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7/15

transformation

Tatsui, Matsumoto and Sukegawa

Sample/(µL)		CompetentCells/(µl)	Total/(µl)	Medium
BBa_J23101(Pcon)	2	20(2/23)	22	LB(CP)
BBa_B0032(RBS)	2	20(2/23)	22	LB(CP)

7/16

transformation

Tatsui

Sample/(µL)		CompetentCells/(µl)	Total/(µl)	Medium
BBa_B0030(1)	2	20(2/23)	22	LB(CP)
BBa_B0030(2)	2	20(bought)	22	LB(CP)
BBa_B0032(1)	2	20(2/23)	22	LB(CP)
BBa_B0032(2)	2	20(bought)	22	LB(CP)

7/17

Colony PCR

Tatsui, Matsumoto, Li, and Yamauchi

NameⅠ	NameⅡ
7/16 BBa_B0030(1) Colony_1	7/16 BBa_B0032(1) Colony_1
7/16 BBa_B0030(1) Colony_2	7/16 BBa_B0032(1) Colony_2
7/16 BBa_B0030(1) Colony_3	7/16 BBa_B0032(1) Colony_3
7/16 BBa_B0030(2) Colony_1	7/16 BBa_B0032(2) Colony_1
7/16 BBa_B0030(2) Colony_2	7/16 BBa_B0032(2) Colony_2
7/16 BBa_B0030(2) Colony_3	7/16 BBa_B0032(2) Colony_3
Negative Control	-
-	-

Liquid Culture

Tatsui, Matsumoto, Li, and Yamauchi

Name	medium
7/16 BBa_B0030(1) Colony_1	Plusgrow medium(CP)
7/16 BBa_B0030(1) Colony_2	Plusgrow medium(CP)
7/16 BBa_B0030(1) Colony_3	Plusgrow medium(CP)
7/16 BBa_B0030(2) Colony_1	Plusgrow medium(CP)
7/16 BBa_B0030(2) Colony_2	Plusgrow medium(CP)
7/16 BBa_B0030(2) Colony_3	Plusgrow medium(CP)
7/16 BBa_B0032(1) Colony_1	Plusgrow medium(CP)
7/16 BBa_B0032(1) Colony_2	Plusgrow medium(CP)
7/16 BBa_B0032(1) Colony_3	Plusgrow medium(CP)
7/16 BBa_B0032(2) Colony_1	Plusgrow medium(CP)
7/16 BBa_B0032(2) Colony_2	Plusgrow medium(CP)
7/16 BBa_B0032(2) Colony_3	Plusgrow medium(CP)

7/18

Miniprep

Tatsui, Matsumoto, Tsujii, Stephane, and Yamauchi

DNA	Concentration/(µg/ml)	260/280	260/230
7/16 BBa_B0030(1) Colony_1	229	1.96	1.40
7/16 BBa_B0030(1) Colony_2	209	1.95	1.50
7/16 BBa_B0030(1) Colony_3	108	2.12	2.12
7/16 BBa_B0030(2) Colony_1	-	-	-
7/16 BBa_B0030(2) Colony_2	174	2.02	1.56
7/16 BBa_B0030(2) Colony_3	158	2.01	1.68
7/16 BBa_B0032(1) Colony_1	179	2.00	2.06
7/16 BBa_B0032(1) Colony_2	-	-	-
7/16 BBa_B0032(1) Colony_3	221	1.90	1.37
7/16 BBa_B0032(2) Colony_1	225	1.89	1.37
7/16 BBa_B0032(2) Colony_2	270	1.98	1.63
7/16 BBa_B0032(2) Colony_3	194	1.89	1.33

7/30

Restriction Enzyme Digestion

No name

Sample	DNA/(µl)	EcoR1/(µl)	Xba1/(µl)	Spe1/(µl)	Pst1/(µl)	Buffur/(µl)	BSA/(µl)	MilliQ/(µl)	TOTAL/(µl)
7/16 BBa_B0030(2) Colony_1	1	0.3	0	0	0	1(H)	0	7.7	10
7/16 BBa_B0030(2) Colony_1	1	0	0.3	0	0	1(M)	1	6.7	10
7/16 BBa_B0030(2) Colony_1	1	0	0	0.3	0	1(M)	0	7.7	10
7/16 BBa_B0030(2) Colony_1	1	0	0	0	0.3	1(H)	0	7.7	10
7/16 BBa_B0030(2) Colony_1(Nega)	1	0	0	0	0	1(M)	0	8	10

