Notebook

E. coli

January

2015/01/21

Transformation

Sakurai

BBa_K1524100

Added 5 µL of antiBBa_E1010 on BBa_K1524100 to 20 µL of thawed competent cells (DH5α) on ice.
Incubated on ice for 30 min.
Heat-shocked for 30 sec at 42℃.
Spread 300 µL of the culture onto plate with LBA.
Incubate 2ml regent with ampicillin at 37℃ for 20 hrs.

2015/01/22

Colony PCR

Sakurai

BBa_K1524100

Reagent	Volume
Single Colony	-
100UP - EX - F 10 µM	0.8 µL
XhoⅠ - RBS - NcoⅠ 10 µM	0.8 µL
KAPA Taq	10 µL
DW	8.4 µL
Total	20 µL

2 Step Cycle (Tm value ≥ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	35 cycle
Cycle 2	68℃	60 sec	Annealing / Elongation	35 cycle
Finish	68℃	60 sec	Final Elongation
Store	4℃	Hold	Store

Electrophoresis

Sakurai

BBa_K1524100

Gel Concentration	Voltage	Time	Buffer
1%	100 V	30 min	1/2 x TBE

Liquid Culture

Sakurai

BBa_K1524100

Reagent	Volume
Single Colony	-
LB	2 µL

Cultured for 16 hrs.

2015/01/26

Colony PCR

Sakurai

BBa_K1524100

Reagent	Volume
Single Colony	-
100UP - EX - F 10 µM	0.4 µL
XhoⅠ - RBS - NcoⅠ 10 µM	0.4 µL
KAPA Taq	5 µL
DW	4.2 µL
Total	10 µL

2 Step Cycle (Tm value ≥ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	35 cycle
Cycle 2	68℃	60 sec	Annealing / Elongation	35 cycle
Finish	68℃	60 sec	Final Elongation
Store	4℃	Hold	Store

Electrophoresis

Sakurai

BBa_K1524100

Gel Concentration	Voltage	Time	Buffer
1%	100 V	30 min	1/2 x TBE

Colony PCR

Sakurai

BBa_K1524100

Reagent	Volume
Single Colony	-
100UP - EX - F 10 µM	0.4 µL
XhoⅠ - RBS - NcoⅠ 10 µM	0.4 µL
KAPA Taq	5 µL
DW	4.2 µL
Total	10 µL

2 Step Cycle (Tm value ≥ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	35 cycle
Cycle 2	68℃	60 sec	Annealing / Elongation	35 cycle
Finish	68℃	60 sec	Final Elongation
Store	4℃	Hold	Store

Colony PCR

Sakurai

BBa_K1524100

Reagent	Volume
Single Colony	-
100UP - EX - F 10 µM	0.4 µL
200DN - PS - R 10 µM	0.4 µL
KAPA Taq	5 µL
DW	4.2 µL
Total	10 µL

2 Step Cycle (Tm value ≥ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	35 cycle
Cycle 2	68℃	60 sec	Annealing / Elongation	35 cycle
Finish	68℃	60 sec	Final Elongation
Store	4℃	Hold	Store

March

2015/03/10

Competent Cells

Tanaka,Sakurai

BL21 (DE3) pLysS

Thawed original competent cells (BL21 (DE3) pLysS) on ice.
Added 5 µL of original competent cells to 2 mL of LB.
Incubated the cells for 16 hrs at 37℃.
Added 5 µL, 50 µL, and 500 µL of original cells to 100 mL of LB.
Incubated the cells at 130 rpm for 14 hrs at 20℃, until OD₆₀₀ reach 0.5.
Took 50 mL of incubated cells to two differnt culture tubes and centrifuged them at 3,000 rpm for 20 min at 4℃.
Removed supernatant and added 75 mL of TB to each tube.
Brought them to a one tube and centrifuged at 3,000 rpm for 20 min at 4℃.
Removed supernatant and added 32 mL of TB.
Added 32 µL of DMSO 10 times.
Took 50 µL and froze with liquid nitrogen.

2015/03/11

PCR

Sakurai

BBa_R0011

Reagent	Volume
BBa_R0011	1 µL
100UP - EX - F 10 µM	1.5 µL
200DN - PS - R 10 µM	1.5 µL
KOD - Plus - Neo	1 µL
10 x PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	32 µL
Total	50 µL

BBa_0030 - BBa_E1010

Reagent	Volume
BBa_0030 - BBa_E1010	1 µL
100UP - EX - F 10 µM	1.5 µL
200DN - PS - R 10 µM	1.5 µL
KOD - Plus - Neo	1 µL
10 x PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	32 µL
Total	50 µL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	30 cycle
Cycle 2	62.6℃	30 sec	Annealing	30 cycle
Cycle 3	68℃	1 min	Elongation	30 cycle
Store	4℃	Hold	Store

PCR Purification

Sakurai

BBa_R0011, BBa_0030 - BBa_E1010
FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
Purification of PCR products

Digestion

Sakurai

BBa_R0011

Reagent	Volume
BBa_R0011	44 µL
XbaⅠ	1 µL
CutSmart Buffer	5 µL
Total	50 µL

BBa_0030 - BBa_E1010

Reagent	Volume
BBa_0030 - BBa_E1010	44 µL
XbaⅠ	1 µL
CutSmart Buffer	5 µL
Total	50 µL

Digestion

Step	Temp.	Time	Process
1	37℃	300 min	Digestion
2	60℃	15 min	Inactivation
Store	4℃	Hold	Store

Electrophoresis

Sakurai

BBa_R0011, BBa_0030 - BBa_E1010

Gel Concentration	Voltage	Time	Buffer
1%	100 V	30 min	1/2 x TBE

Gel Extract

Sakurai

BBa_R0011, BBa_0030 - BBa_E1010
FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel

Electrophoresis

Sakurai

BBa_R0011, BBa_0030 - BBa_E1010

Gel Concentration	Voltage	Time	Buffer
1%	100 V	30 min	1/2 x TBE

May

2015/05/13

Transformation

Onoda

pET15b

Added 1 µL of pET15b to 50 µL of thawed competent cells (DH5α) on ice.
Incubated on ice for 30 min.
Heat-shocked for 30 sec at 42℃.
Spread 50 µL of the culture onto plate with LBA.
Incubated the plate at 37℃ for 16 hrs.

Transformation

Onoda, Sakurai

pET16b

Added 1 µL of pET16b to 50 µL of thawed competent cells (DH5α) on ice.
Incubated on ice for 30 min.
Heat-shocked for 30 sec at 42℃.
Spread 50 µL of the culture onto plate with LBA.
Incubated the plate at 37℃ for 16 hrs.

Competent Cells

Onoda

Rosetta

Thawed original competent cells (Rosetta) on ice.
Added 5 µL of original competent cells to 2 mL of LB.
Incubated the cells for 16 hrs at 37℃.
Added 5 µL, 50 µL, and 500 µL of original cells to 100 mL of LB.
Incubated the cells at 130 rpm for 24 hrs at 20℃, until OD₆₀₀ reach 0.5.
Took 50 mL of incubated cells to two differnt culture tubes and centrifuged them at 3,000 rpm for 20 min at 4℃.
Removed supernatant and added 75 mL of TB to each tube.
Brought them to a one tube and centrifuged at 3,000 rpm for 20 min at 4℃.
Removed supernatant and added 32 mL of TB.
Added 32 µL of DMSO 10 times.
Took 50 µL and froze with liquid nitrogen.

2015/05/27

Transformation

Mimata, Onoda, Nishimura

BBa_E0040

Added 1 µL of BBa_E0040 to thawed competent cells (Rosetta and DH5α) on ice.
Incubated on ice for 30 min.
Heat-shocked for 30 sec at 42℃.
Added 200 µL of LB.
Spread 300 µL of the culture onto plate with LBA.
Incubated the plate at 37℃ for 16 hrs.

Transformation

Mimata, Onoda, Ono, Nishimura

mBBa_R0040

Added 1 µL of mBBa_R0040 to thawed competent cells (Rosetta and DH5α) on ice.
Incubated on ice for 30 min.
Heat-shocked for 30 sec at 42℃.
Added 200 µL of LB.
Spread 300 µL of the culture onto plate with LBA.
Incubated the plate at 37℃ for 16 hrs.

2015/05/29

Mini-prep

Mimata, Onoda, Ono, Nishimura

BBa_E0040, mBBa_R0040
FastGene^TM Plasmid mini kit (Nippon Genetics Co.,Ltd)
standard protocol

PCR

Onoda, Ono

BBa_E0040

Reagent	Volume
BBa_E0040	1 µL
100UP- EX - F 10 µM	1 µL
200DN - PS - R 10 µM	1 µL
KOD - FX - Neo	1 µL
2 x PCR Buffer for KOD - FX - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

mBBa_R0040

Reagent	Volume
mBBa_R0040	1 µL
100UP - EX - F 10 µM	1 µL
200DN - PS - R 10 µM	1 µL
KOD - FX - Neo	1 µL
2 x PCR Buffer for KOD - FX - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

2 Step Cycle (Tm value ≥ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	30 cycles
Cycle 2	68℃	60 sec	Annealing / Elongation	30 cycles
Store	4℃	Hold	Store

2015/05/30

Electrophoresis

Mimata, Onoda, Ono, Nishimura

BBa_E0040, mBBa_R0040

Gel Concentration	Voltage	Time	Buffer
2%	100 V	30 min	1/2 x TBE

Gel Extract

Onoda, Ono, Nishimura

BBa_E0040, mBBa_R0040
FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel

Digestion

Onoda, Ono, Nishimura

BBa_E0040

Reagent	Volume
BBa_E0040	20 µL
DW	5 µL
SpeⅠ	1 µL
EcoRⅠ	1 µL
CutSmart Buffer	3 µL
Total	30 µL

mBBa_R0040

Reagent	Volume
mBBa_R0040	20 µL
DW	5 µL
SpeⅠ	1 µL
EcoRⅠ	1 µL
CutSmart Buffer	3 µL
Total	30 µL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	60℃	15 min	Inactivation
Store	4℃	Hold	Store

Digestion

Onoda, Ono

pET15b

Reagent	Volume
pET15b	10 µL
DW	6 µL
SpeⅠ	1 µL
EcoRⅠ	1 µL
CutSmart Buffer	2 µL
Total	20 µL

pET16b

Reagent	Volume
pET16b	10 µL
DW	6 µL
SpeⅠ	1 µL
EcoRⅠ	1 µL
CutSmart Buffer	2 µL
Total	20 µL

pSB1A3

Reagent	Volume
pSB1A3	10 µL
DW	6 µL
SpeⅠ	1 µL
EcoRⅠ	1 µL
CutSmart Buffer	2 µL
Total	20 µL

pSB4C5

Reagent	Volume
pSB4C5	2 µL
DW	14 µL
SpeⅠ	1 µL
EcoRⅠ	1 µL
CutSmart Buffer	2 µL
Total	20 µL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	60℃	15 min	Inactivation
Store	4℃	Hold	Store

2015/05/31

Electrophoresis

Mimata, Onoda, Ono, Nishimura

BBa_E0040, BBa_R0040, pET15b, pET16b, pSB1A3, pSB4C5

Gel Concentration	Voltage	Time	Buffer
1%	100 V	30 min	1/2 x TBE

Gel Extract

Mimata, Onoda, Ono, Nishimura

BBa_E0040, BBa_R0040, pET15b, pET16b, pSB1A3
FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel

