Template:Kyoto/Notebook/DMS

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(Difference between revisions)

Revision as of 06:23, 13 October 2014

7/31

Restriction Enzyme Digestion

Nakamura and Matsumoto

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
3/24 dddD PCR product	3.2	1	-	1	-	H	3	-	21.8	30
pSB1C3	18.5	1	-	1	-	H	3	-	6.5	30
pSB1C3 control	6.2	-	-	-	-	H	1	-	2.8	10

8/1

Gel Extraction

Murata and Yasuda

Lane	Sample
1	1kbp ladder
2	3/24 dddD PCR product E,S
3	3/24 dddD PCR product E,S
4	Blank
5	pSB1C3 E,S
6	pSB1C3 E,S
7	Blank
8	1kbp ladder
9	3/24 dddD PCR product control
10	pSB1C3 control

Sample	Concentration/(µg/mL)	260/280	260/230
PSB1C3	13.3	1.90	-
dddD	10.8	1.64	0.15

Ligation

Yasuda

Vector/(µL)		Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
8/1 PSB1C3 gel extraction product 2047bp	1.5	8/1 dddD gel extraction product 2545bp	6.9	10xT4Ligase	2	T4Ligase	1	8.6	20

Transformation

Murata

Sample/(µL)		CompetentCells/(µL)		Total/(µL)	Medium
PSB1C3-dddD	3	Self-Making Competent Cells (DH5α)	30	33	SOC

8/2

PCR Cloning

Murata

dddD without Pre-Suf	2x KAPA HiFi Hotstart Ready Mix/(µL)	F Pre-dddD PCR product 62/(µL)	Rv Spe1-dddD PCR product 68/(µL)	MilliQ/(µL)	Total/(µL)
pick up	12.5	0.75	0.75	11	25

Predenature	Denature	Annealing	Extension
95°C	98°C	65°C	72°C
5min	20sec	15sec	50sec

Denature-Annealing-Extension 25cycle

DNA Purification

Murata

8/2 Pre-dddD-Suf PCR product

Colony PCR

Honda,Nakamura and Matsumoto

Master Mix

Quick Taq/(µL)	VF2/(µL)	VR/(µL)	MilliQ/(µL)	Total/(µL)
15	0.6	0.6	13.8	30

Predenature	Denature	Annealing	Extension
94°C	94°C	55°C	68°C
2min	30sec	30sec	24sec

Denature-Annealing-Extension 30cycle

Number	Sample
1	pSB1C3-dddD(1)
2	pSB1C3-dddD(2)
3	pSB1C3-dddD(3)

Concentration Measurement

Nakamura and Matsumoto

Sample	Concentration/(µg/mL)	260/280	260/230
dddD PCR product	245	1.59	2.17

Restriction Enzyme Digestion

Nakamura and Matsumoto

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
8/2 dddD PCR product	8.2	1	-	1	-	H	3	-	16.8	30
8/2 dddD PCR product control	0.41	-	-	-	-	H	1	-	8.6	10
pSB1C3	12.7	1	-	1	-	H	3	-	12.3	30
pSB1C3 control	0.04	-	-	-	-	H	1	-	8.4	10

8/3

Electrophoresis

Murata

Lane	Sample
1	pSB1C3-dddD(1)
2	pSB1C3-dddD(2)
3	pSB1C3-dddD(3)
4	1kb ladder (3µL)
5	1kb ladder (1µL)

Gel Extraction

Tatsui

Lane	Sample
1	1kb ladder
2	Blank
3	pSB1C3 2070bp
4	Blank
5	pSB1C3
6	Blank
7	8/2 dddD PCR product 2544bp
8	Blank
9	8/2 dddD PCR product

Sample	Concentration/(µg/mL)	260/280	260/230
pSB1C3	12.5	1.37	0.42
dddD PCR product	35	1.54	0.85

Ligation

no name

Vector/(µL)		Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
pSB1C3	7	dddD	7	2x Buffer	15	T4 Ligase	1	-	30
pSB1C3	7	dddD	14	2x Buffer	22	T4 Ligase	1	-	44
pSB1C3	7	dddD	-	2x Buffer	15	T4 Ligase	1	7	30

Transformation

no name

Sample	CompetentCells	Medium
(1)	Self-Making Competent Cells (DH5α)	CP
(2)	Self-Making Competent Cells (DH5α)	CP

8/4

Colony PCR

Honda

Master Mix

Quick Taq/(µL)	VF2/(µL)	VR/(µL)	MilliQ/(µL)	Total/(µL)
40	1.6	1.6	36.8	80

Predenature	Denature	Annealing	Extension
94°C	94°C	55°C	68°C
2min	30sec	30sec	24sec

Denature-Annealing-Extension 30cycle

Lane	Sample
1	dddD+pSB1C3(1)-1
2	dddD+pSB1C3(1)-2
3	dddD+pSB1C3(2)-1
4	dddD+pSB1C3(2)-2
5	dddD+pSB1C3(2)-3
6	dddD+pSB1C3(2)-4
7	pSB1C3 control-1
8	control

Electrophoresis

Matsumoto

Lane	Sample
1	1kbp ladder
2	dddD+pSB1C3(1)-1
3	dddD+pSB1C3(1)-2
4	dddD+pSB1C3(2)-1
5	dddD+pSB1C3(2)-2
6	dddD+pSB1C3(2)-3
7	dddD+pSB1C3(2)-4
8	pSB1C3 control-1
9	control
10	1kbp ladder

PCR

Honda

Unexpected bands are confirmed in former Electrophoresis, so contamination of MilliQ or Quicktaq is suspected. For confirming contamination, PCR and Electrophoresis are conducted.

* suspicious samples for contamination

(1)

*Quick Taq/(µL)	VF2/(µL)	VR/(µL)	MilliQ/(µL)	Total/(µL)
5	0.2	0.2	4.6	10

(2)

Quick Taq/(µL)	VF2/(µL)	VR/(µL)	MilliQ/(µL)	Total/(µL)
5	0.2	0.2	4.6	10

(3)

Quick Taq/(µL)	VF2/(µL)	VR/(µL)	*MilliQ/(µL)	Total/(µL)
5	0.2	0.2	4.6	10

(4)

*Quick Taq/(µL)	VF2/(µL)	VR/(µL)	*MilliQ/(µL)	Total/(µL)
5	0.2	0.2	4.6	10

Predenature	Denature	Annealing	Extension
94°C	94°C	55°C	68°C
2min	30sec	30sec	24sec

Denature-Annealing-Extension 30cycle

Electrophoresis

Honda

Lane	Sample
1	1kbp ladder
2	(1)
3	(2)
4	(3)
5	(4)

8/18

PCR

Honda

For confirming contamination of VF2 and VR primers, we pour samples into new tubes and perform PCR and Electrophoresis.

(1)

Quick Taq/(µL)	VF2/(µL)	VR/(µL)	MilliQ/(µL)	Total/(µL)
5	0.2	0.2	4.6	10

control(2)

Quick Taq/(µL)	VF2/(µL)	VR/(µL)	8/2 Pcon3/(µL)	MilliQ/(µL)	Total/(µL)
5	0.2	0.2	0.6	4.6	10

(3)

Quick Taq/(µL)	VF2/(µL)	VR/(µL)	MilliQ/(µL)	Total/(µL)
5	0.2	0.2	4.6	10

(4)

Quick Taq/(µL)	VF2/(µL)	VR/(µL)	8/2 Pcon3/(µL)	MilliQ/(µL)	Total/(µL)
5	0.2	0.2	0.6	4.0	10

Predenature	Denature	Annealing	Extension
94°C	94°C	55°C	68°C
2min	30sec	30sec	24sec

Denature-Annealing-Extension 30cycle

Electrophoresis

Honda

Number	Sample
1	1kbp ladder
2	(1)
3	(2)
4	(3)
5	(4)

Restriction Enzyme Digestion

Honda

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
dddD 8/2 PCR product	5.5	0.7	-	0.7	-	H	2	-	11.1	20
dddD 8/2 PCR product control	0.41	-	-	-	-	H	1	-	8.6	10
pSB1C3	12.7	1	-	1	-	H	3	-	8.4	10
pSB1C3 control	0.64	-	-	-	-	H	1	-	8.4	10

Colony PCR

Honda

Master Mix

Quick Taq/(µL)	VF2/(µL)	VR/(µL)	MilliQ/(µL)	Total/(µL)
40	1.6	1.6	36.8	80

Predenature	Denature	Annealing	Extension
94°C	94°C	55°C	68°C
2min	30sec	30sec	24sec

Denature-Annealing-Extension 30cycle

Number	Sample
1	dddD+pSB1C3(1)-1
2	dddD+pSB1C3(1)-2
3	dddD+pSB1C3(2)-1
4	dddD+pSB1C3(2)-2
5	dddD+pSB1C3(2)-3
6	dddD+pSB1C3(2)-4
7	pSB1C3 control-1
8	control

Electrophoresis

Honda

Lane	Sample
1	1kbp ladder
2	dddD+pSB1C3(1)-1
3	dddD+pSB1C3(1)-2
4	dddD+pSB1C3(2)-1
5	dddD+pSB1C3(2)-2
6	dddD+pSB1C3(2)-3
7	dddD+pSB1C3(2)-4
8	pSB1C3 control-1
9	control

Gel Extraction

Murata

Lane	Sample
1	1kbp ladder
2	dddD control
3	dddD E,S
4	Blank
5	pSB1C3 control
6	pSB1C3 E,S

Sample	Concentration/(µg/mL)	260/280	260/230
8/18 pSB1C3	105	1.30	0.85
8/18 dddD	105	1.27	0.73

8/19

Ligation

Honda,Li,Yamauchi

(1)

Vector/(µL)		Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
pSB1C3	3.6	dddD	1	2xBuffer	5.6	T4Ligase	1	-	11.2

(2)

Vector/(µL)		Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
pSB1C3	3.6	-	-	2xBuffer	5.6	T4Ligase	1	1	11.2

8/20

Transformation

Murata

Sample/(µL)		CompetentCells/(µL)		Total/(µL)	Medium
8/19 Ligation (1)	11.2	Competent Cells (DH5α)	50	61.2	SOC
8/19 Ligation (2)	11.2	Competent Cells (DH5α)	50	61.2	SOC

Ligation

Nakamura,Honda

(1)

Vector/(µL)		Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
pSB1C3	10.8	dddD	3	2xBuffer	16.8	T4Ligase	3	-	33.6

(2)

Vector/(µL)		Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
pSB1C3 control	10.8	-	-	2xBuffer	16.8	T4Ligase	3	3	33.6

Transformation

Nakamura,Honda

Sample/(µL)		CompetentCells/(µL)		Total/(µL)	Medium
dddD+pSB1C3	5	Competent Cells (DH5α)	20	25	SOC
control	5	Competent Cells (DH5α)	20	25	SOC

8/21

Colony PCR

Honda,Nakamura

Master Mix

Quick Taq/(µL)	VF2/(µL)	VR/(µL)	MilliQ/(µL)	Total/(µL)
35	1.4	1.4	32.2	70

Predenature	Denature	Annealing	Extension
94°C	94°C	55°C	68°C
2min	30sec	30sec	24sec

Denature-Annealing-Extension 30cycle

Electrophoresis

Nakamura

Number	Sample
1	8/20 dddD+pSB1C3(1)-1
2	8/20 dddD+pSB1C3(1)-2
3	8/20 dddD+pSB1C3(2)-1
4	8/20 dddD+pSB1C3(2)-2
5	8/20 dddD+pSB1C3(3)-1
6	8/20 dddD+pSB1C3(3)-2
7	control

Number	Sample
1	1kbp ladder
2	8/20 dddD+pSB1C3(1)-1
3	8/20 dddD+pSB1C3(1)-2
4	8/20 dddD+pSB1C3(2)-1
5	8/20 dddD+pSB1C3(2)-2
6	8/20 dddD+pSB1C3(3)-1
7	8/20 dddD+pSB1C3(3)-2
8	control

Gel Extraction

Yasuda

Lane	Sample
1	1kbp ladder
2	8/22 Mp(1) E,S
3	8/22 Mp(1) E,S
4	8/22 Mp(1) E,S

Lane	Sample
1	1kbp ladder
2	8/22 Mp(2) E,S
3	8/22 Mp(2) E,S
4	Blank
5	1kbp ladder
6	8/22 Mp(3) E,S
7	8/22 Mp(3) E,S

8/25

Gel Extraction

Yasuda

Lane	Sample
1	1kbp ladder
2	8/22 Mp(1) E,S
3	8/22 Mp(1) E,S
4	8/22 Mp(1) E,S

Ligation

Yasuda

Vector/(µL)		Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
8/22 Mp(1) E,S	1.3	8/18 dddD gel extraction product	0.71	2x Buffer	5	T4 Ligase	0.3	2.69	10
8/22 Mp(2) E+S	0.95	8/18 dddD gel extraction product	0.71	2x Buffer	5	T4 Ligase	0.3	3.04	10
8/22 Mp(3) E+S	0.69	8/18 dddD gel extraction product	0.71	2x Buffer	5	T4Ligase	0.3	3.3	10

8/26

Transformation

Nakamura,Matsumoto

Sample/(µL)		CompetentCells/(µL)		Total/(µL)	Medium
8/25 Ligation (1)	10	Competent Cells (DH5α)	50	60	SOC
8/25 Ligation(2)	10	Competent Cells (DH5α)	50	60	SOC
8/25 Ligation(3)	10	Competent Cells (DH5α)	30	40	SOC

8/27

Colony PCR

Murata

Master Mix

Quick Taq/(µL)	VF2/(µL)	VR/(µL)	MilliQ/(µL)	Total/(µL)
80	3.2	3.2	73.6	160

Predenature	Denature	Annealing	Extension
94°C	94°C	55°C	68°C
2min	30sec	30sec	140sec

Denature-Annealing-Extension 30cycle

Number	Sample
1	8/26 dddD+pSB1C3(1)-1
2	8/26 dddD+pSB1C3(1)-2
3	8/26 dddD+pSB1C3(1)-3
4	8/26 dddD+pSB1C3(1)-4
5	8/26 dddD+pSB1C3(1)-5
6	8/26 dddD+pSB1C3(2)-1
7	8/26 dddD+pSB1C3(2)-2
8	8/26 dddD+pSB1C3(2)-3
9	8/26 dddD+pSB1C3(2)-4
10	8/26 dddD+pSB1C3(2)-5
11	8/26 dddD+pSB1C3(3)-1
12	8/26 dddD+pSB1C3(3)-2
13	8/26 dddD+pSB1C3(3)-3
14	8/26 dddD+pSB1C3(3)-4
15	8/26 dddD+pSB1C3(3)-5
16	control

Electrophoresis

Yasuda

Lane	Sample
1	1kbp ladder
2	8/26 dddD+pSB1C3(1)-1
3	8/26 dddD+pSB1C3(1)-2
4	8/26 dddD+pSB1C3(1)-3
5	8/26 dddD+pSB1C3(1)-4
6	8/26 dddD+pSB1C3(1)-5

Lane	Sample
1	1kbp ladder
2	8/26 dddD+pSB1C3(2)-1
3	8/26 dddD+pSB1C3(2)-2
4	8/26 dddD+pSB1C3(2)-3
5	8/26 dddD+pSB1C3(2)-4
6	8/26 dddD+pSB1C3(2)-5
7	1kbp ladder
8	8/26 dddD+pSB1C3(3)-1
9	8/26 dddD+pSB1C3(3)-2
10	8/26 dddD+pSB1C3(3)-3
11	8/26 dddD+pSB1C3(3)-4
12	8/26 dddD+pSB1C3(3)-5
13	1kbp ladder
14	control

8/29

PCR

Matsumoto

Master Mix

2x KAPA HiFi HotStart ReadyMix /(µL)	V Pre-dddD 62/(µL)	Rv Spe1-dddD 68/(µL)	MilliQ/(µL)	DNA Purification dddD without Pre-Suf	Total/(µL)
12.5	0.75	0.75	11	pick up	25

Predenature	Denature	Annealing	Extension
95°C	98°C	65°C	72°C
5min	20sec	15sec	50sec

Denature-Annealing-Extension 25cycle

DNA Purification

Matsumoto

Sample	Concentration/(µg/mL)	260/280	260/230
8/29 Pre-dddD-Suf PCR product	335	1.73	1.91

Restriction Enzyme Digestion

Matsumoto

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
8/29 Pre-dddD-Suf PCR product	6.0	1	-	1	-	H	3	-	19	30

DNA Purification

Matsumoto

Sample	Concentration/(µg/mL)	260/280	260/230
8/29 Pre-dddD-Suf Restriction Enzyme Digestion product	60	1.64	0.94

Ligation

Matsumoto

Vector/(µL)		Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
pSB1C3 E,S gel extraction product	4.7	dddD-pSB1C3	4	2x Buffer	9.7	T4 Ligase	1	-	19.4
pSB1C3 E,S gel extraction product control	4.7	-	-	2x Buffer	9.7	T4 Ligase	1	4	19.4

Transformation

Matsumoto

Sample/(µL)		CompetentCells/(µL)		Total/(µL)	Medium
dddD 8/29 ligation product	10	DH5α	40	50	SOC
8/29 ligation product control	10	DH5α	40	50	SOC
pSB1C3 control	1	DH5α	20	21	SOC

9/1

Colony PCR

Honda

Master Mix

Quick Taq/(µL)	VF2/(µL)	VR/(µL)	MilliQ/(µL)	Total/(µL)
30	1.2	1.2	27.6	60

Predenature	Denature	Annealing	Extension
94°C	94°C	55°C	68°C
2min	30sec	30sec	24sec

Denature-Annealing-Extension 30cycle

Number	Sample
1	pSB1C3-dddD(1)
2	pSB1C3-dddD(2)
3	pSB1C3-dddD(3)
4	pSB4A5-(1)
5	pSB4A5-(2)
6	control

Electrophoresis

Matsumoto

Lane	Sample
1	1kb ladder
2	9/1 dddD+pSB1C3-1
3	9/1 dddD+pSB1C3-2
4	9/1 dddD+pSB1C3-3
5	9/1 pSB4A5-1
6	9/1 pSB4A5-2
7	control
8	1kb ladder

Colony PCR

Matsumoto

Master Mix

Quick Taq/(µL)	VF2/(µL)	VR/(µL)	MilliQ/(µL)	Total/(µL)
30	1.2	1.2	27.6	60

Predenature	Denature	Annealing	Extension
94°C	94°C	55°C	68°C
2min	30sec	30sec	24sec