Electrophoresis

No name

Lane	Restriction Enzyme Digestion Products
1	7/30 BBa_B0030(2) Colony_1(E CUT)
2	7/30 BBa_B0030(2) Colony_1(X CUT)
3	7/30 BBa_B0030(2) Colony_1(S CUT)
4	7/30 BBa_B0030(2) Colony_1(P CUT)
5	7/30 BBa_B0030(2) Colony_1(Nega)
6	1kbp ladder

7/31

Transformation

Hashitani, Tatsui, Li and Yamauchi

Sample/(µL)		CompetentCells/(µl)	Total/(µl)	Medium
BBa_E0240(RBS-GFP-DT)	2	20	22	LB(CP)
J23100(Pcon)	2	20	22	LB(AMP)

Liquid Culture

No name

Name	medium
7/31 J23100(Pcon) Colony_1	Plusgrow medium(AMP)
7/31 J23100(Pcon) Colony_2	Plusgrow medium(AMP)
7/31 J23100(Pcon) Colony_3	Plusgrow medium(AMP)
7/31 J23100(Pcon) Colony_4	Plusgrow medium(AMP)
7/31 J23100(Pcon) Colony_5	Plusgrow medium(AMP)
7/31 J23100(Pcon) Colony_6	Plusgrow medium(AMP)

Restriction Enzyme Digestion

Hashitani, Tatsui, Li and Yamauchi

7/18 RBS Sample	DNA/(µl)	EcoR1/(µl)	Spe1/(µl)	Pst1/(µl)	Xba1/(µl)	Buffur/(µl)	BSA/(µl)	MilliQ/(µl)	TOTAL/(µl)
BBa_B0032	5	0.3	0	0	0	1(H)	0	3.7	10
BBa_B0032	5	0	0.3	0	0	1(M)	0	3.7	10
BBa_B0032	5	0	0	0.3	0	1(H)	0	3.7	10
BBa_B0032	5	0	0	0	0.3	1(M)	1	2.7	10
BBa_B0032(Nega)	5	0	0	0	0	1(M)	0	4	10

8/1

Electrophoresis

Honda, Li and Yamauchi

Lane	Restriction Enzyme Digestion Products
1	1kb ladder
2	7/31 BBa_B0032(E CUT)
3	7/31 BBa_B0032(S CUT)Error
4	-
5	-
6	7/31 BBa_B0032(P CUT)
7	7/31 BBa_B0032(X CUT)
8	7/31 BBa_B0032(Nega)

PCR

Tatsui

BBa_E0240(RBS-GFP-DT)	Volume
10×buffur	5μL
2mMdNTPs	5μL
25mM MgSO4	2μL
VF Primer	1.5μL
VR Primer	1.5μL
template	0.5μL
KOD-plus	1μL
MilliQ	33.5μL
TOTAL	50μL

Steps	Temparature	Time	cycle
Pre Denature	94℃	2min	1 time
Denature	94℃	15sec	30 cycle
Annealing	55℃	30sec	30 cycle
Extension	68℃	1min	30 cycle

DNA	Concentration/(µg/ml)	260/280	260/230
Product	80	1.91	1.78

8/2

Miniprep

Tatsui, Honda, Matsumoto, Yamauchi, Li, Tsujii, and Nakamura

DNA	Concentration/(µg/ml)	260/280	260/230
7/31 J23100(Pcon) Colony_1	145	2.11	3.09
7/31 J23100(Pcon) Colony_2	-	-	-
7/31 J23100(Pcon) Colony_3	160	2.01	3.13
7/31 J23100(Pcon) Colony_4	140	1.87	2.70
7/31 J23100(Pcon) Colony_5	105	2.39	5.90
7/31 J23100(Pcon) Colony_6	180	1.90	1.37