Electrophoresis

Mimata, Onoda, Ono, Nishimura

BBa_E0040, mBBa_R0040

Gel Concentration	Voltage	Time	Buffer
1%	100 V	30 min	1/2 x TBE

PCR

Mimata, Onoda

BBa_E0040

Reagent	Volume
BBa_E0040	1 µL
100UP - EX - F 10 µM	1 µL
200DN - PS - R 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

mBBa_R0040

Reagent	Volume
mBBa_R0040	1 µL
100UP - EX - F 10 µM	1 µL
200DN - PS - R 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

2 Step Cycle (Tm value ≥ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	30 cycle
Cycle 2	68℃	60 sec	Annealing / Elongation	30 cycle
Store	4℃	Hold	Store

June

2015/06/10

Digestion

Mimata, Onoda

BBa_E0040

Reagent	Volume
BBa_E0040	16 µL
SpeⅠ	1 µL
EcoRⅠ	1 µL
CutSmart Buffer	2 µL
Total	20 µL

mBBa_R0040

Reagent	Volume
mBBa_R0040	16 µL
SpeⅠ	1 µL
EcoRⅠ	1 µL
CutSmart Buffer	2 µL
Total	20 µL

Digestion

Step	Temp.	Time	Process
1	37℃	60 min	Digestion
2	60℃	15 min	Inactivation
Store	4℃	Hold	Store

2015/06/16

PCR

Mimata

thanatin fragment for TA cloning

Reagent	Volume
TA - F - primer	1 µL
TA - R - primer	1 µL
KAPA Taq	25 µL
DW	23 µL
Total	50 µL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	180 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	35 cycle
Cycle 2	63℃	30 sec	Annealing	35 cycle
Cycle 3	72℃	10 sec	Elongation	35 cycle
Finish	72℃	60 sec	Final Elongation
Store	4℃	Hold	Store

2015/06/17

Electrophoresis

Mimata

thanatin fragment for TA cloning

Gel Concentration	Voltage	Time	Buffer
2%	100 V	30 min	1/2 x TBE

Annealing Oligos and Elongation

Ito

thanatin fragment for TA cloning

Reagent	Volume
TA - F - primer 1 µM	1 µL
TA - R - primer 1 µM	1 µL
KAPA Taq	25 µL
DW	23 µL
Total	50 µL

Annealing Oligos and Elongation

Step	Temp.	Time	Process	Cycle
Start	95℃	1 min	Initialization
Cycle1	95℃ to 23℃	45 min	Annealing	1 cycle
Cycle 2	72℃	1 min	Elongation	1 cycle
Store	4℃	Hold	Store

2015/06/19

Electrophoresis

Ito

thanatin fragment for TA cloning

Gel Concentration	Voltage	Time	Buffer
2%	100 V	30min	1/2 x TBE

Gel Extract

Ito

thanatin fragment for TA cloning
FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel

Ligation

Ito

pGEM - T vector / Thanatin fragment for TA cloning

Reagent	Volume
pGEM - T vector	1.7µL
Thanatin fragment for TA cloning	0.15µL
Mighty Mix	1.85µL
T4 Ligase	0.18µL
DW	6.12µL
Total	10µL

Ligation

Step	Temp.	Time	Process
1	16℃	30 min	Ligation
2	65℃	10 min	Inactivation
Store	4℃	Hold	Store

2015/06/21

Transformation

Ito

pGEM - T vector

Added 1 µL of Thanatin fragment to 50 µL of thawed competent cells (Rosseta/DH5α) on ice.
Incubated on ice for 30 min.
Heat-shocked for 30 sec at 42℃.
Spread 300 µL of the culture onto plate with LBA.
Incubated the plate at 37℃ for 19 hrs.

July

2015/07/25

Transformation

Onoda

BBa_B0015 on pSB1C3

Added 1 µL of BBa_B0015 on pSB1C3 to 50 µL of thawed competent cells (DH5α Turbo) on ice.
Incubated on ice for 30 min.
Heat-shocked for 30 sec at 42℃.
Added 500 µL of LB.
Incubated the cells for 2 hrs at 37℃.
Spread 300 µL of the culture onto plate with LBC.
Incubated the plate at 37℃ for 16 hrs.

Transformation

Onoda

BBa_R0010 - BBa_B0034 on pSB1C3

Added 1 µL of BBa_R0010 - BBa_B0034 on pSB1C3 to 50 µL of thawed competent cells (DH5α Turbo) on ice.
Incubated on ice for 30 min.
Heat-shocked for 30 sec at 42℃.
Added 500 µL of LB.
Incubated the cells for 2 hrs at 37℃.
Spread 300 µL of the culture onto plate with LBC.
Incubated the plate at 37℃ for 16 hrs.

Transformation

Onoda

BBa_I0500 - BBa_B0034 on pSB1C3

Added 1 µL of BBa_I0500 - BBa_B0034 on pSB1C3 to 50 µL of thawed competent cells (DH5α Turbo) on ice.
Incubated on ice for 30 min.
Heat-shocked for 30 sec at 42℃.
Added 500 µL of LB.
Incubated the cells for 2 hrs at 37℃.
Spread 300 µL of the culture onto plate with LBC.
Incubated the plate at 37℃ for 16 hrs.

Transformation

Onoda

BBa_B0034 on pSB1A2

Added 1 µL of BBa_B0034 on pSB1A2 to 50 µL of thawed competent cells (DH5α Turbo) on ice.
Incubated on ice for 30 min.
Heat-shocked for 30 sec at 42℃.
Added 500 µL of LB.
Spread 300 µL of the culture onto plate with LBA.
Incubated the plate at 37℃ for 16 hrs.

PCR

Onoda

BBa_B0015 on pSB1C3

Reagent	Volume
BBa_B0015 on pSB1C3	1 µL
100UP - EX - F 10 µM	1 µL
200DN - PS - R 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

BBa_R0010 - BBa_B0034 on pSB1C3

Reagent	Volume
BBa_R0010 - BBa_B0034 on pSB1C3	1 µL
100UP - EX - F 10 µM	1 µL
200DN - PS - R 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

BBa_I0500 - BBa_B0034 on pSB1C3

Reagent	Volume
BBa_I0500 - BBa_B0034 on pSB1C3	1 µL
100UP - EX - F 10 µM	1 µL
200DN - PS - R 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

BBa_B0034 on pSB1A2

Reagent	Volume
BBa_B0034 on pSB1A2	1 µL
100UP - EX - F 10 µM	1 µL
200DN - PS - R 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	30 cycle
Cycle 2	62.6℃	30 sec	Annealing	30 cycle
Cycle 3	68℃	60 sec	Elongation	30 cycle
Store	4℃	Hold	Store

Electrophoresis

Onoda

BBa_B0015 on pSB1C3, BBa_R0010 - BBa_B0034 on pSB1C3, BBa_I0500 - BBa_B0034 on pSB1C3, BBa_B0034 on pSB1A2

Gel Concentration	Voltage	Time	Buffer
2%	100 V	30 min	1/2 x TBE

2015/07/26

Liquid Culture

Ono

BBa_I0500 - BBa_B0034 on pSB1A2

Reagent	Volume
Single Colony	-
LB	2000 µL
Ampicillin	2 µL

Cultured for 15 hours.

Mini-prep

Ito

BBa_R0010 - BBa_B0034 on pSB1C3, BBa_B0015 on pSB1C3, BBa_B0034 on pSB1A2
FastGene^TM Plasmid mini kit (Nippon Genetics Co.,Ltd)
standard protocol

Electrophoresis

Ito

BBa_I0500 - BBa_B0034 on pSB1C3, BBa_R0010 - BBa_B0034 on pSB1C3, BBa_B0034 on pSB1A3, BBa_B0015 on pSB1C3

Gel Concentration	Voltage	Time	Buffer
2%	100 V	30 min	1/2 x TBE

Gel Extract

Ono

BBa_R0010 - BBa_B0034 on pSB1C3, BBa_I0500 - BBa_B0034 on pSB1C3, BBa_B0034 on pSB1A2, BBa_B0015 on pSB1C3
FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel

2015/07/27

Mini-prep

Ono

BBa_I0500 - BBa_B0034 on pSB1A2
FastGene^TM Plasmid mini kit (Nippon Genetics Co.,Ltd)
standard protocol

August

2015/08/04

Transformation

Ito

pGEM T vector

Added 1 µL of pGEM T vector to 50 µL of thawed competent cells (DH5α) on ice.
Incubated on ice for 30 min.
Heat-shocked for 30 sec at 42℃.
Spread 300 µL of the culture onto plate with LBA.
Incubated the plate at 37℃ for 16 hrs.

2015/08/05

Colony PCR

Ito

pGEM T vector

Reagent	Volume
Single Colony	-
NdeⅠ - F - primer 10 µM	0.4 µL
BamHⅠ - R - primer 10 µM	0.4 µL
KAPA Taq	5.0 µL
DW	4.2 µL
Total	10 µL

2 Step Cycle (Tm value ≥ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	30 sec	Denaturation	35 cycle
Cycle 2	68℃	20 sec	Annealing / Elongation	35 cycle
Store	4℃	Hold	Store

Electrophoresis

Ito

NdeⅠ - Thanatin - BamHⅠ

Gel Concentration	Voltage	Time	Buffer
2%	100V	60 min	1/2 x TBE

Gel Extract

Ito

NdeⅠ - Thanatin - BamHⅠ
FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel

Ligation

Ito

pET vector / NdeⅠ - Thanatin - BamHⅠ

Reagent	Volume
pET vector	1 µL
NdeⅠ - Thanatin - BamHⅠ	3 µL
10 × T4 DNA Ligase Buffer	5 µL
T4 Ligase	1 µL
Total	10 µL

Ligation

Step	Temp.	Time	Process
1	4℃	30 min	Ligation
2	65℃	10 min	Inactivation
Store	4℃	Hold	Store

Transformation

Ito

BBa_E1010 - BBa_B0034 - BBa_E0040 - BBa_B0015 on pSB1C3

Added 1 µL of Thanatin on pET vector to 50 µL of thawed competent cells (Rosetta) on ice.
Incubated on ice for 30 min.
Heat-shocked for 30 sec at 42℃.
Spread 300 µL of the culture onto plate with LBA.
Incubated the plate at 37℃ for 16 hrs.

2015/08/10

Streaking (Single Colony Isolation)

Ito, Mimata, Mitsumoto, Onoda, Sakai

BBa_I0500 - BBa_B0034 - BBa_K759012 (signal and β-domain) - BBa_B0015 on pSB1C3, BBa_E1010 - BBa_B0034 - BBa_R0040 - BBa_B0015 on pSB1C3, BBa_E1010 - BBa_B0034 - BBa_E0400 - BBa_B0015 on pSB1C3

Picked the colony with an inoculating loop from the agar plate．
Draged the loop across on a new agar plate.
Re-sterilised the loop and drag it across again.

