Template:Kyoto/Notebook/Magnetosome

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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/(µl)	5
2mMdNTPs/(µl)	5
25mM MgSO4/(µl)	2
VF Primer/(µl)	1.5
VR Primer/(µl)	1.5
template/(µl)	0.5
KOD-plus/(µl)	1
MilliQ/(µl)	33.5
TOTAL/(µl)	50

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

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 Extraction

Matsumoto

Lane	Restriction Enzyme Digestion ProductⅠ	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
9/3 Ligation Pcon+pSB1C3	3	15	18	LB(CP)
9/3 Ligation pSB1C3(Negative Control)	8	10	18	LB(CP)
9/3 Ligation Pcon+pSB2K3	3	15	18	LB(KAN)
9/3 Ligation pSB2K3(Negative Control)	8	10	18	LB(KAN)
9/3 Ligation Pcon+pSB3T5	3	15	18	LB(TET)
9/3 Ligation 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	-

Restriction Enzyme Digestion

No name

Sample	DNA/(µl)	EcoR1/(µl)	Pst1/(µl)	Buffur/(µl)	MilliQ/(µl)	TOTAL/(µl)
8/27 Miniprep pSB2K3	2	1	1	3(H)	23	30
8/27 Miniprep pSB2K3(Nega)	1	0	0	1(H)	8	10
9/2 Miniprep pSB3T5	10	1	1	3(H)	15	30
9/2 Miniprep pSB3T5(Nega)	3	0	0	1(H)	6	10
9/2 Miniprep pSB4A5	10	1	1	3(H)	15	30
9/2 Miniprep pSB4A5(Nega)	3	0	0	1(H)	6	10
8/22 Miniprep pSB1C3 Colony_2	2	1	1	1(H)	5	10

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

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

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

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	-

Gel Extraction

No Name

Lane	Restriction Enzyme Digestion ProductⅠ	Volume/(µl)
1	1kb ladder	5
2	9/4 pSB2K3(E+P)	5
3	-	-
4	9/4 pSB2K3(E+P)	25
5	-	-
6	9/4 pSB2K3(Nega)	10

Lane	Restriction Enzyme Digestion ProductⅡ	Volume/(µl)
1	1kb ladder	5
2	9/4 pSB3T5(E+P)	5
3	-	-
4	9/4 pSB3T5(E+P)	25
5	-	-
6	9/4 pSB3T5(Nega)	10

Lane	Restriction Enzyme Digestion ProductⅢ	Volume/(µl)
1	1kb ladder	5
2	9/4 pSB4A5(E+P)	5
3	-	-
4	9/4 pSB4A5(E+P)	25
5	-	-
6	9/4 pSB4A5(Nega)	10

Lane	Restriction Enzyme Digestion ProductⅣ	Volume/(µl)
1	1kb ladder	5
2	-	-
3	-	-
4	9/4 pSB1C3(E+P)	10
5	-	-
6	-	-

DNA	Concentration/(µg/ml)	260/280	260/230
9/4 pSB2K3(E+P)	36	1.25	0.24
9/4 pSB3T5(E+P)	10	1.00	-/td>
9/4 pSB4A5(E+P)	40	-	0.01
9/4 pSB1C3(E+P)	75	1.35	0.14

9/8

Cloning PCR

Honda and Li

Target	mamB	mamQ	mamL
2×KAPA HiFi Hotstart Ready Mix/(µl)	12.5	12.5	12.5
Fw Primer/(µl)	1.5(mamB)	1.5(mamQ)	1.5(mamL)
Rv Primer/(µl)	1.5(mamB)	1.5(mamQ)	1.5(mamL)
AMB-1 Genome/(µl)	1	1	1
MilliQ/(µl)	10	10	10
TOTAL/(µl)	25	25	25

Steps	Temparature	Time	cycle
Pre Denature	98℃	5min	1time
Denature	98℃	20sec	25cycle
Annealing	65℃	15sec	25cycle
Extension	72℃	5min	25cycle
Final elongation	72℃	5min	1time
Final hold	4℃	-	-

Ligation

Honda

Insert/(µL)		Vector/(µL)		T4 Ligase/(µl)	2*Buffer/(µl)	MilliQ/(µl)	Total/(µl)
Pcon	1	pSB3T5	10	1	11	0	22
-(Negative Control)	0	pSB1C3	3	0	3	0	6

Electrophoresis

Li

Lane	Cloning PCR Products
1	1kb ladder
2	9/8 Cloning PCR (AMB-1 mamB)
3	9/8 Cloning PCR (AMB-1 mamQ)
4	9/8 Cloning PCR (AMB-1 mamL)
5	1kb ladder

Transformation

Honda

Sample/(µL)		CompetentCells/(µl)	Total/(µl)	Medium
9/8 Ligation Pcon+pSB3T5	3	30	33	LB(TET)
9/8 Ligation pSB3T5(Negative Control)	3	30	33	LB(TET)

Liquid Culture

No name

Name	medium
9/3 Transformation Pcon+pSB1C3 Colony_8	Plusgrow medium(CP)*2

9/9

DNA Purification

Li

DNA	Concentration/(µg/ml)	260/280	260/230
9/8 Cloning PCR (AMB-1 mamB)	165	1.54	1.57
9/8 Cloning PCR (AMB-1 mamQ)	250	1.69	1.92
9/8 Cloning PCR (AMB-1 mamL)	190	1.56	1.79

Miniprep

Li

DNA	Concentration/(µg/ml)	260/280	260/230
9/8 Liquid Culture Pcon+pSB1C3	90	1.65	1.17

Colony PCR

Honda and Li

NameⅠ
-
9/8 Transformation Pcon+pSB3T5 Colony_1
9/8 Transformation Pcon+pSB3T5 Colony_2
9/8 Transformation Pcon+pSB3T5 Colony_3
9/8 Transformation Pcon+pSB3T5 Colony_4
9/8 Transformation Pcon+pSB3T5 Colony_5
9/8 Transformation pSB3T5(Nega) Colony_1
Negative control

Electrophoresis

Honda and Li

Lane	Cloning PCR Products
1	1kb ladder
2	9/8 Colony PCR Pcon+pSB3T5 Colony_1
3	9/8 Colony PCR Pcon+pSB3T5 Colony_2
4	9/8 Colony PCR Pcon+pSB3T5 Colony_3
5	9/8 Colony PCR Pcon+pSB3T5 Colony_4
6	9/8 Colony PCR Pcon+pSB3T5 Colony_5
7	9/8 Colony PCR pSB3T5(Nega) Colony_1
8	9/8 Colony PCR Nega

Restriction Enzyme Digestion

No name

Sample	DNA/(µl)	Pst1/(µl)	Spe1/(µl)	Buffur/(µl)	MilliQ/(µl)	TOTAL/(µl)
9/9 Miniprep Pcon+pSB1C3	22	1	1	3(H)	3	30
9/9 Miniprep Pcon+pSB1C3(Nega)	1	0	0	1(H)	8	10
8/2 Miniprep Pcon Colony_4	14	1	1	3(H)	11	30
8/2 Miniprep Pcon Colony_4(Nega)	1	0	0	1(H)	8	10

9/10

Gel Extraction

Tatsui and Yamaura

Lane	Restriction Enzyme Digestion ProductⅠ	Volume/(µl)
1	1kb ladder	-
2	-	-
3	9/10 Digestion Pcon+pSB1C3(S+P)	-
4	-	-
5	9/10 Digestion Pcon+pSB1C3(Nega)	-
6	-	-
7	Pcon(X+P)	-
8	-	-
9	Pcon(Nega)	-
10	1kb ladder	-
11	-	-

DNA	Concentration/(µg/ml)	260/280	260/230
Pcon(X+P)	70	1.40	0.13

PCR

No Name

Target	E+X+RBS+mamB+S	X+RBS+mamL+His+S+P	X+RBS+mamQ+His+S+P
2×KAPA HiFi Hotstart Ready Mix/(µl)	12.5	12.5	12.5
Fw Primer/(µl)	0.75(E+X+RBS+mamB)	0.75(X+RBS+mamL)	0.75(X+RBS+mamQ)
Rv Primer/(µl)	0.75(S+mamB)	0.75(P+S+His+mamL)	0.75(P+S+His+mamQ)
9/9 DNA Purification Product/(µl)	Trace	Trace	Trace
MilliQ/(µl)	11	11	11
TOTAL/(µl)	25	25	25

Steps	Temparature	Time	cycle
Pre Denature	98℃	5min	1time
Denature	98℃	20sec	25cycle
Annealing	60℃	15sec	25cycle
Extension	72℃	15sec	25cycle
Final elongation	72℃	5min	1time
Final hold	12℃	-	-

Electrophoresis

Li

Lane	Cloning PCR Products
1	1kb ladder
2	9/10 PCR E+X+RBS+mamB+S(922bp)
3	9/10 PCR X+RBS+mamL+His+S+P(340bp)
4	-
5	1kb ladder
6	9/10 PCR X+RBS+mamQ+His+S+P(953bp)

DNA Purification

Honda and Yamaura

DNA	Concentration/(µg/ml)	260/280	260/230
9/10 PCR E+X+RBS+mamB+S	200	1.84	2.15
9/10 PCR X+RBS+mamL+His+S+P	120	1.69	1.84
9/10 PCR X+RBS+mamQ+His+S+P	200	1.70	2.16

Restriction Enzyme Digestion

Shimizu

Sample	DNA/(µl)	Xba1/(µl)	Pst1/(µl)	Buffur/(µl)	MilliQ/(µl)	TOTAL/(µl)
9/10 PCR E+X+RBS+mamB+S	10	1	1	3(H)	15	30
9/10 PCR X+RBS+mamL+His+S+P	16.7	1	1	3(H)	8.3	30
9/10 PCR X+RBS+mamQ+His+S+P	10	1	1	3(H)	15	30