Denature-Annealing-Extension 30cycle

Number	Sample
1	pSB1C3-dddD(1)
2	pSB1C3-dddD(2)
3	pSB1C3-dddD(3)
4	pSB4A5-(1)
5	pSB4A5-(2)
6	control

Electrophoresis

Matsumoto

Lane	Sample
1	1kb ladder
2	9/1 dddD+pSB1C3-1
3	9/1 dddD+pSB1C3-2
4	9/1 dddD+pSB1C3-3
5	9/1 pSB4A5-1
6	9/1 pSB4A5-2
7	control
8	1kb ladder

9/2

Electrophoresis

Mastumoto

Lane	Sample
1	1kb ladder
2	8/29 dddD(DNA purification product)1µL+MilliQ 5µL+looding dye(x6)1µL
3	control

Cloning

Yasuda

2x KAPA HiFi HotStart ReadyMix/(µL) Fw REDOX cloning primer 59/(µL) Rv REDOX primer Ver.2 64/(µL) DNA CCAP 1023/1 /(µL) MilliQ/(µL) Total/(µL)

12.5 0.75 0.75 1 10 25

Predenature Denature Annealing Extension Final Extension Cooling

95°C 98°C 65°C 72°C 72°C 4°C

5min 20sec 15sec 2min10sec 5min Infinity

Denature-Annealing-Extension 25cycle

Restriction Enzyme Digestion

Matsumoto

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
8/29 dddD(DNA purification product)	6	1	-	1	-	-	3	-	19	30

Cloning

Yasuda
Master Mix(x4)

2x KAPA HiFi HotStart ReadyMix/(µL) Fw primer 59/(µL) Rv primer Ver.2 64/(µL) 3/19 dddD(DNA purification) /(µL) MilliQ/(µL) Total/(µL)

12.5 0.75 0.75 0.1 11 25

Predenature Denature Annealing Extension Final Extension Cooling

95°C 98°C 65°C 72°C 72°C 4°C

5min 20sec 15sec 2min10sec 5min Infinity

Denature-Annealing-Extension 25cycle

9/3

Gel Extraction

Yamauchi

Lane	Sample
2	dddD (E,S) 2544bp.
4	dddD (E,S)
6	1kb ladder

DNA Purification

Matsumoto

Sample	Concentration/(µg/mL)	260/280	260/230
Pre-dddD-suf	685	1.66	2.13

Ligation

Honda, Li and Yamauchi

(1)insert: dddD

Vector/(µL)		Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
pSB1C3	1.5	dddD	6.3	2x Buffer	8.8	T4 Ligase	1	-	17.6

(2)control

Vector/(µL)		Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
pSB1C3	1.5	dddD	-	2x Buffer	8.8	T4 Ligase	1	6.3	17.6

Transformation

Yamauchi

Sample/(µL)		CompetentCells/(µL)		Total/(µL)	Medium
9/3 Ligation product (dddD)	3	DH5α	15	18	SOC
9/3 Ligation product (control)	2	DH5α	10	12	SOC

30min on ice
1min heat shock
2min on ice

(1)Adding SOC medium(4 times) to each of them and doing preculture at 37°C for 1 hour.
(2)Adding SOC medium to each of them, up to 100µL.

9/4

Electrophoresis

Honda

Lane	Sample
1	1kb ladder
2	9/3 Ligation dddD + pSB1C3
3	8/20 Ligation dddD + pSB1C3
4	8/1 Ligation dddD + pSB1C3
5	9/3 dddD (Gel extraction product)(E,S)
6	9/3 dddD (DNA purification product)
7	1kb ladder

Cloning

Murata and Honda

(1)

2x KAPA HiFi HotStart ReadyMix/(µL)	Fw primer AT cloning primer 61/(µL)	Rv primer AT cloning primer 61/(µL)	DNA CCAP 1023/1 /(µL)	MilliQ/(µL)	Total/(µL)
12.5	0.75	0.75	1	10	25

(2)

2x KAPA HiFi HotStart ReadyMix/(µL)	Fw SAMmt cloning primer 58/(µL)	Rv SAMmt cloning primer 57/(µL)	DNA CCAP 1023/1 /(µL)	MilliQ/(µL)	Total/(µL)
12.5	0.75	0.75	1	10	25

(3)

2x KAPA HiFi HotStart ReadyMix/(µL)	Fw DECARB cloning primer 56/(µL)	Rv DECARB cloning primer 56/(µL)	DNA CCAP 1023/1 /(µL)	MilliQ/(µL)	Total/(µL)
12.5	0.75	0.75	1	10	25

(4)

2x KAPA HiFi HotStart ReadyMix/(µL)	Fw DiDECARB cloning primer 57/(µL)	Rv DiDECARB cloning primer 57/(µL)	DNA CCAP 1023/1 /(µL)	MilliQ/(µL)	Total/(µL)
12.5	0.75	0.75	1	10	25

(1)(4)

Predenature	Denature	Annealing	Extension	Final Extension	Cooling
98°C	98°C	60°C	72°C	72°C	4°C
5min	20sec	15sec	90sec	5min	Infinity

Denature-Annealing-Extension 25cycle

(2)(3)

Predenature	Denature	Annealing	Extension	Final Extension	Cooling
98°C	98°C	60°C	72°C	72°C	4°C
5min	20sec	15sec	70sec	5min	Infinity

Denature-Annealing-Extension 25cycle

Electrophoresis

Honda

Lane	Sample
1	1kb ladder
2	AT cloning(1)
3	SAMmt cloning(2)
4	DECARB cloning(3)
5	DiDECARB cloning(4)
6	1kb ladder

Restriction Enzyme Digestion

Yasuda

(1)

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
I732095 8/22 miniprep (2)(255ng/µL)	7.8	1	-	1	-	H(x10)	3	-	17.2	30
I732095 8/22 miniprep (2)(255ng/µL)	0.4	-	-	-	-	H(x10)	1	-	8.6	10

incubate at 37°C overnight

(2)

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
I732095 8/22 miniprep (2)(255ng/µL)	7.8	1	-	1	-	H(x10)	3	-	17.2	30
I732095 8/22 miniprep (2)(255ng/µL)	0.4	-	-	-	-	H(x10)	1	-	8.6	10
I732095 8/22 miniprep (1)(430ng/µL)	4.6	1	-	1	-	H(x10)	3	-	20.4	30

9/5

Gel Extraction

Honda and Yamauchi

Lane	Sample
1	1kb ladder
2	9/4 I732095(E,S)-1
3	-
4	9/4 I732095(E,S)-1
5	-
6	9/4 I732095(E,S)-1

Genome Cloning

Honda

(1)

2K KAPA HiFi HotStart ReadyMix/(µL)	Fw Primer AT cloning primer 61/(µL)	Rv Primer AT cloning primer 61/(µL)	DNA CCAP 1023/1 /(µL)	MilliQ/(µL)	Total/(µL)
50	3	3	4	40	100

(2)

2K KAPA HiFi HotStart ReadyMix/(µL)	Fw Primer SAMmt cloning primer 58/(µL)	Rv Primer SAMmt cloning primer 57/(µL)	DNA CCAP 1023/1/(µL)	MilliQ/(µL)	Total/(µL)
50	3	3	4	40	100

(3)

2K KAPA HiFi HotStart ReadyMix/(µL)	Fw Primer DiDECARB cloning primer 57/(µL)	Rv Primer DiDECARB cloning primer 57/(µL)	DNA CCAP 1023/1/(µL)	MilliQ/(µL)	Total/(µL)
50	3	3	4	40	100

Predenature	Denature	Annealing	Extension	Final Extension	Cooling
98°C	98°C	55-65°C	72°C	72°C	12°C
5min	20sec	15sec	90sec	5min	Infinity

Denature-Annealing-Extension 25cycle

Electrophoresis

Honda

(1)AT

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

(2)SAMmt

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

(3)DiDECARB

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

Gel Extraction

Murata

Lane	Sample
1	1kb ladder
2	9/4 I732045(E,S)
3	9/4 I732045(E,S)
4	-
5	-
6	9/4 I732045(E,S)
7	9/4 I732045(E,S)
8	-
9	1kb ladder
10	9/4 control

9/8

Genome Cloning and PCR

Honda and Yamaura

(1)

2K KAPA HiFi HotStart ReadyMix/(µL)	Fw Primer AT cloning primer 61/(µL)	Rv Primer AT cloning primer 61/(µL)	9/5 AT (genome cloning product) (1)-3 /(µL)	MilliQ/(µL)	Total/(µL)
12.5	0.75	0.75	1	10	25

(2)

2K KAPA HiFi HotStart ReadyMix/(µL)	Fw Primer DiDECARB cloning primer 61/(µL)	Rv Primer DiDECARB cloning primer 61/(µL)	9/5 AT (genome cloning product) (3)-3 /(µL)	MilliQ/(µL)	Total/(µL)
12.5	0.75	0.75	1	10	25

(3)

2K KAPA HiFi HotStart ReadyMix/(µL)	Fw Primer Pre-dddD cloning primer/(µL)	Rv Primer suf-dddD cloning primer/(µL)	3/19 dddD (DNA purification product)(121µg/µL)/(µL)	MilliQ/(µL)	Total/(µL)
12.5	0.75	0.75	1	10	25

Predenature	Denature	Annealing	Extension	Final Extension	Cooling
98°C	98°C	60°C	72°C	72°C	4°C
5min	20sec	15sec	90sec	5min	Infinity

Denature-Annealing-Extension 25cycle

Electrophoresis

Honda

Lane	Sample
1	1kb ladder
2	PCR product (AT)
3	PCR product (DIDECARB)
4	PCR product (dddD)
5	PCR product (dddD)
6	1kb ladder

DNA Purification

Matsumoto

Sample	Concentration/(µg/mL)	260/280	260/230
9/8 AT	55	1.37	1.14
9/8 DiDECARB	95	1.69	3.04
9/8 dddD	145	1.52	1.78

Restriction Enzyme Digestion

Matsumoto

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
9/8 dddD (DNA purificaion and PCR product)	13.8	1	-	1	-	H	3	-	11.2	30
control	0.7	-	-	-	-	H	1	-	8.3	10

Genome Cloning and PCR

Yasuda

2K KAPA HiFi HotStart ReadyMix/(µL)	Fw Primer SAMmt cloning primer 58/(µL)	Rv Primer SAMmt cloning primer 57/(µL)	9/5 SAMmt (genome cloning product) No.3/(µL)	MilliQ/(µL)	Total/(µL)
12.5	0.75	0.75	1	10	25

Predenature	Denature	Annealing	Extension	Final Extension	Cooling
98°C	98°C	63°C	72°C	72°C	12°C
5min	20sec	15sec	90sec	5min	Infinity

Denature-Annealing-Extension 25cycle

9/9

DNA purification

Honda

Sample	Concentration/(µg/mL)	260/280	260/230
9/8 dddD(E,S)	1.3(x50)	1.66	1.28

Ligation

Li (1)Sample (2)Control

Vector/(µL)		Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
pSB1C3	4.2	dddD	3.8	(2x)Buffer	9	T4 Ligase	1	-	18
pSB1C3	4.2	-	-	(2x)Buffer	9	T4 Ligase	1	3.8	18

Transformation

Li

Sample/(µL)		CompetentCells/(µL)		Total/(µL)	Medium
9/9 dddD+pSB1C3 ligation product	5	DH5α	50	55	SOC
9/9 dddD+pSB1C3 ligation product control	3	DH5α	30	33	SOC

Liquid Culture

Honda

Sample	Medium
9/8 T7-RBS	Plusgrow

PCR Cloning

Murata

2x KAPA HiFi HotStart ReadyMix/(µL)	Fw REDOX cloning primer 59/(µL)	Rv REDOX primer Ver.2 64/(µL)	DNA CCAP 1023/1 /(µL)	MilliQ/(µL)	Total/(µL)
12.5	0.75	0.75	1	10	25

Predenature	Denature	Annealing	Extension	Final Extension	Cooling
95°C	98°C	64°C	72°C	72°C	12°C
5min	20sec	15sec	15sec	5min	Infinity

Denature-Annealing-Extension 25cycle

Gel Extraction

Murata

Lane	Sample
1	1kb ladder
2	9/8 SAMmt PCR product (5µL)
3	-
4	9/8 SAMmt PCR product (10µL)
5	9/8 SAMmt PCR product (10µL)
6	-

PCR

Honda

2K KAPA HiFi HotStart ReadyMix/(µL)	Fw Primer SAMmt cloning primer/(µL)	Rv Primer SAMmt cloning primer/(µL)	9/9 AT (Gel Extraction product)/(µL)	MilliQ/(µL)	Total/(µL)
12.5	0.75	0.75	1	10	25

Predenature	Denature	Annealing	Extension	Final Extension	Cooling
98°C	98°C	63°C	72°C	72°C	4°C
5min	20sec	15sec	90sec	5min	Infinity

Denature-Annealing-Extension 25cycle

9/10

Electrophoresis

Murata

Lane	Sample
1	-
2	-
3	1kb ladder (5µL)
4	9/9 REDOX PCR product (2µL)
5	9/9 SAMmt PCR product (2µL)
6	1kb ladder (5µL)
7	-
8	-

Miniprep/DNA Purification

Murata

Sample	Concentration/(µg/mL)	260/280	260/230
T7-RBS	290	1.90	1.83

Cloning PCR

Murata

2x KAPA HiFi HotStart ReadyMix/(µL)	Fw REDOX cloning primer 59/(µL)	Rv REDOX primer Ver.2 64/(µL)	DNA CCAP 1023/1 /(µL)	MilliQ/(µL)	Total/(µL)
12.5	0.75	0.75	1	10	25

Electrophoresis

Murata

Lane	Sample
1	-
2	-
3	-
4	1kb ladder
5	SAMmt (x50) (10µL)
6	-
7	-
8	-

Colony PCR

Honda

Master Mix

Quick Taq/(µL)	VF2/(µL)	VR/(µL)	MilliQ/(µL)	Total/(µL)
40	1.6	1.6	36.8	80

Predenature	Denature	Annealing	Extension
94°C	94°C	55°C	68°C
2min	30sec	30sec	24sec

Denature-Annealing-Extension 30cycle

Number	Sample
1	pSB1C3-dddD(1)
2	pSB1C3-dddD(2)
3	pSB1C3-dddD(3)
4	pSB1C3-dddD(4)
5	pSB1C3-dddD(5)
6	pSB1C3-dddD(6)
7	pSB1C3-dddD (control)
8	control

Lane	Sample
1	-
2	-
3	-
4	1kb ladder
5	9/9 dddD+psb1c3 (1)
6	9/9 dddD+psb1c3 (2)
7	9/9 dddD+psb1c3 (3)
8	9/9 dddD+psb1c3 (4)
9	1kb ladder
10	9/9 dddD+psb1c3 (5)
11	9/9 dddD+psb1c3 (6)
12	9/9 dddD+psb1c3 (control)
13	control
14	1kb ladder
15	-
16	-
17	-

Electrophoresis

Yasuda

Lane	Sample
1	-
2	-
3	1kb ladder
4	REDOX PCR product
5	-

Overlap Extension(OE)-PCR

Murata

10x Buffer for KOD-Plus-Ver.2	2mM dNTPs	25mM MgSO4	Primer(10µM each) #1	Template DNA #2	KOD-Plus-(1U/µL)	MilliQ	Total
5µL	5µL	3µL	1.5µL	1µL	1µL	33µL	50µL

(1)
#1: AT OE-A, AT OE-B
#2: 9/8 AT (DNA Purification Product)(55µg/mL)
(2)
#1: AT OE-C, AT OE-D
#2: 9/8 AT (DNA Purification Product)(55µg/mL)
(3)
#1: AT OE-2C, AT OE-D
#2: 9/8 AT (DNA Purification Product)(55µg/mL)
(4)
#1: SAMmt OE-A, SAMmt OE-B
#2: 9/9 SAMmt (Gel Extraction Product)(55µg/mL)
(5)
#1: SAMmt OE-C, SAMmt OE-D
#2: 9/9 SAMmt (Gel Extraction Product)(55µg/mL)
(6)
#1: DECARB OE-A, DECARB OE-B<
#2: 9/4 DECARB Cloning-4 (55µg/mL)
(7)
#1: DECARB OE-C, DECARB OE-D
#2: 9/4 DECARB Cloning-4 (55µg/mL)
(8)
#1: DiDECARB OE-A, DiDECARB OE-B
#2: 9/9 DiDECARB (DNA Purification Product) (55µg/mL)
(9)
#1: DiDECARB OE-C, DiDECARB OE-D
#2: 9/9 DiDECARB (DNA Purification Product) (55µg/mL)
(10)
#1: AT OE-C, AT OE-2B
#2: 9/8 AT (DNA Purification Product)(55µg/mL)

Genome Cloning

Yasuda

2x KAPA HiFi HotStart ReadyMix/(µL)	Fw REDOX cloning primer 59/(µL)	Rv REDOX primer Ver.2 64/(µL)	DNA CCAP 1023/1 /(µL)	MilliQ/(µL)	Total/(µL)
50	3	3	4	40	100

Number	AnnealingTemperature
1	65.0 °C
2	64.3 °C
3	63.0 °C
4	61.1 °C
5	58.8 °C
6	56.9 °C
7	55.7 °C
8	55.0 °C

Gel Extraction

Yasuda

(1)

Lane	Sample
1	1kb ladder
2	9/10 OE-PCR(1)
3	9/10 OE-PCR(1)
4	-
5	9/10 OE-PCR(2)
6	-
7	9/10 OE-PCR(2)
8	-
9	1kb ladder
10	9/10 OE-PCR(3)
11	9/10 OE-PCR(3)

(2)

Lane	Sample
1	1kb ladder
2	9/10 OE-PCR(4)
3	9/10 OE-PCR(4)
4	-
5	9/10 OE-PCR(5)
6	9/10 OE-PCR(5)
7	1kb ladder

(3)

Lane	Sample
1	-
2	1kb ladder
3	9/10 OE-PCR(6)
4	9/10 OE-PCR(6)

(4)

Lane	Sample
1	-
2	1kb ladder
3	9/10 OE-PCR(7)
4	9/10 OE-PCR(7)

(5)

Lane	Sample
1	-
2	1kb ladder
3	9/10 OE-PCR(8)
4	9/10 OE-PCR(8)

(6)

Lane	Sample
1	1kb ladder
2	-
3	9/10 OE-PCR(9)
4	9/10 OE-PCR(9)
5	9/10 OE-PCR(10)
6	9/10 OE-PCR(10)

Ligation

Tatsui and Murata

Vector/(µL)		Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
YC plac111 (SmaI cut)	1	9/8 dddD(DNA purification product)	0.6	(2x)Buffer	5	T4 Ligase	1	2.4	10

