℃,30sec
Annealing 59℃,30sec
Extension 68℃,3min
→25cycles

Restriction

BufferH	tatABCD	EcoR1	Spe1	MilliQ	total
2	5	0.2	0.2	12.6	20

PCR purification
We eluted the product for 30µL MilliQ

Ligation

Insert(tatABCD)	Vector(pSB1C3)	Ligation High	total
10	1	5	16

4℃, overnight

February 29

Transformation

tatABCD	competent cell	total
1	10	11

Checking Restriction enzyme

plasmid seems to be 1-18C promoter	Enzyme	Buffer	MilliQ	total
2	0.2	2	15.8	20

Checking tatABCD

tatABCD	Hind3	Buffer	MilliQ	total
5	0.2	2	12.8	20

PCR

	buffer	dNTPs	MgSO4	Primer-f	Primer-r	ColE1(6.5ng/µL) / TMAO	MilliQ	KOD Plus Neo	total
Kil	2.5	2.5	1.5	0.75	0.75	0.5	16	0.5	25
TMAO	2.5	2.5	1.5	0.75	0.75	0.5	16	0.5	25

Predenature 94℃,2min
Denature 98℃,10sec
Annealing 60℃,30sec
Extension 68℃,3min
→30cycles

Electrophoresis

evernoteに写真なし！至急求む

1. 1kb ladder
2. Kil (649bp)
3. TMAO (2720bp)
4. TMAO (Quick Taq)
5. tatABCD (Quick Taq)
6. tatABCD (Hind3)
7. 1kb ladder

March 1

PCR

TMAO

Template is gDNA and product of colony PCR gel extraction

	Buffer	gNTPs	MgSO4	Primer-f	Primer-r	KOD Plus Neo	Template gDNA	product of gel extraction	DW	total
1	2.5	2.5	1.5	0.75	0.75	0.5	0.5	0	16	25
2	2.5	2.5	1.5	0.75	0.75	0.5	0	2	14.5	25

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 1.5min
→25cycles

Kil

Buffer	dNTPs	MgSO4	primer-f	primer-r	colE1(6.5ng/µL)	KOD Plus Neo	MilliQ	total
2.5	2.5	1.5	0.75	0.75	0.5	16	0.5	25

94℃, 2min
98℃, 10sec
61℃, 30sec
68℃, 1min
→20cycles

Electrophoresis

1. 1kb ladder
2. TMAO1 (gDNA)
3. TMAO2 (product of gel extraction)
4. Kil

PCR

Buffer	dNTPs	MgSO4	primer-f	primer-r	Product of Purification	KOD Plus Neo	MilliQ	total
5	5	3	1.5	1.5	1	32	1	50

94℃, 2min
98℃, 10sec
61℃, 30sec
68℃, 30sec
20cycles
→Purification 230ng/µL

Restriction

Kil	EcoR1	Spe1	BufferH	MilliQ	total
5	0.2	0.2	2	12.6	20

incubate at 37℃, for 1.5 hours

PCR Purification

Ligation

Kil	pSB1C3	Ligation High	total
5	1	3	9

at 4℃, for overnight

March 2

Ligation

Kil	pSB1C3	Ligation High Ver.2	total
5	1	3	9

tatABCD	pSB1C3	Ligation	total
10	1	5	16

at 16℃ for 1 hour

Transformation

Kil	Kil(3/1,Ligation)	tatABCD	competet cell	total
1	0	0	10	11
0	1	0	10	11
0	0	1	10	11

PCR

Quick Taq	primer-r	primer-f	template	MilliQ	total
25	1	1	0.5	22.5	50

Electrophoresis

Restriction

pSB3C5-5	EcoR1	Pst1	BufferH	BSA	MilliQ	total
20	0.5	0.5	3	0.5	5.5	30

at 37℃, for 2 hour

Electrophoresis

1. 1kb ladder 2µL
2. pSB3C5 5µL + 6×Loading Buffer 1µL
・product of gel extraction(about 2700bp)
－30.9µg/mL

Restriction
GFP1,2,3

GFP	EcoR1	Pst1	Buffer	BSA	MilliQ	total
10	0.5	0.5	3	0.5	15.5	30

at 37℃, for 2.5 hours

Restriction

DT	EcoR1	Xba1	BufferM	BSA	MilliQ	total
10	0.2	0.2	3	0.3	16.3	30

Constitutive Promoter	Spe1	Pst1	BufferM	BSA	MilliQ	total
15	0.2	0.2	3	0.3	11.3	30

at 37℃, 2 hours

J23117-1:135ng/µL, J23107-1:115ng/µL
DT3→PCR Purification
Promoter→Gel Extraction

Checking TMAO

something seems to be TMAO	Buffer2	EcoR1	MilliQ	total
10	2	0.5	7.5	20

at 37℃, for 1 hour

Electrophoresis

1. 1kb ladder
2. GFP1 that had been cut by restriction enzyme
3. GFP2 that had been cut by restriction enzyme
4. GFP3 that had been cut by restriction enzyme
5. GFP1
6. GFP2
7. GFP3
8. TMAO (control)
9. TMAO (EcoR1)
10. DT (control)
11. DT (EcoR1, Xba1)
12. 1kb ladder

Checking tatABCD

Quick Taq	primer-f	primer-r	MilliQ	total
25	1	1	23	50

Electrophoresis

March 3

PCR

	template	buffer	dNTPs	MgSO4	VF	VR	KOD plus	MilliQ	total
1	1	5	5	3	1.5	1.5	1	32	50
2	2	5	5	3	1.5	1.5	1	31	50

94℃, 2min
98℃, 10sec
50℃, 30sec
68℃, 1min
→30cycles

Miniprep

March 4

Sequence of tatABCD

Quick Taq	primer-f	promer-r sequence	template	MilliQ	total
25	1	1	1	23	50

Quick Taq	primer-f sequence	primer-r	template	MilliQ	total
25	1	1	1	23	50

Colony PCR of TMAO

buffer	dNTPs	NgSO4	primer-f	primerr-r	KOD plus	MilliQ	total
5	5	4	1.5	1.5	1	32	50

→ethanol precipitation

94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 2.5min
→25cycles

Electrophoresis

1. 1kb ladder
2. tatABCD1
3. tatABCD2
4. TMAO
5. 1kb ladder

Restriction

TMAO	EcoR1	BufferH	BSA	MilliQ	total
10	0.2	3	0.3	16.5	30

TMAO	Xba1	Pst1	BudderM	BSA	MilliQ	total
10	0.2	0.2	3	0.3	16.3	30

at 37℃ for 1 hour

Electrophoresis

Transformation

pSB1C3	competent cell(made at 2/8)	total
5	100	105

March 5

Restriction

pSB1C3(Xba1, Spe1)	Pst1	BufferH	BSA	MilliQ	total
10	0.2	2	0.2	7.6	20
pSB1C3(Xba1, Spe1)	EcoR1	BufferH	BSA	MilliQ	total
10	0.2	2	0.2	7.6	20

at 37℃ for 1 hour
→Then we did ethanol precipitation

Ligation

Kil(EcoR1, Spe1)	pSB1C3(EcoR1)	Ligation High	total
5	1	3	9

at 16℃ for 1 hour

Transformation

Kil	competent cell	total
1	10	11

We used commercially available competent cells in this time.

PCR

TMAO

buffer	dNTPs	MgSO4	Primer-f	Primer-r	Template	KOD plus	MilliQ	total
5	5	3	1.5	1.5	1	1	32	50

Electrophoresis

(31)

Restriction

	Lacp	pSB3C5	EcoR1	Pst1	BufferH	BSA	MilliQ	total
1	20	0	0.5	0.5	3	0.5	5.5	30
2	0	20	0.5	0.5	3	0.5	5.5	30

at 37℃ for 1 hour
1→Ethanol precipitation　45.4µg/mL
2→Gel extraction　38.7µg/mL

Ligation

LacP	pSB3C5	Ligation High	total
10	2	6	18

at 4℃ for overnight

Transformation

Lacp+pSB3C5	competent cell	total
1	10	11

on ice for 30 mins.
heat shock at 42℃ for 60secs
on ice for 2 mins.
After we incubate with 200µL of SOC culture for 1 hour, we did plating on LB culture with CP

Restriction

GFP Plasmid	EcoR1	Spe1	Buffer2	BSA	MilliQ	total
10	0.5	0.5	3	0.5	15.5	30

at 37℃ for 2 hours

Electrophoresis

1. Ladder 2µL
2. GFP Plasmid (already restricted) 2µL + Loading Dye 2µL + MilliQ 8µL
3. Ladder 2µL

PCR

torA signal and pspA pspAはコロニーPCR

Buffer	dNTPs	MgSO4	Primer-f	Primer-r	template(TMAO)	KOD plus	MilliQ	total
2.5	2.5	1.5	0.75	0.75	0.5	0.5	16	25

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 30sec

electrophoresis

1. 100bp Ladder
2. torA signal (272bp)
3. pspA (969bp)
4. 100bp Ladder

March 6

Restriction

GFP	EcoR1	Spe1	BufferM	BSA	MilliQ	total
12	0.5	0.5	3	0.5	13.5	30

We did incubate at 37℃ for 1.5hours.
And we did gel extraction on 3/7 and get 40.0μg/mL GFP.