8/20

Transformation

Honda

Sample/(µL)		CompetentCells/(µl)	Total/(µl)	Medium
7/31 BBa_E0240(RBS-GFD-DT)	2	20	22	LB(CP)
BBa_I732095	2	20	22	LB(CP)

8/21

Colony PCR

Shimazaki

NameⅠ
8/20 BBa_I732095 Colony_1
8/20 BBa_I732095 Colony_2
8/20 BBa_I732095 Colony_3
Negative control

Electrophoresis

Honda

Lane	PCR Product
1	1kb ladder
2	8/21 BBa_I732095 Colony_1
3	8/21 BBa_I732095 Colony_2
4	8/21 BBa_I732095 Colony_3
5	Negative Control

Liquid Culture

Honda and Tatsui

Name	medium
8/20 BBa_I732095 Colony_1	Plusgrow medium(CP)
8/20 BBa_I732095 Colony_2	Plusgrow medium(CP)
8/20 BBa_I732095 Colony_3	Plusgrow medium(CP)

8/22

Miniprep

Honda

DNA	Concentration/(µg/ml)	260/280	260/230
8/20 BBa_I732095 Colony_1	430	1.68	2.13
8/20 BBa_I732095 Colony_2	255	1.87	1.81
8/20 BBa_I732095 Colony_3	215	1.78	1.35

Restriction Enzyme Digestion

Nakamura

Sample	DNA/(µl)	EcoR1/(µl)	Spe1/(µl)	Buffur/(µl)	MilliQ/(µl)	TOTAL/(µl)
8/22 BBa_I732095 Colony_1	4.7	1	1	3(H)	20.3	30
8/22 BBa_I732095 Colony_2	7.8	1	1	3(H)	17.2	30
8/22 BBa_I732095 Colony_3	9.3	1	1	3(H)	15.7	30

Transformation

Tatsui and Yamauchi

Sample/(µL)		CompetentCells/(µl)	Total/(µl)	Medium
BBa_J04450(pSB3C5)	2	20	22	LB(CP)

8/25

Transformation

Tatsui, Kita and Li

Sample/(µL)		CompetentCells/(µl)	Total/(µl)	Medium
BBa_J04450(pSB2K3)	2	20	22	LB(KAN)
BBa_C0051(pSB1A2)	2	20	22	LB(AMP)

Liquid Culture

Tatsui

Name	medium
8/22 BBa_J04450(pSB3C5)	Plusgrow medium(CP)*5

8/26

Miniprep

Tsujii, Honda and Matsumoto

DNA	Concentration/(µg/ml)	260/280	260/230
8/25 BBa_J04450(pSB3C5)	575	1.37	1.37

Liquid Culture

Tatsui, Matsumoto and Nakamura

Name	medium
8/25 BBa_J04450(pSB2K3)	Plusgrow medium(KAN)*5
8/25 BBa_J0051(pSB1A2)	Plusgrow medium(AMP)*2

8/27

Miniprep

No Name

DNA	Concentration/(µg/ml)	260/280	260/230
8/26 BBa_J04450(pSB2K3)	1045	1.43	1.11
8/26 BBa_J0051(pSB1A2)	790	1.39	0.90

Transformation

Tatsui

Sample/(µL)		CompetentCells/(µl)	Total/(µl)	Medium
BBa_J04450(pSB3T5)	2	20	22	LB(TET)

8/28

Liquid Culture

Yamauchi

Name	medium
8/27 BBa_J04450(pSB3T5)	Plusgrow medium(TET)

8/29

Miniprep

Matsumoto

DNA	Concentration/(µg/ml)	260/280	260/230
8/28 BBa_J04450(pSB3T5)	180	1.60	1.67

Transformation

Tatsui

Sample/(µL)		CompetentCells/(µl)	Total/(µl)	Medium
BBa_J04450(pSB4A5)	2	20	22	LB(AMP)