Liquid Culture

Honda

Sample	Medium
9/9 dddD+psB1C3-(1)	CP

Restriction Enzyme Digestion

Shimizu

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
9/9 T7-RBS	6.9	-	-	1	1	H	3	-	18.1	30
control	1	-	-	-	-	H	1	-	8	10

OE-PCR

Shimizu

10x Buffer for KOD-Plus-Ver.2	2mM dNTPs	25mM MgSO4	Primer(10µM each) #1	Template DNA #2	KOD-Plus-(1U/µL)	MilliQ	Total
5µL	5µL	3µL	2µL	0.5µL	1µL	31µL	50µL

(1)
#1: SAMmt OE-A, SAMmt OE-D
#2: 9/10 SAMmt(4)(5) (Gel Extraction Product)
(2)
#1: DECARB OE-A, DECARB OE-D
#2: 9/10 DECARB(6)(7) (Gel Extraction Product)
(3)
#1: DiDECARB OE-A, DiDECARB OE-D
#2: 9/10 DiDECARB(8)(9) (Gel Extraction Product)
(3')
#1: DiDECARB OE-A, DiDECARB OE-D
#2: 9/10 DiDECARB(3)(9) (Gel Extraction Product)

Predenature	Denature	Annealing	Extension	Last Extension	Cooling
98°C	98°C	55°C	72°C	72°C	12°C
5min	20sec	15sec	2min10sec	5min	Infinity

Denature-Annealing-Extension 25cycle

Ligation

Shimizu

Vector/(µL)		Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
YCplac111	1	dddD	1	(2x)Buffer	5	T4 Ligase	1	2	10

9/11

Miniprep

Murata

Sample	Concentration/(µg/mL)	260/280	260/230
9/10 dddD (liquid culture product)	165	1.88	2.83

DNA Purification

Matsumoto

Sample	Concentration/(µg/mL)	260/280	260/230
9/10 REDOX(5)(6) (genome cloning product)	55	1.56	1.32

OE-PCR

Shimizu

10x Buffer for KOD-Plus-Ver.2	2mM dNTPs	25mM MgSO4	Primer(10µM each) #1	Template DNA #2	KOD-Plus-(1U/µL)	MilliQ	Total
5µL	5µL	3µL	1.5µL	1µL	1µL	32µL	50µL

(1)
#1: AT OE-A, AT OE-B
#2: 9/8 AT (DNA Purification Product)(55µg/mL)
(2)
#1: AT OE-C, AT OE-D
#2: 9/8 AT (DNA Purification Product)(55µg/mL)
(3)
#1: AT OE-2C, AT OE-D
#2: 9/8 AT (DNA Purification Product)(55µg/mL)
(4)
#1: REDOX (Forward with prefix), REDOX (Reverse with sufix) #2: 9/11 REDOX (DNA Purification Product)(55µg/mL)
(5)(6)
#1: SAMmt OE-A, SAMmt OE-B
#2: 9/9 SAMmt (Gel Extraction Product)(55µg/mL)
(7)(8)
#1: SAMmt OE-C, SAMmt OE-D
#2: 9/9 SAMmt (Gel Extraction Product)(55µg/mL)
(9)(10)
#1: DECARB OE-A, DECARB OE-B<
#2: 9/4 DECARB Cloning-4 (55µg/mL)
(11)(12)
#1: DECARB OE-C, DECARB OE-D
#2: 9/4 DECARB Cloning-4 (55µg/mL)
(13)(14)
#1: DiDECARB OE-A, DiDECARB OE-B
#2: 9/9 DiDECARB (DNA Purification Product) (55µg/mL)
(15)(16)
#1: DiDECARB OE-C, DiDECARB OE-D
#2: 9/9 DiDECARB (DNA Purification Product) (55µg/mL)

Electrophoresis

Honda and Murata

Lane	Sample
1	-
2	1kb ladder
3	9/10 OE-PCR-(1)
4	9/10 OE-PCR-(2)
5	9/10 OE-PCR-(3)
6	9/10 OE-PCR-(3')
7	1kb ladder

Colony PCR

Murata and Yamauchi

Master Mix (x32)

Quick Taq/(µL)	M13 Fw/(µL)	M13 Rv/(µL)	MilliQ/(µL)	Total/(µL)
160	6.4	6.4	147.2	320

Predenature	Denature	Annealing	Extension
94°C	94°C	55°C	68°C
2min	30sec	30sec	72sec

Denature-Annealing-Extension 30cycle

(1)

Number	Sample
1	LB Amp. 9/10 mamB(1)
2	LB Amp. 9/10 mamB(2)
3	LB Amp. 9/10 mamB(3)
4	LB Amp. 9/10 mamB(4)
5	LB Amp. 9/10 mamB(5)
6	LB Amp. 9/10 mamB(6)
7	LB Amp. 9/10 mamB(7)
8	LB Amp. 9/10 mamB(8)

(2)

Number	Sample
1	LB Amp. 9/10 mamL(1)
2	LB Amp. 9/10 mamL(2)
3	LB Amp. 9/10 mamL(3)
4	LB Amp. 9/10 mamL(4)
5	LB Amp. 9/10 mamL(5)
6	LB Amp. 9/10 mamL(6)
7	LB Amp. 9/10 mamL(7)
8	LB Amp. 9/10 mamL(8)

(3)

Number	Sample
1	LB Amp. 9/10 mamQ(1)
2	LB Amp. 9/10 mamQ(2)
3	LB Amp. 9/10 mamQ(3)
4	LB Amp. 9/10 mamQ(4)
5	LB Amp. 9/10 mamQ(5)
6	LB Amp. 9/10 mamQ(6)
7	LB Amp. 9/10 mamQ(7)
8	LB Amp. 9/10 mamQ(8)

(4)

Number	Sample
1	LB Amp. 9/10 dddD(1)
2	LB Amp. 9/10 dddD(2)
3	LB Amp. 9/10 dddD(3)
4	LB Amp. 9/10 dddD(4)
5	LB Amp. 9/10 dddD(5)
6	LB Amp. 9/10 dddD(6)
7	LB Amp. 9/10 dddD(7)
8	LB Amp. 9/10 dddD(8)

Mutation PCR

Honda

(1)dddD (PstI)

Prime STAR MAX Premix(2x)	Fw dddD PstI mutation primer	Rv dddD PstI mutation primer	9/11 MP dddD (x2000)	MilliQ	Total
25µL	1µL	1µL	1µL	22µL	50µL

(2)T7-RBS-Histag

Prime STAR MAX Premix(2x)	Fw psB1C3-his-F-v2 primer	Rv psB1C3 Histag mutation primer	9/10 MP T7-RBS (x3000)	MilliQ	Total
25µL	1µL	1µL	1µL	22µL	50µL

(3)Control

Prime STAR MAX Premix(2x)	Fw Control primer	Rv Control primer	Control template (x1000)	MilliQ	Total
25µL	0.5µL	0.5µL	1µL	23µL	50µL

Predenature	Denature	Annealing	Extension	Last Extension	Cooling
98°C	98°C	55°C	72°C	72°C	12°C
5min	10sec	15sec	25sec	5min	Infinity

Denature-Annealing-Extension 30cycle

DNA Purification

Honda

Sample	Concentration/(µg/mL)	260/280	260/230
dddD, T7-RBS-His	-	-	-

Gel Extraction

Honda

(1)

Lane	Sample
1	1kb ladder
2	9/11 OE-PCR-(1)
3	9/11 OE-PCR-(1)
4	-
5	-
6	9/11 OE-PCR-(2)
7	9/11 OE-PCR-(2)
8	-
9	1kb ladder
10	9/11 OE-PCR-(3)
11	9/11 OE-PCR-(3)

(2)

Lane	Sample
1	1kb ladder
2	9/11 OE-PCR-(6)
3	9/11 OE-PCR-(6)
4	-
5	-
6	9/11 OE-PCR-(8)
7	9/11 OE-PCR-(8)
8	-
9	1kb ladder
10	9/11 OE-PCR-(10)
11	9/11 OE-PCR-(10)

(3)

Lane	Sample
1	1kb ladder
2	9/11 OE-PCR-(12)
3	9/11 OE-PCR-(12)
4	9/11 OE-PCR (REDOX 2nd PCR)
5	-
6	9/11 OE-PCR-(14)
7	9/11 OE-PCR-(14)
8	-
9	1kb ladder
10	9/11 OE-PCR-(16)
11	9/11 OE-PCR-(16)

DNA Purification

Yamauchi

Sample	Concentration/(µg/mL)	260/280	260/230
9/10 OE-PCR (1) SAMmt	975	1.06	1.08
9/10 OE-PCR (2) DECARB	420	1.67	2.19
9/20 OE-PCR (3) DiDECARB	175	1.66	2.45
9/10 OE-PCR (3') DiDECARB	215	1.57	1.82
9/11 Mutation PCR dddD	275	1.61	1.92
9/11 Mutation PCR T7	140	1.49	1.78
Control C	340	1.62	1.47

Ligation

Tatsui and Yamauchi

Vector/(µL)		Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
YC plac111 (SmaI cut)	1	9/11 SAMmt (DNA purification product)	0.5	(2x) Buufer	5	T4 Ligase	1	2.5	9

YC plac111 (SmaI cut)19/11 DiDECARB (DNA purification product)1(2x) Buufer5T4 Ligase129 YC plac111 (SmaI cut)19/11 DECARB (DNA purification product)1(2x) Buufer5T4 Ligase129 YC plac111 (SmaI cut)19/11 REDOX (DNA purification product)2(2x) Buufer5T4 Ligase119 9/10 Pcon-X(PstI cut, gel extraction product)1.5mamL(X.P)2(2x)Buffer5T4 Ligase1-9.5 9/10 Pcon-X(PstI cut, gel extraction product)1.5mamQ(X.P)2(2x)Buffer5T4 Ligase1-9.5

OE-PCR

Yasuda

10x Buffer for KOD-Plus-Ver.2	2mM dNTPs	25mM MgSO4	Primer(10µM each) #1	Template DNA #2	KOD-Plus-(1U/µL)	MilliQ	Total
5µL	5µL	3µL	2µL	0.5µL	1µL	31µL	50µL

(1)
#1: AT OE-A, AT OE-D
#2: 9/10 OE-PCR(10), 9/11 OE-PCR(1), 9/11 OE-PCR(3)
(2)
#1: SAMmt OE-A, SAMmt OE-D
#2: 9/11 OE-PCR(6), 9/11 OE-PCR(8)
(3)
#1: DECARB OE-A, DECARB OE-D
#2: 9/11 OE-PCR(10), 9/11 OE-PCR(12)
(4)
#1: DiDECARB OE-A, DiDECARB OE-D
#2: 9/11 OE-PCR(14), 9/11 OE-PCR(16)

Predenature	Denature	Annealing	Extension	Last Extension	Cooling
98°C	98°C	55°C	72°C	72°C	12°C
2min	10sec	10sec	1min	5min	Infinity

Denature-Annealing-Extension 25cycle

9/12

Electrophoresis

Li

Lane	Sample
1	1kb ladder
2	9/11 OE-PCR 2nd (1)
3	9/11 OE-PCR 2nd (2)
4	9/11 OE-PCR 2nd (3)
5	9/11 OE-PCR 2nd (4)

DNA Purification

Li

Sample	Concentration/(µg/mL)	260/280	260/230
9/11 OE-PCR 2nd (1)	220	1.65	1.96
9/11 OE-PCR 2nd (2)	240	1.65	2.10
9/11 OE-PCR 2nd (3)	225	1.68	2.03
9/11 OE-PCR 2nd (4)	160	1.64	1.91

Transformation

Yamauchi

Sample/(µL)		CompetentCells/(µL)		Total/(µL)	Medium
9/11 SAMmt (ligation product)	2	DH5&alfa;	10	12	CP
9/11 DiDECARB (ligation product)	2	DH5&alfa;	10	12	CP
9/11 DECARB (ligation product)	2	DH5&alfa;	10	12	CP
9/11 REDOX (ligation product)	2	DH5&alfa;	10	12	CP

Sequence

Yasuda

Bis Dye v3.1 Sequencing RR-24(/µL)	Bis Dye 5x Sequencing Buffer(/µL)	Primer (10µM) #1(/µL)	9/11 dddD miniprep (165ng/nL)(/µL)	MilliQ	Total
4	2	3.2	1.5	9.3	20

1. VF
2. Sequence No.1
3. Sequence No.2
4. Sequence No.3
5. Sequence No.4
6. Sequence No.5
7. Sequence No.6

Liquid Culture

Honda

Sample	Medium
9/10 dddD-YCplac111 (Transformation product)	Amp

Restriction Enzyme Digestion

Murata

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
9/10 OE-PCR SAMmt	2	1	-	-	1	H	3	-	23	30
9/10 OE-PCR DECARB	4.8	-	1	1	-	M	3	3	17.2	30
9/10 OE-PCR DiDECARB	12.4	-	1	1	-	M	3	3	10.6	30
9/10 OE-PCR T7-RBS	6.9	-	-	1	1	H	3	-	18.1	30
Control	2.3	-	-	-	-	H	1	-	5	9.9

Genome Cloning PCR

Honda

(1)

2K KAPA HiFi HotStart ReadyMix/(µL)	Fw Primer SAMmt cloning primer/(µL)	Rv Primer SAMmt cloning primer/(µL)	DNA CCAP 1023/1 /(µL)	MilliQ/(µL)	Total/(µL)
12.5	0.75	0.75	1	10	25

(2)

2K KAPA HiFi HotStart ReadyMix/(µL)	Fw Primer DECARB cloning primer/(µL)	Rv Primer DECARB cloning primer/(µL)	DNA CCAP 1023/1/(µL)	MilliQ/(µL)	Total/(µL)
12.5	0.75	0.75	1	10	25

Predenature	Denature	Annealing	Extension	Final Extension	Cooling
98°C	98°C	60°C	72°C	72°C	12°C
5min	20sec	15sec	90sec	5min	Infinity

Denature-Annealing-Extension 25cycle

Gel Extraction

Honda

Lane	Sample
1	1kb ladder
2	Control(9/11 T7-RBS)
3	-
4	9//11 T7-RBS(S,P)
5	-
6	1kb ladder

Cloning PCR (for Histag)

Murata

2x KAPA HiFi HotStart ReadyMix/(µL)	Fw 9/10 AT OE-A/(µL)	Rv 9/12 AT His/(µL)	Templete 9/11 OE-PCR 2nd (1) 9/12/(µL)	MilliQ/(µL)	Total/(µL)
12.5	0.75	0.75	pick up	11	25

Predenature	Denature	Annealing	Extension	Final Extension	Cooling
95°C	98°C	65°C	72°C	72°C	12°C
5min	20sec	15sec	45sec	5min	Infinity

Denature-Annealing-Extension 25cycle

Miniprep

Tatsui

Sample	Concentration/(µg/mL)	260/280	260/230
dddD-psB1C3 (Liquid culture product)	370	1.75	2.48

DNA Purification

Tatsui

Sample	Concentration/(µg/mL)	260/280	260/230
9/11 DECARB (X,S)	-	-	-
9/11 DiDECARB (X,S)	-	-	-
9/11 SAMmt (X,S)	-	-	-

Restriction Enzyme Digestion

Tatsui

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
9/12 AT (DNA purification product)	9	1	-	-	1	H	3	-	16	30

PCR (for Control)

Murata

(1)

2x KAPA HiFi HotStart ReadyMix/(µL)	Fw AT-A/(µL)	Rv AT-D/(µL)	Templete 9/8 AT (DNA purification product)/(µL)	MilliQ/(µL)	Total/(µL)
12.5	0.75	0.75	1	10	25

(2)

2x KAPA HiFi HotStart ReadyMix/(µL)	Fw SAMmt-A/(µL)	Rv SAMmt-D/(µL)	Templete 9/12 AT (Genome cloning product)/(µL)	MilliQ/(µL)	Total/(µL)
12.5	0.75	0.75	1	10	25

(3)

2x KAPA HiFi HotStart ReadyMix/(µL)	Fw DECARB-A/(µL)	Rv DECARB-D/(µL)	Templete 9/12 DECARB (genome cloning product)/(µL)	MilliQ/(µL)	Total/(µL)
12.5	0.75	0.75	1	10	25

(4)

2x KAPA HiFi HotStart ReadyMix/(µL)	Fw DiDECARB-A/(µL)	Rv DiDECARB-D/(µL)	Templete 9/8 AT (DNA purification product)/(µL)	MilliQ/(µL)	Total/(µL)
12.5	0.75	0.75	1	10	25

Predenature	Denature	Annealing	Extension	Final Extension	Cooling
95°C	98°C	65°C	72°C	72°C	12°C
5min	20sec	15sec	45sec	5min	Infinity

Denature-Annealing-Extension 25cycle

Restriction Enzyme Digestion

Yamauchi

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
9/12 AT-His (DNA purification product)	8.7	-	1	-	1	M	3	3	13.3	30
Control	0.4	-	-	-	-	M	3	0	6.6	10
8/2 Pcon	20	1	-	1	-	H	3	3	2	30
Control	pick up	-	-	-	-	H	3	0	7	10

Ligation

Honda 111.5

Vector/(µL)	Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
YC plac111	AT-His cloning	0.5	(2x)Buffer	5	T4 Ligase	1	3.5	10
YC plac111	AT	0.5	(2x)Buffer	5	T4 Ligase	1	3.5	10
psB1C3 (E,P)	SAMmt (E,P)	1	(2x)Buffer	5	T4 Ligase	1	2.5	10

DNA Purification

Tatsui

Sample	Concentration/(µg/mL)	260/280	260/230
9/11 DECARB (Restriction Enzyme Digestion product)	40	1.74	1.95
9/11 DiDECARB (Restriction Enzyme Digestion product)	30	2.00	1.70
9/11 SAMmt (Restriction Enzyme Digestion product)	20	3.54	2.33
9/11 T7-RBS	60	1.81	0.27
9/10 T7-RBS	50	1.65	1.77

Electrophoresis

Lane	Sample
1	-
2	-
3	-
4	1kb ladder
5	9/11 E-PCR 2nd (1)
6	9/11 Control (PCR product) (1)
7	9/11 E-PCR 2nd (2)
8	9/11 Control (PCR product) (2)
9	9/11 E-PCR 2nd (3)
10	9/11 Control (PCR product) (3)
11	9/11 E-PCR 2nd (4)
12	9/11 Control (PCR product) (4)
13	1kb ladder
14	-
15	-
16	-