PCR

buffer	dNTPs	MgSO4	Primer-f	Primer-r	template	KOD plus neo	MilliQ	total
5	5	3	1.5	1.5	0.5	1	32.5	50

94℃　2min
98℃　10sec
60℃　30sec
68℃　(torA 10sec / pspA 30sec) 25 cycles
We used product of PCR on 3/5 of torA and pspA as template.

Restriction

Kil	EcoR1	Spe1	BufferM	BSA	MilliQ	total
10	0.3	0.3	3	0.3	16.1	30

DT	EcoR1	Xba1	Buffer2	BSA	MilliQ	total
10	0.5	0.5	3	0.5	15.5	30

at 37℃ for 2 hours
→ purification 37.7ng/μL

Ligation

Kil	pSB1C3	Ligation High Ver.2	total
4	2	3	9

Kil : 350fmol
pSB1C3 : 29fmol

at 16℃ for overnight

March 7

Electrophoresis

1. 1kb ladder 2µL
2. pspA (PCR product)2.5µL + Loading Dye 0.5µL
3. GFP 30µL + Loading Dye 6µL
4. 1kb ladder 2µL

The GFP was gel extracted on 3/6 and concentrated by Vacuum in 150µg/mL

Ligation

VectorDNA	GFP	Ligation High Ver.2	total
5	15	10	30

Restriction

torA	EcoR1	Spe1	bufferM	BSA	MilliQ	total
10	0.3	0.3	3	0.3	16.1	30

pspA	EcoR1	Spe1	bufferM	BSA	MilliQ	total
5	0.3	0.3	3	0.3	21.1	30

at 37℃ for 1.5 hours

Purification

torA→31.8ng/µL
pspA→49.3ng/µL

Ligation

torA	pSB1C3	Ligation High Ver.2	total
3	3	3	9

pspA	pSB1C3	Ligation High Ver.2	total
4	2	3	9

at 4℃, for overnight

torA→31.8ng/µL×3µL=95.4ng=0.529pmol
pSB1C3→19.4ng/µL×3µL=58.2ng=0.042pmol
pspA→49.3ng/µL×4µL=197.2ng=0.308pmol
pSB1C3→19.4ng/µL×2µL=38.8ng=0.029pmol

March 8

Restriction

pSB4K5	EcoR1	Spe1	BufferM	BSA	MilliQ	total
20	0.2	0.2	3	0.2	6.4	30

at 37℃ for 1 hour.
→Purification : 36.6ng/µL

Ligation

Kil	pSB4K5	Ligation High Ver.2	total
10	1	5	16

at 4℃ for overnight

Kil→37.7ng/µL×10µL=377ng=879fmol
pSB4K5→36.6ng/µL×1µL=36.6ng=86fmol

Liquid culture

Lacp + pSB3C5 -1, 2

Transformation

torA	pspA	competent cell	total
1	0	10	11
0	1	10	11

We use commercially available competent cells in this time.

March 9

Restriction

tatABCD	Xba1	Pst1	BufferM	BSA	MilliQ	total
10	0.2	0.2	3	0.3	16.3	30

at 37℃ for 1hour
→purification : 75.0μg/mL (1.11 260/280 , 0.81 260/230)

Miniprep

lacP + pSB3C5 1 80.5μg/mL (1.78 260/280 , 2.00 260/230)
lacP + pSB3C5 2 107.2μg/mL (1.83 260/280 , 1.90 260/230)

Colony PCR

Quick Taq	VF	VR	MilliQ	total
25	1	1	23	50

94℃ 2min
94℃ 30sec
55℃ 30sec
68℃ 6sec
25cycles

Ligation

tatABCD	constP J23107	Ligation High Ver.2	total
5	1	3	9

tatABCD : 227fmol
constP J23107 : 21fmol

Transformation

	pspA	torA	Kil	competent cell
1	1	0	0	10
2	0	1	0	10
3	0	0	1	10

Miniprep

4mL of plusgrow which had been cultured for overnight.
pSB4K5 : 80.5μg/mL

March 10

Screening PCR

Quick Taq	VF	VR	MilliQ	total
25	1	1	23	50

Then we did electrophoresis to confirm.
1.1kb ladder
2.kil (649bp)
3,4,5, pspA (969bp)
6,1kb ladder

1, 100bp ladder
2,3,4, torA signal

Restriction

LacP-pSB3C5	Spe1	Pst1	BufferM	BSA	MilliQ	total
10	0.2	0.2	2	0.2	7.4	20

for 2.5 hours at 37℃
→purification 29.0ng/μL

torA	Xba1	Pst1	BufferM	BSA	MilliQ	total
10	0.2	0.2	2	0.2	7.4	20

for 2.5 hours at 37℃
→purification 91.8ng/μL

Ligation

torA	Lacp-pSB3C5	Ligation High Ver.2	total
4	2	3	9

torA : 929fmol
Lacp-pSB3C5 : 284fmol
for overnight at 4℃

March 11

Miniprep

We used 3μL of plus grow that we had cultured for overnight.
torA : 62.8ng/μL

Screening PCR

Quick Taq	VF	VR	MilliQ	total
25	1	1	23	50

Electrophoresis

1. 1kb ladder
2〜11. pspA (969bp)
12. 1kb ladder

The results were shown as photograph in the right.

It seemed that there were shorter sample than expected sample, so we did electrophoresis with pspA which was product of PCR and pspA which had already cut with EcoR1 and Spe1.

1.1kb ladder
2. pspA (PCR product)
3, pspA (Eco, Spe)
4〜6, pspA (colony PCR)
7,1kb ladder

The results were shown as photograph in the right.

March 12

Transformation

DT(1ng/μL)	DT(0.1ng/μL)	Kil	lacP-torA	MilliQ	competent cell	total
1	0	0	0	0	20	21
0	1	0	0	0	20	21
0	0	5	0	0	50	51
0	0	0	5	0	50	51
0	0	0	0	1	20	21

Restriction

pspA	EcoR1	Spe1	BufferM	BSA	MilliQ	total
10	0.5	0.5	3	0.3	15.7	30

at 37℃ for 4 hours
→ We did purification and got 48.3ng/μL pspA.

Ligation

pspA	pSB1C3	MilliQ	Ligation High Ver.2	total
4	2	0	3	9
2	2	0	2	6
0	2	2	2	6

March 13

Miniprep

pSB1C3 74.2µg/mL 1.65 (260/280) 1.31 (260/230)

March 14

Restriction

pSB1C3	EcoR1	Spe1	BufferM	BSA	MilliQ	total
20	0.2	0.2	4	0.4	15.2	40

We did gel extraction and got 47.2ng/µL pSB1C3 but we did not cut out RFP.

Ligation

torA	pSB1C3	Ligation High Ver.2	total
4	2	3	9
MilliQ	pSB1C3	Ligation High Ver.2	total
4	2	3	9

at 16℃, for 1 hour

torA : 0.707pmol
pSB1C3 : 0.068pmol

Liquid culture

We cultured Lacp-pSB3C5 1,2,3,4,5 (CP tolerance)on culture with Amp.
→Only 4 which did not be cultured succeeded.

March 15

Liquid culture

We cultured Lacp-pSB3C5 6,7,8,9,10 on culture with ampicillin.
→6,8,9,10 were succeeded.

Restriction

GFP	EcoR1	Spe1	Buffer2	BSA	MilliQ	total
10	0.5	0.5	3	0.5	15.5	30

DT	EcoR1	Xba1	Buffer2	BSA	MilliQ	total
10	0.5	0.5	3	0.5	15.5	30

for 2 hours at 37℃.

Miniprep

pspA (pSB1C3) 40.5ng/µL

Ligation

pspA	DT	Ligation High Ver.2	total
5	1.5	3	9.5

pspA : 385fmol
DT : 36fmol

Transformation

J23107-tatABCD	DT (0.1ng/µL)	DT (0.01ng/µL)	pspA-DT	competent cells on 3/15	total
2	0	0	0	20	22
0	2	0	0	20	22
0	0	2	0	20	22
0	0	0	2	20	22

Screening PCR

Kil, pspA and torA

Quick Taq	Primer-r	Primer-f	MilliQ	total
25	1	1	23	50

March 16

Miniprep

torA (pSB1C3) 68.8ng/µL
Kil (pSB4K5) 92.7ng/µL
torA was red for some reason. We do not know why.

Colony PCR

Quick Taq	Primer-r	Primer-f	MilliQ	total
25	1	1	23	50

94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 6sec
→25cycles

Electrophoresis

The results were shown as photograph in the right.

Checking Transformation Efficiency

competent cells that were made on March 15.
DNA : 0.02ng → 668 colonies Transformation Efficiency : 3.3×10^7
DNA : 0.2ng → 1739 colonies Transformation Efficiency : 8.7×10^6

Restriction

pSB1C3	EcoR1	Spe1	BSA	BufferM	BufferH	MilliQ	total
20	0.2	0.2	0.3	3	0	6.3	30
5	0.2	0	0.2	0	2	12.6	20

at 37℃, for 2 hours.
We did gel extraction for product with EcoR1, Spe1. We got 46.1ng/µL pSB1C3.