9/1

Colony PCR

NameⅠ
-
-
-
9/1 pSB4A5 Colony_1
9/1 pSB4A5 Colony_2
Negative Control

Restriction Enzyme Digestion

No name

Sample	DNA/(µl)	EcoR1/(µl)	Pst1/(µl)	Buffur/(µl)	MilliQ/(µl)	TOTAL/(µl)
8/28 Miniprep pSB3T5	25	2	2	6(H)	25	60
Negative control_1	3.7	0	0	1(H)	5.3	10
8/22 Miniprep I732095	14	3	3	9(H)	61	90
Negative control_2	1.5	0	0	1(H)	7.5	10
8/27 Miniprep pSB2K3	6	3	3	9(H)	69	90
Negative control_3	1	0	0	1(H)	8	10
8/2 Miniprep Pcon Colony_3	20	1.5	1.5	5(H)	17	45
Negative control_4	4	0	0	1(H)	5	10
8/2 Miniprep Pcon Colony_1	20	1.5	1.5	5(H)	17	45
Negative control_5	4	0	0	1(H)	5	10

Liquid Culture

No name

Name	medium
8/29 BBa_J04450(pSB4A5) Colony_1	Plusgrow medium(AMP)*2
8/1 Pcon Colony_1	Plusgrow medium(AMP)*2
8/27 pSB3T5 Colony_1	Plusgrow medium(TET)*2

9/2

Miniprep

Honda, Yamauchi and Li

DNA	Concentration/(µg/ml)	260/280	260/230
8/29 BBa_J04450(pSB4A5) Colony_1	210	1.61	1.18
8/1 Pcon Colony_1	215	1.79	1.87
8/27 pSB3T5 Colony_1	215	1.58	0.92

Gel Extractions

Matsumoto

Lane	Sample	Volume/(µl)
1	9/1 pSB3T5 Colony_1 (Nega)	10
2	9/1 pSB3T5 Colony_1 (E+P)(Confirmation)	5
3	9/1 pSB3T5 Colony_1 (E+P)(Extraction)	55
4	1kb ladder	3
5	9/1 I732095 (Nega)	10
6	9/1 I732095 (E+P)(Confirmation)	5
7	9/1 I732095 (E+P)(Extraction)	85
8	1kb ladder	3
9	9/1 pSB2K3 (Nega)	10
10	9/1 pSB2K3 (E+P)(Confirmation)	5
11	9/1 pSB2K3 (E+P)(Extraction)	85

Lane	Sample	Volume/(µl)
1	9/1 Pcon Colony_3 (E+P)(Confirmation)	5
2	9/1 Pcon Colony_3 (E+P)(Extraction)	20
3	9/1 Pcon Colony_3 (E+P)(Extraction)	20
4	9/1 Pcon Colony_3 (Nega)	10
5	1kb ladder	3
6	9/1 Pcon Colony_1 (E+P)(Confirmation)	5
7	9/1 Pcon Colony_1 (E+P)(Extraction)	20
8	9/1 Pcon Colony_1 (E+P)(Extraction)	20
9	9/1 Pcon Colony_1 (Nega)	10

9/3

Concentration Measurement

Matsumoto, Yamauchi and Li

DNA	Concentration/(µg/ml)	260/280	260/230
pSB2K3	40	1.48	0.10
Pcon Colony_1	30	1.76	0.62
pSB3T5	50	2.35	0.50
pSB1C3	65	2.14	0.10
Pcon Colony_3	45	1.56	0.58

Ligation

No name

Insert/(µL)		Vector/(µL)		T4 Ligase/(µl)	2*Buffer/(µl)	MilliQ/(µl)	Total/(µl)
Pcon	2.1	pSB1C3	1.5	1	5	0.4	10
-(Negative Control)	0	pSB1C3	1.5	1	5	2.5	10
Pcon	1.3	pSB2K3	2.5	1	5	0.2	10
-(Negative Control)	0	pSB2K3	2.5	1	5	1.5	10
Pcon	10	pSB3T5	2.3	1	5	0.7	10
-(Negative Control)	0	pSB3T5	2.3	1	5	1.7	10