Transformation

Honda

Sample/(µL)		CompetentCells/(µL)		Total/(µL)	Medium
AT-His-YCplac111	1	DH5α	10	11	SOD
AT-YCplac111	1	DH5α	10	11	SOD
SAMmt(E,P)-psB1C3(E,P)	1	DH5α	10	11	SOD

Transformation

Yasuda

Sample/(µL)		CompetentCells/(µL)		Total/(µL)	Medium
9/11 dddD (DNA purification product)	1	DH5α	10	11	SOD
9/11 T7-His (DNA purification product)	1	DH5α	10	11	SOD

Colony PCR

Tatsui and Li

Predenature	Denature	Annealing	Extension	Last Extension	Cooling
94°C	94°C	55°C	68°C	68°C	12°C
2min	30sec	30sec	1min	1sec	Infinity

Denature-Annealing-Extension 25cycle

Lane	Sample
1-8	YCplac111-SAMmt (1)-(8)
9	1kb ladder
10-16	YCplac111-REDOX (1)-(7)

Liquid Culture

Yamauchi

Sample	Medium
9/5 Pcon-psB1C3-(3)	CP
9/5 Pcon-psB1C3-(3)	CP
9/11 SAMmt-(3)	Amp
9/8 T7-RBS-(1)	CP
9/8 T7-RBS-(1)	CP

Colony PCR

Honda

Master Mix

Quick Taq/(µL)	M13 Fw/(µL)	M13 Rv/(µL)	MilliQ/(µL)	Total/(µL)
35	1.4	1.4	32.2	70

Predenature	Denature	Annealing	Extension	Last Extension	Cooling
94°C	94°C	55°C	68°C	68°C	12°C
2min	30sec	30sec	1min	1sec	Infinity

Denature-Annealing-Extension 25cycle

Lane	Sample
1-8	written in "magnetosome"
9	1kb ladder
10-17	REDOX-YCplac111 (1)-(8)

9/13

Miniprep

Honda

Sample	Concentration/(µg/mL)	260/280	260/230
9/12 T7-RBS-psB1C3 (liquid culture product)	95	2.02	2.72
9/12 SAMmt-YCplac111 (liquid culture product)	110	1.70	1.15

Gel Extraction

Honda and Yasuda

Lane	Sample
1	1kb ladder
2	-
3	-
4	psB1C3-Pcon(X,S)
5	-
6	Control (psB1C3-Pcon(X,S))

Miniprep/DNA Purification

Yasuda

Sample	Concentration/(µg/mL)	260/280	260/230
9/12 AT (E,P)	2.3	1.33	1.11
9/12 AT-His(X,P)	2.0	1.33	0.86

Ligation

Honda

Vector/(µL)		Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
9/8 psB1C3(E,P)	3	9/13 AT(E,P)(DNA purification product)	1	(2x)Buffer	5	T4 Ligase	1	0	10
9/13 psB1C3(X,P)	3	9/13 AT-His(X,P)(DNA purification product)	1	(2x)Buffer	5	T4 Ligase	1	0	10

Miniprep

Honda

Sample	Concentration/(µg/mL)	260/280	260/230
9/12 SAMmt-YCplac111	140	1.80	1.90
T7-RBS-psB1C3	290	1.78	1.65

Liquid Culture

Yasuda

Sample	Medium
9/12 dddD-psB1C3-(1)	CP
9/12 dddD-psB1C3-(2)	CP
9/12 dddD-psB1C3-(3)	CP
9/12 T7-RBS-His-(1)	CP
9/12 T7-RBS-His-(2)	CP
9/12 T7-RBS-His-(3)	CP
Control

9/14

Colony PCR

Yasuda

Quick Taq/(µL)	M13 Fw/(µL)	M13 Rv/(µL)	MilliQ/(µL)	Total/(µL)
335	13.4	13.4	368.2	670

Predenature	Denature	Annealing	Extension
94°C	94°C	55°C	68°C
2min	30sec	30sec	160sec

Denature-Annealing-Extension 30cycle

1~8: 9/12 AT-YCplac111(1)~(8)
9~16: 9/12 AT His-YCplac111(1)~(8)
17~24: 9/12 mamB-YCplac111(1)~(8)
25~44: 9/12 DECARB-YCplac111(1)~(20)
45~64: 9/12 DiDECARB-YCplac111(1)~(20)

Gel Extraction

Honda

Lane	Sample
1	1kb ladder
2	9/13 Control (SAMmt)
3	9/13 SAMmt (E,P)
4	-
5	9/13 Control (Pcon-psB1C3)
6	9/13 Pcon-psB1C3(X,S)

DNA Purification

Honda

Sample	Concentration/(µg/mL)	260/280	260/230
Pcon-psB1C3(X,S)	40	0.13	1.27

Electrophoresis

Yasuda

(1)

Lane	Sample
1	9/12 AT-YCplac111(1)
2	9/12 AT-YCplac111(2)
3	9/12 AT-YCplac111(3)
4	9/12 AT-YCplac111(4)
5	9/12 AT-YCplac111(5)
6	9/12 AT-YCplac111(6)
7	9/12 AT-YCplac111(7)
8	9/12 AT-YCplac111(8)
9	1kbp GereRuler
10	9/12 AT His-YCplac111(1)
11	9/12 AT His-YCplac111(2)
12	9/12 AT His-YCplac111(3)
13	9/12 AT His-YCplac111(4)
14	9/12 AT His-YCplac111(5)
15	9/12 AT His-YCplac111(6)
16	9/12 AT His-YCplac111(7)
17	9/12 AT His-YCplac111(8)

(2)

Lane	Sample
1	9/12 mamB-YCplac111(1)
2	9/12 mamB-YCplac111(2)
3	9/12 mamB-YCplac111(3)
4	9/12 mamB-YCplac111(4)
5	9/12 mamB-YCplac111(5)
6	9/12 mamB-YCplac111(6)
7	9/12 mamB-YCplac111(7)
8	9/12 mamB-YCplac111(8)
9	1kbp GereRuler
10	9/12 DECARB-YCplac111(1)
11	9/12 DECARB-YCplac111(2)
12	9/12 DECARB-YCplac111(3)
13	9/12 DECARB-YCplac111(4)
14	9/12 DECARB-YCplac111(5)
15	9/12 DECARB-YCplac111(6)
16	9/12 DECARB-YCplac111(7)
17	9/12 DECARB-YCplac111(8)

(3)

Lane	Sample
1	9/12 DECARB-YCplac111(9)
2	9/12 DECARB-YCplac111(10)
3	9/12 DECARB-YCplac111(11)
4	9/12 DECARB-YCplac111(12)
5	9/12 DECARB-YCplac111(13)
6	9/12 DECARB-YCplac111(14)
7	9/12 DECARB-YCplac111(15)
8	9/12 DECARB-YCplac111(16)
9	1kbp GereRuler
10	9/12 DECARB-YCplac111(17)
11	9/12 DECARB-YCplac111(18)
12	9/12 DECARB-YCplac111(19)
13	9/12 DECARB-YCplac111(20)
14	9/12 DiDECARB-YCplac111(1)
15	9/12 DiDECARB-YCplac111(2)
16	9/12 DiDECARB-YCplac111(3)
17	9/12 DiDECARB-YCplac111(4)

(4)

Lane	Sample
1	9/12 DiDECARB-YCplac111(5)
2	9/12 DiDECARB-YCplac111(6)
3	9/12 DiDECARB-YCplac111(7)
4	9/12 DiDECARB-YCplac111(8)
5	9/12 DiDECARB-YCplac111(9)
6	9/12 DiDECARB-YCplac111(10)
7	9/12 DiDECARB-YCplac111(11)
8	9/12 DiDECARB-YCplac111(12)
9	1kbp GereRuler
10	9/12 DiDECARB-YCplac111(13)
11	9/12 DiDECARB-YCplac111(14)
12	9/12 DiDECARB-YCplac111(15)
13	9/12 DiDECARB-YCplac111(16)
14	9/12 DiDECARB-YCplac111(17)
15	9/12 DiDECARB-YCplac111(18)
16	9/12 DiDECARB-YCplac111(19)
17	9/12 DiDECARB-YCplac111(20)

Miniprep

Honda

Sample	Concentration/(µg/mL)	260/280	260/230
9/13 dddD-psB1C3(1)	645	1.82	1.70
9/13 dddD-psB1C3(2)	660	1,69	1.74
9/13 dddD-psB1C3(3)	635	1.87	1.57
9/13 T7-RBS-His-psB1C3(1)	440	1.65	1.23
9/13 T7-RBS-His-psB1C3(2)	450	1.92	1.46
9/13 T7-RBS-His-psB1C3(3)	190	1.44	1.26

Restriction Enzyme Digestion

Honda

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
9/14 dddD-psB1C3(1)	6	-	2	-	2	M	6	6	38	60
9/14 dddD-psB1C3(2)	6	-	2	-	2	M	6	6	38	60
9/14 dddD-psB1C3(3)	6	-	2	-	2	M	6	6	38	60
9/14 T7-RBS-His-psB1C3(1)	5	-	-	1	1	H	3	-	18	30
9/14 T7-RBS-His-psB1C3(2)	5	-	-	1	1	H	3	-	18	30
9/14 T7-RBS-His-psB1C3(3)	10	-	-	1	1	H	3	-	15	30

Dephosphorylation

Honda

Sample/(µL)		CIAP/(µL)
9/14 psB1C3(X,S)(gel extraction product)	20	0.25

Sample	Concentration/(µg/mL)	260/280	260/230
9/14 psB1C3(X,S)(gel extraction product)	60	2.93	0.16

Ligation

Yasuda

Vector/(µL)		Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
Pcon-psB1C3(X,S)(Dephosphorylation product)	0.83	9/12 DECARB(X,S)(DNA purification product)	2.93	10x Lig Buffer	2	NEB T4 DNA Ligase	1	13.24	20
Pcon-psB1C3(X,S)(Dephosphorylation product)	0.83	9/12 DECARB(X,S)(DNA purification product)	3.66	10x Lig Buffer	2	NEB T4 DNA Ligase	1	12.51	20

9/15

Colony PCR

Tatsui

Master Mix(x24)

Quick Taq/(µL)	VF/(µL)	VR/(µL)	MilliQ/(µL)	Total/(µL)
120	4.8	4.8	110.4	240

Predenature	Denature	Annealing	Extension
94°C	94°C	55°C	68°C
2min	30sec	30sec	24sec

Denature-Annealing-Extension 30cycle

Number	Sample
1~8	9/14 AT-psB1C3

Electrophoresis

Tatsui

Lane	Sample
1	1kb ladder
2~9	9/14 AT-psB1C3

Gel Extraction

Tatsui

Lane	Sample
1	1kb ladder
2	-
3	9/14 dddD(µ)-psB1C3(X,P)-(1)
4	-
5	1kb ladder
6	-
7	9/14 dddD(µ)-psB1C3(X,P)-(2)
8	-
9	1kb ladder
10	-
11	9/14 dddD(µ)-psB1C3(X,P)-(3)

DNA Purification

Tatsui

Sample	Concentration/(µg/mL)	260/280	260/230
9/14 dddD(µ)-psB1C3(X,P)-(1)	1.7 (x50)	1.37	0.33
9/14 dddD(µ)-psB1C3(X,P)-(2)	1.6 (x50)	1.41	0.36
9/14 dddD(µ)-psB1C3(X,P)-(3)	2.2 (x50)	1.23	0.42
T7-RBS-psB1C3(S cut)	2.0 (x50)	2.11	0.34

Restriction Enzyme Digestion

Murata

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
9/13 T7-RBS-psB1C3	7	-	-	1	1	H	3	-	9	30
9/11 REDOX (DNA purification product)	10	1	-	-	1	H	3	-	15	30
9/13 Pcon-psB1C3	25	1	-	-	1	H	3	-	0	30
9/15 T7-RBS-psB1C3	25	-	-	-	1	M	3	-	1	30

PCR Cloning

Murata

2x KAPA HiFi Hotstart Ready Mix/(µL)	9/10 Suf-REDOX Rv/(µL)	9/10 Fw Re-REDOX 59/(µL)	9/11 REDOX 5.6(DNA purification product)	MilliQ/(µL)	Total/(µL)
12.5	0.75	0.75	0.2	11	25.2

Predenature	Denature	Annealing	Extension
95°C	98°C	65°C	72°C
5min	20sec	15sec	50sec

Denature-Annealing-Extension 25cycle

Gel Extraction

Murata

Lane	Sample
1	1kb ladder
2	-
3	9/14 T7-RBS-His-psB1C3(S,P)-(1)
4	-
5	1kb ladder
6	-
7	9/14 T7-RBS-His-psB1C3(S,P)-(1)
8	-
9	1kb ladder
10	-
11	9/14 T7-RBS-His-psB1C3(S,P)-(1)

DNA Purification

Yasuda

Sample	Concentration/(µg/mL)	260/280	260/230
9/15 REDOX (PCR product)	4.9	1.76	0.66

Ligation

Vector/(µL)		Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
YCplac111 (SmaI cut)(x3)	1	9/12 AT His (DNA purification product)	0.3	(x2)Buffer	5	T4 Ligase	1	2.7	10
YCplac111 (SmaI cut)(x3)	1	9/15 REDOX (DNA purification product)	0.2	(x2)Buffer	5	T4 Ligase	1	2.8	10
YCplac111 (SmaI cut)(x3)	1	9/12 DECARB(DNA purification product)	0.4	(x2)Buffer	5	T4 Ligase	1	2.6	10
YCplac111 (SmaI cut)(x3)	1	9/12 DiDECARB(DNA purification product)	0.3	(x2)Buffer	5	T4 Ligase	1	2.7	10

Liquid Culture

Honda

Sample	Medium
9/14 AT-psB1C3 (Transformation product)-(1)	SOD
9/14 AT-psB1C3 (Transformation product)-(2)	SOD
9/14 AT-psB1C3 (Transformation product)-(3)	SOD

Dephosphorylation

Honda

Sample/(µL)		CIAP/(µL)
9/15 T7-RBS-psB1C3(P)(Restriction Enzyme Digestion product)	20	0.25
9/13 psB1C3(E,P)(Gel extraction product)	20	0.25

37°C	50°C	75°C
30min	1h	15min

Gel Extraction

Yasuda

Lane	Sample
1	1kb ladder
2	-
3	9/14 T7-RBS-His-psB1C3(S,P)-(1)
4	-
5	-
6	-
7	9/14 T7-RBS-His-psB1C3(S,P)-(2)
8	-
9	-
10	-
11	9/14 T7-RBS-His-psB1C3(S,P)-(3)

Sample	Concentration/(µg/mL)	260/280	260/230
9/14 T7-RBS-His-psB1C3(S,P)-(1)	25	1.81	0.50
9/14 T7-RBS-His-psB1C3(S,P)-(2)	25	1.13	0.21
9/14 T7-RBS-His-psB1C3(S,P)-(3)	25	1.65	0.09

Restriction Enzyme Digestion

Yasuda

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
9/14 T7-RBS-His-psB1C3 (Miniprep product)	13.63	-	-	3	3	H	9	-	61.37	90

Control0.3----H1-8.910

Transformation

Murata

Sample/(µL)		CompetentCells/(µL)		Total/(µL)	Medium
AT-His-YCplac111	10	DH5α	2	12	CP
REDOX-YCplac111	10	DH5α	2	12	CP
DECARB-pSB1C3	10	DH5α	2	12	CP
DiDECARB-pSB1C3	10	DH5α	2	12	CP

Ligation

Vector/(µL)		Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
9/15 T7-RBS(S,P)(Dephosphorylation product)	2.0	9/15 dddD(µ)(X,P)(Gel extraction product)	2.0	(x2)Buffer	5	T4 Ligase	1	0	10
9/15 psB1C3 (E,P)(Dephosphorylation product)	0.4	9/12 SAMmt (E,P)(DNA purification product)	2.9	(x2)Buffer	5	T4 Ligase	1	0.7	10
9/15 psB1C3 (E,P)(Dephosphorylation product)	1.3	9/15 REDOX (E,P)(DNA purification product)	2.4	(x2)Buffer	5	T4 Ligase	1	0.3	10
9/15 T7-RBS(S,P)(Gel extraction product)	2.6	9/15 AT His (X,P)(DNA purification product)	1.2	(x2)Buffer	5	T4 Ligase	1	0.2	10

Transformation

Murata and Yasuda

Sample/(µL)		CompetentCells/(µL)		Total/(µL)	Medium
DECARB-YCplac111	2	DH5&alfa;	10	12	SOD
DiDECARB-YCplac111	2	DH5&alfa;	10	12	SOD
dddD(µ)-T7-RBS-psB1C3	2	DH5&alfa;	10	12	SOD
SAMmt-psB1C3	2	DH5&alfa;	10	12	SOD
REDOX-psB1C3	2	DH5&alfa;	10	12	SOD
AT-T7-RBS-psB1C3	2	DH5&alfa;	10	12	SOD

DNA Purification

Yasuda

Sample	Concentration/(µg/mL)	260/280	260/230
9/15 REDOX (E,P)	45	-	-

9/16

Miniprep

Yasuda

Sample	Concentration/(µg/mL)	260/280	260/230
9/15 AT-psB1C3-(1)	280	1.81	2.08
9/15 AT-psB1C3-(1)	205	1.64	1.32
9/15 AT-psB1C3-(1)	320	1.66	1.61

PCR (for Sequence)

Tatsui and Nakamura

9/14 dddD-psB1C3(Miniprep product)-(1)
(1)dddD sequence VF2

Templete DNA/(µL)	Big Dye/(µL)	(x5)Buffer/(µL)	Primer	MilliQ/(µL)	Total/(µL)
0.63	0.5	1.75	0.5	6.6	10.5

(2)dddD sequence No.1

Templete DNA/(µL)	Big Dye/(µL)	(x5)Buffer/(µL)	Primer	MilliQ/(µL)	Total/(µL)
0.63	0.5	1.75	0.5	6.6	10.5

(3)dddD sequence No.2

Templete DNA/(µL)	Big Dye/(µL)	(x5)Buffer/(µL)	Primer	MilliQ/(µL)	Total/(µL)
0.63	0.5	1.75	0.5	6.6	10.5

(4)dddD sequence No.3

Templete DNA/(µL)	Big Dye/(µL)	(x5)Buffer/(µL)	Primer	MilliQ/(µL)	Total/(µL)
0.63	0.5	1.75	0.5	6.6	10.5

(5)dddD sequence No.4

Templete DNA/(µL)	Big Dye/(µL)	(x5)Buffer/(µL)	Primer	MilliQ/(µL)	Total/(µL)
0.63	0.5	1.75	0.5	6.6	10.5