Kil(pSB4K5)	EcoR1	Pst1	BSA	BufferH	MilliQ	total
5	0.2	0.2	0.2	2	12.4	20
5	0.2	0	0.2	2	12.6	20

for overnight at 37℃.
We did this to confirm.

pspA (pSB1C3)	EcoR1	Pst1	BSA	BufferH	MilliQ	total
5	0.2	0.2	0.2	2	12.4	20
5	0.2	0	0.2	2	12.6	20

for overnight at 37℃.
We did this to confirm.

Ligation

torA	pSB1C3	Ligation High Ver.2	total
4	2	3	9

MilliQ	pSB1C3	Ligation High Ver.2	total
4	2	3	9

at 16℃, for overnight

torA→767fmol
pSB1C3→68fmol

March 17

Miniprep

J23107-tatABCD 72.7ng/µL
pspA-DT 50.5ng/µL

Checking the Insert

J21037-tatABCD	EcoR1	Pst1	BSA	BufferH	MilliQ	total
5	0.2	0.2	0.2	2	12.4	20
5	0.2	0	0.2	2	12.6	20

Success.

pspA-DT	EcoR1	Pst1	BSA	BufferH	MilliQ	total
5	0.2	0.2	0.2	2	12.4	20
5	0.2	0	0.2	2	12.6	20

Failed.

March 19

Restriction

DT	EcoR1	Xba1	BSA	BufferM	MilliQ	total
10	0.2	0.2	0.3	3	16.3	30

We did Gel extraction and got 17.2ng/µL of DT.

Ligation

Kil	DT	Ligation High Ver.2	total
10	2	6	18

We did this for an hour at 16℃.

Restriction

GFP	EcoR1	Spe1	BSA	BufferM	MilliQ	total
10	0.5	0.5	0.5	3	15.5	30

We did this for 4 hours at 37℃

Ligation

pspA	DT	Ligation High Ver.2	total
5	5	5	15

pspA	pSB1C3	Ligation High Ver.2	total
4	1	3	8

We did these for an hour at 16℃.

pspA (5µL)→377fmol
DT→39fmol
pspA (4µL)→339fmol
pSB1C3→34fmol

Transformation

pspA-DT, pspA (pSB1C3), torA (pSB1C3) and GFP-DT

March 20

Screaning PCR

Quick Taq	Primer-R	Primer-F	MilliQ	total
25	1	1	23	50

pspA→○
pspA-DT→○
GFP-DT→○
torA→×
Kil-DT 6 of 8 sumples→○

Quick Taq	VR	VF	MilliQ	total
25	1	1	23	50

pspA→○
pspA-DT→×

Restriction

torA	EcoR1	Spe1	BSA	BufferM	MilliQ	total
10	0.3	0.3	0.3	3	16.1	30

DT	EcoR1	Xba1	BSA	BufferM	MilliQ	total
10	0.2	0.2	0.3	3	16.3	30

We did this for overnight at 37℃. And we did purification.
torA 34.2ng/µL
DT 28.0ng/µL

March 21

Miniprep

GFP-DT-1 : 77.7ng/µL
GFP-DT-2 : 67.6ng/µL

Restriction

pSB1C3	EcoR1	Spe1	BSA	BufferM	MilliQ	total
10	0.2	0.2	0.3	3	16.3	30
5	0.2	0	0.2	2	12.6	20

We did this for 3 hours at 37℃, and then we did gel extraction. We got 25.1ng/µL pSB1C3

GFP-DT	EcoR1	Pst1	Xba1	BSA	BufferM	MilliQ	total
5	0.2	0.2	0	0.2	2	12.4	20
5	0.2	0	0	0.2	2	12.6	20
10	0.2	0	0.2	0.3	3	16.3	30

We did this for 3 hours at 37℃, and then we did Purification. We got 30.7ng/µL GFP-DT.

Ligation

	torA	pSB1C3	pspA	DT	GFP-DT	Ligation High Ver.2	total
1	4	1	0	0	0	3	8
2	0	1	7	0	0	4	12
3	0	0	5	3	0	4	12
4	3	0	0	0	5	4	12

We did this for an hour at16℃.

March 22

PCR

We did PCR to amplify torA_signal that was product of PCR at March 5 with redesigned primers.

Buffer	dNTPs	MgSO4	Primer-F	Primer-R	Template	MilliQ	KOD plus neo	total
5	5	3	1.5	1.5	0.5	32.5	1	50

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 10sec
→30cycles
→Purification 110.7ng/µL

Restriction

torA	EcoR1	Spe1	BSA	BufferM	MilliQ	total
10	0.2	0.2	0.3	3	16.3	30

Ligation

	torA	pSSB1C3	pspA	DT	GFP-DT	Ligation high	total
1	4	3	0	0	0	4	11
2	3	0	0	0	3	3	9
3	0	0	5	5	0	5	15

torA (4µL)→512fmol
pSB1C3→54fmol
torA (3µL)→384fmol
GFP-DT→36fmol
pspA→377fmol
DT→65fmol

March 23

Screening PCR

torA (pSB1C3), torA-GFP-DT and pspA-DT

Quick Taq	VF	VR	MilliQ	total
25	1	1	23	50

E.coli in liquid culture that had pspA(pSB1C3) also expressed RFP.

Restriction

pspA	EcoR1	Spe1	BufferM	BSA	MilliQ	total
5	0.3	0.3	3	0.3	21.1	30

And then we did ethanol precipitation

Ethanol precipitation

pspA 11.5ng/µL.

Miniprep

Lacp+pSB3C5-8 77.9µg/mL
Lacp+pSB3C5-10 69.0µg/mL
Kil+DT-4 58.0µg/mL
Kil+DT-7 15.2µg/mL

Restriction

Lacp+pSB3C5-8	Spe1	Pst1	Buffer2	BSA	MilliQ	total
20	0.5	0.5	3	0.5	5.5	30

Kil+DT-4	Xba1	Pst1	Buffer2	BSA	MilliQ	total
20	0.5	0.5	3	0.5	5.5	30

at 37℃ for 1.5 hours
And then we did Gel extraction.

Gel Extraction

Lacp+pSB3C5-8 40.4µg/mL
Kil+DT-4 26.8µg/mL

March 26

Miniprep

torA(pSB1C3) 18.5[ng/µL]
torA-GFP-DT 20.0[ng/µL]

Restriction

torA(pSB1C3)	EcoR1	Pst1	BufferH	BSA	MilliQ	total
5	0.2	0.2	2	0.2	12.4	20
5	0.2	0	2	0.2	12.6	20

torA-GFP-DT	EcoR1	Xba1	Pst1	BufferH	BufferM	BSA	MilliQ	total
5	0.2	0	0.2	2	0	0.2	12.4	20
5	0.2	0	0	2	0	0.2	12.6	20
20	0	0.2	0.2	0	3	0.3	6.3	30

We did Gel extraction and then got ??? 28.7[ng/µL]

Ligation

Lacp (pSB3C5)	torA-GFP-DT	Ligation High Ver.2	total
1	5	3	9

Lacp : 22fmol
torA-GFP-DT : 197fmol

pspA	pSB1C3	Ligation High Ver.2	total
10	1	5	16

pspA	DT	Ligation High Ver.2	total
10	1	5	16

pspA : 180fmol
pSB1C3 : 18fmol
DT : 16fmol

LacP(pSB3C5)	Kil-DT	Ligation High Ver.2	total
1	5	3	9

for 2 hours at 16℃

Transformation

Lacp-Kil-DT	competent cell	total
1	10	11

March 27

Miniprep

We retryed miniprep of torA(pSB1C3).
We got torA and its concentration was 39.3[ng/µL].

Transformation

Name	Well	Sample	Competent Cells	Total	Plate	Colony
BBa_K117004	14J(2011 plate2)	5	20	?	?	?

We added 100[µL] of culture medium before we started culturing the E.coli.

Screening PCR

Quick Taq	VF2	VR	MilliQ	Total
25	1	1	23	50

Lacp-torA-GFP-DT 1, 3, 5, 6, 8, 9 was successful.
pspA-DT was failed.
E.coli that had pspA(pSB1C3) did not make any colony.
Lacp-Kil-DT 1,2,3,4,5,6,7,8 was failed.