Ligation

Tatsui and Murada

Insert/(µL)		Vector/(µL)		T4 Ligase/(µl)	2*Buffer/(µl)	MilliQ/(µl)	Total/(µl)
9/10 DNA Purification mamQ	1	YCplac111	1	1	5	2	10
9/10 DNA Purification mamL	1	YCplac111	1	1	5	2	10
9/10 DNA Purification mamB	1	YCplac111	1	1	5	2	10
Control	0	YCplac111	1	1	5	2	9

Transformation

Tatsui and Murada

Sample/(µL)		CompetentCells/(µl)	Total/(µl)	Medium
9/10 Ligation mamQ+YCplac111	-	-	-	-
9/10 Ligation mamL+YCplac111	-	-	-	-
9/10 Ligation mamB+YCplac111	-	-	-	-

9/11

DNA Purification

No Name

DNA	Concentration/(µg/ml)	260/280	260/230
9/10 Digestion mamB(X+P)	65	1.68	0.51
9/10 Digestion mamL(X+P)	30	1.95	0.81
9/10 Digestion mamQ(X+P)	60	1.65	1.22

Electrophoresis

Murada

Lane	Colony PCR Products

Lane	Colony PCR Products

Lane	Colony PCR Products

Lane	Colony PCR Products

Lane	Colony PCR Products

Ligation

No Name

Insert/(µL)		Vector/(µL)		T4 Ligase/(µl)	2*Buffer/(µl)	MilliQ/(µl)	Total/(µl)
-	-	-	-	-	-	-	-

Transformation

Yamauchi

Sample/(µL)		CompetentCells/(µl)	Total/(µl)	Medium
9/11 Ligation mamL	2	10	12	-
9/11 Ligation mamQ	1	5	6	-

Liquid Culture

Honda

Name	medium
9/8 Master Pcon+pSB1C3 Colony_3	Plusgrow medium(CP)
9/10 Transformation mamL+YCplac111	Plusgrow medium(AMP)
9/10 Transformation mamQ+YCplac111	Plusgrow medium(AMP)

9/12

Colony PCR

Yamaura

NameⅠ

Electrophoresis

Yamaura

Lane	Colony PCR Products

Miniprep

Honda

DNA	Concentration/(µg/ml)	260/280	260/230
9/11 Liquid Culture Pcon+pSB1C3	110	1.83	2.63
9/11 Liquid Culture mamL+YCplac111	300	1.75	2.38
9/11 Liquid Culture mamQ+YCplac111	160	1.81	2.37

Restriction Enzyme Digestion

Yasuda

Sample	DNA/(µl)	Xba1/(µl)	Pst1/(µl)	Spe1/(µl)	Buffur/(µl)	BSA	MilliQ/(µl)	TOTAL/(µl)
9/12 Miniprep mamL+YCplac111	6.7	1	1	0	3(H)	3	15.3	30
9/12 Miniprep mamQ+YCplac111	12.5	1	1	0	3(H)	3	9.5	30
9/12 Miniprep Pcon+pSB1C3	18.1	0	1	1	3(H)	0	6.9	30

Ligation

Honda

Insert/(µL)		Vector/(µL)		T4 Ligase/(µl)	2*Buffer/(µl)	MilliQ/(µl)	Total/(µl)
9/12 Digestion mamB(E+S)	3	9/8 Digestion pSB1C3(E+S)	1	1	5	0	10
9/10 mamB	0.5	YCplac111	1	1	5	2.5	10

DNA Purification

Tatsui

DNA	Concentration/(µg/ml)	260/280	260/230
mamB(E+S)	50	1.75	2.04

Transformation

Honda

Sample/(µL)		CompetentCells/(µl)	Total/(µl)	Medium
9/12 Ligation mamB+pSB1C3	1	10	11	LB(CP)
9/12 Ligation mamB+YCplac111	1	10	11	LB(AMP)

Colony PCR

Li

NameⅠ	NameⅡ
9/11 Transformation mamL+pSB1A2 Colony_1	9/11 Transformation mamL+pSB1A2 Colony_9
9/11 Transformation mamL+pSB1A2 Colony_2	9/11 Transformation mamL+pSB1A2 Colony_10
9/11 Transformation mamL+pSB1A2 Colony_3	9/11 Transformation mamQ+pSB1A2 Colony_1
9/11 Transformation mamL+pSB1A2 Colony_4	9/11 Transformation mamQ+pSB1A2 Colony_2
9/11 Transformation mamL+pSB1A2 Colony_5	9/11 Transformation mamQ+pSB1A2 Colony_3
9/11 Transformation mamL+pSB1A2 Colony_6	9/11 Transformation mamQ+pSB1A2 Colony_4
9/11 Transformation mamL+pSB1A2 Colony_7	9/11 Transformation mamQ+pSB1A2 Colony_5
9/11 Transformation mamL+pSB1A2 Colony_8	9/11 Transformation mamQ+pSB1A2 Colony_6

Electrophoresis

Li

Lane	Colony PCR ProductsⅠ	Lane	Colony PCR ProductsⅠ
1	9/12 mamL+pSB1A2 Colony_1	10	9/12 mamL+pSB1A2 Colony_9
2	9/12 mamL+pSB1A2 Colony_2	11	9/12 mamL+pSB1A2 Colony_10
3	9/12 mamL+pSB1A2 Colony_3	12	9/12 mamQ+pSB1A2 Colony_1
4	9/12 mamL+pSB1A2 Colony_4	13	9/12 mamQ+pSB1A2 Colony_2
5	9/12 mamL+pSB1A2 Colony_5	14	9/12 mamQ+pSB1A2 Colony_3
6	9/12 mamL+pSB1A2 Colony_6	15	9/12 mamQ+pSB1A2 Colony_4
7	9/12 mamL+pSB1A2 Colony_7	16	9/12 mamQ+pSB1A2 Colony_5
8	9/12 mamL+pSB1A2 Colony_8	17	9/12 mamQ+pSB1A2 Colony_6
9	1kb ladder

Colony PCR

Honda

NameⅠ
9/11 Transformation mamL+pSB1A2 Green_1
9/11 Transformation mamL+pSB1A2 Green_2
9/11 Transformation mamL+pSB1A2 Green_3
9/11 Transformation mamL+pSB1A2 Green_4
9/11 Transformation mamL+pSB1A2 Green_5
9/11 Transformation mamL+pSB1A2 Black_7
9/11 Transformation mamL+pSB1A2 Black_8
9/11 Transformation mamQ+pSB1A2 Black_1

Electrophoresis

Tatsui

Lane	Colony PCR ProductsⅠ
1	9/12 mamL+pSB1A2 Green_1
2	9/12 mamL+pSB1A2 Green_2
3	9/12 mamL+pSB1A2 Green_3
4	9/12 mamL+pSB1A2 Green_4
5	9/12 mamL+pSB1A2 Green_5
6	9/12 mamL+pSB1A2 Black_7
7	9/12 mamL+pSB1A2 Black_8
8	9/12 mamQ+pSB1A2 Black_1
9	1kb ladder

Liquid Culture

Yamauchi

Name	medium
9/11 Transformation mamQ+pSB1A2 Colony_1	Plusgrow medium(AMP)
9/11 Transformation mamQ+pSB1A2 Colony_2	Plusgrow medium(AMP)
9/11 Transformation mamQ+pSB1A2 Colony_3	Plusgrow medium(AMP)
9/11 Transformation mamQ+pSB1A2 Colony_4	Plusgrow medium(AMP)
9/11 Transformation mamQ+pSB1A2 Colony_5	Plusgrow medium(AMP)
9/11 Transformation mamQ+pSB1A2 Colony_6	Plusgrow medium(AMP)

9/13

Miniprep

Tatsui

DNA	Concentration/(µg/ml)	260/280	260/230
9/12 Liquid Culture mamQ+pSB1A2 Colony_1 Error	-	-	-
9/12 Liquid Culture mamQ+pSB1A2 Colony_2 Error	-	-	-
9/12 Liquid Culture mamQ+pSB1A2 Colony_3 Error	-	-	-
9/12 Liquid Culture mamQ+pSB1A2 Colony_4	80	2.02	2.76
9/12 Liquid Culture mamQ+pSB1A2 Colony_5	70	2.13	3.41
9/12 Liquid Culture mamQ+pSB1A2 Colony_6	110	1.96	1.70
9/12 Liquid Culture Pcon+pSB1C3	110	1.96	1.70

PCR Labeling and Sequence

No Name

Target	9/13 mamQ+pSB1A2 Colony_4	9/13 mamQ+pSB1A2 Colony_5	9/13 mamQ+pSB1A2 Colony_6	Pcon+pSB1A2	9/13 mamQ+pSB1A2 Colony_4	9/13 mamQ+pSB1A2 Colony_5	9/13 mamQ+pSB1A2 Colony_6
Template/(µl)	5	4	6	4	5	6	4
Big dye/(µl)	0.5	0.5	0.5	0.5	0.5	0.5	0.5
5×Buffer/(µl)	1.75	1.75	1.75	1.75	1.75	1.75	1.75
Primer/(µl)	0.5(VF)	0.5(VF)	0.5(VF)	0.5(VF)	0.5(VR)	0.5(VR)	0.5(VR)
MilliQ/(µl)	2.75	1.75	3.75	3.75	2.75	1.75	3.75
TOTAL/(µl)	10.5	10.5	10.5	10.5	10.5	10.5	10.5

Target	9/12 mamQ+YCplac111	9/12 mamL+YCplac111	9/12 mamQ+YCplac111	9/12 mamL+YCplac111	9/12 mamQ+YCplac111
Template/(µl)	3	1.5	3	1.5	3
Big dye/(µl)	0.5	0.5	0.5	0.5	0.5
5×Buffer/(µl)	1.75	1.75	1.75	1.75	1.75
Primer/(µl)	0.5(M13F)	0.5(M13F)	0.5(M13R)	0.5(M13R)	0.5(seqpLQB-2)
MilliQ/(µl)	4.75	6.25	4.75	6.25	4.75
TOTAL/(µl)	10.5	10.5	10.5	10.5	10.5