Transformation

Matsumoto

Sample/(µL)		CompetentCells/(µl)	Total/(µl)	Medium
Pcon+pSB1C3	3	15	18	LB(CP)
pSB1C3(Negative Control)	8	10	18	LB(CP)
Pcon+pSB2K3	3	15	18	LB(KAN)
pSB2K3(Negative Control)	8	10	18	LB(KAN)
Pcon+pSB3T5	3	15	18	LB(TET)
pSB3T5(Negative Control)	2	10	12	LB(TET)

9/4

Colony PCR

Honda and Yamauchi

NameⅠ	NameⅡ	NameⅢ	NameⅣ
-	9/3 pSB1C3(Nega) Colony_7	9/3 Pcon+pSB3T5 Colony_5	9/3 Pcon+pSB2K3 Colony_1
-	9/3 pSB1C3(Nega) Colony_8	9/3 Pcon+pSB3T5 Colony_6	9/3 Pcon+pSB2K3 Colony_2
9/3 pSB1C3(Nega) Colony_1	9/3 Pcon+pSB3T5 Colony_1	9/3 Pcon+pSB3T5 Colony_7	9/3 Pcon+pSB2K3 Colony_3
9/3 pSB1C3(Nega) Colony_2	9/3 Pcon+pSB3T5 Colony_2	9/3 Pcon+pSB3T5 Colony_8	9/3 Pcon+pSB2K3 Colony_4
9/3 pSB1C3(Nega) Colony_3	9/3 Pcon+pSB3T5 Colony_3	9/3 Pcon+pSB3T5 Colony_9	9/3 Pcon+pSB2K3 Colony_5
9/3 pSB1C3(Nega) Colony_4	9/3 Pcon+pSB3T5 Colony_4	9/3 Pcon+pSB3T5 Colony_10	9/3 Pcon+pSB2K3 Colony_6
9/3 pSB1C3(Nega) Colony_5	9/3 pSB1C3(Nega) Colony_9	9/3 pSB3T5(Nega) Colony_1	9/3 Pcon+pSB2K3 Colony_7
9/3 pSB1C3(Nega) Colony_6	9/3 pSB1C3(Nega) Colony_10	9/3 pSB3T5(Nega) Colony_2	9/3 Pcon+pSB2K3 Colony_8

NameⅤ	NameⅥ	NameⅦ	NameⅧ
9/3 Pcon+pSB2K3 Colony_9	9/3 Pcon+pSB1C3 Colony_2	9/3 Pcon+pSB1C3 Colony_10	9/3 pSB1C3(Nega) Colony_8
9/3 Pcon+pSB2K3 Colony_10	9/3 Pcon+pSB1C3 Colony_3	9/3 pSB1C3(Nega) Colony_1	9/3 pSB1C3(Nega) Colony_9
9/3 pSB2K3(Nega) Colony_1	9/3 Pcon+pSB1C3 Colony_4	9/3 pSB1C3(Nega) Colony_2	9/3 pSB1C3(Nega) Colony_10
9/3 pSB2K3(Nega) Colony_2	9/3 Pcon+pSB1C3 Colony_5	9/3 pSB1C3(Nega) Colony_3	9/3 pSB1C3(Nega) Colony_11
9/3 pSB2K3(Nega) Colony_3	9/3 Pcon+pSB1C3 Colony_6	9/3 pSB1C3(Nega) Colony_4	-
9/3 pSB2K3(Nega) Colony_4	9/3 Pcon+pSB1C3 Colony_7	9/3 pSB1C3(Nega) Colony_5	-
9/3 pSB2K3(Nega) Colony_5	9/3 Pcon+pSB1C3 Colony_8	9/3 pSB1C3(Nega) Colony_6	-
9/3 Pcon+pSB1C3 Colony_1	9/3 Pcon+pSB1C3 Colony_9	9/3 pSB1C3(Nega) Colony_7	-

NameⅨ	NameⅩ
9/3 Pcon+pSB1C3 Colony_11	8/22 Miniprep I732095 Colony_3
Negative Control	-