Liquid culture

Lacp-torA-GFP-DT

July 23

Transformation

DT(64.4ng/μl)	Competent cell	Plating in SOC medium	Total
1	20	100	121

July 24

Plating in SOC medium

July 25

Transformation

BBa_J23113 from iGEM Kit

LacP from iGEM Kit

July 25

July 26

July 27

Restriction Enzyme Processing

Kil +DT (44.0 ng/μl)	10xBuffer2	BSA	Xbal	MilliQ	Total
13.6	3.0	0.5	0.5	11.9	30.0

LacP(28.7ng/μl)	10xBuffer2	BSA	Spe1	Pst1	Total
20.9	3.0	0.5	0.5	4.6	30.0

July 30

Ligation

kil+DT(XbaI,PstI)	LacP(SpeI,PstI)	Ligation High	Total
30	6	36	72

July 31

Restriction Enzyme Processing

torAGFP+DT	10×M Buffer	BSA	MilliQ	XbaI	PstI	Total
2.5	3.0	0.5	23.4	0.3	0.3	30.0

Liquid Culture

J23113(backbone J61002)

August 1

Restriction Enzyme Processing

lacP+kil+DT	BSA	10×H Buffer	EcoRI	PstI	MilliQ	Total
5.0	0.5	3.0	0.5	0.5	20.5	30.0

37℃ 2h

lacP middle copy	BSA	10×H Buffer	EcoRI	PstI	MilliQ	Total
10.0	0.5	3.0	0.5	0.5	15.5	30.0

37℃ 2h

MIniprep

J23113(backbone J61002)

Liquid culture

J23113(backboneJ61002) Three test tubes of 4 mL LB medium with ampicillin 37℃ overnight

August 2

August 3

Restriction Enzyme processing

pSB4K5	BSA	10×H Buffer	EcoRI	PstI	MilliQ	Total
8.0	0.5	3.0	0.5	0.5	17.5	30.0

B0034	BSA	10×H Buffer	SpeI	PstI	MilliQ	Total
12.0	0.5	3.0	0.5	0.5	13.5	30.0

37℃ 2h

DNA purification

Ligation

lacP+kil+DT	pSB4K5	ligation high	Total
15	15	15	45

16℃ 1h

Miniprep

J23113(backbone J61002) 218.0μg/mL
J23113(backbone J61002) 252.5μg/mL
J23113(backbone J61002) 201.0μg/mL
pSB3C5 45.0μg/ml 1.60 260/280 1.80 260/230
pSB4C5 213.0μg/mL 1.77 260/280 4.40 260/230

August 4

August 5

August 6

August 7

August 8

August 9

August 10

Ligation

B0034 SpeI PstI	GFP+DT XbaI PstI	ligation high	Total
2	3	4	9

16℃ 1h

Transformation

RBS+GFP+DT
lacP+kil+DT
RBS（for control）

To put 2ng DNA in 20μL competent cell and leave it on ice for 30min

Heat shock for 60s at 42℃

To leave on ice for 2min

To spread on ampicillin LB plate

August 11

August 12

August 13

Liquid culture

B0034 3mL ×3 　　　

37℃　overnight

August 14

Miniprep

B0034 0μg/mL

B0034 235.5μg/mL

B0034 76.5μg/mL

Colony PCR

lacP+kil+DT ×5

2×Quick Taq	VF	VF	MilliQ	Total
25	1	1	23	50

25cycles

pspA

10×Buffer for KOD-Plus-Ver2	2mM dNTPs	25mM MgSO4	Primer PsPA f-p	Primer PsPA r-s	KOD-Plus-	MilliQ	Total
5	5	3	1.5	1.5	1	33	50

Electrophoresis

写真貼ってください

positive control(GFP+DT)

negative control(MilliQ)

① ～⑥ colony PCR lacP+kil+DT

次の写真

① 　　　1kb ladder

② 　　　Positive control (GFP+DT)

③ 　　　Negative control (MilliQ)

④ ～⑨　lacP+kil+DT　①～⑥

⑩～⑫　 pspA 10μL ,6×buffer 2μL

⑬　　　　1kb ladder

August 15

Colony PCR

pspA

2×Quick Taq	pspA r-s	pspA f-p	MilliQ	Total
25	1	1	23	50

94℃, 2min
94℃, 30sec
56℃, 30sec
68℃, 1min
→35cycles

August 16

August 17

Colony PCR

pspA

10×Buffer for KOD-Plus-Ver2	2mM dNTPs	25mM MgSO4	Primer PsPA f	Primer PsPA r	KOD-Plus-	MilliQ	Total
5	3	3	1.5	1.5	1	35	50
5	3	5	1.5	1.5	1	33	50

negative control(MilliQ 50μL)

94℃, 2min
94℃, 15sec
55℃, 30sec
68℃, 1min
→35cycles

Electrophoresis

写真貼ってください

①1kb ladder

②, ③　　MgSO4 3μL, pspA

④, ⑤　　MgSO4 5μL, pspA

⑥ negative control(MilliQ)

⑧ 1kb ladder

August 18

August 19

August 20

Restriction

J23113(201.0ng/μL)	Xba1	Pst1	BufferM	BSA	MilliQ	total
10	1	1	3	0.5	14.5	30

August 21

Colony PCR

pspA

10×Buffer	2mM dNTPs	25mM MgSO4	Primer PsPA f	Primer PsPA r	KOD Plus Neo	MilliQ	Total
5	3	5	1.5	1.5	1	33	50
5	3	7	1.5	1.5	1	31	50
5	3	10	1.5	1.5	1	28	50

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 30sec
→25cycles

Restriction

J23113(218ng/μL)	Spe1	Pst1	BufferH	BSA	MilliQ	total
10	1	1	3	0.5	14.5	30

torA-GFP-DT(20ng/μL)	Xba1	Pst1	BufferM	BSA	MilliQ	total
5	0.6	0.6	3	0.5	20.3	30

Electrophoresis

写真貼ってください

① 　　　1kb ladder

②, ③　　torA-GFP-DT(XbaI,PstI)

④, ⑤　　J23113(SpeI,PstI)

Transformation

lacP-torA-GFP-DT(Backbone pSB3C5)
BBa_K11704(Backbone pSB1A2)

Colony PCR

pspA

10×Buffer	2mM dNTPs	25mM MgSO4	Primer PsPA f	Primer PsPA r	KOD Plus Neo	MilliQ	Total
5	5	3	1.5	1.5	1	33	50

10×Buffer	2mM dNTPs	25mM MgSO4	Primer PsPA f-p	Primer PsPA r-s	KOD Plus Neo	MilliQ	Total
5	5	3	1.5	1.5	1	33	50

negative control for pspA

We did PCR without a template.

GFP-DT

10×Buffer	2mM dNTPs	25mM MgSO4	VF	VR	KOD Plus Neo	MilliQ	Total
5	5	3	1.5	1.5	1	33	50

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 30sec

Transformation

Lacp-torA-GFP-DT(Backbone pSB3C5)
J23107-tatABCD

PCR

kil, torA-GFP

10×Buffer	2mM dNTPs	25mM MgSO4	VF	VR	KOD Plus Neo	MilliQ	DNA	Total
5	5	3	1.5	1.5	1	32	1	50

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 30sec
→25cycles

August 22

Restriction

pSB3C5	EcoR1	Pst1	BufferH	BSA	MilliQ	total
13.3	0.5	0.5	3	0.5	12.2	30

Gel extraction

pSB3C5(EcoR1, Pst1)
27.2μg/mL

Transformation(the second time)

Lacp-torA-GFP-DT(Backbone pSB3C5)
J23107-tatABCD

Electrophoresis

写真お願いします。
See "August 21 -Colony PCR-"
1. 1kb ladder
2. pspA(Primer pspA f&r)
3. negative control for 2
4. GFP-DT
5. pspA(Primer pspA f-p&r-s)
6. pspA(Primer pspA f-p&r-s)
7. negative control for 5&6

PCR

Lacp-GFP-DT(a kit of biological parts)

10×Buffer	2mM dNTPs	25mM MgSO4	VF	VR	KOD Plus Neo	MilliQ	DNA	Total
5	5	3	1.5	1.5	1	32	1	50

Colony PCR

pspA

10×Buffer	2mM dNTPs	25mM MgSO4	Primer pspA f	Primer pspA r	KOD Plus Neo	MilliQ	Total
5	5	3	1.5	1.5	1	33	50

10×Buffer	2mM dNTPs	25mM MgSO4	Primer pspA f-p	Primer pspA r-s	KOD Plus Neo	MilliQ	Total
5	5	3	1.5	1.5	1	33	50

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 30sec
→30cycles

Electrophoresis

写真お願いします。
1. 1kb ladder
2. pspA(Primer pspA f&r)
3. pspA(Primer pspA f-p&r-s)
4. negative control for pspA(Primer pspA f&r)
5. negative control for pspA(Primer pspA f-p&r-s)

Transformation

Kil

August 23

Colony PCR

J23107-tatABCD
VF and VR were used for amplifying approximately 2800bp

10×Buffer	2mM dNTPs	25mM MgSO4	VF	VR	KOD Plus Neo	MilliQ	DNA	Total
5	5	3	1.5	1.5	1	32.5	0.5	50

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 90sec
→25cycles

August 24

Purification of PCR products

pspA(Primer pspA f-p&r-s)
84.1μg/mL
→See "August 22 -Colony PCR-"

Restriction

pspA	EcoR1	Pst1	BufferH	BSA	MilliQ	total
20	0.5	0.5	3	0.5	5.5	30

pspA	Xba1	Pst1	BufferM	BSA	MilliQ	total
20	0.5	0.5	3	0.5	5.5	30

at 37℃ for 1h

Gel extraction

pspA(EcoR1, Pst1) 30.7μg/mL
pspA(Xba1, Pst1) 44.0μg/mL

Restriction

pSB1C3(82.9μg/mL)	EcoR1	Pst1	BufferH	BSA	MilliQ	total
20	0.5	0.5	3	0.5	5.5	30

→Gel extraction

Electrophoresis

写真お願いします。
1. 1kb ladder
2. J23107-tatABCD →See "August 23 -PCR-"
3. negative control for J23107-tatABCD
4. pSB1C3(EcoR1, Pst1)

Ligation

pSB3C5(EcoR1, Pst1)	pspA(EcoR1, Pst1)	Ligation High Ver.2	total
1	5	3	9

16℃, overnight

August 25

August 26

August 27

Ligation

pSB1C3(EcoR1, Pst1)	pspA(EcoR1, Pst1)	Ligation High Ver.2	total
1	5	3	9

16℃, overnight

Colony PCR

Kil (1-7)