Gel Extraction

No Name

Lane	Restriction Enzyme Digestion ProductⅠ	Volume/(µl)
1	1kb ladder	-
2	-	-
3	9/12 Digestion mamQ+YCpla111(X+P)(992bp)	-
4	-	-
5	-	-
6	-	-
7	9/12 Digestion mamL+YCpla111(X+P)(340bp)	-
8	-	-
9	9/12 Digestion Pcon+pSB1C3(Nega)(5600bp)	-
10	-	-
11	9/12 Digestion Pcon+pSB1C3(S+P)(2070bp)	-

DNA	Concentration/(µg/ml)	260/280	260/230
9/12 Digestion mamL+YCpla111(X+P)	10	1.00	0.70
9/12 Digestion Pcon+pSB1C3(S+P)	150	1.25	0.71

Restriction Enzyme Digestion

Li

Sample	DNA/(µl)	Xba1/(µl)	Pst1/(µl)	Buffur/(µl)	BSA	MilliQ/(µl)	TOTAL/(µl)
9/13 Miniprep mamQ+pSB1A2 Colony_4	12.5	0.5	0.5	1.7(M)	1.6	0	16.8
9/13 Miniprep mamQ+pSB1A2 Colony_5	14.3	0.5	0.5	1.9(M)	1.86	0	19.06
9/13 Miniprep mamQ+pSB1A2 Colony_6	9.1	0.5	0.5	1.5(M)	1.5	1.9	15
9/13 Miniprep mamQ+pSB1A2 Colony_6(Nega)	3	0	0	0.5(M)	0.5	1	5

Ligation

Yasuda

Insert/(µL)		Vector/(µL)		T4 Ligase/(µl)	2*Buffer/(µl)	MilliQ/(µl)	Total/(µl)
9/13 Gel Extraction mamL(X+P)	3	Pcon+pSB1C3(S+P)	1	1	5	0	10

Miniprep

Honda

DNA	Concentration/(µg/ml)	260/280	260/230
9/12 Liquid Culture Pcon+pSB1C3	240	1.78	1.59

9/14

Gel Extraction

Honda

Lane	Restriction Enzyme Digestion ProductⅠ	Volume/(µl)
1	1kb ladder	3
2	-	-
3	9/13 mamQ Colony_4(X+P)	16.8
4	-	-
5	1kb ladder	3
6	-	-
7	9/13 mamQ Colony_5(X+P)	19.06
8	-	-
9	9/13 mamQ Colony_6(Nega)	5
10	-	-
11	9/13 mamQ Colony_6(X+P)	15

Ligation

Tatsui

Insert/(µL)		Vector/(µL)		T4 Ligase/(µl)	2*Buffer/(µl)	MilliQ/(µl)	Total/(µl)
mamB(E+S)	4	pSB1C3(E+S)	3	1	8	0	16
mamL(X+P)	3.3	pSB1C3(E+S)	0.7	1	5	0	10

PCR

Tatsui

Target	E+X+RBS+mamB+S
2×KAPA HiFi Hotstart Ready Mix/(µl)	12.5
Fw Primer/(µl)	0.75(E+X+RBS+mamB)
Rv Primer/(µl)	0.75(S+mamB)
9/9 DNA Purification Product/(µl)	Trace
MilliQ/(µl)	11
TOTAL/(µl)	25

Steps	Temparature	Time	cycle
Pre Denature	98℃	5min	1time
Denature	98℃	20sec	25cycle
Annealing	60℃	15sec	25cycle
Extension	72℃	15sec	25cycle
Final elongation	72℃	5min	1time
Final hold	12℃	-	-

Transformation

Tatsui

Sample/(µL)		CompetentCells/(µl)	Total/(µl)	Medium
9/14 Ligation Pcon+mamL+pSB1C3	1	10	11	LB(CP)
9/14 Ligation Pcon+mamL+pSB1C3(Nega)	1	10	11	LB(CP)
9/14 Ligation mamB+pSB1C3	1	10	11	LB(CP)

9/15

Colony PCR

Tatsui

NameⅠ	NameⅡ
9/14 Transformation mamB+pSB1C3 Colony_1	9/14 Transformation mamB+pSB1C3 Colony_9
9/14 Transformation mamB+pSB1C3 Colony_2	9/14 Transformation mamB+pSB1C3 Colony_10
9/14 Transformation mamB+pSB1C3 Colony_3	9/14 Transformation mamB+pSB1C3 Colony_11
9/14 Transformation mamB+pSB1C3 Colony_4	9/14 Transformation mamB+pSB1C3 Colony_12
9/14 Transformation mamB+pSB1C3 Colony_5	9/14 Transformation mamB+pSB1C3 Colony_13
9/14 Transformation mamB+pSB1C3 Colony_6	9/14 Transformation mamB+pSB1C3 Colony_14
9/14 Transformation mamB+pSB1C3 Colony_7	9/14 Transformation mamB+pSB1C3 Colony_15
9/14 Transformation mamB+pSB1C3 Colony_8	-

Electrophoresis

Tatsui

Lane	Colony PCR ProductsⅠ
1	9/14 mamB+pSB1C3 Colony_1
2	9/14 mamB+pSB1C3 Colony_2
3	9/14 mamB+pSB1C3 Colony_3
4	9/14 mamB+pSB1C3 Colony_4
5	9/14 mamB+pSB1C3 Colony_5
6	9/14 mamB+pSB1C3 Colony_6
7	9/14 mamB+pSB1C3 Colony_7
8	9/14 mamB+pSB1C3 Colony_8
9	1kb ladder
10	9/14 mamB+pSB1C3 Colony_9
11	9/14 mamB+pSB1C3 Colony_10
12	9/14 mamB+pSB1C3 Colony_11
13	9/14 mamB+pSB1C3 Colony_12
14	9/14 mamB+pSB1C3 Colony_13
15	9/14 mamB+pSB1C3 Colony_14
16	9/14 mamB+pSB1C3 Colony_15

Colony PCR

No Name

NameⅠ
9/14 Transformation Pcon+mamL+SB1C3 Colony_1
9/14 Transformation mamB+pSB1C3 Colony_9

Transformation

Tatsui

Sample/(µL)		CompetentCells/(µl)	Total/(µl)	Medium
mamB+YCplac111	1	10	11	LB(AMP)
Pcon+mamL+pSB1C3	1	10	11	LB(CP)

Miniprep

Honda

DNA	Concentration/(µg/ml)	260/280	260/230
mamB+pSB1C3 Colony_9	-	-	-
mamB+pSB1C3 Colony_10	-	-	-
mamB+pSB1C3 Colony_11	-	-	-
mamL+pSB1A2 Colony_1	170	1.74	1.41
mamL+pSB1A2 Colony_2	140	1.95	1.43
mamL+pSB1A2 Colony_3	135	1.95	1.60
mamL+pSB1A2 Colony_4	-	-	-
mamL+pSB1A2 Colony_5	-	-	-
mamL+pSB1A2 Colony_6	-	-	-

Liquid Culture

Honda

Name	medium
9/14 Transformation Pcon+mamL+pSB1C3 Colony_1	Plusgrow medium(CP)
9/14 Transformation mamB+pSB1C3 Colony_9	Plusgrow medium(CP)
9/14 Transformation mamB+pSB1C3 Colony_10	Plusgrow medium(CP)
9/14 Transformation mamB+pSB1C3 Colony_11	Plusgrow medium(CP)

Restriction Enzyme Digestion

Li

Sample	DNA/(µl)	EcoR1/(µl)	Xba1/(µl)	Spe1/(µl)	Pst1/(µl)	BSA	MilliQ/(µl)	Buffur/(µl)	TOTAL/(µl)
9/15 DNA Purification mamB	25	1	0	1	0	0	0	3(H)	30
9/15 Mniprep mamL+pSB1A2 Colony_1	11.8	0	1	0	1	3	10.2	3(M)	30
9/15 Mniprep mamL+pSB1A2 Colony_1(Nega)	0.6	0	0	0	0	1	8.4	1(M)	10
9/15 Mniprep mamL+pSB1A2 Colony_2	14.3	0	1	0	1	3	7.7	3(M)	30
9/15 Mniprep mamL+pSB1A2 Colony_2(Nega)	0.7	0	0	0	0	1	8.3	1(M)	10
9/15 Mniprep mamL+pSB1A2 Colony_3	14.8	0	1	0	1	3	7.2	3(M)	30
9/15 Mniprep mamL+pSB1A2 Colony_3(Nega)	0.7	0	0	0	0	1	8.3	1(M)	10
9/15 Mniprep mamL+pSB1A2 Colony_4	13.3	0	1	0	1	3	8.7	3(M)	30
9/15 Mniprep mamL+pSB1A2 Colony_4(Nega)	0.7	0	0	0	0	1	8.3	1(M)	10
9/13 Pcon+pSB1C3	20	0	0	1	1	0	5	3(H)	30
9/13 Pcon+pSB1C3(Nega)	1	0	0	0	0	0	6	3(H)	10

PCR

Honda

Target	mamB Colony_10	mamB Colony_11
2×PrimeSTAR Max Premix/(µl)	25	25
Fw mamB Mutation Primer/(µl)	1	1
Rv mamB Mutation Primer/(µl)	1	1
9/15 Miniprep mamB+pSB1C3/(µl)	Colony_10(Trace)	Colony_11(Trace)
MilliQ/(µl)	23	23
TOTAL/(µl)	50	50

Steps	Temparature	Time	cycle
Pre Denature	98℃	2min	1time
Denature	98℃	10sec	30cycle
Annealing	55℃	15sec	30cycle
Extension	72℃	15sec	30cycle
Final elongation	72℃	5min	1time
Final hold	12℃	-	-