2015/08/11

Mini-prep

Ito, Mimata, Mitsumoto, Onoda, Sakai

BBa_I0500 - BBa_B0034 - BBa_K759012 (Signal - β domain) - BBa_B0015 on pSB1C3
FastGene^TM Plasmid mini kit (Nippon Genetics Co.,Ltd)
Fast protocol

Digestion

Ito, Mimata, Onoda, Sakai, Kusumi

BBa_I0500 - BBa_B0034 - BBa_K759012 (Signal - β domain) - BBa_B0015 on pSB1C3

Reagent	Volume
pSB1C3	20 µL
DW	23 µL
Bgl Ⅱ	2 µL
3.1 Buffer	5 µL
Total	50 µL

Digestion

Step	Temp.	Time	Process
1	37℃	60 min	Digestion
2	70℃	15 min	Inactivation
Store	4℃	Hold	Store

Electrophoresis

Ito, Mimata, Onoda, Sakai, Nishimura, Kusumi

BBa_I0500 - BBa_B0034 - BBa_K759012 (signal and β-domain) - BBa_B0015 on pSB1C3

Gel Concentration	Voltage	Time	Buffer
1%	100 V	60 min	1/2 x TBE

2015/08/12

Gel Extract

Nishimura, Sakai

BBa_I0500 - BBa_B0034 - BBa_K759012 (Signal - β domain) - BBa_B0015 on pSB1C3
FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel

Electrophoresis

Ito, Sakai, Fujita

BBa_I0500 - BBa_B0034 - BBa_K759012 (Signal - β domain) - BBa_B0015 on pSB1C3 (Gel Extract Poduct)

Gel Concentration	Voltage	Time	Buffer
1%	100 V	60 min	1/2 x TBE

2015/08/13

Sequencing

Ito, Onoda, Nishimura, Fujita

BBa_I0500 - BBa_B0034 - BBa_K759012 (signal and β-domain) - BBa_B0015 on pSB1C3 (Gel Extract Product)

Reagent	Volume
pSB1C3	1 µL
T7 promoter primer / SP6 promoter primer	1.5 µL
Ready Reaction Premix	1 µL
5 x Sequencing Buffer	1.5 µL
DW	5 µL
Total	10 µL

Sequencing

Step	Temp.	Time	Process	Cycle
Start	96℃	10 sec	Denaturation
Cycle 1	50℃	5 sec	-	25 cycle
Cycle 2	60℃	240 sec	-	25 cycle
Store	4℃	Hold	Store

Ethanol Precipitation

Ito, Onoda, Nishimura, Fujita, Mimata

BBa_I0500 - BBa_B0034 - BBa_K759012 (signal and β-domain) - BBa_B0015 on pSB1C3 (Sequencing PCR product)

Added 2 µL of NaOAc, 1.5 µL of glycogen and 50 µL of 100% ethanol.
Left it at -80℃ for 1 hr.
Centrifuged at 15,000 rpm for 15 min at 4℃.
Removed supernatant and added 100 µL of 70% ethanol.
Centrifuged at 15,000 rpm for 10 min at 4℃.
Removed supernatant and air-dried at room temperature with light shield.
Suspended with 10 µL of DW.

PCR

Ito, Onoda, Tanaka, Nishimura, Mimata

XbaⅠ - BBa_B0034 - XbaⅠ / SpeⅠ scar - Met - 10 × His tag - Thanatin - SpeⅠ

Reagent	Volume
XbaⅠ - BBa_B0034 - XbaⅠ / SpeⅠ scar - Met - 10 × His tag - Thanatin - SpeⅠ	1 µL
BamHⅠ - Thanatin forward Neo 10 µM	1 µL
BglⅡ - Asp - Thanatin reverese Neo 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

2 Step Cycle (Tm value ≥ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	25 cycle
Cycle 2	68℃	20 sec	Annealing / Elongation	25 cycle
Store	4℃	Hold	Store

Digestion

Mimata

NdeⅠ - Thanatin - BamHⅠ on pET vector

Reagent	Volume
NdeⅠ - Thanatin - BamHⅠ on pET vector	10 µL
NdeⅠ	1 µL
BamHⅠ	1 µL
CutSmart Buffer	5 µL
DW	33 µL
Total	50 µL

Digestion

Step	Temp.	Time	Process
1	37℃	60 min	Digestion
2	70℃	15 min	Inactivation
Store	4℃	Hold	Store

2015/08/14

PCR

Fujita, Nishimura, Onoda

Thanatin (Mini-prep product)

Reagent	Volume
Thanatin fragment	1 µL
T7 - promoter primer 10 µM	1 µL
SP6 - promoter primer 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 × PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

3 Step Cycle (Tm value ≤ 66℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	94℃	10 sec	Denaturation	25 cycle
Cycle 2	50℃	10 sec	Annealing	25 cycle
Cycle 3	68℃	30 sec	Elongation	25 cycle
Store	4℃	Hold	Store

Electrophoresis

Fujita, Nishimura, Onoda, Mimata

BamHⅠ - Thanatin - BglⅡ, Thanatin fragment(PCR product)

Gel Concentration	Voltage	Time	Buffer
2%	100 V	60 min	1/2 x TBE

Annealing of Oligonucleotides

Onoda, Nishimura, Fujita

Thanatin fragment (derived from annealing TA primers)

Reagent	Volume
TA - F - primer 10 µM	1 µL
TA - R - primer 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	34 µL
Total	50 µL

Annealing of Oligonucleotides

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	94℃	10 sec	Denaturation	10 cycle
Cycle 2	60℃	10 sec	Annealing	10 cycle
Cycle 3	68℃	30 sec	Elongation	10 cycle
Store	4℃	Hold	Store

PCR

Nishimura, Onoda

Thanatin fragment (derived from annealing TA primers)

Reagent	Volume
Thanatin fragment (derived from annealing TA primers)	1 µL
BamHⅠ - Thanatin Neo 10 µM	1 µL
BglⅡ - Asp - thanatin Neo 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

2 Step Cycle (Tm value ≥ 66℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	35 cycle
Cycle 2	68℃	60 sec	Annealing / Elongation	35 cycle
Store	4℃	Hold	Store

PCR Purification

Nishimura, Onoda

Thanatin fragment from last 2 step PCR
FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
Purification of PCR products

Electrophoresis

Nishimura, Onoda

Thanatin frament (TA-primer), Thanatin fragment (BamHⅠ/BglⅡ), Thanatin fragment(PCR Purification)

Gel Concentration	Voltage	Time	Buffer
2%	100 V	60 min	1/2 x TBE

Transformation

Fujita, Mitsumoto

BBa_B0031 on pSB1A2, BBa_E0040 on pSB1A2

Added 1 µL of BBa_B0031 on pSB1A2, BBa_E0040 on pSB1A2 to 20 µL of thawed competent cells (DH5α) on ice.
Incubated on ice for 30 min.
Heat-shocked for 30 sec at 42℃.
Spread 300 µL of the culture onto plate with LBA.
Incubated the plate at 37℃ for 12 hours.

Transformation

Fujita, Mitsumoto

BBa_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0030

Added 1 µL of BBa_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0030 to 20 µL of thawed competent cells (DH5α) on ice.
Incubated on ice for 30 min.
Heat-shocked for 30 sec at 42℃.
Added 200 µL of LB.
Incubated the cells for 2 hrs at 37℃.
Spread 300 µL of the culture onto plate with LBC.
Incubated the plate at 37℃ for 12 hours.

PCR

Fujita, Mitsumoto

BBa_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0031, BBa_B0030, BBa_E0040

Reagent	Volume
Ba_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0031, BBa_B0030, BBa_E0040	1 µL
100UP - EX - F 10 µM	1 µL
200DN - PS - R 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 × PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

2 Step Cycle (Tm value ≥ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	35 cycle
Cycle 2	68℃	60 sec	Annealing / Elongation	35 cycle
Store	4℃	Hold	Store

2015/08/15

Electrophoresis

Fujita, Nishimura

BBa_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0031, BBa_B0030, BBa_E0040

Gel Concentration	Voltage	Time	Buffer
2%	100 V	60 min	1/2 x TBE

PCR

Fujita, Nishimura, Ono

BBa_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0031, BBa_B0030, BBa_E0040

Reagent	Volume
BBa_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0031, BBa_B0030, BBa_E0040	1 µL
100UP - EX - F 1 µM	1 µL
200DN - PS - R 1 µM	1 µL
KOD - Plus - Neo	1 µL
10 × PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

2 Step Cycle (Tm value ≥ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	35 cycle
Cycle 2	68℃	60 sec	Annealing / Elongation	35 cycle
Store	4℃	Hold	Store

Electrophoresis

Fujita, Nishimura

BBa_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0031, BBa_B0030, BBa_E0040

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

PCR

Nishimura, Ono

Thanatin fragment (derived from annealing TA primers)

Reagent	Volume
Thanatin fragment (derived from annealing TA primers)	1 µL
NdeI - F - primer 10 µM	1 µL
BamHI - R - primer 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 × PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

2 Step Cycle (Tm value ≥ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	25 cycle
Cycle 2	68℃	30 sec	Annealing / Elongation	25 cycle
Store	4℃	Hold	Store

PCR

Sakai, Ono

Thanatin fragment (PCR 2STEP product)

Reagent	Volume
Thanatin fragment (PCR 2STEP product)	1 µL
BamHI - Thanatin - F - Neo 10 µM	1 µL
BglⅡ - Tanatin - R - Neo 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

2 Step Cycle (Tm value ≥ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	25 cycle
Cycle 2	68℃	30 sec	Annealing / Elongation	25 cycle
Store	4℃	Hold	Store

Annealing Oligos and Elongation

Onoda

Thanatin fragment (derived from annealing TA primers)

Reagent	Volume
TA - F - primer 10 µM	1 µL
TA - R - primer 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	34 µL
Total	50 µL

Annealing Oligos and Elongation

Step	Temp.	Time	Process	Cycle
Start	95℃	1 min	Initialization
Cycle1	95℃ to 23℃	45 min	Annealing	1 cycle
Cycle 2	68℃	30 sec	Elongation	1 cycle
Store	4℃	Hold	Store

Electrophoresis

Ono, Onoda

Thanatin fragment (Annealing and Elongation product), Thanatin fragment(PCR 2STEP product)

Gel Concentration	Voltage	Time	Buffer
2%	100 V	30 min	1/2 x TBE

Gel Extract

Ono

BBa_B0031, BBa_B0030, BBa_E0040
FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel

Mini-prep

Ono

BBa_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0031, BBa_B0030, BBa_E0040
FastGene^TM Plasmid mini kit (Nippon Genetics Co.,Ltd)
Standard protocol

2015/08/16

PCR

Nishimura, Ono

Thanatin fragment (derived from annealing TA primers)

Reagent	Volume
Thanatin fragment (derived from annealing TA primers)	1 µL
NdeI - F - primer 10 µM	1 µL
BamHI - R - primer 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

2 Step Cycle (Tm value ≥ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	25 cycle
Cycle 2	68℃	60 sec	Annealing / Elongation	25 cycle
Store	4℃	Hold	Store

Electrophoresis

Nishimura, Ono

Thanatin fragment (PCR product)

Gel Concentration	Voltage	Time	Buffer
2%	100 V	60 min	1/2 x TBE

Colony PCR

Onoda, Ono, Nishimura

Thanatin fragment (derived from annealing TA primers) into DH5α, nothing (as a negative control)

Reagent	Volume
Single Colony	-
NdeI - F - primer 10 µM	0.4 µL
BamHI - R - primer 10 µM	0.4 µL
KAPA Taq	5 µL
DW	4.2 µL
Total	10 µL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	40 cycle
Cycle 2	62.9℃	30 sec	Annealing	40 cycle
Cycle 3	72℃	40 sec	Elongation	40 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Colony PCR