10×Buffer	2mM dNTPs	25mM MgSO4	VF	VR	KOD Plus Neo	MilliQ	Total
5	5	3	1.5	1.5	1	33	50

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 30sec
→30cycles
写真お願いします。

Purification of PCR products

torA-GFP →See "August 21 -PCR-"

Restriction

Lacp(Backbone:pSB3C5)	Spe1	Pst1	BufferH	BSA	MilliQ	total
20	0.5	0.5	3	0.5	5.5	30

at 37℃ for 1h
→Gel extraction

torA-GFP	Xba1	Pst1	BufferM	BSA	MilliQ	total
20	0.5	0.5	3	0.5	5.5	30

at 37℃ for 1h
→Purification

Purification of PCR products

J23107-tatABCD
42.0μg/mL
→See "August 23 -PCR-"

Restriction

J23107-tatABCD	Spe1	EcoR1	BufferM	BSA	MilliQ	total
20	0.5	0.5	3	0.5	5.5	30

August 28

Colony PCR

Kil (1-16)

10×Buffer	2mM dNTPs	25mM MgSO4	VF	VR	KOD Plus Neo	MilliQ	Total
5	5	3	1.5	1.5	1	33	50

写真お願いします。

Transformation

pspA-pSB1C3
pspA-pSB3C5

Liquid culture

Kil-3 →Miniprep 100.4μg/mL
Kil-6
Kil-7 →Miniprep 77.7μg/mL
Lacp-torA-GFP-1 →Miniprep 88.3μg/mL
Lacp-torA-GFP-2 →Miniprep 85.0μg/mL
Lacp-torA-GFP-3 →Miniprep +++

August 29

Kil assay

evernoteに結果のグラフがあります。

Liquid culture

pspA-pSB1C3(1-3)
pspA-pSB3C5(1-3)

Restriction

Lacp-Kil-DT(134.7ng/μL)	EcoR1	Spe1	BufferM	MilliQ	total
10	1	1	3	15	30

37℃, overnight

Colony PCR

Lacp-torA-GFP-DT (1-5)
pspA-pSB1C3 (1-5)
pspA-pSB3C5 (1-6)

10×Buffer	2mM dNTPs	25mM MgSO4	VF	VR	KOD Plus Neo	MilliQ	Total
5	5	3	1.5	1.5	1	33	50

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 40sec
→30cycles

Gel extraction

J23107-tatABCD(EcoR1, Pst1) -5.9μg/mL

August 30

Ligation

J23107-tatABCD(EcoR1, Spe1)22.2μg/mL	DT(EcoR1, Xba1)37.5μg/mL	Ligation High Ver.2	total
10	1	9	20

at 16℃ for 1h

Transformation

J23107-tatABCD-DT (Amp resistance)

Electrophoresis

写真お願いします。
1. Lacp-torA-GFP-DT-1
2. Lacp-torA-GFP-DT-2
3. Lacp-torA-GFP-DT-3
4. Lacp-torA-GFP-DT-4
5. Lacp-torA-GFP-DT-5
6. pspA-pSB1C3-1
7. pspA-pSB1C3-2
8. pspA-pSB1C3-3
9. pspA-pSB1C3-4
10.pspA-pSB1C3-5
11.pspA-pSB3C5-1
12.pspA-pSB3C5-2
13.pspA-pSB3C5-3
14.pspA-pSB3C5-4
15.pspA-pSB3C5-5
16.pspA-pSB3C5-6

Miniprep

pspA-pSB3C5-1 176μg/mL
pspA-pSB3C5-2 214μg/mL
pspA-pSB3C5-3 461μg/mL
pspA-pSB1C3-2 79μg/mL

Colony PCR

Lacp-torA-GFP-DT (6-13)

2×Quick Taq	VF	VR	MilliQ	Total
25	1	1	23	50

94℃, 2min
94℃, 30sec
56℃, 30sec
68℃, 1min
→30cycles

Restriction

pspA-pSB1C3	EcoR1	Spe1	BufferM	BSA	MilliQ	total
15	0.5	0.5	5	0.5	28.5	50

at 37℃ for 1h

Electrophoresis

写真お願いします。
1. 1kb ladder
3. pspA-pSB1C3(EcoR1, Spe1)

August 31

Gel extraction

写真お願いします。
pspA-pSB1C3(EcoR1, Spe1) 8.0μg/mL

Purification

Lacp-GFP-DT -0.7μg/mL

Ligation

pspA(EcoR1, Spe1)	DT(EcoR1, Xba1)	Ligation High Ver.2	total
25	1	13	39

at 16℃ for 1h

Transformation

pspA-DT (Amp resistance)

September 1

September 2

Colony PCR

J23107-tatABCD-DT (1-8)

2×Quick Taq	VF	VR	MilliQ	Total
25	1	1	23	50

94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 3min
→25cycles

pspA-DT (2-8)

2×Quick Taq	VF	VR	MilliQ	Total
25	1	1	23	50

Extension, 10sec
→25cycles

Restriction

LacP-torA-GFP-DT	EcoR1	Spe1	BufferH	MilliQ	total
10	1	1	2	6	20

at 4℃ for overnight

Electrophoresis

写真お願いします。
1. 1kb ladder
3. J23107-tatABCD-DT-1
4. J23107-tatABCD-DT-3
5. J23107-tatABCD-DT-4
6. J23107-tatABCD-DT-5
7. J23107-tatABCD-DT-6
8. J23107-tatABCD-DT-7
9. J23107-tatABCD-DT-8

写真お願いします。
1. 1kb ladder
3. J23107-tatABCD-DT-1
4. J23107-tatABCD-DT-3
5. J23107-tatABCD-DT-4
6. J23107-tatABCD-DT-5
7. J23107-tatABCD-DT-6
8. J23107-tatABCD-DT-7
9. J23107-tatABCD-DT-8
11. LacP-pSB3C5(SpeI, PstI)

写真お願いします。
1. 1kb ladder
3. pspA-DT-2
4. pspA-DT-3
5. pspA-DT-4
6. pspA-DT-5
7. pspA-DT-6
8. pspA-DT-7
9. pspA-DT-8

September 3

PCR

tatABCD-DT

10×Buffer	2mM dNTPs	25mM MgSO4	VF	VR	KOD Plus Neo	MilliQ	DNA	Total
5	5	3	1.5	1.5	1	32	1	50

Gel extraction

写真お願いします。
LacP-torA-GFP-DT →See "September 5 -Gel extraction-"

Restriction

torA-GFP-DT	XbaI	PstI	BufferM	MilliQ	total
20	2	2	4	12	40

Purification

J23107-tatABCD 29.6μg/mL 1.56(260/280) 1.60(260/230)

Electrophoresis

写真お願いします。
1. 1kb ladder
2. J23107-tatABCD

Liquid culture

LacP-kil-DT(1)
pspA-DT(4, 7)

PCR

J23107-tatABCD

10×Buffer	2mM dNTPs	25mM MgSO4	VF	VR	KOD Plus Neo	MilliQ	DNA	Total
5	5	3	1.5	1.5	1	29	4	50

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 10sec
→30cycles

Gel extraction

写真お願いします。
LacP-torA-GFP-DT 31μg/mL 1.28(260/280) 0.49(260/230) LacP-torA-GFP-DT 8μg/mL 1.32(260/280) 0.83(260/230)

Transformation

J23107-tatABCD (Amp resistance)
J23113-kil-DT (Amp resistance)
J23100-kil-DT (CP resistance)
J23101-kil-DT (CP resistance)
J23106-kil-DT (CP resistance)
J23115-kil-DT (CP resistance)→We didn't transform because of lack of culture medium.
J23105-kil-DT (CP resistance)→We didn't transform because of lack of culture medium.