9/16

PCR Labeling and Sequence

No Name

Target	9/15 Pcon+mamL+pSB1C3	9/15 mamL+pSB1A2 Colony_1	9/15 mamL+pSB1A2 Colony_2	9/15 mamL+pSB1A2 Colony_3	9/15 mamL+pSB1A2 Colony_4	9/15 mamB+pSB1C3 Colony_9	9/15 mamB+pSB1C3 Colony_10	9/15 mamB+pSB1C3 Colony_11
Template/(µl)	1.8	2.35	2.9	3.0	3.0	1.14	1.2	1.2
Big dye/(µl)	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5
5×Buffer/(µl)	1.75	1.75	1.75	1.75	1.75	1.75	1.75	1.75
Primer(VF)/(µl)	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5
MilliQ/(µl)	5.95	5.4	4.85	4.75	4.75	6.61	6.55	6.55
TOTAL/(µl)	10.5	10.5	10.5	10.5	10.5	10.5	10.5	10.5

Target	9/15 Pcon+mamL+pSB1C3	9/15 mamL+pSB1A2 Colony_1	9/15 mamL+pSB1A2 Colony_2	9/15 mamL+pSB1A2 Colony_3	9/15 mamB+pSB1C3 Colony_9	9/15 mamB+pSB1C3 Colony_10	9/15 mamB+pSB1C3 Colony_11
Template/(µl)	1.8	2.35	2.9	3.0	1.14	1.2	1.2
Big dye/(µl)	0.5	0.5	0.5	0.5	0.5	0.5	0.5
5×Buffer/(µl)	1.75	1.75	1.75	1.75	1.75	1.75	1.75
Primer(VR)/(µl)	0.5	0.5	0.5	0.5	0.5	0.5	0.5
MilliQ/(µl)	5.95	5.4	4.85	4.75	6.61	6.55	6.55
TOTAL/(µl)	10.5	10.5	10.5	10.5	10.5	10.5	10.5

Steps	Temparature	Time	cycle
Pre Denature	96℃	2min	1time
Denature	96℃	10sec	30cycle
Annealing	55℃	5sec	30cycle
Extension	72℃	4min	30cycle
Final elongation	72℃	1min	1time
Final hold	4℃	-	-

Gel Extraction

Honda

Lane	Restriction Enzyme Digestion ProductⅠ	Volume/(µl)
1	1kb ladder	-
2	1kb ladder	-
3	9/15 mamL Colony_1(X+P)	-
4	-	-
5	9/15 mamL Colony_2(Nega)	-
6	-	-
7	9/15 mamL Colony_2(X+P)	-
8	1kb ladder	-
9	1kb ladder	-
10	9/15 mamL Colony_3(Nega)	-
11	9/15 mamL Colony_3(X+P)	-

Lane	Restriction Enzyme Digestion ProductⅡ	Volume/(µl)
1	1kb ladder	-
2	-	-
3	-	-
4	9/15 mamQ(X+P)	-
5	-	-
6	9/15 mamQ(Nega)	-

DNA	Concentration/(µg/ml)	260/280	260/230
mamL Colony_1	45	1.69	0.60
mamL Colony_2	60	1.33	0.70
mamL Colony_3	40	1.76	0.08
mamQ	65	1.35	0.83

Dephosphorylation

Honda

Sample/(µL)
9/15 Digestion Pcon+pSB1C3(S+P)	-
9/15 Gel Extraction Pcon+pSB1C3(S+P)	-

DNA Purification

Honda

DNA	Concentration/(µg/ml)	260/280	260/230
9/15 Mutation PCR mamB+pSB1C3 Colony_10	225	1.64	1.74
9/15 Mutation PCR mamB+pSB1C3 Colony_11	205	1.59	1.76

Gel Extraction

Yamauchi

Lane	SampleⅠ	Volume/(µl)
1	1kb ladder	-
2	-	-
3	9/15 Digestion Pcon+pSB1C3(S+P)	-
4	-	-
5	1kb ladder	-
6	-	-
7	9/15 Gel Extraction Pcon+pSB1C3(S+P)	-
8	-	-
9	1kb ladder	-
10	-	-
11	-	-

Transformation

Honda and Tatsui

Sample/(µL)		CompetentCells/(µl)	Total/(µl)	Medium
9/16 DNA Purification mamB+pSB1C3 Colony_10	1	10	11	LB(CP)
9/16 DNA Purification mamB+pSB1C3 Colony_11	1	10	11	LB(CP)

Restriction Enzyme Digestion

No Name

Sample	DNA/(µl)	Xba1/(µl)	Spe1/(µl)	Pst1/(µl)	BSA	MilliQ/(µl)	Buffur/(µl)	TOTAL/(µl)
9/15 mamQ+pSB1A2 Colony_4	26.7	2	0	2	6	17.3	6(M)	60
9/15 mamQ+pSB1A2 Colony_4(Nega)	0.7	0	0	0	1	7.3	1(M)	10
9/15 Pcon+mamL+pSB1C3	15	0	2	2	0	35	6(H)	60
9/15 Pcon+mamL+pSB1C3(Nega)	0.5	0	0	0	0	8.5	1(H)	10

PCR

Tatsui

Target	E+Pcon+X+RBS+mamB+His+S(*2)
2×KAPA HiFi Hotstart Ready Mix/(µl)	12.5
Fw E+Pcon+X+RBS+mamB/(µl)	0.75
Rv S+His+mamB/(µl)	0.75
9/9 DNA Purification mamB/(µl)	Trace
MilliQ/(µl)	11
TOTAL/(µl)	25

Miniprep

Honda

DNA	Concentration/(µg/ml)	260/280	260/230
9/15 mamB+pSB1C3 Colony_9	350	1.68	1.81
9/15 mamB+pSB1C3 Colony_10	325	1.65	1.50
9/15 mamB+pSB1C3 Colony_11	340	1.67	1.68
Pcon+mamL+pSB1C3	220	1.75	2.09

9/17

Colony PCR

Tatsui

NameⅠ
9/16 Transformation Mutation mamB Colony_11_1
9/16 Transformation Mutation mamB Colony_11_2
9/16 Transformation Mutation mamB Colony_11_3
9/16 Transformation Mutation mamB Colony_11_4
9/16 Transformation Mutation mamB Colony_10_1
9/16 Transformation Mutation mamB Colony_10_2
9/16 Transformation Mutation mamB Colony_10_3
9/16 Transformation Mutation mamB Colony_10_4

Miniprep

Tatsui and Yamauchi

DNA	Concentration/(µg/ml)	260/280	260/230
9/15 Pcon+pSB1C3 Colony_3	110	1.66	1.48

Gel Extraction

Yamauchi

Lane	SampleⅠ	Volume/(µl)
1	9/16 Mutation PCR Pcon+mamB+His	-
2	Pcon+mamL(Nega)	-
3	Pcon+mamL(S+P)	-
4	1kb ladder	-
5	9/16 Mutation PCR Pcon+mamB+His	-
6	-	-
7	mamQ+pSB1A2 Colony_4(X+P)	-
8	1kb ladder	-
9	mamQ(Nega)	-
10	-	-
11	mamQ+pSB1A2 Colony_4(X+P)	-

DNA	Concentration/(µg/ml)	260/280	260/230
Pcon+mamB+His	245	1.87	2.10
mamQ+pSB1A2 Colony_4(X+P)	35	1.87	0.580
Pcon+mamL(S+P)	285 Error	1.14	0.85

Ligation

Tatsui

Insert/(µL)		Vector/(µL)		T4 Ligase/(µl)	2*Buffer/(µl)	MilliQ/(µl)	Total/(µl)
mamQ(X+P)	3	Pcon+mamL(S+P)	1	1	5	0	10

Transformation

Tatsui

Sample/(µL)		CompetentCells/(µl)	Total/(µl)	Medium
9/17 Ligation Pcon+mamL+mamQ+pSB1C3	1	10	11	LB(CP)

Restriction Enzyme Digestion

Tatsui

Sample	DNA/(µl)	EcoR1/(µl)	Spe1/(µl)	Pst1/(µl)	Buffur/(µl)	MilliQ/(µl)	TOTAL/(µl)
9/17 Gel Extraction Pcon+mamB+His	16.3	2	2	0	6(H)	33.7	60
9/17 Gel Extraction Pcon+mamB+His(Nega)	1	0	0	0	1(H)	8	10
9/17 Miniprep Pcon+pSB1C3	36.4	0	2	2	6(H)	13.6	60
9/17 Miniprep Pcon+pSB1C3(Nega)	1	0	0	0	1(H)	8	10

Liquid Culture

Tatsui

Name	medium
Mutation mamB Colony_10_3	Plusgrow medium(CP)
Mutation mamB Colony_10_4	Plusgrow medium(CP)
Mutation mamB Colony_11_3	Plusgrow medium(CP)
Mutation mamB Colony_11_4	Plusgrow medium(CP)
mamQ+pSB1A2 Colony_4	Plusgrow medium(AMP)*2
Pcon+mamL Colony_1	Plusgrow medium(CP)*3.5

9/18

Miniprep

Tatsui and Honda

DNA	Concentration/(µg/ml)	260/280	260/230
9/17 Liquid Culture Mutation mamB Colony_10_3	355	1.69	2.02
9/17 Liquid Culture Mutation mamB Colony_10_4	265	1.52	1.55
9/17 Liquid Culture Mutation mamB Colony_11_3	355	1.62	1.77
9/17 Liquid Culture Mutation mamB Colony_11_4	250	1.62	1.51
9/17 Liquid Culture mamQ+pSB1A2 Colony_4	290	1.41	1.10
9/17 Liquid Culture Pcon+mamL+pSB1C3 Colony_1	85	1.47	1.56

PCR Labeling and Sequence

No Name

Target	9/18 Miniprep Mutation mamB Colony_10_3	9/18 Miniprep Mutation mamB Colony_10_4	9/18 Miniprep Mutation mamB Colony_11_3	9/18 Miniprep Mutation mamB Colony_11_4	9/18 Miniprep Mutation mamB Colony_10_3	9/18 Miniprep Mutation mamB Colony_10_4	9/18 Miniprep Mutation mamB Colony_11_3	9/18 Miniprep Mutation mamB Colony_11_4
Template/(µl)	1.1	1.5	1.1	1.6	1.1	1.5	1.1	1.6
Big dye/(µl)	2	2	2	2	2	2	2	2
5×Buffer/(µl)	1	1	1	1	1	1	1	1
Primer/(µl)	0.16(VF)	0.16(VF)	0.16(VF)	0.16(VF)	0.16(VR)	0.16(VR)	0.16(VR)	0.16(VR)
MilliQ/(µl)	5.74	5.34	5.74	5.24	5.74	5.34	5.74	5.24
TOTAL/(µl)	10	10	10	10	10	10	10	10