Onoda, Ono, Nishimura

BBa_B0031 on pSB1A2 into DH5α (as positive control)

Reagent	Volume
Single Colony	-
100UP - EX - F 10 µM	0.4 µL
200DN - PS - R 10 µM	0.4 µL
KAPA Taq	5 µL
DW	4.2 µL
Total	10 µL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	30 cycle
Cycle 2	57.2℃	30 sec	Annealing	30 cycle
Cycle 3	72℃	30 sec	Elongation	30 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Electrophoresis

Onoda, Ono

Thanatin fragment derived from annealing TA primer (colony PCR product)

Gel Concentration	Voltage	Time	Buffer
2%	100 V	60 min	1/2 x TBE

Electrophoresis

Ono, Mitsumoto, Fujita

BamHI - Thanatin - BglⅡ, NdeI - Thanatin - BamHI

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

PCR

Onoda

Thanatin fragment (Mini-prep product)

Reagent	Volume
Thanatin fragment (Mini-prep product)	1 µL
BamHI - Thanatin - F - Neo 10 µM	1 µL
BglⅡ - Asp - Thanatin - R - Neo 10 µM	1 µL
KOD - Plus - Neo	1 µL
10　× PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	45 cycle
Cycle 2	65.1℃	30 sec	Annealing	45 cycle
Cycle 3	68℃	30 sec	Elongation	45 cycle
Store	4℃	Hold	Store

PCR

Onoda

Thanatin fragment (Mini-prep product)

Reagent	Volume
Thanatin fragment (Mini-prep product)	1 µL
NdeI - F - primer 10 µM	1 µL
BamHI - R - primer 10 µM	1 µL
KOD - Plus - Neo	1 µL
10　× PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	45 cycle
Cycle 2	66.5℃	30 sec	Annealing	45 cycle
Cycle 3	68℃	30 sec	Elongation	45 cycle
Store	4℃	Hold	Store

Electrophoresis

Onoda

Thanatin fragment for TA cloning and last PCR prduct

Gel Concentration	Voltage	Time	Buffer
2%	100 V	60 min	1/2 x TBE

PCR

Fujita, Mimata

Ba_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0031

Reagent	Volume
Ba_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0031	1 µL
100UP - EX - F 10 µM	1 µL
200DN - PS - R 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

2 Step Cycle (Tm value ≥ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	35 cycle
Cycle 2	68℃	60 sec	Annealing / Elongation	35 cycle
Store	4℃	Hold	Store

Electrophoresis

Fujita, Mimata

Ba_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0031

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

Electrophoresis

Mitsumoto, Fujita

BamHI - Thanatin - BglⅡ, NdeI - Thanatin - BamHI

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

Colony PCR

Ono, Onoda

Thanatin fragment on pGEM - T vector into DH5α

Reagent	Volume
Single Colony	-
NdeI - F - primer 10 µM	0.4 µL
BamHI - R - primer 10 µM	0.4 µL
KAPA Taq	5 µL
DW	4.2 µL
Total	10 µL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	40 cycle
Cycle 2	62.9℃	30 sec	Annealing	40 cycle
Cycle 3	72℃	40 sec	Elongation	40 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Colony PCR

Ono, Onoda

Thanatin fragment on pGEM - T vector into DH5α

Reagent	Volume
Single Colony	-
T7 promoter primer 10 µM	0.4 µL
SP6 promoter primer 10 µM	0.4 µL
KAPA Taq	5 µL
DW	4.2 µL
Total	10 µL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	40 cycle
Cycle 2	51℃	30 sec	Annealing	40 cycle
Cycle 3	72℃	40 sec	Elongation	40 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Colony PCR

Ono, Onoda

BBa_B0031 on pSB1A2 into DH5α (as positive control)

Reagent	Volume
Single Colony	-
100UP - EX - F 10 µM	0.4 µL
200DN - PS - R 10 µM	0.4 µL
KAPA Taq	5 µL
DW	4.2 µL
Total	10 µL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	40 cycle
Cycle 2	57℃	30 sec	Annealing	40 cycle
Cycle 3	72℃	40 sec	Elongation	40 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Electrophoresis

Onoda

Thanatin fragment (Colony PCR product)

Gel Concentration	Voltage	Time	Buffer
1%	100 V	60 min	1/2 x TBE

Annealing Oligos and Elongation

Mitsumoto

Thanatin fragment (derived from annealing TA primers)

Reagent	Volume
TA - F - primer 10 µM	1 µL
TA - R - primer 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	34 µL
Total	50 µL

Annealing Oligos and Elongation

Step	Temp.	Time	Process	Cycle
Start	95℃	1 min	Initialization
Cycle1	95℃ to 23℃	60 sec	Annealing	45 cycle
Cycle 2	68℃	30 sec	Elongation	45 cycle
Store	4℃	Hold	Store

Annealing Oligos and Elongation

Mitsumoto

Thanatin fragment (derived from annealing TA primers)

Reagent	Volume
TA - F - primer 10 µM	1 µL
TA - R - primer 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	34 µL
Total	50 µL

Annealing Oligos and Elongation

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	94℃	10 sec	Denaturation	10 cycle
Cycle 2	60℃	10 sec	Annealing	10 cycle
Cycle 3	68℃	30 sec	Elongation	10 cycle
Store	4℃	Hold	Store

Annealing Oligos and Elongation

Mitsumoto

Thanatin fragment (derived from annealing TA primers)

Reagent	Volume
TA - F - primer 10 µM	1 µL
TA - R - primer 10 µM	1 µL
KOD - FX - Neo	1 µL
10 × PCR Buffer for KOD - FX - Neo	25 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	14 µL
Total	50 µL

Annealing Oligos and Elongation

Step	Temp.	Time	Process	Cycle
Start	95℃	1 min	Initialization
Cycle1	95℃ to 23℃	60 sec	Annealing	45 cycle
Cycle 2	68℃	30 sec	Elongation	45 cycle
Store	4℃	Hold	Store

Annealing Oligos and Elongation

Mitsumoto

Thanatin fragment (derived from annealing TA primers)

Reagent	Volume
TA - F - primer 10 µM	1 µL
TA - R - primer 10 µM	1 µL
KOD - FX - NEO	1 µL
2 × PCR Buffer for KOD - FX - Neo	25 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	14 µL
Total	50 µL

Annealing Oligos and Elongation

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	94℃	10 sec	Denaturation	10 cycle
Cycle 2	60℃	10 sec	Annealing	10 cycle
Cycle 3	68℃	30 sec	Elongation	10 cycle
Store	4℃	Hold	Store

Annealing Oligos and Elongation

Mitsumoto

Thanatin fragment (derived from annealing TA primers)

Reagent	Volume
Single Colony	-
100UP - EX - F 10 µM	1 µL
200DN - PS - R 10 µM	1 µL
KAPA Taq	25 µL
DW	23 µL
Total	50 µL

Annealing Oligos and Elongation

Step	Temp.	Time	Process	Cycle
Start	95℃	1 min	Initialization
Cycle 1	95℃ to 23℃	60 sec	Annealing	45 cycle
Cycle 2	68℃	30 sec	Elongation	45 cycle
Store	4℃	Hold	Store

Annealing Oligos and Elongation

Mitsumoto

Thanatin fragment (derived from annealing TA primers)

Reagent	Volume
Single Colony	-
100UP - EX - F 10 µM	1 µL
200DN - PS - R 10 µM	1 µL
KAPA Taq	25 µL
DW	23 µL
Total	50 µL

Annealing Oligos and Elongation

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	94℃	10 sec	Denaturation	10 cycle
Cycle 2	60℃	10 sec	Annealing	10 cycle
Cycle 3	68℃	30 sec	Elongation	10 cycle
Store	4℃	Hold	Store

Electrophoresis

Mitsumoto

Thanatin fragment (derived from annealing TA primers)

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

2015/08/17

Annealing Oligos and Elongation

Nishimura, Onoda

Thanatin fragment (Mini-prep product)

Reagent	Volume
TA - F - primer 10 µM	1 µL
TA - R - primer 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	34 µL
Total	50 µL

Annealing Oligos and Elongation

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	94℃	10 sec	Denaturation	10 cycle
Cycle 2	60℃	10 sec	Annealing	10 cycle
Cycle 3	68℃	30 sec	Elongation	10 cycle
Store	4℃	Hold	Store

Annealing Oligos and Elongation

Nishimura, Onoda

Thanatin fragment (Mini-prep product)

Reagent	Volume
TA - F - primer 10 µM	1 µL
TA - R - primer 10 µM	1 µL
KAPA Taq	25 µL
DW	23 µL
Total	50 µL

(Tm value ≤ -℃)

Annealing Oligos and Elongation

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	94℃	10 sec	Denaturation	10 cycle
Cycle 2	60℃	10 sec	Annealing	10 cycle
Cycle 3	72℃	30 sec	Elongation	10 cycle
Store	4℃	Hold	Store

PCR

Nishimura, Ono, Onoda, Mimata

NdeⅠ - Thanatin - BamHⅠ

Reagent	Volume
NdeⅠ - Thanatin - BamHⅠ	1 µL
NdeⅠ - F - primer 10 µM	1 µL
BamHⅠ - R - primer 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

2 Step Cycle (Tm value ≥ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	25 cycle
Cycle 2	68℃	30 sec	Annealing / Elongation	25 cycle
Store	4℃	Hold	Store

PCR

Nishimura, Ono, Onoda, Mimata

BamHI - Thanatin - BglⅡ

Reagent	Volume
BamHI - Thanatin - BglⅡ	1 µL
BamHI - Asp - Thanatin - R - Neo 10 µM	1 µL
BglⅡ - D - Tanatin - R - Neo 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

2 Step Cycle (Tm value ≥ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	25 cycle
Cycle 2	68℃	30 sec	Annealing / Elongation	25 cycle
Store	4℃	Hold	Store

PCR Purification

Ono, MImata, Nishimura

Thanatin fragment (PCR 2STEP product)
FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
Purification of PCR products

Electrophoresis

Nishimura, Ono

Thanatin fragment (PCR 2STEP product)

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

PCR

Ito, Ono, Onoda

Thanatin fragment (derived from annealing TA cloning)

Reagent	Volume
Thanatin fragment (derived from annealing TA cloning)	1 µL
NdeⅠ - F - primer 10 µM	1 µL
BamHⅠ - R - primer 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	45 cycle
Cycle 2	65.1℃	30 sec	Annealing	45 cycle
Cycle 3	68℃	30 sec	Elongation	45 cycle
Store	4℃	Hold	Store

PCR

Ito, Ono, Onoda

BamHⅠ - Thanatin - BglⅡ

Reagent	Volume
Thanatin fragment (derived from annealing TA cloning)	1 µL
BamHⅠ - Asp - Thanatin - R - Neo 10 µM	1 µL
BglⅡ - D - Tanatin - R - Neo 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	45 cycle
Cycle 2	66.5℃	30 sec	Annealing	45 cycle
Cycle 3	68℃	30 sec	Elongation	45 cycle
Store	4℃	Hold	Store