Electrophoresis

写真お願いします。
1. 1kb ladder
2-7. LacP-torA-GFP-DT

Colony PCR

LacP-torA-GFP-DT (14-21) J23113-kil-DT (Amp resistance, 1-4) J23113-kil-DT (CP resistance, 1-4)

2×Quick Taq	primer-f	primer-r	MilliQ	Total
25	1	1	23	50

94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 1min
→25cycles

September 4

Liquid culture

LacP-kil-DT(4, 5)(Amp resistance)
pspA-DT(3, 6, 7)(Amp resistance)

PCR

We did PCR 10 more cycles to amplify products of PCR that we had done yesterday because we could not amplify J23107-tatABCD on September 3. 94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 1.5min
→30cycles

Electrophoresis

写真お願いします。
1-8. Lacp-torA-GFP-DT

写真お願いします。
1-4. J23113-kil-DT (Amp resistance)
5-8. J23113-kil-DT (CP resistance)

Liquid culture

LacP-torA-GFP-DT
J23113-kil-DT
LacP-kil-DT(with IPTG)
LacP-kil-DT(without IPTG)

After 20 hour, we quantified OD600.

plasmid	OD600
LacP-kil-DT(with IPTG)	2.116
LacP-kil-DT(without IPTG)	2.320

We cultured CP tolerance plasmid and give an antibiotic(Amp or Kan or none) after 2 hours. We quantify OD600.

time	1	2	3
2hours	0.131	0.100	0.606
add an antibiotic	Amp	Kan	none
7hours	0.491	0.988	2.634
19hours	2.253	0.815	2.742

September 5

Colony PCR

J23107-tatABCD (1-7)

buffer	2mM dNTPs	25mM MgSO4	primer-f	primer-r	KOD Plus Neo	DW	Total
5	5	3	1.5	1.5	1	33	50

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 1.25min
→30cycles

Gel extraction

by Kato
See "September 3 -Gel extraction-" LacP-torA-GFP-DT 15.9μg/mL 1.29(260/280) 0.38(260/230) LacP-torA-GFP-DT 17.2μg/mL 1.23(260/280) 0.48(260/230)

Miniprep

J23113-kil-DT 85.7μg/mL 1.76(260/280) 1.97(260/230)
LacP-torA-GFP-DT 126.6μg/mL 1.81(260/280) 2.09(260/230)
LacP-kil-DT-4 62.5μg/mL 1.79(260/280) 1.99(260/230)
LacP-kil-DT-5 56.0μg/mL 1.79(260/280) 2.01(260/230)
pspA-DT-3 322μg/mL 1.45(260/280) 1.42(260/230)
pspA-DT-6 147.6μg/mL 1.25(260/280) 1.37(260/230)
pspA-DT-7 145.5μg/mL 1.24(260/280) 1.27(260/230)

Restriction

pspA-DT-6	XbaI	PstI	BufferM	BSA	MilliQ	total
20	0.5	0.5	3	0.5	5.5	30

at 37℃ for 1h

Electrophoresis

写真お願いします。
J23107-tatABCD (1-7)

Colony PCR

J23107-tatABCD (8-23)

2×Quick Taq	VF2	VR	MilliQ	Total
25	1	1	23	50

94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 1.5min
→30cycles

Gel extraction

pspA-DT 10.2μg/mL 1.42(260/280) 0.76(260/230)

Ligation

pspA-DT(XbaI, PstI)	LacP(pSB3C5)(SpeI, PstI)	Ligation High Ver.2	total
5	1	3	9

at 16℃

Electrophoresis

写真お願いします。
Lacp-torA-GFP-DT(8-15)

写真お願いします。
Lacp-torA-GFP-DT(9-23)

Ligation

Lacp-torA-GFP-DT(EcoRI, SpeI)	DT 17.2μg/mL(XbaI, PstI)	Ligation High Ver.2	total
15	25	20	60

Liquid culture

J23107-tatABCD-8
J23107-tatABCD-20
Lacp-torA-GFP-DT-21

September 6

Miniprep

J23107-tatABCD-8 82.4μg/mL 2.06(260/280) 2.95(260/230)
J23107-tatABCD-20 102.9μg/mL 2.04(260/280) 2.94(260/230)

Restriction

J23107-tatABCD 102.9μg/mL	SpeI	PstI	BufferH	BSA	MilliQ	total
10	0.5	0.5	3	0.5	15.5	30

at 37℃ for 1h

Transformation

LacP-pspA-DT(pSB3C5)

DNA	Competent cell	total
2	20	22

Gel extraction

写真お願いします。
J23107-tatABCD(SpeI, PstI) →We failed gel extraction because we mistook steps of experiment.

Electrophoresis

写真お願いします。
LacP-torA-GFP-DT-pspA-DT

Restriction

by Terasaka

J23107-tatABCD	SpeI	PstI	BufferH	BSA	MilliQ	total
10	0.5	0.5	3	0.5	15.5	30

Gel extraction

写真お願いします。
J23107-tatABCD(SpeI, PstI) →See "September 7 -Gel extraction-"

September 7

Liquid culture

Lacp-torA-GFP-DT-1 (CP resistance)
Lacp-torA-GFP-DT-21 (CP resistance)

preculture for 1 hour

Colony PCR

Lacp-torA-GFP-DT (1-8) Lacp-pspA-DT (1-8)

2×Quick Taq	VF2	VR	MilliQ	Total
25	1	1	23	50

94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 1.5min
→30cycles

Restriction

by Terasaka

J23107-tatABCD	EcoRI	SpeI	BufferM	BSA	MilliQ	total
10	0.5	0.5	3	0.5	15.5	30

GFP-DT	XbaI	PstI	BufferM	BSA	MilliQ	total
15	0.5	0.5	3	0.5	10.5	30

Electrophoresis

写真お願いします。
① -⑧. LacP-pspA-DT
1-8. LacP-torA-GFP-DT

Gel extraction

写真お願いします。

J23107-tatABCD(EcoR1, Spe1)
J23107-tatABCD(Pst1, Spe1) ①57.6μg/mL ②23.4μg/mL
GFP-DT(Xba1, Pst1)

Restriction

pspA-DT(145.5μg/mL)	Xba1	Pst1	BufferM	BSA	MilliQ	total
20	0.5	0.5	3	0.5	5.5	30

37℃, overnight

Liquid culture

Lacp-torA-GFP-DT-7 (LB 2mL)

+IPTG 0mM, 0.05mM, 0.1mM, 0.15mM, 0.2mM, 0.3mM

Lacp-torA-GFP-DT-7 (LB 5mL)

September 8

Check the effect of Amp

We used 5mL of the liquid culture medium with Lacp-torA-GFP-DT-7.
→See "September 7 -Liquid culture-"
We diluted it with LB until OD600=0.444, and dispensed it.
The dispense volume was 1.3mL.
We added 0/13/130μL of Amp(50mg/mL) to it.

5.5h after incubating at 37℃

Amp	0μL	13μL	130μL
OD600	2.457	2.696	0.310

Observation through a confocal microscope

We used the liquid culture media with Lacp-torA-GFP-DT + 13μL of Amp(50mg/mL) + IPTG 0/0.1/0.15mM.
Their OD600 was 0.47.
3h after incubating at 37℃, we eliminated each supernatant using a centrifuge, and diluted them with LB to which Amp was added.
2.5h after incubating at 37℃, we observed them through a confocal microscope.
We failed to observe it.

Gel extraction

pspA-DT -5.5μg/mL

Restriction

pspA-DT-3(322μg/mL)	Xba1	Pst1	BufferM	BSA	MilliQ	total
10	1	1	3	3	12	30

at 37℃ for 1h

September 9

Restriction

J23107-tatABCD(102.9μg/mL)	EcoR1	Spe1	BufferM	MilliQ	total
10	1	1	3	15	30

Electrophoresis

写真お願いします。
1. J23107-tatABCD(EcoR1, Spe1)
2. J23107-tatABCD(before restriction)
3. pspA-DT(Xba1, Pst1)
4. pspA-DT(before restriction)

Gel extraction

写真(2枚)お願いします。

J23107-tatABCD(EcoR1, Spe1) 26μg/mL
pspA-DT(Xba1, Pst1) 199μg/mL

Liquid culture

J23100/J23101/J23106-1
pspA-DT-5 ×2
Lacp-torA-GFP-DT-21
GFP generator-2
Lacp-pspA-DT-6

September 10

Miniprep

J23100-1 105.5μg/mL
J23101-1 76.0μg/mL
J23106-1 90.9μg/mL
pspA-DT-5 ①104.1μg/mL ②80.0μg/mL
Lacp-torA-GFP-DT-21 79.6μg/mL
GFP generator-2 84.1μg/mL
Lacp-pspA-DT-6 99.8μg/mL

Ligation

J23107-tatABCD-DT

J23107-tatABCD(EcoR1, Spe1)	DT(EcoR1, Xba1)	Ligation High Ver.2	total
10	5	7.5	22.5

Negative control for J23107-tatABCD-DT

DT(EcoR1, Xba1)	Ligation High Ver.2	total
5	5	10

J23107-tatABCD-pspA-DT

J23107-tatABCD(Spe1, Pst1)	pspA-DT(Xba1, Pst1)	Ligation High Ver.2	total
3	6	9	18

Negative control for J23107-tatABCD-pspA-DT

J23107-tatABCD(Spe1, Pst1)	Ligation High Ver.2	total
3	3	6

Restriction

写真お願いします。

DNA	Spe1	Pst1	BufferH	MilliQ	total
15	1	1	3	10	30

DNA=J23100(105.5μg/mL), J23101(76.0μg/mL), J23106(90.9μg/mL)
at 37℃

Gel extraction

写真（２まい）お願いします。
J23100 27.1μg/mL
J23101 50.2μg/mL
J23106 16.1μg/mL

Ligation

J23100/J23101/J23106-kil-DT

J23100/J23101/J23106(Spe1, Pst1)	kil-DT(Xba1, Pst1)	Ligation High Ver.2	total
1	5	3	9

Negative control for J23100/J23101/J23106-kil-DT

J23100/J23101/J23106(Spe1, Pst1)	MilliQ	Ligation High Ver.2	total
1	5	3	9

Transformation

J23100/J23101/J23106-kil-DT

September 11

Colony PCR

J23107-tatABCD-DT (1-8)
J23107-tatABCD-pspA-DT (1-8)

2×Quick Taq	VF2	VR	MilliQ	Total
25	1	1	23	50

94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 3.5min
→30cycles

Electrophoresis

写真お願いします。

J23107-tatABCD-DT (1-8)
J23107-tatABCD-pspA-DT (1-8)

Restriction

写真お願いします。

GFP generator(84.1μg/mL)	Xba1	Pst1	BufferM	BSA	MilliQ	total
15	1	1	3	3	7	30

Lacp-torA-GFP-DT(85.0μg/mL)	Spe1	Pst1	BufferH	MilliQ	total
15	1	1	3	10	30

Lacp-pspA-DT(99.8μg/mL)	Xba1	Pst1	BufferM	BSA	MilliQ	total
15	1	1	3	3	7	30

Liquid culture

pSB1C3(self-ligation) ×5 in LB 3mL+CP 3μL
When OD600=approximately 0.6, we added 0/30/60/150/300μL of Amp(50mg/mL) to each liquid culture medium.
1h and 5h after incubating at 37℃, we measured OD600.