(6)dddD sequence No.5

Templete DNA/(µL)	Big Dye/(µL)	(x5)Buffer/(µL)	Primer	MilliQ/(µL)	Total/(µL)
0.63	0.5	1.75	0.5	6.6	10.5

(7)dddD sequence No.6

Templete DNA/(µL)	Big Dye/(µL)	(x5)Buffer/(µL)	Primer	MilliQ/(µL)	Total/(µL)
0.63	0.5	1.75	0.5	6.6	10.5

Predenature	Denature	Annealing	Extension	Final Extension	Cooling
96°C	96°C	50°C	60°C	4°C	4°C
2min	10sec	5sec	4min	1min	Infinity

Denature-Annealing-Extension 30cycle

9/14 dddD-psB1C3(Miniprep product)-(2)

(1)dddD sequence VF2

Templete DNA/(µL)	Big Dye/(µL)	(x5)Buffer/(µL)	Primer	MilliQ/(µL)	Total/(µL)
0.63	0.5	1.75	0.5	6.6	10.5

(2)dddD sequence No.1

Templete DNA/(µL)	Big Dye/(µL)	(x5)Buffer/(µL)	Primer	MilliQ/(µL)	Total/(µL)
0.63	0.5	1.75	0.5	6.6	10.5

(3)dddD sequence No.2

Templete DNA/(µL)	Big Dye/(µL)	(x5)Buffer/(µL)	Primer	MilliQ/(µL)	Total/(µL)
0.63	0.5	1.75	0.5	6.6	10.5

(4)dddD sequence No.3

Templete DNA/(µL)	Big Dye/(µL)	(x5)Buffer/(µL)	Primer	MilliQ/(µL)	Total/(µL)
0.63	0.5	1.75	0.5	6.6	10.5

(5)dddD sequence No.4

Templete DNA/(µL)	Big Dye/(µL)	(x5)Buffer/(µL)	Primer	MilliQ/(µL)	Total/(µL)
0.63	0.5	1.75	0.5	6.6	10.5

(6)dddD sequence No.5

Templete DNA/(µL)	Big Dye/(µL)	(x5)Buffer/(µL)	Primer	MilliQ/(µL)	Total/(µL)
0.63	0.5	1.75	0.5	6.6	10.5

(7)dddD sequence No.6

Templete DNA/(µL)	Big Dye/(µL)	(x5)Buffer/(µL)	Primer	MilliQ/(µL)	Total/(µL)
0.63	0.5	1.75	0.5	6.6	10.5

9/15 AT-psB1C3(Miniprep product)-(1)

(1)VF

Templete DNA/(µL)	Big Dye/(µL)	(x5)Buffer/(µL)	Primer	MilliQ/(µL)	Total/(µL)
1.43	0.5	1.75	0.5	5.8	10.5

(2)AT for Sequence

Templete DNA/(µL)	Big Dye/(µL)	(x5)Buffer/(µL)	Primer	MilliQ/(µL)	Total/(µL)
1.43	0.5	1.75	0.5	5.8	10.5

Predenature	Denature	Annealing	Extension	Final Extension	Cooling
96°C	96°C	50°C	60°C	4°C	4°C
2min	10sec	5sec	4min	1min	Infinity

Denature-Annealing-Extension 30cycle

Dephosphorylation

Honda

Sample/(µL)
1.*(written in magnetosome)	*
2.*	*
3.9/15 T7-RBS-psB1C3(P)	30
4.9/15 T7-RBS-psB1C3(S,P)	30
5.9/15 T7-RBS-psB1C3(S,P)	30
6.9/15 T7-RBS-psB1C3(S,P)	30
7.9/15 T7-RBS-psB1C3(S,P)	30

Colony PCR

Yasuda

Master Mix

Quick Taq/(µL)	M13 Ew/(µL)	M13 Rv/(µL)	MilliQ/(µL)	Total/(µL)
300	12	12	276	600

Electrophoresis

Yasuda

(1)

Lane	Sample
1-8	9/15 REDOX-YCplac111
9	1kb ladder
10-17	9/15 DECARB-YCplac111

(2)

Lane	Sample
1-8	9/15 DiDECARB-YCplac111
9	1kb ladder
10-17	9/15 AT-His-YCplac111

(3)

Lane	Sample
1	1kb ladder
2-8	9/15 AT-His-YCplac111

(4)

Lane	Sample
1	1kb ladder
2-6	9/15 AT-His-YCplac111

Gel Extraction

Yamauchi

(1)

Lane	Sample
1	1kb ladder
2	-
3	(written in "magnetosome")
4	-
5	1kb ladder
6	-
7	(written in "magnetosome")
8	-
9	1kb ladder
10	-
11	9/15 psB1C3(E,P)

(2)

Lane	Sample
1	1kb ladder
2	-
3	9/15 T7-RBS-psB1C3(P)
4	-
5	1kb ladder
6	-
7	9/15 T7-RBS-psB1C3(S,P)
8	-
9	1kb ladder
10	-
11	9/15 T7-RBS-psB1C3(S,P)

Sample	Concentration/(µg/mL)	260/280	260/230
7. 9/15 T7-RBS-psB1C3(S,P)	90	1.40	0.44

(3)

Lane	Sample
1	-
2	1kb ladder
3	-
4	9/15 T7-RBS-psB1C3(S,P)
5	-
6	1kb ladder

Sample	Concentration/(µg/mL)	260/280	260/230
4. 9/15 T7-RBS-psB1C3(S,P)	80	1.30	1.02

DNA Purification

Yasuda

Sample	Concentration/(µg/mL)	260/280	260/230
9/15 T7-RBS-psB1C3(S,P)(Dephosphorylation product)	0.7(x50)	1.81	1.13
9/15 psB1C3(E,P)(Dephosphorylation product)	1.1(x50)	1.59	1.25

Ligation

Yasuda

Vector/(µL)		Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
9/16 T7-RBS(S,P)(DNA purification product)	1.7	9/15 dddD(µ)(X,P)(gel extraction product)	2	(x2)Buffer	5	T4 Ligase	1	0.3	10
9/16 psB1C3 (E,P)(DNA purification product)	0.55	9/15 SAMmt (E,P)(gel extraction product)	2.9	(x2)Buffer	5	T4 Ligase	1	0.55	10
9/16 psB1C3 (E,P)(DNA purification product)	1.6	9/15 REDOX (E,P)(gel extraction product)	2.2	(x2)Buffer	5	T4 Ligase	1	0.2	10
9/16 T7-RBS-His(S,P)(DNA purification product)	2.6	9/15 AT His(X,P)(gel extraction product)	1.2	(x2)Buffer	5	T4 Ligase	1	0.2	10

Dephosphorylation

Murata

Sample/(µL)		CIAP	Total
9/13 psB1C3 (X,S)(gel extraction product)	16	0.2	16.2

Transformation

Yasuda

Sample/(µL)		CompetentCells/(µL)		Total/(µL)	Medium
9/16 AT-His-T7-RBS-His	-	DH5α	-	-	-
9/16 REDOX-psB1C3	-	DH5α	-	-	-
9/16 SAMmt-psB1C3	-	DH5α	-	-	-
9/16 dddD(µ)-T7-RBS	-	DH5α	-	-	-
9/16 AT-His-YCplac111	-	DH5α	-	-	-
9/16 REDOX-YCplac111	-	DH5α	-	-	-
9/16 DECARB-YCplac111	-	DH5α	-	-	-
9/16 DiDECARB-YCplac111	-	DH5α	-	-	-

DNA Purification

Yasuda

Sample	Concentration/(µg/mL)	260/280	260/230
9/16 psB1C3(X,S)(Dephosphorylation product)	35	1.69	0.56

Liquid Culture

Yasuda

Sample	Medium
9/16 AT RBS His-T7 RBS His(1)	-
9/16 AT RBS His-T7 RBS His(2)	-

PCR

Yasuda

(1)

9/11 OE-PCR SAMmt 2nd-(2)	2x KAPA HiFi Hotstart Ready Mix/(µL)	Fw Primer/(µL)	Rv Primer/(µL)	MilliQ/(µL)	Total/(µL)
pick up	12.5	0.75	0.75	11	25

9/11 OE-PCR DECARB 2nd-(3)	2x KAPA HiFi Hotstart Ready Mix/(µL)	Fw Primer/(µL)	Rv Primer/(µL)	MilliQ/(µL)	Total/(µL)
pick up	12.5	0.75	0.75	11	25

Predenature	Denature	Annealing	Extension
95°C	98°C	65°C	72°C
5min	20sec	15sec	50sec

Denature-Annealing-Extension 25cycle

Restriction Enzyme Digestion

Yasuda

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
9/11 OE-PCR SAMmt 2nd-(2)	8.2	0.5	-	-	0.5	H	1.5	0	4.3	15
9/15 AT-psB1C3-(3)(Miniprep product)	9.8	-	1	-	1	M	3	3	12.2	30
Control	0.5	-	-	-	-	M	1	1	7.5	10

Ligation

Honda

Vector/(µL)		Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
psB1C3	1.1	9/12 DECARB(X,E)(DNA purification product)	2.9	(x2)Buffer	5	T4 Ligase	1	0	10
psB1C3	0.85	9/12 DiDECARB(X,E)(DNA purification product)	2.2	(x2)Buffer	5	T4 Ligase	1	0.95	10

Transformation

Honda

Sample/(µL)		CompetentCells/(µL)		Total/(µL)	Medium
9/16 DECARB-psB1C3	1	DH5α	10	11	-
9/16 DiDECARB-psB1C3	1	DH5α	10	11	-

15min on ice - 1min heat shock(42°C) - 45min preculture (42°C)

9/17

Cycle PCR

Murata and Nakamura

16 Cycle

BigDye Terminator v3.1 Cycle Sequencing RR-24/(µL)	BigDye Terminator v3.1 v1.1 5x Sequencing Buffer/(µL)	Primer #1/(pmol)	Templete DNA #2/(ng)	MilliQ /(µL)	Total/(µL)
2	1	1.6	75-150	up to 10	10

(1)
#1: VF2
#2: 9/11 dddD (miniprep product) 1µL

#1: Sequence No.5
#2: 9/11 dddD (miniprep product) 1µL

(2)
#1: VF2
#2: 9/14 dddD(µ) (miniprep product) 0.25µL

#1: VR
#2: 9/14 dddD(µ) (miniprep product) 0.25µL

(3)
#1: VF2
#2: 9/14 T7-RBS-His-psB1C3 (miniprep product) 0.34µL

#1: Sequence No.5
#2: 9/14 T7-RBS-His-psB1C3 (miniprep product) 0.34µL

(4)
#1: VR
#2: 9/16 AT-psB1C3-(1) (miniprep product) 0.5µL

#1: VF2
#2: 9/16 AT-psB1C3-(1) (miniprep product) 0.5µL

#1: Sequence No.1
#2: 9/16 AT-psB1C3-(1) (miniprep product) 0.5µL

#1: Secuence No.2
#2: 9/16 AT-psB1C3-(1) (miniprep product) 0.5µL

Predenature	Denature	Annealing	Extension	Final Extension	Cooling
96°C	96°C	50°C	60°C	4°C	4°C
2min	10sec	5sec	4min	1min	Infinity

Denature-Annealing-Extension 30cycle

Gel Extraction

Murata

Lane	Sample
1	1kb ladder
2	Control
3	-
4	9/16 AT-psB1C3(X,P)
5	-
6	-

DNA Purification

Murata and Yasuda

Sample	Concentration/(µg/mL)
9/12 SAMmt (E,P)	75
9/16 SAMmt (PCR product)	140
9/16 DECARB (PCR product)	230

PCR

Yasuda

10x Buffer for KOD-Plus-Ver.2	2mM dNTPs	25mM MgSO4	Primer(10µM each) #1	Template DNA #2	KOD-Plus-(1U/µL)	MilliQ	Total
5µL	5µL	3µL	2µL	0.5µL	1µL	31.5µL	50µL

(1)
#1: Pre-REDOX, Suf-REDOX
#2: 9/11 REDOX (DNA purification product)

(2)
#1: Pre-REDOX, Rv-REDOX cloning for His
#2: 9/11 REDOX (DNA purification product)

(3)
#1: DiDECARB OE-A, DiDECARB OE-D
#2: 9/11 DiDECARB (for OE-PCR 2nd)(9/12 DNA purification product)

Predenature	Denature	Annealing	Extension	Final Extension	Cooling
98°C	98°C	55°C	72°C	72°C	12°C
2min	10sec	10sec	1min	5min	Infinity

Denature-Annealing-Extension 25cycle

DNA Purification

Yasuda

Sample	Concentration/(µg/mL)
9/17 REDOX PCR	435
9/17 REDOX-His PCR	365
9/17 DiDECARB PCR	230

Colony PCR

Honda,Nakamura and Matsumoto

Master Mix

Quick Taq/(µL)	VF2/(µL)	VR/(µL)	MilliQ/(µL)	Total/(µL)
85	3.4	3.4	78.2	170

Predenature	Denature	Annealing	Extension
94°C	94°C	55°C	68°C
2min	30sec	30sec	2min30sec

Denature-Annealing-Extension 30cycle

Number	Sample
1-2	9/15 AT-His-T7-RBS-His (1)-(2)
3-7	9/16 AT-His-T7-RBS-His (1)-(5)
8-9	9/16 dddD(µ)-T7-RBS (1)-(2)
10-16	9/16 SAMmt-psB1C3 (1)-(7)

Restriction Enzyme Digestion

Murata

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
9/17 REDOX-His (DNA purification product)	5.5	-	1	-	1	M	3	3	16.5	30
9/17 REDOX (DNA purification product)	4.6	-	1	-	1	M	3	3	17.4	30

Ligation

Yasuda

Vector/(µL)		Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
9/16 T7-RBS(S,P) (DNA purification product)	1.4	9/17 AT(X,P) (DNA purification product)	2.3	(x2)Buffer	5	T4 Ligase	1	0.3	10
YCplac111(SmaI cut)	1	9/17 REDOX-His (DNA purification product)	0.6	(x2)Buffer	5	T4 Ligase	1	2.39	10
9/16 psB1C3(E,P) (DNA purification product)	0.8	9/17 SAMmt(E,P) (DNA purification product)	1.9	(x2)Buffer	5	T4 Ligase	1	0.8	10
YCplac111(SmaI cut)	1	-	-	(x2)Buffer	5	T4 Ligase	1	3	10

Transformation

Yasuda

Sample/(µL)		CompetentCells/(µL)		Total/(µL)	Medium
9/17 AT-T7-RBS (Ligation product)	2	DH5α	10	12	SOC
9/17 REDOX-His-YCplac111 (Ligation product)	2	DH5α	10	12	SOC
9/17 SAMmt-psB1C3 (Ligation product)	2	DH5α	10	12	SOC
9/17 Control (SmaX3, Ligation product)	2	DH5α	10	12	SOC

Make Control (for 9/18 UV-Spectrum)

Murata

Add MTOB (1.6mg) to ethanenitrile (313.4µL)

Electrophoresis

Yasuda and Honda

(1)

Lane	Sample
1-2	9/15 AT-His-T7-RBS His (1)(2)
3-7	9/15 AT-His-T7-RBS His (1)-(5)
8	9/16 dddD(µ)-T7-RBS(1)
9	1kb ladder

(2)

Lane	Sample
1	1kb ladder
2	9/16 dddD(µ)-T7-RBS(2)
3-8	9/16 SAMmt-psB1C3 (1)-(6)

(3)

Lane	Sample
1	1kb ladder
2	9/16 SAMmt-psB1C3 (7)

Liquid Culture

Yasuda

Sample
9/16 AT His-T7-RBS His (2)-(4)
9/16 SAMmt-psB1C3 (2)(4)

DNA Purification

Murata

Sample	Concentration/(µg/mL)	260/280	260/230
9/17 REDOX His (X,P)	1.6	1.74	-
9/17 REDOX (X,P)(PCR product)	1.3	2.43	-

9/18

Miniprep

Murata

Sample	Concentration/(µg/mL)	260/280	260/230
9/15 T7-AT-His (1)	200	1.54	1.60
9/15 T7-AT-His (2)	180	1.64	2.02
9/16 T7-AT-His (2)	140	1.84	2.09
9/16 T7-AT-His (3)	140	1.71	1.82
9/16 T7-AT-His (4)	145	1.83	2.16
9/16 SAMmt-psB1C3 (2)	225	1.64	1.71
9/16 SAMmt-psB1C3 (4)	190	1.75	2.12

Colony PCR

Honda

Master Mix

Quick Taq/(µL)	Fw M13/(µL)	Rv M13/(µL)	MilliQ/(µL)	Total/(µL)
45	1.8	1.8	41.4	90

Predenature	Denature	Annealing	Extension	Cooling
94°C	94°C	55°C	68°C	12°C
2min	30sec	30sec	90sec	Infinity

Denature-Annealing-Extension 30cycle

Number	Sample
1	9/16 REDOX-(1)-1
2	9/16 DiDECARBO-(1)-1
3	9/16 DiDECARBO-(1)-2
4	9/16 DiDECARBO-(1)-3
5	9/16 DiDECARBO-(1)-4
6	9/17 AT-T7-RBS-1
7	9/17 REDOX-His-YCplac111-1
8	9/17 REDOX-His-YCplac111-2
9	9/17 REDOX-His-YCplac111-3

Lane	Sample
1	1kb ladder
2	9/16 REDOX-YCplac111 (1)
3-6	9/16 DiDECARB-YCplac111 (1)-(4)

Lane	Sample
1	-
2	1kb ladder
3	9/17 AT-T7-RBS (1)
4-6	REDOX-His=YCplac111 (1)-(3)

Liquid Culture

Honda

Sample	Medium
9/17 REDOX-His-YCplac111-(1)	CP
9/17 REDOX-His-YCplac111-(2)	CP
9/14 AT-psB1C3-(1)	CP

Restriction Enzyme Digestion

Honda

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
9/15 AT-pSB1C3-(3)	15	2	-	-	2	H	6	6	51.5	82.5
9/15 AT-pSB1C3-(2)	19	-	2	2	-	MC	6	6	45	80
9/9 T7-RBS-pSB1C3	14	-	-	2	2	B	6	6	50	80
9/18 T7-RBS-AT-His-pSB1C3	33	-	-	3	3	B	9	9	63	120

UV-spequtrum measurement@

Yasuda and Murata Ethanenitrile solution of saturated MTOB (x10)

HPLC@

Yasuda and Murata

Mutation PCR

Honda

SAMmt (SpeI)

Prime STAR MAX Premix(2x)	Fw SAMmt SpeI mutation primer	Rv SAMmt SpeI mutation primer	9/18 SAMmt-pSB1C3-4	MilliQ	Total
25µL	1µL	1µL	pick upµL	23µL	50µL