Target	9/18 Miniprep Mutation mamB Colony_10_3	9/18 Miniprep Mutation mamB Colony_10_4	9/18 Miniprep Mutation mamB Colony_11_3	9/18 Miniprep Mutation mamB Colony_11_4
Template/(µl)	1.1	1.5	1.1	1.6
Big dye/(µl)	2	2	2	2
5×Buffer/(µl)	1	1	1	1
Primer/(µl)	0.16(pQLB seq_2)	0.16(pQLB seq_2)	0.16(pQLB seq_2)	0.16(pQLB seq_2)
MilliQ/(µl)	5.74	5.34	5.74	5.24
TOTAL/(µl)	10	10	10	10

Steps	Temparature	Time	cycle
Pre Denature	96℃	2min	1time
Denature	96℃	10sec	30cycle
Annealing	50℃	5sec	30cycle
Extension	60℃	4min	30cycle
Final elongation	4℃	1min	1time
Final hold	4℃	-	-

Colony PCR

Tatsui

NameⅠ	NameⅡ
9/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony_1	9/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony_9
9/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony_2	9/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony_10
9/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony_3	9/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony_11
9/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony_4	9/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony_12
9/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony_5	9/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony_13
9/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony_6	9/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony_14
9/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony_7	9/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony_15
9/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony_8	9/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony_16

Electrophoresis

Tatsui

Lane	Colony PCR ProductsⅠ
1	9/18 Pcon+mamL+mamQ+pSB1C3 Colony_1
2	9/18 Pcon+mamL+mamQ+pSB1C3 Colony_2
3	9/18 Pcon+mamL+mamQ+pSB1C3 Colony_3
4	9/18 Pcon+mamL+mamQ+pSB1C3 Colony_4
5	9/18 Pcon+mamL+mamQ+pSB1C3 Colony_5
6	9/18 Pcon+mamL+mamQ+pSB1C3 Colony_6
7	9/18 Pcon+mamL+mamQ+pSB1C3 Colony_7
8	9/18 Pcon+mamL+mamQ+pSB1C3 Colony_8
9	1kb ladder
10	9/18 Pcon+mamL+mamQ+pSB1C3 Colony_9
11	9/18 Pcon+mamL+mamQ+pSB1C3 Colony_10
12	9/18 Pcon+mamL+mamQ+pSB1C3 Colony_11
13	9/18 Pcon+mamL+mamQ+pSB1C3 Colony_12
14	9/18 Pcon+mamL+mamQ+pSB1C3 Colony_13
15	9/18 Pcon+mamL+mamQ+pSB1C3 Colony_14
16	9/18 Pcon+mamL+mamQ+pSB1C3 Colony_15
17	9/18 Pcon+mamL+mamQ+pSB1C3 Colony_16

DNA Purification

Tatsui

DNA	Concentration/(µg/ml)	260/280	260/230
9/17 Digestion Pcon+mamB+His(E+S)	165	-	-

Gel Extraction

Tatsui

Lane	SampleⅠ	Volume/(µl)
1	-	-
2	1kb ladder	-
3	9/17 Digestion Pcon+pSB1C3(Nega)	-
4	9/17 Digestion Pcon+pSB1C3(S+P)	-
5	-	-
6	-	-

Lane	SampleⅡ	Volume/(µl)
1	-	-
2	-	-
3	1kb ladder	-
4	9/17 Digestion Pcon+pSB1C3(S+P)	-
5	-	-
6	-	-

DNA	Concentration/(µg/ml)	260/280	260/230
9/17 Digestion Pcon+pSB1C3	-	-	-

Ligation

Tatsui

Insert/(µL)		Vector/(µL)		T4 Ligase/(µl)	10*Buffer/(µl)	MilliQ/(µl)	Total/(µl)
mamQ(X+P)	6	9/18 Gel Extraction Pcon+pSB1C3(S+P)	Trace	0.5	1	2.5	10
9/18 DNA Purification Pcon+mamB+His(E+S)	5	9/14 pSB3T5(E+S)	2	0.5	1	1.5	10

Transformation

Tatsui

Sample/(µL)		CompetentCells/(µl)	Total/(µl)	Medium
9/18 Ligation Pcon+mamQ+pSB1C3	1	10	11	LB(CP)
9/18 Ligation Pcon+mamB+His+pSB3T5	1	10	11	LB(TET)

Liquid Culture

Tatsui

Name	medium
pSB3T5	Plusgrow medium(TET)*2
9/17 Transformation Pcon+mamL+mamQ+pSB1C3	Plusgrow medium(CP)

Restriction Enzyme Digestion

No Name

Sample	DNA/(µl)	Xba1/(µl)	Spe1/(µl)	Pst1/(µl)	BSA	Buffur/(µl)	MilliQ/(µl)	TOTAL/(µl)
9/18 Miniprep Mutation mamB Colony_10_3	11.2	2	0	2	0.8	8(D)	56	80
9/18 Miniprep Mutation mamB Colony_10_3(Nega)	1	0	0	0	0	1(D)	8	10
9/18 Miniprep Mutation mamB Colony_10_4	15.1	2	0	2	0.8	8(D)	52.1	80
9/18 Miniprep Mutation mamB Colony_10_4(Nega)	1	0	0	0	0	1(D)	8	10
9/18 Miniprep Mutation mamB Colony_11_3	11.2	2	0	2	0.8	8(D)	56	80
9/18 Miniprep Mutation mamB Colony_11_3(Nega)	1	0	0	0	0	1(D)	8	10
9/18 Miniprep Mutation mamB Colony_11_4	16	2	0	2	0.8	8(D)	51.2	80
9/18 Miniprep Mutation mamB Colony_11_4(Nega)	1	0	0	0	0	1(D)	8	10
9/18 Miniprep mamQ+pSB1A2	34.5	5	0	5	2	20(D)	133.5	200
9/18 Miniprep mamQ+pSB1A2(Nega)	1	0	0	0	0	1(D)	8	10
9/17 Miniprep Pcon+pSB1C3	40	0	2	2	0.8	8(B)	27.2	80
9/17 Miniprep Pcon+pSB1C3(Nega)	1	0	0	0	0	1(B)	8	10
9/18 Miniprep Pcon+mamL+pSB1C3	47	0	2	2	0.8	8(B)	20.2	80
9/18 Miniprep Pcon+mamL+pSB1C3(Nega)	1	0	0	0	0	1(B)	8	10

9/19

Miniprep

Tatsui

DNA	Concentration/(µg/ml)	260/280	260/230
9/18 Liquid Culture pSB3T5	115	1.52	1.22
9/18 Liquid Culture Pcon+mamL+mamQ+pSB1C3	75	1.59	1.20

PCR Labeling and Sequence

No Name

Target	9/19 Miniprep Pcon+mamL+mamQ+pSB1C3	9/19 Miniprep Pcon+mamL+mamQ+pSB1C3	9/19 Miniprep Pcon+mamL+mamQ+pSB1C3	9/19 Miniprep Pcon+mamL+mamQ+pSB1C3
Template/(µl)	3.5	3.5	3.5	3.5
Big dye/(µl)	0.5	0.5	0.5	0.5
5×Buffer/(µl)	1.75	1.75	1.75	1.75
Primer/(µl)	0.5(VF)	0.5(VR)	0.5(seq_1)	0.5(seq_2)
MilliQ/(µl)	4.25	4.25	4.25	4.25
TOTAL/(µl)	10.5	10.5	10.5	10.5

Steps	Temparature	Time	cycle
Pre Denature	96℃	2min	1time
Denature	96℃	10sec	30cycle
Annealing	50℃	5sec	30cycle
Extension	60℃	4min	30cycle
Final elongation	4℃	1min	1time
Final hold	4℃	-	-

Gel Extraction

Tatsui

Lane	SampleⅠ	Volume/(µl)
1	-	-
2	-	-
3	9/18 Digestion Mutation mamB Colony_10_3(X+P)	40
4	9/18 Digestion Mutation mamB Colony_10_3(Nega)	10
5	1kb ladder	-
6	-	-
7	9/18 Digestion Mutation mamB Colony_10_3(X+P)	40
8	-	-
9	1kb ladder	-
10	9/18 Digestion Mutation mamB Colony_10_4(Nega)	10
11	9/18 Digestion Mutation mamB Colony_10_4(X+P)	40

Lane	SampleⅡ	Volume/(µl)
1	9/18 Digestion mamQ+pSB1A2(X+P)	200
2	-	-
3	-	-
4	-	-
5	-	-
6	-	-

Lane	SampleⅢ	Volume/(µl)
1	100b ladder	-
2	9/18 Digestion Pcon+pSB1C3(Nega)	10
3	9/18 Digestion Pcon+pSB1C3(S+P)	80

DNA	Concentration/(µg/ml)	260/280	260/230
mamB Colony_10_3(X+P)	-	-	-
mamB Colony_10_4(X+P)	-	-	-
mamQ+pSB1A2(X+P)	-	-	-
Pcon+pSB1C3(S+P)	-	-	-

Colony PCR

Tatsui

NameⅠ
9/18 Transformation Pcon+mamQ+pSB1C3 Colony_1
9/18 Transformation Pcon+mamQ+pSB1C3 Colony_2
9/18 Transformation Pcon+mamQ+pSB1C3 Colony_3
9/18 Transformation Pcon+mamQ+pSB1C3 Colony_4
9/18 Transformation Pcon+mamQ+pSB1C3 Colony_5

Electrophoresis

Tatsui

Lane	Colony PCR ProductsⅠ
1	-
2	-
3	Pcon+mamQ+pSB1C3 Colony_1
4	Pcon+mamQ+pSB1C3 Colony_2
5	Pcon+mamQ+pSB1C3 Colony_3
6	Pcon+mamQ+pSB1C3 Colony_4
7	Pcon+mamQ+pSB1C3 Colony_5
8	-