	1h	5h
0	-0.020	0.403
30	-0.046	0.544
60	0.263	0.007
150	0.061	0.839
300	0.093	1.441

Ligation

pSB3C5(EcoR1, Pst1)	pspA-DT(Xba1, Pst1)	J23107-tatABCD(EcoR1, Spe1)	Ligation High Ver.2	total
1	3	5	4.5	13.5

4℃, overnight

Colony PCR

J23100-kil-DT (1-6)
J23101-kil-DT (1-7)
J23106-kil-DT (1-2)

2×Quick Taq	VF	VR	MilliQ	Total
23	1	1	25	50

Gel extraction

写真お願いします。

GFP generator (Xba1, Pst1) 14.1μg/mL
lacp-torA-GFP-DT (Spe1, Pst1) 42.9μg/mL 1.13(260/280) 1.19(260/230)
lacp-pspA-DT (Xba1, Pst1) 20.0μg/mL 1.01(260/280) 1.30(260/230)

Electrophoresis

写真お願いします。

J23107-tatABCD-DT (1-8)
J23106-kil-DT (1, 2)
J23100-kil-DT (1-4)

Liquid culture

Lacp-torA-GFP-DT
pSB1C3

Colony PCR

We did PCR 10 more cycle to amplify products of PCR on 9/11 of J23107-tatABCD-pspA-DT and J23107-tatABCD-DT.

September 12

Colony PCR

J23107-tatABCD-pspA-DT (9-15) 94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 4min
→26cycles

Check the effect of Amp

We cultured pSB1C3 for overnight.
→See "September 7 -Liquid culture-"
We diluted it with LB, and dispensed it.
We added Amp(50mg/mL) to it until density of Amp was 1/10, 1/30, 1/50 .

Amp	start	5 hours
1/10	0.557	0.077
1/30	0.628	0.166
1/50	0.472	0.055
0	0.594	2.585

Electrophoresis

写真お願いします。

J23107-tatABCD-pspA-DT

写真お願いします。

J23101-kil-DT (1-7)

Restriction

pspA(3C5) (106μg/mL)	EcoR1	Pst1	BufferH	MilliQ	total
15	1	1	3	10	30

pspA-DT-3	Xba1	Pst1	BufferM	BSA	MilliQ	total
15	1	1	3	3	7	30

J23107-tatABCD (102.4μg/mL)	EcoR1	Spe1	BufferM	MilliQ	total
15	1	1	3	10	30

Electrophoresis

写真お願いします。

pspA(3C5)(EcoR1, Pst1)
pspA-DT(Xba1, Pst1)
J23107-tatABCD(EcoR1, Spe1)
pSB1C3(EcoR1, Spe1)

Transformation

J23107-tatABCD-pspA-DT(pSB3C5)

DNA	Competent cell	total
2	20	22

on ice for 30 minites heatshock at 42℃ for 1 hour on ice 2 minites then add SOC medium 200μl preculture at 37℃ for 1 hour incubate CP culture plate

Gel extraction

写真お願いします。

pspA(3C5)(EcoR1, Pst1) 26.8μg/mL 1.27(260/280) 0.32(260/230)
pspA-DT(Xba1, Pst1) 26.1μg/mL 1.13(260/280) 0.55(260/230)
J23107-tatABCD(EcoR1, Spe1) 1.5μg/mL 1.99(260/280) 0.06(260/230)

Ligation

pSB3C5(EcoR1, Pst1) 26.8μg/mL	pspA-DT(Xba1, Pst1) 26.1μg/mL	J23107-tatABCD(EcoR1, Spe1) 1.5μg/mL	Ligation High Ver.2	total
1	3	6	5	15

pSB3C5(EcoR1, Pst1) 26.8μg/mL	MilliQ	Ligation High Ver.2	total
1	9	5	15

LacP(pSB3C5)(Spe1, Pst1) 10μg/mL	GFP-DT(Xba1, Pst1) 7.0μg/mL	Ligation High Ver.2	total
1	5	3	9

LacP(pSB3C5)(Spe1, Pst1) 10μg/mL	MilliQ	Ligation High Ver.2	total
1	5	3	9

4℃, overnight

Observation through a confocal microscope

Evernoteに上がっている写真をお願いします。

September 13

Miniprep

J23107-tatABCD-pspA-DT(pSB3C5)-1 130μg/mL 1.37(260/280) 0.79(260/230)

Restriction

J23107-tatABCD-pspA-DT(pSB3C5) (130μg/mL)	Spe1	Pst1	BufferH	MilliQ	total
10	1	1	3	15	30

Transformation

LacP-GFP generater
LacP
J23107-tatABCD-pspA-DT(pSB3C5)
J23107-tatABCD

Colony PCR

J23107-tatABCD-pspA-DT
→See "September 14 -Colony PCR-"

September 14

Colony PCR

写真お願いします。
See "September 13 -Colony PCR-"
J23107-tatABCD-pspA-DT

Colony PCR

J23107-tatABCD-pspA-DT (1-6)

2×Quick Taq	VF	VR	MilliQ	Total
23	1	1	25	50

94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 4min
→30cycles

September 15

September 16

September 17

September 18

Restriction enzyme processing

J23101-Kil-DT	BufferM	XbaI	Pst	BSA	MilliQ	total
15	3	1	1	3	7	30

J23101-Kil-DT	BufferM	XbaI	BSA	MilliQ	total
3	1	0.5	1	4.5	10

J23101-Kil-DT	BufferH	Pst	MilliQ	total
3	1	0.5	5.5	10

37℃

September 19

GeneClean II

LacP-torA-GFP-DT
4k5

Ligation

LacP-torA-GFP-DT(X,P) 9.7μg/mL	pSB4K5(E,P) 9.6μg/mL	J23107-tatABCD-PsPA-DT(E,S) 14.2μg/mL	Ligation High Ver.2	total
7	2	7	8	24μL

pSB4K5(E,P) 9.6μg/mL	Ligation High Ver.2	MilliQ	total
2	1	21	24μL

incubate at 37℃ for 2h

Electrophoresis

J23101-kil-DT(X,P) (X) (P)
Lane1: 1kb ladder
Lane2: empty
Lane3: plasmid
Lane4: X
Lane5: P
Lane6: X,P

Liquid Culture

We did liquid culture J23106-kil-DT(9/10, Amp)'s 6,7 at LB medium+Amp.

Transformation

J2-tat-psp-DT-LacP-torAGFP-DT
pSB4K5 negative control

Liquid culture

J2-tat-psp-DT-LacP-torAGFP-DT:1~5

Colony PCR

J2-tat-psp-DT-LacP-torAGFP-DT

VF2	VR	Quick tag	MilliQ	total
1	1	25	23	50

Predenature 94℃ 2min
Denature 94℃ 30sec
Annealing 55℃ 30sec
Extension 68℃ 5min
→30cycles

Restriction enzyme processing

LacP-kil-DT(4) 62.5μg/mL	XbaI	PstI	10xBSA	MilliQ	BufferM	total
10	1	1	3	12	3	30

LacP-kil-DT(4)	EcoRI	PstI	MilliQ	BufferH	total
10	1	1	15	3	30

LacP-kil-DT	XbaI	10xBSA	MilliQ	BufferM	total
2	0.5	1	5.5	1	10

LacP-kil-DT	EcoRI	MilliQ	BufferH	total
2	0.5	6.5	1	10

LacP-kil-DT	PstI	MilliQ	BufferH	total
2	0.5	6.5	1	10

at 37℃, 2 hours

Electrophoresis

Lane1: 1kb ladder
Lane2: empty
Lane3: plasmid
Lane4: E
Lane5: X
Lane6: P
Lane7: E,P
Lane8: X,P

Electrophoresis

J2-tat-psp-DTLacP-torAGFP-DT

Lane1: ladder
Lane2: 1
Lane3: 2
Lane4: 3
Lane5: 4
Lane6: 5

Ligation

LacP-torA-GFP-DT(X,P) 9.7μg/mL	pSB4K5(E,P) 9.6μg/mL	J23107-tatABCD-PsPA-DT(E,S) 14.2μg/mL	Ligation High Ver.2	total
7	4	7	9	27μL