Digestion

Ono, Onoda

NdeⅠ - Thanatin - BamHⅠ

Reagent	Volume
NdeⅠ - Thanatin - BamHⅠ	20 µL
NdeⅠ	1 µL
BamHⅠ	1 µL
10 × K Buffer	2 µL
DW	5 µL
Total	30 µL

Digestion

Step	Temp.	Time	Process
1	37℃	60 min	Digestion
2	70℃	15 min	Inactivation
Store	4℃	Hold	Store

Digestion

Ono, Onoda

BamHⅠ - Thanatin - BglⅡ

Reagent	Volume
BamHⅠ - Thanatin - BglⅡ	20 µL
BamHⅠ	1 µL
BglⅡ	1 µL
10 × K Buffer	2 µL
DW	5 µL
Total	30 µL

Digestion

Step	Temp.	Time	Process
1	37℃	60 min	Digestion
2	70℃	15 min	Inactivation
Store	4℃	Hold	Store

2015/08/18

Electrophoresis

Nishimura, Ono

Thanatin fragment (PCR 2STEP product)

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

PCR Purification

Ono, Mimata, Nishimura

Thanatin fragment (PCR 2STEP product)
FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
Purification of PCR products

Annealing Oligos and Elongation

Mimata

Thanatin fragment (derived from annealing TA cloning)

Reagent	Volume
TA - F - primer 10 µM	5 µL
TA - R - primer 10 µM	5 µL
TE 0.8 M NaCl	10 µL
Total	50 µL

Annealing Oligos and Elongation

1

Step	Temp.	Time	Process
Start	94℃	2 min	Initialization
Step1	95℃ to 25℃	20 min	Annealing
Store	4℃	Hold	Store

PCR

Mimata

Thanatin fragment (derived from annealing)

Reagent	Volume
Thanatin fragment (derived from annealing)	1 µL
NdeⅠ - F - primer 10 µM	1 µL
BamHⅠ - R - primer 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

2 Step Cycle (Tm value ≥ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	25 cycle
Cycle 2	68℃	30 sec	Annealing / Elongation	25 cycle
Store	4℃	Hold	Store

PCR

Mimata

Thanatin fragment (derived from annealing)

Reagent	Volume
Thanatin fragment (derived from annealing)	1 µL
BamHⅠ - Asp - Thanatin - R - Neo 10 µM	1 µL
BglⅡ - D - Tanatin - R - Neo 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

2 Step Cycle (Tm value ≥ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	25 cycle
Cycle 2	68℃	30 sec	Annealing / Elongation	25 cycle
Store	4℃	Hold	Store

PCR Purification

Ono, Mimata, Nishimura

Thanatin fragment (PCR 2STEP product)
FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
Purification of PCR products

Digestion

Ono, Onoda, Nishimura

NdeⅠ - Thanatin - BamHⅠ

Reagent	Volume
NdeⅠ - Thanatin - BamHⅠ	20 µL
NdeⅠ	1 µL
BamHⅠ	1 µL
10 × K Buffer	2 µL
DW	5 µL
Total	30 µL

Digestion

Step	Temp.	Time	Process
1	37℃	60 min	Digestion
2	70℃	15 min	Inactivation
Store	4℃	Hold	Store

Digestion

Ono, Onoda, Nishimura

BamHⅠ - Thanatin - BglⅡ

Reagent	Volume
BamHⅠ - Thanatin - BglⅡ	20 µL
BamHⅠ	1 µL
BglⅡ	1 µL
10 × K Buffer	2 µL
DW	5 µL
Total	30 µL

Digestion

Step	Temp.	Time	Process
1	37℃	60 min	Digestion
2	70℃	15 min	Inactivation
Store	4℃	Hold	Store

Ligation

Onoda, Mimata

Thanatin fragment on pGEM T - vector / Thanatin fragment (derived from annealing TA cloning)

Reagent	Volume
pGEM T - vector	1 µL
Thanatin fragment	3 µL
2 × Ligation Buffer	5 µL
T4 Ligase	1 µL
Total	10 µL

Ligation

Step	Temp.	Time	Process
1	4℃	6 hour	Ligation
2	65℃	10 min	Inactivation
Store	4℃	Hold	Store

2015/08/19

PCR

Ono

Thanatin fragment (derived from annealing)

Reagent	Volume
Thanatin fragment (derived from annealing)	1 µL
NdeⅠ - F - primer 10 µM	1 µL
BamHⅠ - R - primer 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	94℃	10 sec	Denaturation	40 cycle
Cycle 2	60℃	30 sec	Annealing	40 cycle
Cycle 3	68℃	30 sec	Elongation	40 cycle
Store	4℃	Hold	Store

PCR

Ono

Thanatin fragment (derived from annealing)

Reagent	Volume
Thanatin fragment (derived from annealing)	1 µL
BamHⅠ - Thanatin - F 10 µM	1 µL
BglⅡ - Tanatin - R 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	94℃	10 sec	Denaturation	40 cycle
Cycle 2	60℃	30 sec	Annealing	40 cycle
Cycle 3	68℃	30 sec	Elongation	40 cycle
Store	4℃	Hold	Store

Ethanol Precipitation

Mimata, Ono

NdeⅠ - Thanatin - BamHⅠ (PCR 3STEP product), BamHⅠ - Thanatin - BglⅡ (PCR 3STEP product)

Added 2 µL of NaOAc, 1 µL of glycogen and 50 µL of 100% ethanol.
Left it at -80℃ for 30 min.
Centrifuged at 15,000 rpm for 15 min at 4℃.
Removed supernatant and added 100 µL of 70% ethanol.
Centrifuged at 15,000 rpm for 10 min at 4℃.
Removed supernatant and air-dried at room temperature with light shield.
Suspended with 10 µL of TE.

Electrophoresis

Mimata

NdeⅠ - Thanatin - BamHⅠ, BamHⅠ - Thanatin - BglⅡ

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

PCR

Ono

BamHⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - BglⅡ

Reagent	Volume
BamHⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - BglⅡ	1 µL
BamHⅠ - Thanatin - F 10 µM	1 µL
BglⅡ - Thanatin - R 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

2 Step Cycle (Tm value ≥ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	40 cycle
Cycle 2	68℃	30 sec	Annealing / Elongation	40 cycle
Store	4℃	Hold	Store

PCR

Ono

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ

Reagent	Volume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ	1 µL
XbaⅠ - Thanatin - F 10 µM	1 µL
NdeⅠ - Thanatin - R 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

2 Step Cycle (Tm value ≥ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	40 cycle
Cycle 2	68℃	30 sec	Annealing / Elongation	40 cycle
Store	4℃	Hold	Store

Ethanol Precipitation

Ono

NdeⅠ - Thanatin - BamHⅠ (PCR 2STEP product), BamHⅠ - Thanatin - BglⅡ (PCR 2STEP product)

Added 2 µL of NaOAc, 1 µL of glycogen, 7 µL of DW and 50 µL of 100% ethanol.
Left it at room temperature for 15 min.
Centrifuged at 15,000 rpm for 15 min at 4℃.
Removed supernatant and added 100 µL of 70% ethanol.
Centrifuged at 15,000 rpm for 10 min at 4℃.
Removed supernatant and air-dried at room temperature with light shield.
Suspended with 10 µL of DW.

Digestion

Ono, Onoda, Mimata, Sakai

NdeⅠ - Thanatin - BamHⅠ

Reagent	Volume
NdeⅠ - Thanatin - BamHⅠ	20 µL
NdeⅠ	1 µL
BamHⅠ	1 µL
10 × K Buffer	3 µL
DW	5 µL
Total	30 µL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	60℃	15 min	Inactivation
Store	4℃	Hold	Store

Digestion

Ono, Onoda, Mimata, Sakai

BamHⅠ - Thanatin - BglⅡ

Reagent	Volume
BamHⅠ - Thanatin - BglⅡ	20 µL
BamHⅠ	1 µL
BglⅡ	1 µL
10 × K Buffer	3 µL
DW	5 µL
Total	30 µL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	60℃	15 min	Inactivation
Store	4℃	Hold	Store

Digestion

Ono, Onoda, Mimata, Sakai

pET - 15b vector, pET - 16b vector

Reagent	Volume
pET - 15b vector, pET - 16b vector	20 µL
NdeⅠ	1 µL
BamHⅠ	1 µL
10 × K Buffer	3 µL
DW	5 µL
Total	30 µL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	60℃	15 min	Inactivation
Store	4℃	Hold	Store

Transformation

Onoda

Thanatin fragment on pGEM - T vector

Added 1 µL of Thanatin fragment on pGEM - T vector to 50 µL of thawed competent cells (DH5α) on ice.
Incubated on ice for 30 min.
Heat-shocked for 30 sec at 42℃.
Spread 300 µL of the culture onto plate with LBA.
Incubated the plate at 37℃ for 18 hours.

2015/08/20

Electrophoresis

Ono, Nishimura, Mimata

NdeⅠ - Thanatin - BamHⅠ (digestion product), BamHⅠ - Thanatin - BglⅡ digestion product), BamHⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - BglⅡ (PCR 2STEP product), XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ (PCR 2STEP product)

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

Electrophoresis

Ono, Nishimura, Mimata

pET - 15b vector (digestion product), pET - 16b vector (digestion product)

Gel Concentration	Voltage	Time	Buffer
1%	100 V	60 min	1/2 x TBE

Gel Extract

Nishimura, Ono

pET - 15b vector, pET - 16b vector
FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel

Digestion

Ono, Nishimura, Ito

BamHⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - BglⅡ(PCR 2STEP poduct)

Reagent	Volume
BamHⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - BglⅡ (PCR 2STEP poduct)	9 µL
BamHⅠ	5 µL
BglⅡ	5 µL
10 × K Buffer	10 µL
DW	71 µL
Total	100 µL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	70℃	15 min	Inactivation
Store	4℃	Hold	Store

Digestion

Ono, Nisimura, Ito

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ (PCR 2STEP poduct)

Reagent	Volume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ (PCR 2STEP poduct)	9 µL
XbaⅠ	5 µL
SpeⅠ	5 µL
10 × K Buffer	10 µL
DW	71 µL
Total	100 µL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	80℃	20 min	Inactivation
Store	4℃	Hold	Store

Digestion

Ono, Nishimura, Ito

BBa_B0015 on pSB1C3

Reagent	Volume
BBa_B0015 on pSB1C3	20 µL
SpeⅠ	1 µL
CutSmart Buffer	3 µL
DW	6 µL
Total	30 µL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	80℃	20 min	Inactivation
Store	4℃	Hold	Store

Ethanol Precipitation

Ito

BamHⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - BglⅡ (digestion product), XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ (digestion product)

Added 9 µL of NaOAc, 1.5 µL of glycogen and 270 µL of 100% ethanol.
Left it at -80℃ for 1 hr.
Centrifuged at 15,000 rpm for 15 min at 4℃.
Removed supernatant and added 220 µL of 70% ethanol.
Centrifuged at 15,000 rpm for 10 min at 4℃.
Removed supernatant and air-dried at room temperature with light shield.
Suspended with 10 µL of TE.