Restriction Enzyme Digestion

Tatsui

Sample	DNA/(µl)	EcoR1/(µl)	Spe1/(µl)	Pst1/(µl)	BSA	Buffur/(µl)	MilliQ/(µl)	TOTAL/(µl)
9/19 Miniprep Pcon+mamL+mamQ+pSB1C3	34.7	0	2	2	0.8	8(B)	32.5	80
9/19 Miniprep pSB3T5	53.3	2	2	0	0.8	8(B)	14	80
9/19 Miniprep pSB3T5(Nega)	1	0	0	0	0	1(B)	8	10

PCR

Tatsui

Target	Pcon+mamB+His
KOD Plus/(µl)	1
10×Buffer/(µl)	5
2mM dNTPS/(µl)	5
25mM MgSO4/(µl)	3
Fw Primer(E+Pcon+X+RBS+mamB)/(µl)	2
Rv Primer(S+His+mamB)/(µl)	2
Template/(µl)	0.5
MilliQ/(µl)	31.5
Total/(µl)	50

Steps	Temparature	Time	cycle
Pre Denature	98℃	2min	1time
Denature	98℃	10sec	25cycle
Annealing	55℃	15sec	25cycle
Extension	72℃	1min	25cycle
Final elongation	72℃	5min	1time
Final hold	12℃	-	-

DNA Purification

Honda

DNA	Concentration/(µg/ml)	260/280	260/230
9/18 Digestion mamL+pSB1C3(S+P)	55	1.66	1.66
9/19 PCR Pcon+mamB+His	150	1.63	1.52

Restriction Enzyme Digestion

Honda

Sample	DNA/(µl)	EcoR1/(µl)	Xba1/(µl)	Spe1/(µl)	Pst1/(µl)	BSA	Buffur/(µl)	MilliQ/(µl)	TOTAL/(µl)
9/19 DNA Purification Pcon+mamB+His	20	1	0	1	0	0.8	8(B)	49.2	80

Liquid Culture

Tatsui

Name	medium
9/18 Transformation Pcon+mamB+His+pSB3T5 Colony_1	Plusgrow medium(TET)
9/18 Transformation Pcon+mamB+His+pSB3T5 Colony_2	Plusgrow medium(TET)
9/18 Transformation Pcon+mamB+His+pSB3T5 Colony_3	Plusgrow medium(TET)
9/18 Transformation Pcon+mamB+His+pSB3T5 Colony_4	Plusgrow medium(TET)
9/18 Transformation Pcon+mamB+His+pSB3T5 Colony_5	Plusgrow medium(TET)

Colony PCR

Tatsui

NameⅠ
9/18 Transformation Pcon+mamQ+pSB1C3 Colony_1
9/18 Transformation Pcon+mamQ+pSB1C3 Colony_2
9/18 Transformation Pcon+mamQ+pSB1C3 Colony_3
9/18 Transformation Pcon+mamQ+pSB1C3 Colony_4
9/18 Transformation Pcon+mamQ+pSB1C3 Colony_5

9/20

Electrophoresis

Tatsui

Lane	Colony PCR ProductsⅠ
1	-
2	100b ladder
3	Pcon+mamQ+pSB1C3 Colony_1
4	Pcon+mamQ+pSB1C3 Colony_2
5	Pcon+mamQ+pSB1C3 Colony_3
6	Pcon+mamQ+pSB1C3 Colony_4
7	Pcon+mamQ+pSB1C3 Colony_5

Miniprep

Tatsui

DNA	Concentration/(µg/ml)	260/280	260/230
9/19 Liquid Culture Pcon+mamQ+pSB1C3 Colony_3	-	-	-
9/19 Liquid Culture Pcon+mamQ+pSB1C3 Colony_5	-	-	-

Colony PCR

Tatsui

NameⅠ
Pcon+mamB+His+pSB3T5 Colony_1(VF+VR)
Pcon+mamB+His+pSB3T5 Colony_1(seq_4+VR)

Electrophoresis

Yasuda

Lane	Colony PCR ProductsⅠ
1	-
2	100b ladder
3	100b ladder
4	Pcon+mamB+His+pSB3T5 Colony_1(VF+VR)
5	Pcon+mamB+His+pSB3T5 Colony_1(seq_4+VR)
6	100b ladder

Restriction Enzyme Digestion

Honda

Sample	DNA/(µl)	EcoR1/(µl)	Xba1/(µl)	Spe1/(µl)	Pst1/(µl)	BSA	Buffur/(µl)	MilliQ/(µl)	TOTAL/(µl)
9/18 Miniprep Mutation mamB Colony_10_3	11.2	0	2	0	2	0.8	8(D)	56	80
9/18 Miniprep Mutation mamB Colony_10_3(Nega)	1	0	0	0	0	0	1(D)	8	10
9/18 Miniprep Mutation mamB Colony_10_4	15.1	0	2	0	2	0.8	8(D)	56	83.9
9/18 Miniprep Mutation mamB Colony_10_4(Nega)	1	1	0	1	0	0	1(D)	8	10

Gel Extraction

Tatsui

Lane	SampleⅠ	Volume/(µl)
1	-	-
2	100b ladder	-
3	Digestion Mutation mamB Colony_10_3(X+P)	-
4	-	-
5	-	-
6	-	-
7	Digestion Mutation mamB Colony_11_3(X+P)	-
8	Digestion Mutation mamB Colony_11_3(Nega)	-
9	1kb ladder	-
10	-	-
11	Digestion Mutation mamB Colony_11_3(X+P)	-

Lane	SampleⅡ	Volume/(µl)
1	-	-
2	100b ladder	-
3	9/19 Digestion pSB3T5(Nega)	-
4	9/19 Digestion pSB3T5(E+S)	-

Lane	SampleⅢ	Volume/(µl)
1	-	-
2	100b ladder	-
3	Digestion Mutation mamB Colony_11_4(X+P)	-
4	Digestion Mutation mamB Colony_11_4(Nega)	-

DNA	Concentration/(µg/ml)	260/280	260/230
mamB Colony_10_3(X+P)	60	-	-
mamB Colony_11_3(X+P)	85	1.38	2.04
mamB Colony_11_4(X+P)	60	0.58	2.32
pSB3T5(E+S)	70	0.44	2.04

DNA Purification

Tatsui

DNA	Concentration/(µg/ml)	260/280	260/230
Pcon+mamL+mamQ(S+P)	-	-	-

Ligation

Tatsui

Insert/(µL)		Vector/(µL)		T4 Ligase/(µl)	2*Buffer/(µl)	Total/(µl)
mamB Colony_10_3(X+P)	1.7	Pcon+mamL+mamQ(S+P)	2.3	1	5	10
mamB Colony_10_4(X+P)	1.7	Pcon+mamL+mamQ(S+P)	2.3	1	5	10
mamB Colony_11_3(X+P)	1.7	Pcon+mamL+mamQ(S+P)	2.3	1	5	10
mamB Colony_11_4(X+P)	1.7	Pcon+mamL+mamQ(S+P)	2.3	1	5	10
pSB3T5(E+S)	1.4	Pcon+mamB+His(E+S)	2.6	1	5	10

Transformation

Tatsui

Sample/(µL)		CompetentCells/(µl)	Total/(µl)	Medium
9/20 Ligation Pcon+mamL+mamQ+mamB Colony_10_3	1	10	11	LB(CP)
9/20 Ligation Pcon+mamL+mamQ+mamB Colony_10_4	1	10	11	LB(CP)
9/20 Ligation Pcon+mamL+mamQ+mamB Colony_11_3	1	10	11	LB(CP)
9/20 Ligation Pcon+mamL+mamQ+mamB Colony_11_4	1	10	11	LB(CP)
9/20 Ligation Pcon+mamB+His	1	10	11	LB(TET)

9/22

Colony PCR

Tatsui

NameⅠ	NameⅡ	NameⅢ	NameⅣ
9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_3_1	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_4_1	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_3_1	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_4_1
9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_3_2	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_4_2	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_3_2	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_4_2
9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_3_3	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_4_3	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_3_3	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_4_3
9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_3_4	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_4_4	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_3_4	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_4_4
9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_3_5	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_4_5	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_3_5	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_4_5
9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_3_6	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_4_6	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_3_6	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_4_6
9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_3_7	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_4_7	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_3_7	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_4_7
9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_3_8	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_4_8	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_3_8	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_4_8

NameⅤ	NameⅥ
9/20 Transformation Pcon+mamB+His Colony_1	9/19 Transformation Pcon+mamB+His Colony_1
9/20 Transformation Pcon+mamB+His Colony_2	9/19 Transformation Pcon+mamB+His Colony_2
9/20 Transformation Pcon+mamB+His Colony_3	9/19 Transformation Pcon+mamB+His Colony_3
9/20 Transformation Pcon+mamB+His Colony_4	-
9/20 Transformation Pcon+mamB+His Colony_5	-