(Negative control)

pSB4K5(E,P) 9.6μg/mL	Ligation High Ver.2	MilliQ	total
4	9	14	27μL

Restriction enzyme processing

torA-R9 101.8μg/mL	bufferH	MilliQ	EcoRI	Pst	total
10	3	15	1	1	30

torA-R9 101.8μg/mL	bufferH	MilliQ	EcoRI	Pst	total
3	1	5.5	0.5	0	10

torA-R9 101.8μg/mL	bufferH	MilliQ	EcoRI	Pst	total
3	1	5.5	0	0.5	10

PCR

torA-R9	MilliQ	10xPCR buffer for KOD plus neo	2mM dNTP3	25mM MgSO4	M13	KOD plus neo	total
0.5	34	5	5	3	1.5	1	50

Predenature 94℃ 2min
Denature 98℃ 10sec
Annealing 60℃ 30sec
Extension 68℃ 20sec
→35cycles

Electrophoresis

torA-R9,plasmid,(E),(P),(E,P)

Lane1: 1kb ladder
Lane2: empty
Lane3: plasmid
Lane4: E
Lane5: P
Lane6: E,P
Lane7: empty
Lane8: 1kb ladder

Liquid culture

LacP-tatA9FT-DT (9/4)

PCR purification

torA-R9 68.0μg/mL

September 20

Gel extraction

LacP-kil-DT (E,P) (X,P)

Restriction enzyme processing

torA-R9 68.0μg/mL	bufferM	SpeI	MilliQ	Total
20	3	1	6	30

at 37℃, 2h

Ligation

LacP-kil-DT(X,P) 12.4μg/mL	pSB1C3(E,P) 34.2μg/mL	J23107-tatABCD-PsPA-DT(E,S) 14.2μg/mL	Ligation High Ver.2	total
7	4	7	9	27μL

16℃, 1h

(Negative control)

pSB1C3(E,P)	Ligation High Ver.2	MilliQ	total
4	9	14	27μL

Transformation

Using above Ligation and Negative control's product and torA-R9.

Miniprep

J23106-kil-DT 6; 12.0μg/mL, (260/280): 1.78, (260/230): 1.73
J23106-kil-DT 7; 13.6μg/mL, (260/280): 1.85, (260/230): 1.73

Restriction enzyme processing

GFP-DT 84.1μg/mL	bufferM	10xBSA	BbaI	MilliQ	total
15	3	3	1	8	30

at 37℃, 2 hours

Colony PCR

We made J2-tat-psp-DT-LacP-torAGFP-DT's 1~8 samples.

Quick tag	tat f primer	VR	MilliQ	total
25	1	1	23	50

Predenature 94℃ 2min
Denature 94℃ 30sec
Annealing 54℃ 30sec
Extension 68℃ 4min
→30cycles

Liquid culture

We did liquid culture J2-tat-psp-DT-LacP-torAGFP-DT's 1~4 samples.

Electrophoresis

J2-tat-psp-DT-LacP-torAGFP-DT

Lane1: 1kb ladder
Lane2: 1
Lane3: 2
Lane4: 3
Lane5: 4
Lane6: 5
Lane7: 6
Lane8: 7
Lane9: 8
Lane10: 1kb ladder
Lane11: empty
Lane12: empty

torA-R9 (E,P): 31μg/mL, (280/280): 0.80, (280/230): 0.39
1C3(E,P): 22μg/mL, (280/280):1.33, (280/230):0.6
torA-R9 (S): 56μg/mL, (280/280): 1.11, (280/230): 0.69
GFP-DT(X): 45μg/mL, (280/280): 1.15, (280/230): 0.88

Ligation

torA-R9 (E,P)	1C3(E,P)	Ligation High Ver.2	total
8	2	5	15μL

torA-R9 (S)	1C3(X)	Ligation High Ver.2	total
3	3	3	9μL

at 16℃, 1hour

Colony PCTR

J23107-tat-psp-DT-lacP-torA-GFP-DT (4k5)
numbering 9~15

Transformation

J23107-tat-psp-DT-lacP-torA-GFP-DT (4k5)
J23107-tat-psp-DT-lacP-torA-kil-DT(1C3)
Negative control (1C3)
Negative control(4K5)
torA-R9(1C3)

Liquid culture

J23107-tat-pspA-DT-LacP-torA-GFP-DT: colony PCR(9~15)

2xQuick tag	VF	VR	MilliQ	total
25	1	1	23	50

Predenature 94℃ 2min
Denature 94℃ 30sec
Annealing 55℃ 30sec
Extension 68℃ 5min
→30cycles

PCR

torA-R9-GFP-DT	MilliQ	KOD plus ver.2 buffer	dNTP3	MgSO4	M13	VR	KOD plus	total
1	35	5	5	2	1.5	1.5	1	52

Predenature 94℃ 2min
Denature 94℃ 15sec
Annealing 55℃ 30sec
Extension 68℃ 5min
→30cycles

Miniprep

J2-tat-psp-DT-LacP-torA-GFP-DT:2,3
2: 10.4μg/mL, (250/280): 1.45, (260/230): 0.95
3: 6.0μg/mL, (250/280): 1.32, (260/230): 0.79
(*)100μL

Electrophoresis

torA-R9-GFP-DT(after PCR

September 21

September 22

September 23

Electrophoresis

写真お願いします。

LacP-torA-GFP-DT(EcoR1, Pst1)

lane_1 1kb ladder lane_3 DNA lane_4 DNA lane_5 DNA

Gel extraction

写真お願いします。

J23107-tatABCD-pspA-DT(pSB1C3)(Spe1, Pst1)
LacP-torA-GFP-DT(Xba1, Pst1)

lane_1 1kb ladder lane_3 J23107-tatABCD-pspA-DT(pSB1C3)(Spe1, Pst1) lane_5 LacP-torA-GFP-DT(Xba1, Pst1)

Restriction

LacP-torA-GFP-DT (78.4μg/mL)	Xba1	Pst1	BufferM	BSA	MilliQ	total
15	1	1	3	3	7	30

LacP-torA-GFP-DT (78.4μg/mL)	Xba1	BufferM	BSA	MilliQ	total
3	0.5	1	1	4.5	10

J23107-tatABCD-pspA-DT(pSB1C3) (78μg/mL)	Spe1	Pst1	BufferH	MilliQ	total
9	1	1	2	7	20

Ligation

LacP-torA-GFP-DT(EcoR1, Pst1) (5.5μg/mL)	pSB4K5 (9.6μg/mL)	Ligation High Ver.2	total
8	4	6	18

at 16℃ for 1 hour

Liquid culture

LacP-tatABCD-pspA-DT (2, 3, 5, 6)

Colony PCR

LacP-tatABCD-pspA-DT (1-6)

2×Quick Taq	VF2	VR	MilliQ	Total
25	1	1	23	50

94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 4min
→30cycles

Electrophoresis

写真お願いします。
①, LacP-torA-GFP-DT
②, LacP-torA-GFP-DT(Xba1, Pst1)
③, LacP-torA-GFP-DT(Xba1)
④, LacP-torA-GFP-DT(Pst1)
⑤, J23107-tatABCD-pspA-DT(Spe1, Pst1)

Liquid culture

LacP-torA-GFP-DT (4, 7)
LacP-GFP generater (1-3)
T7-6His-GFP-DT (1, 2)

Gel extraction

写真お願いします。

LacP-torA-GFP-DT (Xba1, Pst1) 6.8μg/mL 1.27(260/280) 0.40(260/230)

Purification

J23107-tatABCD-pspA-DT(Spe1, Pst1) 15.1μg/mL 1.49(260/280) 0.80(260/230)

Ligation

J23107-tatABCD-pspA-DT(Spe1, Pst1) (15.1μg/mL)	LacP-torA-GFP-DT (Xba1, Pst1) (6.8μg/mL)	Ligation High Ver.2	total
10	4	7	21

J23107-tatABCD-pspA-DT(Spe1, Pst1) (15.1μg/mL)	LacP-kil-DT (Xba1, Pst1) (12.4μg/mL)	Ligation High Ver.2	total
8	2	5	15

at 16℃ for 1 hour

Transformation

LacP-torA-GFP-DT(pSB4K5)
J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT
J23107-tatABCD-pspA-DT-LacP-kil-DT

DNA	competent cell	total
2	10	12

preculture at 37℃ for 1 hour culture at 37℃ for 16 hours

Electrophoresis

写真お願いします。
J23107-tatABCD-pspA-DT(pSB1C3) (1-6)
lane_1 1kb ladder lane_2~7 DNA lane_8 1kb ladder

Team:Kyoto/Secretion/Notebook

From 2012.igem.org

Contents

Secretion Notebook

February 7

February 8

February 9

February 10

February 11

February 13

February 14

February 15

February 16

February 17

February 18

February 20

February 21

February 22

February 23

February 27

February 28

February 29

March 1

March 2

March 3

PCR

Miniprep

March 4

Sequence of tatABCD

Colony PCR of TMAO

Electrophoresis

Restriction

Electrophoresis

Transformation

March 5

Restriction

Ligation

Transformation

PCR

Electrophoresis

Restriction

Ligation

Transformation

Restriction

Electrophoresis

PCR

electrophoresis

March 6

Restriction

PCR

Restriction

Ligation

March 7

Electrophoresis

Ligation

Restriction

Purification

Ligation

March 8

Restriction

Ligation

Liquid culture

Transformation

March 9

Restriction

Miniprep

Colony PCR

Ligation

Transformation

Miniprep

March 10

Screening PCR

Restriction

Ligation

March 11

Miniprep

Screening PCR

Electrophoresis

March 12

Transformation