Ito

Electrophoresis

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ (digestion product, Ethanol Precipitation product), BamHⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - BglⅡ (digestion product, Ethanol Precipitation product)

Gel Concentration	Voltage	Time	Buffer
1%	100 V	60 min	1/2 x TBE

2015/08/21

PCR

Ono, Nishimura, Onoda

BamHⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - BglⅡ

Reagent	Volume
BamHⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - BglⅡ	1 µL
BamHⅠ - Thanatin - F 10 µM	1 µL
BglⅡ - Thanatin - R 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

2 Step Cycle (Tm value ≥ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	40 cycle
Cycle 2	68℃	30 sec	Annealing / Elongation	40 cycle
Store	4℃	Hold	Store

PCR

Ono, Nishimura, Onoda

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ

Reagent	Volume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ	1 µL
XbaⅠ - Thanatin - F 10 µM	1 µL
SpeⅠ - Thanatin - R 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

2 Step Cycle (Tm value ≥ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	40 cycle
Cycle 2	68℃	30 sec	Annealing / Elongation	40 cycle
Store	4℃	Hold	Store

Electrophoresis

Ono, Onoda, Nishimura

XbaⅠ - Thanatin - SpeⅠ, BamHⅠ- Thanatin - BglⅡ

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

Ethanol Precipitation

Ono, Nishimura

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ, BamHⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - BglⅡ

Added 3 µL of NaOAc, 1 µL of glycogen and 90 µL of 100% ethanol.
Left it at -80℃ for 10 min.
Centrifuged at 15,000 rpm for 5 min at 4℃.
Removed supernatant and added 100 µL of 70% ethanol.
Centrifuged at 15,000 rpm for 5 min at 4℃.
Removed supernatant and air-dried at room temperature.
Suspended with 10 µL of TE.

Electrophoresis

Ono, Nishimura

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ (after Ethanol Prescipitation) BamHⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - BglⅡ (after Ethanol Precipitation)

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

Ligation

Ono, Nishimura, Ito

BBa_K759012 on pSB1C3 / BamHⅠ - Thanatin - BglⅡ

Reagent	Volume
BBa_K759012 on pSB1C3	5 µL
BamHⅠ - Thanatin - BglⅡ	1 µL
10 × T4 DNA Ligase Buffer	7 µL
T4 Ligase	1 µL
Total	14 µL

Ligation

Step	Temp.	Time	Process
1	16℃	30 min	Ligation
Store	4℃	Hold	Store

Ligation

Ono, Nishimura, Ito

BBa_B0015 on pSB1C3 / XbaⅠ - Thanatin - SpeⅠ

Reagent	Volume
BBa_B0015 on pSB1C3	5 µL
XbaⅠ - Thanatin - SpeⅠ	1 µL
10 × T4 DNA Ligase Buffer	7 µL
T4 DNA Ligase	1 µL
Total	14 µL

Ligation

Step	Temp.	Time	Process
1	16℃	30 min	Ligation
Store	4℃	Hold	Store

Transformation

Onoda

Thanatin - BBa_K759012 on pSB1C3, Thanatin - BBa_B0015 on pSB1C3

Added 5 µL of Thanatin - BBa_K759012 on pSB1C3, Thanatin - BBa_B0015 on pSB1C3 to 50 µL of thawed competent cells (DH5α) on ice.
Incubated on ice for 30 min.
Heat-shocked for 30 sec at 42℃.
Added 200 µL of LB.
Incubated the cells for 2 hrs at 37℃.
Spread 300 µL of the culture onto plate with LBC.
Incubated the plate at 37℃ for 16 hours.

Digestion

Onoda, Ono

BBa_B0033 on pSB1C3, XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ, XbaⅠ - BBa_K168000

Reagent	Volume
BBa_B0033 on pSB1C3, XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ, XbaⅠ - BBa_K168000	20 µL
XbaⅠ	1 µL
SpeⅠ	1 µL
10 × M Buffer	3 µL
DW	5 µL
Total	30 µL

Digestion

Step	Temp.	Time	Process
1	37℃	60 min	Digestion
2	80℃	20 min	Inactivation
Store	4℃	Hold	Store

Digestion

Onoda

BBa_E0040 on pSB1C3, BBa_R0040 on pSB1C3

Reagent	Volume
BBa_E0040 on pSB1C3, BBa_R0040 on pSB1C3	20 µL
XbaⅠ	1 µL
CutSmart Buffer	3 µL
DW	6 µL
Total	30 µL

Digestion

Step	Temp.	Time	Process
1	37℃	60 min	Digestion
2	60℃	15 min	Inactivation
Store	4℃	Hold	Store

2015/08/22

Electrophoresis

Ono, Nishimura

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ (digestion product)

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

Digestion

Ono, Onoda

BBa_R0010 - BBa_B0034 on pSB1C3

Reagent	Volume
BBa_R0010 - BBa_B0034 on pSB1C3	20 µL
SpeⅠ - HF	1 µL
10 × M Buffer	5 µL
DW	24 µL
Total	50 µL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
Store	4℃	Hold	Store

Electrophoresis

Ono, Onoda

BBa_R0010 - BBa_B0034 on pSB1C3 (Digestion product)

Gel Concentration	Voltage	Time	Buffer
1%	100 V	45 min	1/2 x TBE

Ethanol Precipitation

Ono

BBa_R0010 - BBa_B0034 on pSB1C3 (Digestion product)

Added 5 µL of NaOAc, 1.5 µL of glycogen and 150 µL of 100% ethanol.
Left it at -80℃ for 1 hr.
Centrifuged at 15,000 rpm for 5 min at 4℃.
Removed supernatant and added 100 µL of 70% ethanol.
Centrifuged at 15,000 rpm for 10 min at 4℃.
Removed supernatant and air-dried at room temperature with light shield.
Suspended with 10 µL of TE.

Colony PCR

Ono, Onoda

Thanatin - BBa_K759012 on pSB1C3, nothing (as a negative control)

Reagent	Volume
Single Colony	-
AGSP - BamHⅠ - Spidroin 10 µM	0.4 µL
BglⅡ - D - Thanatin 10 µM	0.4 µL
KAPA Taq	5 µL
DW	4.2 µL
Total	10 µL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	30 sec	Denaturation	40 cycle
Cycle 2	65℃	40 sec	Annealing	40 cycle
Cycle 3	72℃	40 sec	Elongation	40 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Colony PCR

Ono, Onoda

BBa_B0031 on pSB1A2 (as Positive Control)

Reagent	Volume
Single Colony	-
100UP - EX - F 10 µM	0.4 µL
200DN - PS - R 10 µM	0.4 µL
KAPA Taq	5 µL
DW	4.2 µL
Total	10 µL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	40 cycle
Cycle 2	57℃	30 sec	Annealing	40 cycle
Cycle 3	72℃	40 sec	Elongation	40 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Electrophoresis

Ono, Onoda

Thanatin - BBa_K759012 on pSB1C3 (Colony PCR product), BBa_B0031 on pSB1A2 (Colony PCR product)

Gel Concentration	Voltage	Time	Buffer
1%	100 V	45 min	1/2 x TBE

Ligation

Ono

BBa_R0010 - BBa_B0034 on pSB1C3 / XbaⅠ - Thanatin - SpeⅠ

Reagent	Volume
BBa_R0010 - BBa_B0034 on pSB1C3	5 µL
XbaⅠ - Thanatin - SpeⅠ	4 µL
Mighty Mix	10 µL
DW	1 µL
Total	20 µL

Ligation

Step	Temp.	Time	Process
1	16℃	30 min	Ligation
Store	4℃	Hold	Store

Transformation

Ono

BBa_R0010 - BBa_B0034 on pSB1C3

Added 1 µL of BBa_R0010 - BBa_B0034 on pSB1C3 to 50 µL of thawed competent cells (DH5α) on ice.
Incubated on ice for 30 min.
Heat-shocked for 30 sec at 42℃.
Added 200 µL of LB.
Incubated the cells for 2 hrs at 37℃.
Spread 300 µL of the culture onto plate with LBC.
Incubated the plate at 37℃ for 16 hours.

PCR

Onoda

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ

Reagent	Volume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ	1 µL
XbaI - B0034 - XS scar - F 10 µM	1 µL
SpeⅠ - Thanatin - R 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

2 Step Cycle (Tm value ≥ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	30 cycle
Cycle 2	68℃	30 sec	Annealing / Elongation	30 cycle
Store	4℃	Hold	Store

Electrophoresis

Onoda

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ

Gel Concentration	Voltage	Time	Buffer
2%	50 V	50 min	1/2 x TBE

PCR

Onoda

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ

Reagent	Volume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ	1 µL
XbaI - B0034 - XS scar - F 10 µM	1 µL
SpeⅠ - Thanatin - R 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

2 Step Cycle (Tm value ≥ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	30 cycle
Cycle 2	68℃	30 sec	Annealing / Elongation	30 cycle
Store	4℃	Hold	Store

Transformation

Mitsumoto

BBa_K206000 on pSB1C3, BBa_K206001 on pSB1C3, BBa_I0500 - BBa_B0034 - BBa_R0040 on pSB1C3, BBa_B0034 - BBa_E0040 on pSB1C3, BBa_B0034 - BBa_R0040 on pSB1C3, BBa_B0034 - BBa_E0040 - BBa_B0015 on pSB1C3, BBa_I0500 on pSB1C3, BBa_B0034 - BBa_R0040 - BBa_B0015 on pSB1C3

Added 5 μL of BBa_K206000 on pSB1C3, BBa_K206001 on pSB1C3, BBa_I0500 - BBa_B0034 - BBa_R0040 on pSB1C3, BBa_B0034 - BBa_E0040 on pSB1C3, BBa_B0034 - BBa_R0040 on pSB1C3, BBa_B0034 - BBa_E0040 - BBa_B0015 on pSB1C3, BBa_I0500 on pSB1C3, BBa_B0034 - BBa_R0040 - BBa_B0015 on pSB1C3 to 50 μL of thawed competent cells (DH5α) on ice.
Incubated on ice for 30 min.
Heat-shocked for 30 sec at 42℃.
Added 200 μL of LB.
Incubated the cells for 2 hrs at 37℃.
Spread 200 μL of the culture onto plate with LBC.
Incubated the plate at 37℃ for 15 hours.

Transformation

Mitsumoto

BBa_B0034 - BBa_R0040 - BBa_B0015 on pSB1A2

Added 5 μL of BBa_B0034 - BBa_R0040 - BBa_B0015 on pSB1A2 to 50 μL of thawed competent cells (DH5α) on ice.
Incubated on ice for 30 min.
Heat-shocked for 30 sec at 42℃.
Spread 200 μL of the culture onto plate with LBA.
Incubated the plate at 37℃ for 18 hours.

Liquid Culture

Mitsumoto

BBa_K206000 on pSB1C3, BBa_K206001 on pSB1C3, BBa_I0500 - BBa_B0034 - BBa_R0040 on pSB1C3, BBa_B0034 - BBa_E0040 on pSB1C3, BBa_B0034 - BBa_R0040 on pSB1C3, BBa_B0034 - BBa_E0040 - BBa_B0015 on pSB1C3, BBa_I0500 on pSB1C3, BBa_B0034 - BBa_R0040 - BBa_B0015 on pSB1C3

Reagent	Volume
Single Colony	-
LBC	2000 μL
Chloramphenicol	2 μL

Cultured for 12 hours.

Liquid Culture

Mitsumoto

BBa_B0034 - BBa_R0040 - BBa_B0015 on pSB1A2

Reagent	Volume
Single Colony	-
LBA	2000 μL
Ampicillin	2 μL

Cultured for 12 hours.