Predenature	Denature	Annealing	Extension	Cooling
98°C	98°C	55°C	72°C	12°C
5min	10sec	15sec	30sec	Infinity

Denature-Annealing-Extension 30cycle

Quality check of preligation

Murata

Sample	Concentration(ng/µL)	A260	A280	260/280	260/230
DiDECARB	538.36	10.767	5.734	1.88	2.07
DECARB	223.37	4.467	2.361	1.89	1.99
SAMmt-psB1C3	185.24	3.705	1.896	1.95	1.87
REDOX	454.07	9.081	4.800	1.89	2.17
AT-psB1C3	299.73	5.995	2.993	2.00	1.99
T7-RBS	306.31	6.126	2.901	2.11	1.97
T7-RBS-His	307.59	6.152	3.129	1.97	1.50
Pcon-psB1C3	245.88	4.918	2.423	2.03	1.65

PCR

Murata

DiDECARB	10x Buffer	2mM dNTPs	25mM MgSO4	#1	#2	KOD(1U/µL)	MilliQ	Total
1µL	2.5µL	2.5µL	1.5µL	1.25µL	1.25µL	0.5µL	14.5µL	25

25µL x4 (100µL)

(1)
#1: DiDECARB OE-A (10 µM)
#2: DiDECARB Mutation Rv (10 µM)

Predenature	Denature	Annealing	Extension	Cooling
94°C	98°C	50-65°C	68°C	15°C
2min	10sec	30sec	48sec	Infinity

Denature-Annealing-Extension 30cycle

(2)
#1: DiDECARB OE-D (10 µM)
#2: DiDECARB Mutation Fw (10 µM)

(3)
#1: RV-DiDECARB His (10 µM)
#2: DiDECARB Mutation Fw (10 µM)

Predenature	Denature	Annealing	Extension	Cooling
94°C	98°C	50-65°C	68°C	15°C
2min	10sec	30sec	78sec	Infinity

Denature-Annealing-Extension 30cycle

(4)
#1: DiDECARB OE-A (10 µM)
#2: DiDECARB MutationP Rv (10 µM)

(5)
#1: DiDECARB MutationP-Fw (10 µM)
#2: DiDECARB MutationE-Rv (10 µM)

Predenature	Denature	Annealing	Extension	Cooling
94°C	98°C	50-65°C	68°C	15°C
2min	10sec	30sec	48sec	Infinity

Denature-Annealing-Extension 30cycle

(6)
#1: DiDECARB OE-D (10 µM)
#2: DiDECARB Mutation-E-Fw (10 µM)

(7)
#1: RV-DiDECARB His (10 µM)
#2: DiDECARB Mutation E-Fw (10 µM)

Predenature	Denature	Annealing	Extension	Cooling
94°C	98°C	50-65°C	68°C	15°C
2min	10sec	30sec	78sec	Infinity

Denature-Annealing-Extension 30cycle

(8)
#1: SAMmt OE-A (10 µM)
#2: SAMmt Mutation-Rv (10 µM)

Predenature	Denature	Annealing	Extension	Cooling
94°C	98°C	50-65°C	68°C	15°C
2min	10sec	30sec	48sec	Infinity

Denature-Annealing-Extension 30cycle

(9)
#1: SAMmt OE-D (10 µM)
#2: SAMmt Mutation-Fw (10 µM)

(10)
#1: RV-SAMmt His (10 µM)
#2: SAMmt Mutation-Fw (10 µM)

Predenature	Denature	Annealing	Extension	Cooling
94°C	98°C	50-65°C	68°C	15°C
2min	10sec	30sec	78sec	Infinity

Denature-Annealing-Extension 30cycle

(11)
#1: Fw-Pre-REDPX (10 µM)
#2: Suf-REDOX-RV (10 µM)

(12)
#1: Fw-Pre-REDPX (10 µM)
#2: RV-REDOX-His (10 µM)

Predenature	Denature	Annealing	Extension	Cooling
94°C	98°C	50-65°C	68°C	15°C
2min	10sec	30sec	48sec	Infinity

Denature-Annealing-Extension 30cycle

Restriction Enzyme Digestion

Murata

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	XhoI	DraI	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
AT-pSB1C3 (299ng/µL)	4	-	1	-	1	1	1	M	5	5	32	50
AT-pSB1C3 (299ng/µL)	6.8	-	1	-	1	-	-	M	3	3	15.20	30
pSB1C3-T7-RBS (310ng/µL)	7	-	-	1	1	-	-	H	5	1*	35	50
Pcon-pSB1C3 (245ng/µL)	4	1	-	-	1	-	-	H	5	1*	38	50

The cell marked * is not BSA but BAP.

9/19

Sequence PCR

Honda

Number/(µL)	Sample	Template/(µL)	BigDye/(µL)	5x Buffer/(µL)	Primer/(µL)		MilliQ/(µL)	Conc/(µL)
1	T7-RBS-AT-His(9/16-(2))	2.8	0.5	1.75	VF	0.5	4.95	10.5
2	T7-RBS-AT-His(9/16-(3))	2.8	0.5	1.75	VF	0.5	4.95	10.5
3	SAM-1C3(4)	2.8	0.5	1.75	VF	0.5	4.95	10.5
4	T7-RBS-AT-His(9/16-(2))	2.8	0.5	1.75	VR	0.5	4.95	10.5
5	T7-RBS-AT-His(9/16-(3))	2.8	0.5	1.75	VR	0.5	4.95	10.5
6	SAM-1C3(4)	2.8	0.5	1.75	VR	0.5	4.95	10.5
7	T7-RBS-AT-His(9/16-(2))	2.8	0.5	1.75	seg-1	0.5	4.95	10.5
8	T7-RBS-AT-His(9/16-(3))	2.8	0.5	1.75	seg-1	0.5	4.95	10.5
9	SAM-1C3(4)	2.8	0.5	1.75	OE-B	0.5	4.95	10.5
10	T7-RBS-AT-His(9/16-(2))	2.8	0.5	1.75	seg-2	0.5	4.95	10.5
11	9/23M PQLB	2.8	0.5	1.75	seg-2	0.5	4.95	10.5

Predenature	Denature	Annealing	Extension	Cooling	Cooling
96°C	96°C	50°C	60°C	4°C	4°C
2min	10sec	5sec	4min	1min	infinity

Denature-Annealing-Extension 30cycle

Gel Extraction

Honda

Lane	Sample
1,2,4,5,8	1kbp ladder
3	T7-RBS-AT-His-1C3 S,P
6	T7-RBS-1C3 S,P

Miniprep

Honda

Sample	Concentration/(µg/mL)	260/280	260/230
9/18 T7-RBS-AT-His(2)	305	1.66	1.49
9/18 T7-RBS-His-1C3(1)	280	1.68	1.67
9/18 REDOX(His)-YC Plac3(1)	985	1.74	2.39
9/18 REDOX(His)-YC Plac3(2)	1025	1.72	2.28

Gel Extraction

Yasuda

Lane	Sample
1,2	9/18 AT-1C3 X,S
3	100bp ladder
5,6	9/18 AT-1C3 E,P

Restriction Enzyme Digestion

Yasuda

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
9/19 T7-RBS-His Miniprep product	20	-	-	3	3	B	12	0	82	120
9/19 REDOX-3 M(1)	10.1	5	-	-	5	H	20	0	159.9	200
9/19 REDOX-3 M(1) control	0.1	-	-	-	-	H	1	-	8.9	10

Electrophoresis

Honda

Lane	Sample
1	DiDECARB-OE-A 52.1°C
2	DiDECARB-OE-A 55.4°C
3	DiDECARB-OE-A 59.4°C
4	DiDECARB-OE-A 64.9°C
5	DiDECARB-OE-A 52.1°C
6	DiDECARB-OE-A 55.4°C
7	DiDECARB-OE-A 59.4°C
8	DiDECARB-OE-A 64.9°C
9	DiDECARB-Mutation PCR product Pst1 Fwd 52.1°C
10	DiDECARB-Mutation PCR product Pst1 Fwd 55.4°C
11	DiDECARB-Mutation PCR product Pst1 Fwd 59.4°C
12	DiDECARB-Mutation PCR product Pst1 Fwd 64.9°C
13	REDOX-Fwd 52.1°C
14	REDOX-Fwd 55.4°C
15	REDOX-Fwd 59.4°C
16	REDOX-Fwd 64.9°C
17	REDOX-Fwd-His 52.1°C
18	REDOX-Fwd-His 55.4°C
19	REDOX-Fwd-His 59.4°C
20	REDOX-Fwd-His 64.9°C
21	DiDECARB-Mutation PCR product Fwd-His 52.1°C
22	DiDECARB-Mutation PCR product Fwd-His 55.4°C
23	DiDECARB-Mutation PCR product Fwd-His 59.4°C
24	DiDECARB-Mutation PCR product Fwd-His 64.9°C
25	DiDECARB-Mutation PCR product Fwd 52.1°C
26	DiDECARB-Mutation PCR product Fwd 55.4°C
27	DiDECARB-Mutation PCR product Fwd 59.4°C
28	DiDECARB-Mutation PCR product Fwd 64.9°C
29	SAM-Mutation PCR product Fwd 52.1°C
30	SAM-Mutation PCR product Fwd 55.4°C
31	SAM-Mutation PCR product Fwd 59.4°C
32	SAM-Mutation PCR product Fwd 64.9°C
33	1kbp ladder
34	SAM-Mutation PCR product Fwd 52.1°C
35	SAM-Mutation PCR product Fwd 55.4°C
36	SAM-Mutation PCR product Fwd 59.4°C
37	SAM-Mutation PCR product Fwd 64.9°C
38	SAM-Mutation PCR product Fwd-His 52.1°C
39	SAM-Mutation PCR product Fwd-His 55.4°C
40	SAM-Mutation PCR product Fwd-His 59.4°C
41	SAM-Mutation PCR product Fwd-His 64.9°C
42	1kbp ladder
43	DiDECARB-Mutation PCR product E-His 52.1°C
44	DiDECARB-Mutation PCR product E-His 55.4°C
45	DiDECARB-Mutation PCR product E-His 59.4°C
46	DiDECARB-Mutation PCR product E-His 64.9°C
47	DiDECARB-Mutation PCR product E-Fwd 52.1°C
48	DiDECARB-Mutation PCR product E-Fwd 55.4°C
49	DiDECARB-Mutation PCR product E-Fwd 59.4°C
50	DiDECARB-Mutation PCR product E-Fwd 64.9°C

PCR

Murata

Template;DiDECARB-OE-A,DiDECARB Mutation product Fw

10x Buffer/(µL)	2.0mM dNTP/(µL)	25.mM MgSO4/(µL)	10.0µM DiDECARB-OE-A/(µL)	10.0µM DiDECARB-OE-D/(µL)	1.0U/µL KOD/(µL)	H2O	Total/(µL)
10	10	6	5	5	2	up tp 100	100

Template;DiDECARB-OE-A,DiDECARB Mutation product Fw-His

10x Buffer/(µL)	2.0mM dNTP/(µL)	25.mM MgSO4/(µL)	10.0µM DiDECARB-OE-A/(µL)	10.0µM Rv DiDECARB-His/(µL)	1.0U/µL KOD/(µL)	H2O	Total/(µL)
10	10	6	5	5	2	up tp 100	100

Template;DiDECARB-OE-A,DiDECARB Mutation produc Pst1t Fw,DiDECARB Mutation produc Eco1

10x Buffer/(µL)	2.0mM dNTP/(µL)	25.mM MgSO4/(µL)	10.0µM DiDECARB-OE-A/(µL)	10.0µM DiDECARB-OE-D/(µL)	1.0U/µL KOD/(µL)	H2O	Total/(µL)
10	10	6	5	5	2	up tp 100	100

Template;DiDECARB-OE-A,DiDECARB Mutation product Pst1 Fw,DiDECARB Mutation product Eco1-His

10x Buffer/(µL)	2.0mM dNTP/(µL)	25.mM MgSO4/(µL)	10.0µM DiDECARB-OE-A/(µL)	10.0µM Rv DiDECARB-His/(µL)	1.0U/µL KOD/(µL)	H2O	Total/(µL)
10	10	6	5	5	2	up tp 100	100

Template;SAM-OE-A,SAM Mutation product Fw

10x Buffer/(µL)	2.0mM dNTP/(µL)	25.mM MgSO4/(µL)	10.0µM SAM-OE-A/(µL)	10.0µM SAM-OE-D/(µL)	1.0U/µL KOD/(µL)	H2O	Total/(µL)
10	10	6	5	5	2	up tp 100	100

Template;SAM-OE-A,SAM Mutation product Fw-His

10x Buffer/(µL)	2.0mM dNTP/(µL)	25.mM MgSO4/(µL)	10.0µM SAM-OE-A/(µL)	10.0µM SAM His/(µL)	1.0U/µL KOD/(µL)	H2O	Total/(µL)
10	10	6	5	5	2	up tp 100	100

Sample	Concentration/(µg/mL)	260/280	260/230
pSB1C3-AT/Pst1,Xba1,Xhol1,Dra1	24.75	1.90	1.92
T7RBS/Spe1,Pst1	8.48	1.91	1.01
T7RBS-His/Spe1,Pst1	35.98	1.88	1.78

Gel Extraction/Electrophoresis

Murata

Lane	Sample
2~5	DiDECARB His
6~8,10	DiDECARB
11~14	SAM
16~20	SAM His
21~23,25	DE His
26~30	DE
1,9,15,24	Blank

DNA Purification

Murata

DiDECARB His

DiDECARB

SAM

SAM His

Restriction Enzyme Digestion

Murata

Sample/(ng)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
DiDECARB His X,P	2000	-	1	-	1	M	3	3	up tp 30	30
DiDECARB X,P	2000	-	1	-	1	M	3	3	up to 30	30
DiDECARB E,P	2000	1	-	-	1	H	3	-	up to 30	30
SAM X,P	2000	-	1	-	1	M	3	3	-	30
SAM E,P	2000	1	-	-	1	H	3	-	up to 30	30
SAM His X,P	2000	-	1	-	1	M	3	3	up to 30	30

9/20

Electrophoresis

Murata

Lane	Sample
1	SAM His X,P
2	Di X,P
3	Di His X,P
4	SAM E,P
5	SAM X,P
6	Di E,P
7	AT X,P 2

Lane	Sample
1	AT X,P 3
2	T7-RBS S,P
3	dddD Mutation PCR product (2)
4	dddD Mutation PCR product (1)
5	T7-RBS 1C3
6	REDOX PCR product
7	REDOX His PCR product

Transformation

Murata

Sample/(µL)		CompetentCells/(µL)		Total/(µL)	Medium
7-2 DiDECARB T7-RBS	2	DH5α	10	12	SOC
7-5 SAMmt T7-RBS	2	DH5α	10	12	SOC
7-11 T7-RBS dddD(1) Mutation PCR product	2	DH5α	10	12	SOC
7-12 T7-RBS dddD(3) Mutation PCR product	2	DH5α	10	12	SOC
7-13 T7-RBS AT	2	DH5α	10	12	SOC
T7-RBS control	2	DH5α	10	12	SOC
10-1 DiDECARB pSB1C3	2	DH5α	10	12	SOC
10-4 SAMmt pSB1C3	2	DH5α	10	12	SOC
10-8 REDOX pSB1C3	2	DH5α	10	12	SOC
pSB1C3 control	2	DH5α	10	12	SOC
14-3 T7-RBS SAM-His	2	DH5α	10	12	SOC
T7-RBS DiDECARB His	2	DH5α	10	12	SOC
REDOX T7 RBS His(14)-9	2	DH5α	10	12	SOC
T7-RBS-His control	2	DH5α	10	12	SOC

9/22

Colony PCR

Yasuda

Quick Tag/(µL)	VF2/(µL)	VR/(µL)	MilliQ/(µL)	Total/(µL)
5	0.2	0.2	4.6	10

Predenature	Denature	Annealing	Extension	Cooling
94°C	94°C	55°C	68°C	12°C
2min	30sec	30sec	150sec	infinity

Denature-Annealing-Extension 30cycle

1~8	REDOX-T7-RBS-His
Number	Sample
9~16	T7-RBS-SAM-His
17~24	T7-RBS control
25~32	T7-RBS-dddD
33~40	DiDECARB-1C3
41~48	1C3 control
49~56	T7-RBS-DiDECARB
57~64	T7-RBS-His
65~72	REDOX-1C3
73~80	DiDECARB-T7-RBS
81~88	SAM-1C3
89~96	T7-RBS-sssS
97~104	T7-RBS-SAM
105	T7-RBS-AT
106~123	pBC345-AT

Colony PCR

Honda

Master Mix

Quick Tag/(µL)	VF2/(µL)	VR/(µL)	MilliQ/(µL)	Total/(µL)
225	9	9	217	450

Predenature	Denature	Annealing	Extension
94°C	94°C	55°C	68°C
2min	30sec	30sec	150sec

Denature-Annealing-Extension 30cycle

Number	Sample
1~7	9/20 T7-RBS-REDOX-His
8~14	9/20 SAMmt-1C3
15~21	9/20 T7-RBS-SAMmt-His
22~28	9/20 DiDECARB-1C3
29~35	9/20 T7-RBS-DiDECARB
36~42	9/20 T7-RBS-DiDECARB-His
43	9/20 T7-RBS-dddD
44	9/20 DiDECARB-1C3

Miniprep

Tatsui

Sample	Concentration/(µg/mL)	260/280	260/230
T7-RBS-His	55	1.58	0.80

Concentration Measurement

Yasuda

Sample	Concentration/(µg/mL)	260/280	260/230
T7-RBS-His S,P	1.0	1.54	1.94
T7-RBS-His S,P	1.3	1.59	2.01
T7-RBS E,S	1.1	1.40	2.01
HT-1C3 X,P	4.4	1.52	1.08
AT-1C3 X,P	4.0	1.65	1.06

Cut Check

Yasuda

Lane	Sample
1	Blank
2	100bp GeneRuler
3	T7 RBS-His S,P
4	T7-RBS S,P
5	T7-RBS E,P
6	AT-1C3 X,P
7	AT-1C3 X,P

Electrophoresis

Honda

(1)

Lane	Sample
1	100b ladder
2~8	T7-RBS-REDOX-His

(2)

Lane	Sample
1	100b ladder
2~8	SAMmt-1C3

(3)

Lane	Sample
1	100b ladder
2~8	T7-RBS-SAMmt-His

(4)

Lane	Sample
1	100b ladder
2~8	DiDECARB-1C3

(5)

Lane	Sample
1	100b ladder
2~8	T7-RBS-DiDECARB

(6)