Electrophoresis

Tatsui

Lane	Colony PCR ProductsⅠ	Colony PCR ProductsⅡ
1	Pcon+mamL+mamQ+mamB Colony_10_3_1	Pcon+mamL+mamQ+mamB Colony_11_3_1
2	Pcon+mamL+mamQ+mamB Colony_10_3_2	Pcon+mamL+mamQ+mamB Colony_11_3_2
3	Pcon+mamL+mamQ+mamB Colony_10_3_3	Pcon+mamL+mamQ+mamB Colony_11_3_3
4	Pcon+mamL+mamQ+mamB Colony_10_3_4	Pcon+mamL+mamQ+mamB Colony_11_3_4
5	Pcon+mamL+mamQ+mamB Colony_10_3_5	Pcon+mamL+mamQ+mamB Colony_11_3_5
6	Pcon+mamL+mamQ+mamB Colony_10_3_6	Pcon+mamL+mamQ+mamB Colony_11_3_6
7	Pcon+mamL+mamQ+mamB Colony_10_3_7	Pcon+mamL+mamQ+mamB Colony_11_3_7
8	Pcon+mamL+mamQ+mamB Colony_10_3_8	Pcon+mamL+mamQ+mamB Colony_11_3_8
9	100b ladder	100b ladder
10	Pcon+mamL+mamQ+mamB Colony_10_4_1	Pcon+mamL+mamQ+mamB Colony_11_4_1
11	Pcon+mamL+mamQ+mamB Colony_10_4_2	Pcon+mamL+mamQ+mamB Colony_11_4_2
12	Pcon+mamL+mamQ+mamB Colony_10_4_3	Pcon+mamL+mamQ+mamB Colony_11_4_3
13	Pcon+mamL+mamQ+mamB Colony_10_4_4	Pcon+mamL+mamQ+mamB Colony_11_4_4
14	Pcon+mamL+mamQ+mamB Colony_10_4_5	Pcon+mamL+mamQ+mamB Colony_11_4_5
15	Pcon+mamL+mamQ+mamB Colony_10_4_6	Pcon+mamL+mamQ+mamB Colony_11_4_6
16	Pcon+mamL+mamQ+mamB Colony_10_4_7	Pcon+mamL+mamQ+mamB Colony_11_4_7
17	Pcon+mamL+mamQ+mamB Colony_10_4_8	Pcon+mamL+mamQ+mamB Colony_11_4_8

Ligation

No Name

Insert/(µL)		Vector/(µL)		T4 Ligase/(µl)	10*Buffer/(µl)	MilliQ/(µl)	Total/(µl)
mamB Colony_10_3(X+P)	1	Pcon+mamL+mamQ(S+P)	7.5	0.5	1	0	10

Restriction Enzyme Digestion

Tatsui

Sample	DNA/(µl)	EcoR1/(µl)	Xba1/(µl)	Spe1/(µl)	Pst1/(µl)	BSA	Buffur/(µl)	CLAP/(µl)	MilliQ/(µl)	TOTAL/(µl)
9/19 Miniprep Pcon+mamL+mamQ+pSB1C3	34.8	0	0	2	2	0.8	8(B)	1	31.4	80

Colony PCR

Honda

NameⅠ	NameⅡ	NameⅢ	NameⅣ
9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_3_1	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_4_1	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_3_1	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_4_1
9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_3_2	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_4_2	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_3_2	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_4_2
9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_3_3	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_4_3	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_3_3	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_4_3
9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_3_4	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_4_4	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_3_4	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_4_4
9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_3_5	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_4_5	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_3_5	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_4_5
9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_3_6	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_4_6	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_3_6	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_4_6
9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_3_7	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_4_7	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_3_7	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_4_7
9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_3_8	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_4_8	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_3_8	9/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_4_8

NameⅤ
9/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_3_2(VF+VR)
9/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_3_2(seq_4+VR)
9/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_3_2(L_F+B_R)
9/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_3_2(Q_F+B_R)

Electrophoresis

Tatsui

Lane	Colony PCR ProductsⅠ	Colony PCR ProductsⅡ	Colony PCR ProductsⅢ
1	Pcon+mamL+mamQ+mamB Colony_10_3_1	Pcon+mamL+mamQ+mamB Colony_11_3_1	-
2	Pcon+mamL+mamQ+mamB Colony_10_3_2	Pcon+mamL+mamQ+mamB Colony_11_3_2	-
3	Pcon+mamL+mamQ+mamB Colony_10_3_3	Pcon+mamL+mamQ+mamB Colony_11_3_3	-
4	Pcon+mamL+mamQ+mamB Colony_10_3_4	Pcon+mamL+mamQ+mamB Colony_11_3_4	-
5	Pcon+mamL+mamQ+mamB Colony_10_3_5	Pcon+mamL+mamQ+mamB Colony_11_3_5	-
6	Pcon+mamL+mamQ+mamB Colony_10_3_6	Pcon+mamL+mamQ+mamB Colony_11_3_6	-
7	Pcon+mamL+mamQ+mamB Colony_10_3_7	Pcon+mamL+mamQ+mamB Colony_11_3_7	-
8	Pcon+mamL+mamQ+mamB Colony_10_3_8	Pcon+mamL+mamQ+mamB Colony_11_3_8	-
9	100b ladder	100b ladder	100b ladder
10	Pcon+mamL+mamQ+mamB Colony_10_4_1	Pcon+mamL+mamQ+mamB Colony_11_4_1	9/19 Transformation Pcon+mamB+His Colony_1
11	Pcon+mamL+mamQ+mamB Colony_10_4_2	Pcon+mamL+mamQ+mamB Colony_11_4_2	9/19 Transformation Pcon+mamB+His Colony_2
12	Pcon+mamL+mamQ+mamB Colony_10_4_3	Pcon+mamL+mamQ+mamB Colony_11_4_3	9/19 Transformation Pcon+mamB+His Colony_3
13	Pcon+mamL+mamQ+mamB Colony_10_4_4	Pcon+mamL+mamQ+mamB Colony_11_4_4	Pcon+mamL+mamQ+mamB Colony_11_3_2(VF+VR)
14	Pcon+mamL+mamQ+mamB Colony_10_4_5	Pcon+mamL+mamQ+mamB Colony_11_4_5	Pcon+mamL+mamQ+mamB Colony_11_3_2(seq_4+VR)
15	Pcon+mamL+mamQ+mamB Colony_10_4_6	Pcon+mamL+mamQ+mamB Colony_11_4_6	Pcon+mamL+mamQ+mamB Colony_11_3_2(L_F+B_R)
16	Pcon+mamL+mamQ+mamB Colony_10_4_7	Pcon+mamL+mamQ+mamB Colony_11_4_7	Pcon+mamL+mamQ+mamB Colony_11_3_2(Q_F+B_R)
17	Pcon+mamL+mamQ+mamB Colony_10_4_8	Pcon+mamL+mamQ+mamB Colony_11_4_8	-

Miniprep

Tatsui

DNA	Concentration/(µg/ml)	260/280	260/230
Pcon+mamQ+pSB1C3 Colony_3	85	1.51	0.79
Pcon+mamQ+pSB1C3 Colony_5	60	1.94	1.12

9/27

Western Blotting

Honda

Electrophoresis:200V 25mA 30min

Transcription:50V 200mA 73min

Make the 20% acrilamid gel apray the following

Lane	Sample
1-5 9-12	Sample buffer
6	T7-RBS-1C3(control)
7	pL(1C3)
8	protein marker

Liquid Culture

Sample
pLQB(9/22 master)
pLQ(9/22 master)

9/28

Fixation

Honda

25% Glyceraldehyde/(ml)	Phosphate buffer/(ml)	MilliQ/(ml)	Total/(ml)
1.25	5.0	3.75	10.0

9/29

Liquid Culture

Honda

Sample
9/22 master pLQB
9/22 master pLQ
9/22 master pL
9/22 master pQ-3
9/22 master pQ-5
9/20 master pB-22
9/20 master pB-23

Fixation

Honda

Osmic acid/(ml)	Phosphate buffer/(ml)	Total/(ml)
4	4	8

9/30

Miniprep

Tatsui

DNA	Concentration/(µg/ml)	260/280	260/230
9/29 Liquid Culture pLQB	-	-	-
9/29 Liquid Culture pLQ	-	-	-

Western Blotting

Honda

Electrophoresis:200V 25mA 1h30min

Transcription:50V 200mA 73min

Make the 20% acrilamid gel apray the following

Lane	Sample
2	Marker
3	pLQB
4	pLQ
5	pL
6	pQ-3
7	pQ-5
8	pB
9	GTP-His
10	T7-RBS-1C3(control)
11	Sample buffer

Liquid Culture

Honda

Sample
9/22 master pLQB
9/22 master pQ(3)

10/1

Ethanol Precipitation

Tatsui

Sample
9/28 M pL(1C3)(VF)
9/28 M pL(1C3)(VR)
9/22 M pB(22)(VF)
9/22 M pB(22)(VR)
9/22 pB(23)(VF)
9/22 pB(23)(VR)
10/1 M pQ(3)(VF)
10/1 M pQ(3)(VR)
10/1 M pLQB(VF)
10/1 M pLQB(seq-1)
10/1 M pLQB(seq-2)
10/1 M pLQB(seq-3)
10/1 M pLQB(seq-4)
10/1 M pLQB(VR)

Liquid Culture

Honda

Sample
9/22 master Plate pLQB
9/22 master Plate pQ(3)

Restriction Enzyme Digestion

Sample	DNA/(µl)	Xba1/(µl)	Spe1/(µl)	Pst1/(µl)	BSA	Buffer/(µl)	MilliQ/(µl)	TOTAL/(µl)
9/23 pQ	30	3	0	3	1	9	44	90
pQ(control)	1	0	0	0	0	1	8	10
9/2 Pcon(1C3)	30	0	3	3	1	9	44	90
9/2 Pcon(1C3)(control)	1	0	0	0	0	1	8	10

Miniprep

Honda

DNA	Concentration/(µg/ml)	260/280	260/230
9/29 Liquid Culture pLQB	-	-	-
9/29 Liquid Culture pQ(3)	-	-	-
9/29 Liquid Culture pQ(22)	-	-	-
9/29 Liquid Culture pQ(23)	-	-	-

Sequence PCR

Target	9/28 M pL(1C3)(VF)	9/28 M pL(1C3)(VR)	9/22 M pB(22)(VF)	9/22 M pB(22)(VR)	9/22 pB(23)(VF)	9/22 pB(23)(VR)	10/1 M pQ(3)(VF)	10/1 M pQ(3)(VR)	10/1 M pLQB(VF)	10/1 M pLQB(seq-1)	10/1 M pLQB(seq-2)	10/1 M pLQB(seq-3)	10/1 M pLQB(seq-4)	10/1 M pLQB(VR)
Template/(µl)	4.7	4.7	1.6	1.6	3.2	3.2	5	5	5	5	5	5	5	5
Big dye/(µl)	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5
5×Buffer/(µl)	1.75	1.75	1.75	1.75	1.75	1.75	1.75	1.75	1.75	1.75	1.75	1.75	1.75	1.75
Primer(VF)/(µl)	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5
MilliQ/(µl)	3.05	3.05	6.15	6.15	4.55	4.55	2.75	2.75	2.75	2.75	2.75	2.75	2.75	2.75
TOTAL/(µl)	10.5	10.5	10.5	10.5	10.5	10.5	10.5	10.5	10.5	10.5	10.5	10.5	10.5	10.5