2015/08/23

Electrophoresis

Ono

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ (PCR 2STEP product)

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

Gel Extract

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ
FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel

Colony PCR

Ito, Ono, Onoda

BBa_R0010 - BBa_B0034 - Thanatin, nothing (as a negative control)

Reagent	Volume
Single Colony	-
100UP - EX - F 10 µM	0.4 µL
SpeⅠ - Thanatin - R 10 µM	0.4 µL
KAPA Taq	5 µL
DW	4.2 µL
Total	10 µL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	40 cycle
Cycle 2	57℃	30 sec	Annealing	40 cycle
Cycle 3	72℃	40 sec	Elongation	40 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Colony PCR

Ito, Ono, Onoda

BBa_B0030 on pSB1A2 (as a positive control)

Reagent	Volume
Single Colony	-
100UP - EX - F 10 µM	0.4 µL
200DN - PS - R 10 µM	0.4 µL
KAPA Taq	5 µL
DW	4.2 µL
Total	10 µL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	40 cycle
Cycle 2	57℃	30 sec	Annealing	40 cycle
Cycle 3	72℃	40 sec	Elongation	40 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

2015/08/24

Mini-prep

Ono, Nishimura

Thanatin - BBa_K759012 on pSB1C3
FastGene^TM Plasmid mini kit (Nippon Genetics Co.,Ltd)
standard protocol

Colony PCR

Ono, Nishimura

BBa_R0010 - BBa_B0034 - Thanatin on pSB1C3, nothing (as a negative control), BBa_B0030 on pSB1A2 (as a positive control)

Reagent	Volume
Single Colony	-
100UP - EX - F 10 µM	0.4 µL
SpeⅠ - Thanatin - R 10 µM	0.4 µL
KAPA Taq	5 µL
DW	4.2 µL
Total	10 µL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	40 cycle
Cycle 2	57℃	30 sec	Annealing	40 cycle
Cycle 3	72℃	40 sec	Elongation	40 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Colony PCR

Ono, Nishimura

Thanatin - BBa_K759012 on pSB1C3

Reagent	Volume
Single Colony	-
AGSP - BamHⅠ - Spidroin 10 µM	0.4 µL
BBa_K759012 - R 10 µM	0.4 µL
KAPA Taq	5 µL
DW	4.2 µL
Total	10 µL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	30 sec	Denaturation	40 cycle
Cycle 2	65℃	40 sec	Annealing	40 cycle
Cycle 3	72℃	40 sec	Elongation	40 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Electrophoresis

Ono, Nishimura

BBa_R0010 - BBa_B0034 - Thanatin on pSB1C3, Thanatin - BBa_K759012 on pSB1C3

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

Digestion

Ono, Nishimura

BBa_R0010 - BBa_B0034 - Thanatin on pSB1C3

Reagent	Volume
BBa_R0010 - BBa_B0034 - Thanatin on pSB1C3	20 µL
SpeⅠ - HF	1 µL
CutSmart Buffer	3 µL
DW	6 µL
Total	30 µL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	80℃	20 min	Inactivation
Store	4℃	Hold	Store

Electrophoresis

Ono, Nishimura

BBa_R0010 - BBa_B0034 - Thanatin on pSB1C3 (digestion product)

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

Liquid Culture

Ono, Nishimura

Thanatin - BBa_K759012 on pSB1C3 into DH5α

Reagent	Volume
Single Colony	-
LB	2000 µL
Chloramphenicol	2 µL

Cultured for 16 hours.

2015/08/25

Liquid Culture

Ono, Nishimura

Thanatin - BBa_K759012 on pSB1C3 into DH5α, BBa_R0010 - BBa_B0034 - Thanatin on pSB1C3 into DH5α

Reagent	Volume
Single Colony	-
LB	2000 µL
Chloramphenicol	2 µL

Cultured for 16 hours.

Sequencing

Ono, Nishimura

Thanatin - BBa_K759012 on pSB1C3

Reagent	Volume
Thanatin - BBa_K759012 on pSB1C3	1 µL
pbad - F2 / 200 - βdomain BBa_K759012 - R	1.5 µL
BigDye Terminator	1 µL
5 x Sequencing Buffer	1.5 µL
DW	5 µL
Total	10 µL

Sequencing

Step	Temp.	Time	Process	Cycle
Start	96℃	10 sec	Denaturation
Cycle 1	50℃	5 sec	-	30 cycle
Cycle 2	60℃	240 sec	-	30 cycle
Store	4℃	Hold	Store

Ethanol Precipitation

Ono, Nishimura

Thanatin - BBa_K759012 on pSB1C3

Added 5 µL of NaOAc, 1.5 µL of glycogen and 150 µL of 100% ethanol.
Left it at -80℃ for 1 hr.
Centrifuged at 15,000 rpm for 15 min at 4℃.
Removed supernatant and added 220 µL of 70% ethanol.
Centrifuged at 15,000 rpm for 10 min at 4℃.
Removed supernatant and air-dried at room temperature with light shield.
Suspended with 10 µL of DW.

PCR

Ono, Nishimura

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ

Reagent	Volume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ	1 µL
XbaⅠ - BBa_B0034 - XS scar - F 10 µM	1 µL
SpeⅠ - Thanatin - R 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	40 cycle
Cycle 2	55℃	10 sec	Annealing	40 cycle
Cycle 3	68℃	30 sec	Elongation	40 cycle
Store	4℃	Hold	Store

Ethanol Precipitation

Fujita, Nishimura

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ

Added 5 µL of NaOAc, 1.5 µL of glycogen and 150 µL of 100% ethanol.
Left it at -80℃ for 1 hr.
Centrifuged at 15,000 rpm for 15 min at 4℃.
Removed supernatant and added 220 µL of 70% ethanol.
Centrifuged at 15,000 rpm for 10 min at 4℃.
Removed supernatant and air-dried at room temperature with light shield.
Suspended with 10 µL of TE.

Electrophoresis

Fujita

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

2015/08/26

PCR

Ono、Nishimura

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ

Reagent	Volume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ	1 µL
XbaⅠ - BBa_B0034 - XS scar - F 10 µM	1 µL
SpeⅠ - Thanatin - R 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	40 cycle
Cycle 2	55℃	10 sec	Annealing	40 cycle
Cycle 3	68℃	30 sec	Elongation	40 cycle
Store	4℃	Hold	Store

PCR

Ono, Nishimura

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ

Reagent	Volume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ	1 µL
XbaⅠ - BBa_B0034 - XS scar - F 10 µM	1 µL
SpeⅠ - Thanatin - R 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	40 cycle
Cycle 2	53℃	30 sec	Annealing	40 cycle
Cycle 3	68℃	30 sec	Elongation	40 cycle
Store	4℃	Hold	Store

PCR

Ono, Nishimura, Fujita

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ

Reagent	Volume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ	1 µL
XbaⅠ - BBa_B0034 - XbaⅠ / SpeⅠ scar - F 10 µM	1 µL
SpeⅠ - Thanatin - R 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

2 Step Cycle (Tm value ≥ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	30 cycle
Cycle 2	68℃	30 sec	Annealing / Elongation	30 cycle
Store	4℃	Hold	Store

Electrophoresis

Ono, Nishimura, Fujita

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ (PCR 2STEP, 3STEP products)

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

Ethanol Precipitation

Fujita, Nishimura, Ono

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ (PCR 2STEP product)

Added 4 µL of NaOAc, 1.5 µL of glycogen and 120 µL of 100% ethanol.
Left it at -80℃ for 1 hr.
Centrifuged at 15,000 rpm for 5 min at 4℃.
Removed supernatant and added 100 µL of 70% ethanol.
Centrifuged at 15,000 rpm for 10 min at 4℃.
Removed supernatant and air-dried at room temperature with light shield.
Suspended with 10 µL of TE.

Mini-prep

Nishimura, Ono

BBa_R0010 - BBa_B0034 - Thanatin on pSB1C3
FastGene^TM Plasmid mini kit (Nippon Genetics Co.,Ltd)
standard protocol

Sequencing

Fujita, Nishimura, Ono

BBa_R0010 - BBa_B0034 - Thanatin on pSB1C3 (Mini-prep product)

Reagent	Volume
BBa_R0010 - BBa_B0034 - Thanatin on pSB1C3 (Mini-prep producrt)	1 µL
100UP - EX - F / 200DN - PS - R	1.5 µL
Ready Reaction Premix	1 µL
5 x Sequencing Buffer	1.5 µL
DW	5 µL
Total	10 µL

Sequencing

Step	Temp.	Time	Process	Cycle
Start	96℃	10 sec	Denaturation
Cycle 1	50℃	5 sec	-	30 cycle
Cycle 2	60℃	240 sec	-	30 cycle
Store	4℃	Hold	Store

Ethanol Precipitation

Fujita, Nishimura, Ono

BBa_R0010 - BBa_B0034 - Thanatin on pSB1C3 (Sequencing PCR product)

Added 5 µL of NaOAc, 1.5 µL of glycogen and 150 µL of 100% ethanol.
Left it at -80℃ for 1 hr.
Centrifuged at 15,000 rpm for 15 min at 4℃.
Removed supernatant and added 220 µL of 70% ethanol.
Centrifuged at 15,000 rpm for 10 min at 4℃.
Removed supernatant and air-dried at room temperature with light shield.
Suspended with 10 µL of TE.

2015/08/27

Ligation

Ono, Ito

BBa_K759012 on pSB1C3 (dephosphorylated) / BamHⅠ - Thanatin - BglⅡ

Reagent	Volume
BBa_K759012 on pSB1C3	5 µL
BamHⅠ - Thanatin - BglⅡ	4 µL
Mighty Mix	10 µL
DW	1 µL
Total	20 µL

Ligation

Step	Temp.	Time	Process
1	16℃	30 min	Ligation
Store	4℃	Hold	Store

Ligation

Ono, Ito

BBa_K759012 on pSB1C3 (not phosphorylated)

Reagent	Volume
BBa_K759012 on pSB1C3	2 µL
Mighty Mix	2 µL
DW	6 µL
Total	10 µL

Ligation

Step	Temp.	Time	Process
1	16℃	30 min	Ligation
Store	4℃	Hold	Store

Ligation

Ono, Ito

BBa_K759012 on pSB1C3 (phosphorylated)

Reagent	Volume
BBa_K759012 on pSB1C3	2 µL
Mighty Mix	2 µL
DW	6 µL
Total	10 µL

Ligation

Step	Temp.	Time	Process
1	16℃	30 min	Ligation
Store	4℃	Hold	Store

Transformation

Ono, Ito

Thanatin - BBa_K759012 on pSB1C3, self ligated BBa_K759012 on pSB1C3, linearized BBa_K759012 on pSB1C3, self ligated BBa_K759012 on pSB1C3 (phosphorylated), linearized BBa_K759012 on pSB1C3 (phosphorylated)

Added 1 µL of Thanatin - BBa_K759012 on pSB1C3, self ligated BBa_K759012 on pSB1C3, linearized BBa_K759012 on pSB1C3, self ligated BBa_K759012 on pSB1C3 (phosphorylated), linearized BBa_K759012 on pSB1C3 (phosphorylated) or linearized BBa_K759012 on pSB1C3 (phosphorylated) to 50 µL of thawed competent cells (DH5α) on ice.
Incubated on ice for 30 min.
Heat-shocked for 30 sec at 42℃.
Added 200 µL of LB.
Incubated the cells for 2 hrs at 37℃.
Spread 300 µL of the culture onto plate with LBC.
Incubated the plate at 37℃ for 12 hours.