Lane	Sample
1	100b ladder
2~8	T7-RBS-DiDECARB-His

(7)

Lane	Sample
1	Blank
2	Blank
3	100b ladder
4	T7-RBS-dddD-1
5	DiDECARB-1C3-1

Liquid Culture

Yasuda

Sample	Medium
T7-RBS-dddD	CP

Sample	Medium
REDOX-1C3	CP

Sample	Medium
T7-RBS-SAMmt	CP

Colony PCR

Honda

Master Mix

Quick Taq/(µL)	VF2/(µL)	VR/(µL)	MilliQ/(µL)	Total/(µL)
240	9.6	9.6	220.8	480

Predenature	Denature	Annealing	Extension
94°C	94°C	55°C	68°C
2min	30sec	30sec	90sec

Denature-Annealing-Extension 30cycle

Number	Sample
1~16	T7-RBS-DiDECARB-His
19~32	T7-RBS-SMAmt-His
33~48	T7-RBS-REDOX-His

9/23

Miniprep

Sample	Concentration/(µg/mL)	260/280	260/230
pB-22	250	1.61	1.69
pB-23	125	2.07	2.90
pQ-3	185	1.68	1.87
pQ-5	210	1.72	1.86
pLQB	90	2.40	3.09

Electrophoresis

Honda

1

Number	Sample
1	100bp ladder
2~8	T7-RBS-REDOX-His 2~7

2

Number	Sample
1	100bp ladder
2~8	T7-RBS-REDOX-His 8~14

3

Number	Sample
1	100bp ladder
2,3	T7-RBS-REDOX-His 15,16
4~8	T7-RBS-SAMmt-His 1~5

4

Number	Sample
1	100bp ladder
2~8	T7-RBS-SAMmt-His 6~12

5

Number	Sample
1	100bp ladder
2~5	T7-RBS-SAMmt-His 13~16
6~8	T7-RBS-DiDECARB-His 1~3

6

Number	Sample
1	100bp ladder
2~8	T7-RBS-DiDECARB-His 4~10

7

Number	Sample
1	100bp ladder
2~7	T7-RBS-DiDECARB-His 11~16

9/24

Dephosphorylation

Yasuda

T7-RBS-His S,P

Miniprep

Murata

Sample	Concentration/(µg/mL)	260/280	260/230
T7-RBS-SAM 6	10.3	1.67	1.15
9/22 DiDECARB 1C3-4	11.3	1.54	1.11
9/22 DiDECARB 1C3-2	10.5	1.67	0.83
9/20 T7-RBS-dddD Mutation PCR product(3)-(4)	14.2	1.71	1.38
REDOX-1C3(2)	9.8	1.71	0.83
9/20 T7-RBS-dddD Mutation PCR product(3)-(5)	12.7	1.66	1.42
9/22 DiDECARB 1C3-5	10.5	1.69	1.15

Cycle PCR

Yasuda

Master Mix

Number/(µL)	Sample	Template/(µL)	BigDye/(µL)	5x Buffer/(µL)	Primer/(µL)		MilliQ/(µL)	Conc/(µL)
1	9/23M PQLB	4.4	0.5	1.75	VF2	0.5	2.8	90
2	9/23M PQLB	4.4	0.5	1.75	pQLB seq-1	0.5	2.8	90
3	9/23M PQLB	4.4	0.5	1.75	pQLB seq-2	0.5	2.8	90
4	9/23M PQLB	4.4	0.5	1.75	pQLB seq-3	0.5	2.8	90
5	9/23M PQLB	4.4	0.5	pQLB seq-4	VF2	0.5	2.8	90
6	9/23M PQLB	4.4	0.5	1.75	VR	0.5	2.8	90
7	9/23M PQ-3	2.2	0.5	1.75	VF2	0.5	5.0	185
8	9/23M PQ-3	2.2	0.5	1.75	VR	0.5	5.0	185
9	9/23M PQ-5	1.9	0.5	1.75	VF2	0.5	5.3	210
10	9/23M PQ-5	1.9	0.5	1.75	VR	0.5	5.3	210
11	9/22M pB-23	3.2	0.5	1.75	VF2	0.5	4.0	125
12	9/22M pB-23	3.2	0.5	1.75	VR	0.5	4.0	125
13	9/23M pB-22	1.6	0.5	1.75	VF2	0.5	5.7	250
14	9/23M pB-22	1.6	0.5	1.75	VR	0.5	5.7	250
15	9/15 MP AT-1C3 1	1.3	0.5	1.75	VF2	0.5	6.0	299
16	9/15 MP AT-1C3 1	1.3	0.5	1.75	AT Seq1	0.5	6.0	299
17	9/15 MP AT-1C3 1	1.3	0.5	1.75	AT Seq2	0.5	6.0	299
18	9/15 MP AT-1C3 1	1.3	0.5	1.75	VR	0.5	6.0	299
19	9/22 M REDOX 1C3	0.8	0.5	1.75	VF2	0.5	6.4	490
20	9/22 M REDOX 1C3	0.8	0.5	1.75	REDOX Seq1	0.5	6.4	490
21	9/22 M REDOX 1C3	0.8	0.5	1.75	VR	0.5	6.4	490
22	9/18 MP SAM-1C3	2.1	0.5	1.75	VF2	0.5	5.0	185
23	9/18 MP SAM-1C3	2.1	0.5	1.75	SAM Seq1	0.5	5.0	185
24	9/18 MP SAM-1C3	2.1	0.5	1.75	VR	0.5	5.0	185
25	9/22 DiDECARB 1C3-4	0.7	0.5	1.75	VF	0.5	6.5	565
26	9/22 DiDECARB 1C3-4	0.7	0.5	1.75	DiDECARB Seq1	0.5	6.5	565
27	9/22 DiDECARB 1C3-4	0.7	0.5	1.75	DiDECARB Seq2	0.5	6.5	565
28	9/22 DiDECARB 1C3-4	0.7	0.5	1.75	VR	0.5	6.5	565

DNA Purification

Honda

Sample	Concentration/(µg/mL)	260/280	260/230
9/24 PCR product AT	265	1.67	2.10

Restriction Enzyme Digestion

Yasuda

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
9/24 REDOX(His) column	7.5	-	1	-	1	M	3	3	14.5	30

9/25

Colony PCR

Honda

Quick Taq/(µL)	VF2/(µL)	VR/(µL)	MilliQ/(µL)	Total/(µL)
5	0.2	0.2	4.6	10

Predenature	Denature	Annealing	Extension	Cooling
94°C	94°C	55°C	68°C	12°C
2min	30sec	30sec	1min	infinity

Denature-Annealing-Extension 30cycle

Number	Sample
1	T7-RBS-REDOX-His(1)
2	T7-RBS-REDOX-His(2)
3	T7-RBS-REDOX-His(3)

Electrophoresis

Honda

Lane	Sample
1,2	Blank
3	100b ladder
4~6	T7-RBS-REDOX-His(1)~(3)

Colony PCR

Yasuda

Master Mix

Quick Taq/(µL)	VF2/(µL)	VR/(µL)	MilliQ/(µL)	Total/(µL)
130	5.2	5.2	119.6	260.0

Predenature	Denature	Annealing	Extension
94°C	94°C	55°C	68°C
2min	30sec	30sec	90sec

Denature-Annealing-Extension 30cycle

Number	Sample
1~8	T7-RBS-REDOX-His (1)~(8)
9~16	T7-RBS-SAMmt-His (1)~(8)
17~24	T7-RBS-DiDECARB-His (1)~(8)

Electrophoresis

Yasuda

1

Lane	Sample
1	100bp GeneRuler
2~8	T7-RBS-REDOX-His(1)~(7)

2

Lane	Sample
1	100bp GeneRuler
2	T7-RBS-REDOX-His(8)
3~7	T7-RBS-SAMmt-His(1)~(5)
8	Blank

3

Lane	Sample
1	100bp GeneRuler
2~4	T7-RBS-SAMmt-His(6)~(8)
5~8	T7-RBS-DiDECARB-His(1)~(4)

4

Lane	Sample
1	100bp GeneRuler
2,3,5,6	T7-RBS-DiDECARB-His(5)~(8)
4	Blank

Transformation

Yasuda

Sample/(µL)		CompetentCells/(µL)		Total/(µL)	Medium
T7-RBS-AT-His 9/19 M(3)	1	Zip BL21(DE3)	10	11	CP
T7-RBS-dddD(n) 9/22 M (3)-5	1	Zip BL21(DE3)	10	11	CP

9/26

Colony PCR

Yasuda

Quick Taq/(µL)	VF2/(µL)	VR/(µL)	MilliQ/(µL)	Total/(µL)
5	0.2	0.2	4.6	10

Predenature	Denature	Annealing	Extension	Cooling
94°C	94°C	55°C	68°C	12°C
5min	30sec	30sec	2min	infinity

Denature-Annealing-Extension 30cycle

Number	Sample
1~8	T7-RBS-REDOX-His
9~16	T7-RBS-SAMmt-His
17~24	T7-RBS-DiDECARB-His

Electrophoresis

Yasuda

Lane	Sample
1~8	T7-RBS-REDOX-His(1)~(8)
9~12,14~17	T7-RBS-SAMmt-His(1)~(8)
18~25	T7-RBS-DiDECARB-His(1)~(8)
13	100bp ladder

9/27

Colony PCR

Yasuda

Quick Taq/(µL)	VF2/(µL)	VR/(µL)	MilliQ/(µL)	Total/(µL)
5	0.2	0.2	4.6	10

Predenature	Denature	Annealing	Extension
94°C	94°C	55°C	68°C
2min	30sec	30sec	160sec

Denature-Annealing-Extension 30cycle

Number	Sample
1~7	T7-RBS-REDOX-His
8~14	T7-RBS-SAMmt-His
15~20	T7-RBS-DiDECARB-His
21,22	T7-RBS-AT-His 9/25 Transformation(3),(4)
23,24	T7-RBS-dddD 9/25 Transformation(3),(4)

Cloning PCR

Yasuda

Master Mix

KOD Plus/(µL)	DNA CCAP 1023/11/(µL)	2mMdNTPs/(µL)	10xBuffer forKDDplusV2/(µL)	25mM MgSO4/(µL)	Fw AT cloning primer 61/(µL)	Rv AT cloning primer 61/(µL)	MilliQ/(µL)	Total/(µL)
1	2	5	5	3	1.5	1.5	31	50

Predenature	Denature	Annealing	Extension	Cooling
94°C	98°C	56°C	68°C	12°C
2min	10sec	30sec	90sec	infinity

Denature-Annealing-Extension 30cycle

9/28

OE-PCR 1st

Yasuda

KOD Plus/(µL)	9/27 AT PCR product/(µL)	10xBuffer for KODPlus/(µL)	2mMdNTPS/(µL)	25mM MgSO4/(µL)	Primer #1/(µL)	MilliQ/(µL)	Total/(µL)
1	pick up	5	5	3	1.5(each)	up to 50	50

1. #1 AT OE-A/AT OE-B

2. #1 AT OE-C/AT2 OE-B

3. #1 AT2 OE-C/AT OE-D

Predenature	Denature	Annealing	Extension	Final Extension	Cooling
98°C	98°C	55°C	72°C	72°C	12°C
2min	10sec	10sec	1min	5min	infinity

Denature-Annealing-Extension 25cycle

Restriction Enzyme Digestion

Yasuda

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
8/27 Miniprep product 1A2	7.6	-	-	3	3	B	12	1.2	93.2	120
control	0.5	-	-	-	-	B	1	0.1	8.4	10

Gel Extraction

Yasuda

Lane	Sample
1	9/28 OE-(3)
2	9/28 OE-(3)
3	Blank
4	9/28 OE-(1)
5	100bp ladder

Lane	Sample
1	9/28 OE-(3)
4	9/28 OE-(2)
6	100bp ladder
2,3,5	Blank

OE-PCR 2nd

Yasuda

Master Mix

KOD Plus/(µL)	10xBuffer for KODPlus ver2/(µL)	2mMdNTPS/(µL)	25mM MgSO4/(µL)	Primer #1/(µL)	Template #2/(µL)	MilliQ/(µL)	Total/(µL)
1	5	5	3	2(each)	0.5(each)	up to 50	50

1. #1 AT OE-A/AT OE-D #2 9/28 OE-(1) gel extraction product/9/28 OE-(2) gel extraction product/9/28 OE-(3) gel extraction product

2. #1 AT OE-A/AT OE-D #2 9/27 AT PCR product 9/28 column

Predenature	Denature	Annealing	Extension	Final Extension	Cooling
98°C	98°C	55°C	72°C	72°C	12°C
2min	10sec	10sec	1min	5min	infinity

Denature-Annealing-Extension 25cycle

9/29

Mutation PCR

Yasuda

PrimeStar Max Premix/(µL)	SAMmt Mutation Fw/(µL)	SAMmt Mutation Rv/(µL)	9/18 MP SAM-1C3/(µL)	MilliQ/(µL)	Total/(µL)
25	1	1	0.5	22.5	50

Denature	Annealing	Extension
98°C	55°C	72°C
10sec	15sec	55sec

Denature-Annealing-Extension 30cycle

Gel Extraction

Yasuda

Lane	Sample
5	100bp ladder
3,7	1A2 9/28 S,P
9	9/28 Digestion control
1,2,4,5,6,8	Blank

Restriction Enzyme Digestion

Yasuda

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
8/27 M 1A2	7.6	-	3	-	3	D	12	1.2	93.2	10
control	0.5	-	-	-	-	D	1	0.1	8.4	10

Gel Extraction

Yasuda

Lane	Sample
1	100bp ladder
4	1A2 9/29 X,P
6	9/29 Digestion control
2,3,5	Blank

Concentration Measurement

Yasuda

Sample	Concentration/(µg/mL)	260/280	260/230
1. 9/28 OE(1) 9/29 gel extraction product	17.9	1.92	0.94
2. 9/28 OE(2) 9/29 gel extraction product	122.2	1.86	1.64
3. 9/28 OE(3) 9/29 gel extraction product	78.8	1.89	1.67
1A2 9/29 X,P	26.1	1.37	0.76

Transformation

Murata

Sample/(µL)		CompetentCells/(µL)		Total/(µL)	Medium
9/29 Ligation DiDECARB(His)-1A2	1	Zip BL21(DE3)	10	11	CP
9/29 Ligation REDOX(His)-1A2	1	Zip BL21(DE3)	10	11	CP
9/29 SAMmt mutation	1	Zip BL21(DE3)	10	11	CP

9/30

Mutation PCR

Yasuda

PrimeStar Max PreMix/(µL)	Template #1/(µL)	Primers #2/(µL)	MilliQ/(µL)	Total/(µL)
25	0.5	1(each)	up to 50	50

1. #1 9/15 Miniprep AT-1C3(1) #2 Fw:AT Mutation Insert/Rv:AT Mutation Insert

2. #1 9/19 Miniprep T7-RBS-AT(His) #2 Fw Add His to T7RBS/Rv Add His toT7 RBS

3. #1 9/13 Miniprep T7-RBS-1C3 #2 Fw Add His to T7RBS/Rv Add His to T7RBS

Denature	Annealing	Extension	Cooling
98°C	55°C	72°C	12°C
10sec	15sec	20sec	infinity

Denature-Annealing-Extension 35cycle

DNA Purification

Yasuda

Sample	Concentration/(µg/mL)	260/280	260/230
SAMmt-1C3 9/30 column	136.3	1.87	1.68

OE-PCR 2nd

Yasuda

Master Mix

KOD Plus/(µL)	10xBuffer for KODPlus ver2/(µL)	2mMdNTPS/(µL)	25mM MgSO4/(µL)	Primer #1/(µL)	Template #2/(µL)	MilliQ/(µL)	Total/(µL)
1	5	5	3	2(each)	0.5(each)	up to 50	50

1. #1 AT OE-A/AT OE-D #2 9/28 OE-(1) gel extraction product/9/28 OE-(2) gel extraction product/9/28 OE-(3) gel extraction product

2. #1 AT OE-A/AT OE-D #2 9/27 AT PCR product 9/28 column

Predenature	Denature	Annealing	Extension	Final Extension	Cooling
98°C	98°C	55°C	72°C	72°C	12°C
2min	10sec	10sec	1min	5min	infinity

Denature-Annealing-Extension 30cycle

Transformation

Yasuda

Sample/(µL)		CompetentCells/(µL)		Total/(µL)	Medium
T7-RBS-AT-His	1	self-making DH5α	10	11	CP
SAMmt-1C3 9/29 Mutation PCR product	1	self-making DH5α	10	11	CP
AT-1C3 9/30 insertion Mutation PCR product	1	self-making DH5α	10	11	CP
T7-RBS-His 9/30 Mutation PCR product	1	self-making DH5α	10	11	CP

DNA Purification

Yasuda

Sample	Concentration/(µg/mL)	260/280	260/230
9/30 OE-PCR 2nd product(Pre-AT-Suf)	217	1.85	1.22

Restriction Enzyme Digestion

Yasuda

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
Pre-At-Suf 9/30 Column	9.2	1	-	-	1	H	4	0.4	14.4	40

10/1

DNA Purification

Yasuda

Sample	Concentration/(µg/mL)	260/280	260/230
AT 9/30 E,P	38	1.84	0.46
1C3 E,P	5	1.78	0.27

Transformation

Yasuda

Sample/(µL)		CompetentCells/(µL)		Total/(µL)	Medium
9/30 AT-1C3	1	BL21(DE3)	10	11	CP
9/30 T7-RBS-His	1	self-making DH5α 2/23	10	11	CP
9/30 T7-RBS-AT-His	1	self-making DH5α 2/23	10	11	CP
9/29 SAMmt-1C3	1	self-making DH5α 2/23	10	11	CP
9/22 T7-RBS-dddD(3)-5	1	self-making DH5α 2/23	10	11	CP

10/2

Kination

Yasuda

Add His to T7RBS Fw

ATOE-D

PCR

Yasuda

PrimeSTAR Premix/(µL)	Template #1/(ng)	Primer #2/(µL)	MilliQ/(µL)	Total/(µL)
50	400	2(each)	up to 100	100

#1 AT-1C3(without (1))

#2 Fw:Add His to T7RBS/Rv:AT OE-D

!