Steps	Temparature	Time	cycle
Pre Denature	96℃	2min	1time
Denature	96℃	10sec	30cycle
Annealing	55℃	5sec	30cycle
Extension	60℃	4min	30cycle
Final elongation	4℃	1min	1time
Final hold	4℃	infinity	-

Gel Extraction

(1)

Lane	SampleⅠ	Volume/(µl)
1	blank	-
2	1400b ladder	-
3	His-OE-1	-
4	1kb ladder	-
5	750b ladder	-
6	His-OE-2	-

(2)

Lane	SampleⅡ	Volume/(µl)
3,6	pQ(X,P)	-
4	100b ladder	-

(3)

Lane	SampleⅢ	Volume/(µl)
3,6	Pcon 1C3(S,P)	-
4	1kb ladder	-

(4)

Lane	SampleⅣ	Volume/(µl)
3	DiDECARB-4	-
6	DiDECARB-5	-
2	DiDECARB-4(control)	-
5	DiDECARB-5(control)	-
4	1kb ladder	-

10/2

Miniprep

DNA	Concentration/(µg/ml)	260/280	260/230
pLQB	390	1.70	1.20
pLQ	319	1.64	0.92
j04450	255	1.59	1.04

10/3

PCR

Honda

Target	Volume
10×buffur for KOD Plus Ver.2/(µl)	5
2mMdNTPs/(µl)	5
25mM MgSO4/(µl)	3
VF Primer/(µl)	1.5
VR Primer/(µl)	1.5
template/(µl)	1
KOD-plus/(µl)	1
MilliQ/(µl)	33
TOTAL/(µl)	50

DNA Purification

DNA	Concentration/(µg/ml)	260/280	260/230
10/3 PB	39.8	1.75	0.92

Digestion

Sample	DNA/(µl)	EcoRI/(µl)	Spe1/(µl)	BSA	Buffer/(µl)	MilliQ/(µl)	TOTAL/(µl)
10/3 Column pB	15.8	1	1	0.2	2	-	20

Restriction Enzyme Digestion

Honda

Sample	DNA/(µl)	Xba1/(µl)	Pst1/(µl)	BSA	Buffer/(µl)	MilliQ/(µl)	TOTAL/(µl)
DiDECARB-4	10	2	2	0.4	4	21.6	40
DiDECARB-4(control)	1	0	0	0	1	8	10
DiDECARB-5	10	2	2	0.4	4	21.6	40
DiDECARB-5(control)	1	0	0	0	1	8	10

Gel Extraction

Tatsui

DNA	Concentration/(µg/ml)	260/280	260/230
pSB1C3(X,P)-4	942	1.89	1.26
pSB1C3(X,P)-5	518	1.87	1.26

10/4

Digestion

Tatsui

Sample	DNA/(µl)	Xba1/(µl)	Pst1/(µl)	BSA	Buffer/(µl)	MilliQ/(µl)	TOTAL/(µl)
9/18 MP pL-1C3(85)	24	1	1	0.4	4	9.6	40
9/18 MP pL-1C3(85)(control)	1	0	0	0	1	8	10
9/18 MP Bµ-1C3-11-3(355)	6	1	1	0.4	4	27.6	40
9/18 MP Bµ-1C3-11-3(355)(control)	1	0	0	0	1	8	10

Gel Extraction

(1)

Lane	SampleⅠ	Volume/(µl)
1	10/4 Bµ 1C3-11-3(control)	-
3,4	10/4 Bµ 1C3-11-3(XP)	-
6	ladder	-

(2)

Lane	SampleⅡ	Volume/(µl)
1	ladder	-
3,4	10/4 pL-1C3(XP)	-
6	10/4 pL-1C3(control)	-

Ligation

No name

Sample	Vector/(µL)	Insert/(µL)	Ligase/(µl)	Buffer/(µl)	MilliQ/(µl)	Total/(µl)
9/20 Q-1C3(B)	1	3	1	5	0	10
9/16 Q-1C3(B)	1	3	1	5	0	10
9/20 Q-1C3(B)(control)	1	0	1	5	3	10
9/16Q-1C3(B)(control)	1	0	1	5	3	10

Transformation

Sample	Plasmid/(µl)	CompetentCells(DH5α)/(µl)
E1	2	10
E2	2	10
E3	2	10
E4	2	10

10/5

Ethanol Precipitation

Honda

10/3 Gel Extraction Pcon1C3(SP)

Ligation

Honda

sample	Vector/(µL)	Insert/(µL)	Toyobo Ligation high/(µl)
10/5 Pcon1C3(SP)+9/20pQ(3)(XP)	1	1	2
10/5 Pcon1C3(SP)(control)	1	1	2

Transformation

Honda

Sample	Plasmid/(µl)	CompetentCells(DH5α)/(µl)
F1	2	10
F2	2	10

Colony PCR

Lane	Colony PCR ProductsⅠ
9-14	E1 1-6
15-20	E2 1-6

Liquid Culture

Sample
E1 1-6
E2 1-6

DNA Purification

Honda

DNA	Concentration/(µg/ml)	260/280	260/230
pB PCR	57	2.06	2.39

Digestion

Sample	DNA/(µl)	EcoRI/(µl)	Xba1/(µl)	Spe1/(µl)	Pst1/(µl)	Buffer/(µl)	BSA	MilliQ/(µl)	TOTAL/(µl)
9/18 MP Bµ(1C3)11-4(ES)	10	1	0	1	0	4	0.4	23.6	40
9/18 MP Bµ(1C3)11-4(control)	1	0	0	0	0	1	0	8	10
9/22 MP pQ-3(XP)	40	0	4	0	4	16	1.6	94.4	160
9/22 MP pQ-3(control)	1	0	0	0	0	1	0	8	10
9/22 Pcon Master(SP)	10	0	0	1	1	4	0.4	23.6	40
9/22 Pcon Master(control)	1	0	0	0	0	1	0	8	10
9/22 pB(PCR Product)(ES)	20	1	0	1	0	4	0.4	13.6	40
9/22 pB(PCR Product)(control)	1	0	0	0	10/td>	1	0	8	10

PCR

No Name

Target	Volume
2×KAPA HiFi Hotstart Ready Mix/(µl)	12.5
Fw Primer/(µl)	0.75
Rv Primer/(µl)	0.75
Template(9/22 Mini Pcon-mamQ-3)	pick up
MilliQ/(µl)	11
TOTAL/(µl)	25

Steps	Temparature	Time	cycle
Pre Denature	98℃	5min	1time
Denature	98℃	20sec	30cycle
Annealing	60℃	15sec	30cycle
Extension	72℃	30sec	30cycle
Final elongation	72℃	5min	1time
Final hold	12℃	-	-

10/6

Sequence PCR

Target	10/6 M T7-RBS-his(1C3)-1-VF	10/6 M T7-RBS-his(1C3)-1-VR	10/6 M T7-RBS-his(1C3)-2-VF	10/6 M T7-RBS-his(1C3)-2-VR	10/6 M T7-RBS-his(1C3)-3-VF	10/6 M T7-RBS-his(1C3)-3-VR	10/6 M Q(1C3)E1-1 VF	10/6 M Q(1C3)E1-1 VR	10/6 M Q(1C3)E1-2-VF	10/6 M Q(1C3)E1-2-VR	10/6 M Q(1C3)E1-3-VF	10/6 M Q(1C3)E1-3-VR	10/6 M Q(1C3)E2-1-VF	10/6 M Q(1C3)E2-1-VR	10/6 M Q(1C3)E2-2-VF	10/6 M Q(1C3)E2-2-VR
Template/(µl)	2.2	2.2	2.5	2.5	2.5	2.5	1.85	1.85	1.1	1.1	1.7	1.7	1.8	1.8	1.7	1.7
Big dye/(µl)	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5
5×Buffer/(µl)	1.75	1.75	1.75	1.75	1.75	1.75	1.75	1.75	1.75	1.75	1.75	1.75	1.75	1.75	1.75	1.75
Primer/(µl)	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5
MilliQ/(µl)	5.55	5.55	5.25	5.25	5.25	5.25	5.9	5.9	6.65	6.65	6.05	6.05	5.95	5.95	6.05	6.05
TOTAL/(µl)	10.5	10.5	10.5	10.5	10.5	10.5	10.5	10.5	10.5	10.5	10.5	10.5	10.5	10.5	10.5	10.5

Steps	Temparature	Time	cycle
Pre Denature	96℃	2min	1time
Denature	96℃	10sec	30cycle
Annealing	50℃	5sec	30cycle
Extension	60℃	4min	30cycle
Final elongation	4℃	1min	1time
Final hold	4℃	infinity	-

Miniprep

DNA	Concentration/(µg/ml)	260/280	260/230
T7-RBS-his(1C3)-1	180	1.84	1.76
T7-RBS-his(1C3)-2	160	1.92	1.92
T7-RBS-his(1C3)-3	156	1.69	1.64
E1-1	216	1.84	2.04
E1-2	355	1.82	2.31
E1-3	238	1.81	2.11
E2-1	225	1.83	2.00
E2-2	230	1.76	1.84

Gel Extraction

(1)

Lane	SampleⅠ	Volume/(µl)
1	10/5 B&micro(1C3)(control)	-
3	10/5 B&micro(1C3)(ES)	-
6	10/5 pB(ES)	-

(2)

Lane	SampleⅡ	Volume/(µl)
1	pQ-3(control)	-
3	pQ-3(XP)	-
4	Pcon(control)	-
6	Pcon(SP)	-