9/5

Electrophoresis

Honda and Matsumoto

Lane	Colony PCR ProductsⅠ	Lane	Colony PCR ProductsⅠ
1	1kb ladder	10	9/4 pSB1C3(Nega) Colony_7
2	-	11	9/4 pSB1C3(Nega) Colony_8
3	-	12	12	9/4 pSB1C3(Nega) Colony_9
4	9/4 pSB1C3(Nega) Colony_1	13	9/4 pSB1C3(Nega) Colony_10
5	9/4 pSB1C3(Nega) Colony_2	14	9/4 Pcon+pSB2K3 Colony_1
6	9/4 pSB1C3(Nega) Colony_3	15	9/4 Pcon+pSB2K3 Colony_2
7	9/4 pSB1C3(Nega) Colony_4	16	9/4 Pcon+pSB2K3 Colony_3
8	9/4 pSB1C3(Nega) Colony_5	17	1kb ladder
9	9/4 pSB1C3(Nega) Colony_6

Lane	Colony PCR ProductsⅡ
1	1kb ladder
2	9/4 Pcon+pSB2K3 Colony_4
3	9/4 Pcon+pSB2K3 Colony_5
4	9/4 Pcon+pSB2K3 Colony_6
5	9/4 Pcon+pSB2K3 Colony_7
6	9/4 Pcon+pSB2K3 Colony_8
7	9/4 Pcon+pSB2K3 Colony_9
8	9/4 Pcon+pSB3T5 Colony_10
9	9/4 pSB3T5(Nega) Colony_1
10	9/4 pSB3T5(Nega) Colony_2
11	9/4 Pcon+pSB2K3 Colony_1
12	9/4 Pcon+pSB2K3 Colony_2
13	9/4 Pcon+pSB2K3 Colony_3
14	9/4 Pcon+pSB2K3 Colony_4
15	9/4 Pcon+pSB2K3 Colony_5
16	9/4 Pcon+pSB2K3 Colony_6
17	1kb ladder

Lane	Colony PCR ProductsⅢ
1	1kb ladder
2	9/4 Pcon+pSB2K3 Colony_7
3	9/4 Pcon+pSB2K3 Colony_8
4	9/4 Pcon+pSB2K3 Colony_9
5	9/4 Pcon+pSB2K3 Colony_10
6	9/4 pSB2K3(Nega) Colony_1
7	9/4 pSB2K3(Nega) Colony_2
8	9/4 pSB2K3(Nega) Colony_3
9	9/4 pSB2K3(Nega) Colony_4
10	9/4 pSB2K3(Nega) Colony_5
11	9/4 pSB1C3(Nega) Colony_1
12	9/4 pSB1C3(Nega) Colony_2
13	9/4 pSB1C3(Nega) Colony_3
14	9/4 pSB1C3(Nega) Colony_4
15	9/4 pSB1C3(Nega) Colony_5
16	9/4 pSB1C3(Nega) Colony_6
17	1kb ladder

Lane	Colony PCR ProductsⅣ
1	1kb ladder
2	9/4 pSB1C3(Nega) Colony_7
3	9/4 pSB1C3(Nega) Colony_8
4	9/4 pSB1C3(Nega) Colony_9
5	9/4 pSB1C3(Nega) Colony_10
6	9/4 Pcon+pSB1C3 Colony_1
7	9/4 Pcon+pSB1C3 Colony_2
8	9/4 Pcon+pSB1C3 Colony_3
9	9/4 Pcon+pSB1C3 Colony_4
10	9/4 Pcon+pSB1C3 Colony_5
11	9/4 Pcon+pSB1C3 Colony_6
12	9/4 Pcon+pSB1C3 Colony_7
13	9/4 Pcon+pSB1C3 Colony_8
14	9/4 Pcon+pSB1C3 Colony_9
15	9/4 Pcon+pSB1C3 Colony_10
16	8/22 Miniprep BBa_I732095 Colony_3
17	1kb ladder

Lane	Colony PCR ProductsⅤ
1	1kb ladder
2	9/4 Pcon+pSB1C3 Colony_11
3	9/4 Negative Control
4	8/22 Miniprep BBa_I732095 Colony_3
5	-
6	-
7	-
8	-

9/8

9/9

9/10

9/11

9/12

9/13

9/14

9/15

9/16

9/17

9/18

9/19

9/20

9/22