10/2

Kination

Yasuda

Add His to T7RBS Fw

ATOE-D

PCR

Yasuda

PrimeSTAR Premix/(µL)	Template #1/(ng)	Primer #2/(µL)	MilliQ/(µL)	Total/(µL)
50	400	2(each)	up to 100	100

#1 AT-1C3(without (1))

#2 Fw:Add His to T7RBS/Rv:AT OE-D

DNA Purification

Murata

Sample
PCR product (1),(2) His-pSB1C3-AT
PCR product(3) AT-1C3 (AT Mota I)
PCR product(4) AT-1C3 (AT Mota D)
PCR product(5) T7-RBS-His-1C3

Restriction Enzyme Digestion

Murata

Dpn1/(µL)	10/2 PCR AT-1C3 miniprep product(3)/ (µL)	10x Buffer(H)/(µL)	MilliQ/(µL)	Total/(µL)
1	8	5	36	50

Electrophoresis

Yasuda

Lane	Sample
4	His 1C3-AT 10/2 DNA Purification
5	AT-1C3 Mutation Deletion 10/2 DNA Purification
6	1kbp GeneRuler
7	AT-1C3 Mutation Insert 10/2 DNA Purification
8	T7-RBS AT His 10/2 DNA Purification
1~3,9~12	Blank

!

10/3

Miniprep

Yasuda

Sample	Concentration/(µg/mL)	260/280	260/230
9/14 AT-1C3(1)	119	2.00	2.19
10/1 SAMmt(Mutated)-1C3(1)	275	1.86	1.75
10/1 SAMmt(Mutated)-1C3(2)	227	1.93	1.90
10/1 T7-RBS-His(1)	147	2.00	1.64
10/1 T7-RBS-His(2)	107	1.94	1.74

Mutation PCR

Murata

Master Mix

PrimeSTARPremix/(µL)	Template 10/3 Miniprep AT-1C3(1)/(µL)	Primer AT Mutation Insert Rv/(µL)	AT Mutation Insert Fw/(µL)	MilliQ/(µL)	Total/(µL)
12.5	119	0.5	0.5	up to 25	25

Denature	Annealing	Extension
94°C	55°C	68°C
10sec	15sec	20sec

Denature-Annealing-Extension 35cycle

Gel Extraction

Yasuda

Lane	Sample
1	His-OE-(1)
2	His-OE-(2)

Electrophoresis

Yasuda

Lane	Sample
1	AT-1C3 Mutation insert 50.2°C
2	AT-1C3 Mutation insert 52.8°C
3	AT-1C3 Mutation insert 54.2°C
4	AT-1C3 Mutation insert 55.7°C
5	AT-1C3 Mutation insert 57.1°C
6	10/2 DNA Purification His 1C3-AT
7	AT-1C3 Mutation Deletion 50.2°C
8	AT-1C3 Mutation Deletion 52.8°C
9	AT-1C3 Mutation Deletion 54.2°C
10	AT-1C3 Mutation Deletion 55.7°C
11	AT-1C3 Mutation Deletion 57.1°C
12	100bp ladder

OE-PCR 1st

Murata

(1)

KOD Plus/(µL)	10xBuffer for KOD Plus/(µL)	2mM dNTPs/(µL)	25mM MgSO4/(µL)	10/2 MP J04450(1C3)/(µL)	VF2/(µL)	Rv mutation primer T7-RBS-His/(µL)	MilliQ/(µL)	Total/(µL)
1	5	5	3	1	1.5	1.5	32	50

(2)

KOD Plus/(µL)	10xBuffer for KOD Plus/(µL)	2mM dNTPs/(µL)	25mM MgSO4/(µL)	10/2 MP J04450(1C3)/(µL)	Fw mutation primer T7-RBS-His/(µL)	Present from Santa/(µL)	MilliQ/(µL)	Total/(µL)
1	5	5	3	1	1.5	1.5	32	50

Denature	Annealing	Extension	Cooling	Cooling
94°C	98°C	55°C	68°C	4°C
2min	10sec	30sec	3min	infinity

Denature-Annealing-Extension 35cycle

OE-PCR 2nd

Yausda

KOD Plus/(µL)	10xBuffer for KOD Plus/(µL)	2mM dNTPs/(µL)	25mM MgSO4/(µL)	His OE-(1) 10/3 gel extraction product/ (µL)	His OE-(2) 10/3 gel extraction product/ (µL)	VF2/(µL)	Present from Santa/(µL)	MilliQ/(µL)	Total/(µL)
1	5	5	3	1	1	1.5	1.5	31	50

Denature	Annealing	Extension	Cooling	Cooling
94°C	98°C	55°C	68°C	4°C
2min	10sec	30sec	3min	infinity

Denature-Annealing-Extension 35cycle

Restriction Enzyme Digestion

Yasuda

Sample/(µL)		Xba1/(µL)	ApnL1/(µL)	10XT/(µL)	BSA/(µL)	CIAP/(µL)	MilliQ/(µL)	Total/(µL)
AT-1C3(1) 10/3 Miniprep	10	1	1	10	4	4	1	19	40
OE-PCR 2nd(T7 RBS His) without CIAP	10	1	1	10	4	4	-	20	40

Ligation

Yasuda

A

Vector/(µL)		Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
1C3 10/3 XA	2	T7 RBS His 10/3 XA	2	2xRapidLigBuffer	5	Promega T4 Ligase	1	-	10
1C3 10/3 XA	2	T7 RBS His 10/3 XA	-	2xRapidLigBuffer	5	Promega T4 Ligase	1	2	10

B

Vector/(µL)		Insert/(µL)		Buffer/(µL)		MilliQ/(µL)	Total/(µL)
1C3 10/3 XA	2	T7 RBS His 10/3 XA	2	2xRapidLigBuffer	4	-	8
1C3 10/3 XA	1	T7 RBS His 10/3 XA	-	2xRapidLigBuffer	4	1	6

Transformation

Yasuda

Sample	CompetentCells	Medium
10/3 Ligation products	DH5α	CP

!

10/4

@

!

10/5

OE-PCR 2nd

Yasuda

KOD Plus/(µL)	10xBuffer for KOD Plus/(µL)	2mM dNTPs/(µL)	25mM MgSO4/(µL)	Primers #1/(µL)	Templates #2/(µL)	MilliQ/(µL)	Total/(µL)
2	10	5	10	6	3(each)	2(each)	62	100

1. #1 VF2/Present from Santa #2 RFP OE(1)/RFP OE(2)

2. #1 VF2/Present from Santa #2 RFP OE(1)/His OE(2)

Denature	Annealing	Extension	Final Extension
94°C	98°C	55°C	68°C
2min	10sec	30sec	100min

Denature-Annealing-Extension 35cycle

Restriction Enzyme Digestion

Yasuda

Sample/(µL)		Xba1/(µL)	Spe1/(µL)	CIAP/(µL)	10XT/(µL)	BSA/(µL)	MilliQ/(µL)	Total/(µL)
10/5 RPFHis(1) 10/5 OE2(1)	5	1	1	-	4	4	20	40
10/5 RPFHis(2) 10/5 OE2(2)	5	1	1	-	4	4	20	40
10/5 RPFHis(3) 10/4 OE2 RPF His	5	1	1	-	4	4	20	40
10/5 1C3 XA (1) 9/18 Mutated Bn-1C310-3	5	1	1	1	4	4	19	40
10/5 1C3 XA (2) 9/14 Mutated dddD(mutated)-1C3(2)	5	1	1	1	4	4	19	40

Gel Extraction

Yasuda

Lane	Sample
(1)-1,(2)-6	SKY
(1)-2,3	12FP His 10/5 XA(1)
(1)-4,5	1C3 10/5 XA(2)
(1)-6	RFP His 10/5 XA(2)
(2)-1	RFP His 10/5 XA(2)
(2)-2,3	RFP His 10/5 XA(3)
(2)-4,5	1C3 10/5 XA(1)

!

10/6

Miniprep

Honda

Sample	Concentration/(µg/mL)	260/280	260/230
10/5 Pcon RPF-His A	133	1.93	2.24
10/5 Pcon RPF-His B	133	1.90	1.83
10/5 Pcon RPF-His C	137	1.80	1.72
10/5 Pcon RPF-His D	142	1.98	2.61

PCR

Honda

10/6 Pcon RPFHisA VF

Template/(µL)	Bigdye/(µL)	5xBuffer/(µL)	Primer/(µL)	MilliQ/(µL)	Total/(µL)
2.9	0.5	1.75	0.5	4.85	10.5

10/6 Pcon RPFHisA VR

Template/(µL)	Bigdye/(µL)	5xBuffer/(µL)	Primer/(µL)	MilliQ/(µL)	Total/(µL)
2.9	0.5	1.75	0.5	4.85	10.5

10/6 Pcon RPFHisB VF

Template/(µL)	Bigdye/(µL)	5xBuffer/(µL)	Primer/(µL)	MilliQ/(µL)	Total/(µL)
3.0	0.5	1.75	0.5	4.75	10.5

10/6 Pcon RPFHisB VR

Template/(µL)	Bigdye/(µL)	5xBuffer/(µL)	Primer/(µL)	MilliQ/(µL)	Total/(µL)
3.0	0.5	1.75	0.5	4.75	10.5

10/6 Pcon RPFHisC VF

Template/(µL)	Bigdye/(µL)	5xBuffer/(µL)	Primer/(µL)	MilliQ/(µL)	Total/(µL)
2.9	0.5	1.75	0.5	4.85	10.5

10/6 Pcon RPFHisC VR

Template/(µL)	Bigdye/(µL)	5xBuffer/(µL)	Primer/(µL)	MilliQ/(µL)	Total/(µL)
2.9	0.5	1.75	0.5	4.85	10.5

10/6 Pcon RPFHisD VF

Template/(µL)	Bigdye/(µL)	5xBuffer/(µL)	Primer/(µL)	MilliQ/(µL)	Total/(µL)
2.8	0.5	1.75	0.5	4.95	10.5

10/6 Pcon RPFHisD VR

Template/(µL)	Bigdye/(µL)	5xBuffer/(µL)	Primer/(µL)	MilliQ/(µL)	Total/(µL)
2.8	0.5	1.75	0.5	4.95	10.5

Predenature	Denature	Annealing	Extension	Cooling	Cooling
96°C	96°C	50°C	60°C	4°C	4°C
2min	10sec	5sec	4min	1min	infinity

Denature-Annealing-Extension 30cycle

!

10/7

PCR

Honda

AT(1)

Template/(µL)	Bigdye/(µL)	5xBuffer/(µL)	Primer/(µL)	MilliQ/(µL)	Total/(µL)
4.0	0.5	1.75	0.5	3.25	10.5

AT(2)

Template/(µL)	Bigdye/(µL)	5xBuffer/(µL)	Primer/(µL)	MilliQ/(µL)	Total/(µL)
2.0	0.5	1.75	0.5	5.25	10.5

AT(3)

Template/(µL)	Bigdye/(µL)	5xBuffer/(µL)	Primer/(µL)	MilliQ/(µL)	Total/(µL)
2.0	0.5	1.75	0.5	5.25	10.5

AT(4)

Template/(µL)	Bigdye/(µL)	5xBuffer/(µL)	Primer/(µL)	MilliQ/(µL)	Total/(µL)
2.5	0.5	1.75	0.5	4.75	10.5

Predenature	Denature	Annealing	Extension	Cooling	Cooling
96°C	96°C	50°C	60°C	4°C	4°C
2min	10sec	5sec	4min	1min	infinity

Denature-Annealing-Extension 30cycle

Restriction Enzyme Digestion

Honda

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
10/6 T7-RBS-His(2)	12	-	-	1	1	B	4	0.4	21.6	40
10/6 T7-RBS-His(2) control	1	-	-	-	-	B	1	-	8	10
10/6 T7-RBS-His(3)	20	-	-	2	2	B	8	0.8	47.2	80
10/6 T7-RBS-His(3) control	1	-	-	-	-	B	1	-	8	10

Gel Extraction

Honda

Lane	Sample
1	T7RBSHis(3) 10/7 S,P control
2	T7RBSHis(3) 10/7 S,P Experimental
3	100bp ladder
4	Blank
5	7RBSHis(2) 10/7 S,P Experimental
6	7RBSHis(2) 10/7 S,P control

Lane	Sample
3	T7RBSHis(3) 10/7 S,P Experimental
1,2,4,5,6	Blank

Sample	Concentration/(µg/mL)	260/280	260/230
T7RBSHis(3) low	38	1.52	1.30
T7RBSHis(2)	46	1.49	1.53
T7RBSHis(3) high	28	2.04	0.38

!

10/8

Liquid Culture

Honda

Sample	Medium
BL21	LB

Liquid Culture

Yasuda

Sample	Medium
9/30 AT-1C3 insertion mutated	CP
9/30 SAMmt-1C3 mutated	CP

!

10/10

Restriction Enzyme Digestion

Yasuda

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
AT(Mutation Insert)-1C3(2)	20	-	1	-	1	D	4	0.4	3.6	40

Dephosphorylation

Yasuda

10/8 T7 RBSHis(2)S,P/(µL)	CIAP/(µL)	10xBuffer/(µL)	MilliQ/(µL)	Total/(µL)
12	1	2	5	20

37°C	50°C	15°C
30min	15min	30min

Miniprep

Yasuda

Sample
Liquid Culture product SAM(Mutated)-1C3(1)~(4)

Cloning PCR

Yasuda

2X KAPA HiFI HotStart ReadyMix/(µL)	AT OE-A/(µL)	AT cloning for His/(µL)	10/7 M AT(Mutation Insert)-1C3(2)/(µL)	MilliQ/(µL)	Total/(µL)
12.5	0.75	0.75	up to 25	11	25

Predenature	Denature	Annealing	Extension	Final Extension
95°C	98°C	60°C	72°C	72°C
5min	20sec	15sec	90sec	5sec

Denature-Annealing-Extension 35cycle

Gel Extraction

Yasuda

Lane	Sample
1	AT 1C3 (Mutation Insert)(2)10/3
2	Blank
3	100bp ladder

Restriction Enzyme Digestion

Yasuda

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
10/10 Miniprep product SAMmt(Mutated)-1C3(1)	10	-	-	-	1	E	4	0.4	24.6	40
10/10 Miniprep product SAMmt(Mutated)-1C3(2)	10	-	-	-	1	E	4	0.4	24.6	40
10/10 Miniprep product SAMmt(Mutated)-1C3(3)	10	-	-	-	1	E	4	0.4	24.6	40
10/10 Miniprep product SAMmt(Mutated)-1C3(4)	10	-	-	-	1	E	4	0.4	24.6	40

Ligation

Yasuda

Vector/(µL)		Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
T7 RBS-1C3 S,P	2	10/10 gel extraction product(true)XP	2	2xRapidLigaseBuffer	5	Promega T4 Ligase	1	-	10
T7 RBS-1C3 S,P	1	10/10 gel extraction product(true)XP	3	2xRapidLigaseBuffer	5	Promega T4 Ligase	1	-	10
T7 RBS-1C3 S,P	2	10/10 gel extraction product(true)XP	0	2xRapidLigaseBuffer	5	Promega T4 Ligase	1	2	10
T7 RBS-1C3 S,P	2	10/10 gel extraction product(true)XP	2	2xRapidLigaseBuffer	5	Promega T4 Ligase	1	-	10
T7 RBS-1C3 S,P	1	10/10 gel extraction product(true)XP	3	2xRapidLigaseBuffer	5	Promega T4 Ligase	1	-	10
T7 RBS-1C3 S,P	2	10/10 gel extraction product(true)XP	0	2xRapidLigaseBuffer	5	Promega T4 Ligase	1	2	10

Transformation

Yasuda

Sample	CompetentCells	Medium
10/10 Ligation product	BL21(DE3)	CP

!

10/11

Miniprep

Honda

Sample	Concentration/(µg/mL)	260/280	260/230
T7RBSHis(2)	217.4	1.92	2.16
T7RBSHis(3)	418.0	1.95	2.30
AT-1C3(2)	258.0	1.96	2.25

Restriction Enzyme Digestion

Yasuda

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	CIAP/(µL)	MilliQ/(µL)	Total/(µL)
10/11 miniprep product T7RBSHis(2)	10	-	-	2	2	B	8	0.8	47.2	80
10/11 miniprep product T7RBSHis(3)	10	-	-	2	2	B	8	0.8	57.2	80
10/11 miniprep product AT-1C3(2)	10	-	1	-	1	B	4	0.4	0	23.6	40
10/10 miniprep product SAM(mutated)-1C3-(1)	10	-	-	1	-	B	4	0.4	0	24.6	40
10/10 miniprep product SAM(mutated)-1C3-(2)	10	-	-	1	-	B	4	0.4	0	24.6	40
10/10 miniprep product SAM(mutated)-1C3-(3)	10	-	-	1	-	B	4	0.4	0	24.6	40
10/10 miniprep product SAM(mutated)-1C3-(4)	10	-	-	1	-	B	4	0.4	0	24.6	40

DNA Purification

Yasuda

Sample	Concentration/(µg/mL)	260/280	260/230
10/10 PCR Cloning product AT	207.6	1.88	2.00

Restriction Enzyme Digestion

Yasuda

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
10/11 DNA Purification product AT(His)	10	0	1	0	1	D	4	0.4	24.6	40

Transformation

Yasuda

Sample	CompetentCells	Total/(µL)	Medium
10/10 Ligation	BL21(DE3)	CP

@@ Line 1,518: / Line 1,518: @@
 			</table>
 			<img src="https://static.igem.org/mediawiki/2014/6/6f/Kyoto-dms-0825_electrophoresis_No1.jpg">
+			<img src="https://static.igem.org/mediawiki/2014/2/2a/Kyoto-dms-0825_Gel_Extraction_No1_after.jpg">
 </div>
@@ Line 1,549: / Line 1,550: @@
 				</tr>
 			</table>
-			<img src="https://static.igem.org/mediawiki/2014/2/2a/Kyoto-dms-0825_Gel_Extraction_No1_after.jpg">
 			<img src="https://static.igem.org/mediawiki/2014/5/5f/Kyoto-dms-0825_Gel_Extraction_No2_after.jpg">
 			<img src="https://static.igem.org/mediawiki/2014/2/25/Kyoto-dms-0825_Gel_Extraction_No2_before.jpg">
@@ Line 1,566: / Line 1,566: @@
 					<td>1.3</td>
 					<td>8/18 dddD gel extraction product</td>
-					<td>0.81</td>
+					<td>0.71</td>
 					<td>2x Buffer</td>
 					<td>5</td>
@@ Line 1,680: / Line 1,680: @@
 					<td>30sec</td>
 					<td>30sec</td>
-					<td>2h20min</td>
+					<td>140sec</td>
 				</tr>
 			</table>
@@ Line 1,986: / Line 1,986: @@
 				</tr>
 				<tr>
-					<td>pSB1C3 E,S gel extraction product</td>
+					<td>pSB1C3 E,S gel extraction product control</td>
 					<td>4.7</td>
 					<td>-</td>