Colony PCR

Ito, Ono

Thanatin - BBa_K759012 on pSB1C3

Reagent	Volume
Single Colony	-
Agsp - BamHⅠ - Spidroin 10 µM	0.4 µL
BBa_K759012 - bunit - R / BglⅡ - D - Thanatin - R 10 µM	0.4 µL
KAPA Taq	5 µL
DW	4.2 µL
Total	10 µL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	40 cycle
Cycle 2	65℃	30 sec	Annealing	40 cycle
Cycle 3	72℃	60 sec	Elongation	40 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

2015/08/28

Electrophoresis

Fujita, Ono

BamHⅠ - Thanatin - BglⅡ, XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

Ethanol Precipitation

Fujita

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ

Added 5 µL of NaOAc, 1.5 µL of glycogen and 150 µL of 100% ethanol.
Left it at -80℃ for 1 hr.
Centrifuged at 15,000 rpm for 15 min at 4℃.
Removed supernatant and added 220 µL of 70% ethanol.
Centrifuged at 15,000 rpm for 10 min at 4℃.
Removed supernatant and air-dried at room temperature with light shield.
Suspended with 10 µL of TE.

Electrophoresis

Fujita, Ono

BamHⅠ - Thanatin - BglⅡ

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

Gel Extract

Fujita

BamHⅠ - Thanatin - BglⅡ
FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel

PCR

Fujita, Ono

XbaⅠ - Thanatin - SpeⅠ

Reagent	Volume
XbaⅠ - Thanatin - SpeⅠ	1 µL
XbaⅠ - Thanatin - F 10 µM	1 µL
SpeⅠ - Thanatin - R 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

2 Step Cycle (Tm value ≥ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	30 cycle
Cycle 2	68℃	30 sec	Annealing / Elongation	30 cycle
Store	4℃	Hold	Store

PCR

Fujita, Ono

XbaⅠ - Thanatin - SpeⅠ

Reagent	Volume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ	1 µL
XbaⅠ - Thanatin - F 10 µM	1 µL
SpeⅠ - Thanatin - R 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	30 cycle
Cycle 2	67℃	30 sec	Annealing	30 cycle
Cycle 3	68℃	30 sec	Elongation	30 cycle
Store	4℃	Hold	Store

PCR

Fujita, Ono

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ

Reagent	Volume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ	1 µL
XbaⅠ - Thanatin - F 10 µM	1 µL
SpeⅠ - Thanatin - R 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

2 Step Cycle (Tm value ≥ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	30 cycle
Cycle 2	68℃	30 sec	Annealing / Elongation	30 cycle
Store	4℃	Hold	Store

PCR

Fujita, Ono

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ

Reagent	Volume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ	1 µL
XbaⅠ - Thanatin - F 10 µM	1 µL
SpeⅠ - Thanatin - R 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	30 cycle
Cycle 2	67℃	30 sec	Annealing	30 cycle
Cycle 3	68℃	30 sec	Elongation	30 cycle
Store	4℃	Hold	Store

PCR

Fujita, Ono

BamHⅠ - Thanatin - BglⅡ

Reagent	Volume
BamHⅠ - Thanatin - BglⅡ	1 µL
BamHⅠ - Thanatin - F 10 µM	1 µL
BglⅡ - Thanatin - R 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

2 Step Cycle (Tm value ≥ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	30 cycle
Cycle 2	68℃	30 sec	Annealing / Elongation	30 cycle
Store	4℃	Hold	Store

PCR

Fujita, Ono

BamHⅠ - Thanatin - BglⅡ

Reagent	Volume
BamHⅠ - Thanatin - BglⅡ	1 µL
BamHⅠ - Thanatin - F 10 µM	1 µL
BglⅡ - Thanatin - R 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	30 cycle
Cycle 2	67℃	30 sec	Annealing	30 cycle
Cycle 3	68℃	30 sec	Elongation	30 cycle
Store	4℃	Hold	Store

Electrophoresis

Fujita, Ono

XbaⅠ - Thanatin - SpeⅠ, XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ, BamHⅠ - Thanatin - BglⅡ

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

Digestion

Ono, Fujita

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ

Reagent	Volume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ	20 µL
SpeⅠ	1 µL
XbaⅠ	1 µL
10 × M Buffer	3 µL
0.1%BSA	3 µL
DW	2 µL
Total	30 µL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
Store	4℃	Hold	Store

Digestion

Ono, Fujita

BamHⅠ - Thanatin - BglⅡ

Reagent	Volume
BamHⅠ - Thanatin - BglⅡ	20 µL
BamHⅠ	1 µL
BglⅡ	1 µL
10 × K Buffer	3 µL
DW	5 µL
Total	30 µL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
Store	4℃	Hold	Store

2015/08/29

Electrophoresis

Fujita, Ono

XbaⅠ - Thanatin - SpeⅠ

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

Gel Extract

Fujita, Ono

XbaⅠ - Thanatin - SpeⅠ

FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel

Ethanol Precipitation

Fujita, Ono

XbaⅠ - Thanatin - SpeⅠ

Added 20 µL of NaOAc, 1.5 µL of glycogen and 600 µL of 100% ethanol.
Left it at -80℃ for 1 hr.
Centrifuged at 15,000 rpm for 15 min at 4℃.
Removed supernatant and added 100 µL of 70% ethanol.
Centrifuged at 15,000 rpm for 10 min at 4℃.
Removed supernatant and air-dried at room temperature with light shield.
Suspended with 10 µL of TE.

Electrophoresis

Fujita, Ono

XbaⅠ - Thanatin - SpeⅠ

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

PCR

Ono

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ

Reagent	Volume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ	1 µL
XbaⅠ - Thanatin - F 10 µM	1 µL
SpeⅠ - Thanatin - R 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

2 Step Cycle (Tm value ≥ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	40 cycle
Cycle 2	68℃	30 sec	Annealing / Elongation	40 cycle
Store	4℃	Hold	Store

Ethanol Precipitation

Ono

BamHⅠ - Thanatin - BglⅡ (Digestion product), XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ (Digestion product)

Added 3 µL of NaOAc, 1.5 µL of glycogen and 90 µL of 100% ethanol.
Left it at -80℃ for 10 min.
Centrifuged at 15,000 rpm for 5 min at 4℃.
Removed supernatant and added 100 µL of 70% ethanol.
Centrifuged at 15,000 rpm for 5 min at 4℃.
Removed supernatant and air-dried at room temperature with light shield.
Suspended with 10 µL of TE.

Electrophoresis

Fujita, Mimata

BBa_B0033, Thanatin - BBa_K759012, BBa_R0010 - Thanatin, BBa_R0040, BBa_E0040

Gel Concentration	Voltage	Time	Buffer
1%	100 V	60 min	1/2 x TBE

Electrophoresis

Fujita, Mimata

BamHⅠ - Thanatin - BglⅡ, XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ, XbaⅠ - Thanatin - SpeⅠ

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

Digestion

Fujita

BBa_R0010 - BBa_B0034 on pSB1C3

Reagent	Volume
BBa_R0010 - BBa_B0034 on pSB1C3	20 µL
SpeⅠ - HF	1 µL
CutSmart Buffer	3 µL
DW	6 µL
Total	30 µL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	80℃	20 min	Inactivation
Store	4℃	Hold	Store

Transformation

Mitsumoto

BBa_B0015 on pSB1C3

Added 5 μL of BBa_B0015 on pSB1C3 to 50 μL of thawed competent cells (DH5α) on ice.
Incubated on ice for 30 min.
Heat-shocked for 30 sec at 42℃.
Added 200 μL of LB.
Incubated the cells for 2 hrs at 37℃.
Spread 200 μL of the culture onto plate with LBC.
Incubated the plate at 37℃ for 16 hours.

Transformation

Mitsumoto

BBa_I0500 on pSB2K3

Added 5 μL of BBa_I0500 on pSB2K3 to 50 μL of thawed competent cells (DH5α) on ice.
Incubated on ice for 30 min.
Heat-shocked for 30 sec at 42℃.
Added 200 μL of LB.
Incubated the cells for 2 hrs at 37℃.
Spread 200 μL of the culture onto plate with LBK.
Incubated the plate at 37℃ for 16 hours.

PCR (2 STEP)

Mitsumoto

HLA on pCOLA

Reagent	Volume
HLA on pCOLA	1 μL
XbaI - HLA - F - primer 10 μM	1 μL
SpeI - HLA - R - primer 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	5 μL
2 mM dNTPs	5 μL
25 mM MgSO₄	3 μL
DW	33 μL
Total	50 μL

2 Step Cycle (Tm value ≥ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	35 cycle
Cycle 2	68℃	elongation time	Annealing / Elongation	35 cycle
Store	4℃	Hold	Store

PCR (3 STEP)

Mitsumoto

HLZ on pCOLA

Reagent	Volume
HLZ on pCOLA	1 μL
XbaI - HLZ - F - primer 10 μM	1 μL
SpeI - HLZ - R - primer 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	5 μL
2 mM dNTPs	5 μL
25 mM MgSO₄	3 μL
DW	33 μL
Total	50 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	35 cycle
Cycle 2	62.7℃	30 sec	Annealing	35 cycle
Cycle 3	68℃	elongation time	Elongation	35 cycle
Store	4℃	Hold	Store

PCR (3 STEP)

Mitsumoto

BLA on pCOLA

Reagent	Volume
BLA on pCOLA	1 μL
XbaI - BLA - F - primer 10 μM	1 μL
SpeI - BLA - R - primer 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	5 μL
2 mM dNTPs	5 μL
25 mM MgSO₄	3 μL
DW	33 μL
Total	50 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	35 cycle
Cycle 2	60.9℃	30 sec	Annealing	35 cycle
Cycle 3	68℃	elongation time	Elongation	35 cycle
Store	4℃	Hold	Store

PCR (3 STEP)

Mitsumoto

BBa_B0015, BBa_B0032, BBa_B0033, BBa_B0034 on pSB1C3

Reagent	Volume
BBa_B0015, BBa_B0032, BBa_B0033, BBa_B0034 on pSB1C3	1 μL
100UP - EX - F 10 μM	1 μL
200DN - PS - R 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	5 μL
2 mM dNTPs	5 μL
25 mM MgSO₄	3 μL
DW	33 μL
Total	50 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	35 cycle
Cycle 2	62.6℃	30 sec	Annealing	35 cycle
Cycle 3	68℃	elongation time	Elongation	35 cycle
Store	4℃	Hold	Store

Electrophoresis

Mitsumoto

BBa_B0015, BBa_B0032, BBa_B0033, BBa_B0034 on pSB1C3, BLA on pCOLA, HLA on pCOLA

Gel Concentration	Voltage	Time	Buffer
2%	100 V	30 min	1/2 x TBE

Dephosphorylation

Mitsumoto

BBa_R0040 on pSB1C3, BBa_E0040 on pSB1A2 XbaI (Digestion product)

Reagent	Volume
BBa_R0040 on pSB1C3, BBa_E0040 on pSB1A2 XbaI (Digestion product)	10 μL
Antarctic Phosphatase	1 μL
Antarctic Phosphatase Buffer	2 μL
DW	7 μL
Total	20 μL

Dephosphorylation

Step	Temp.	Time	Process
1	37℃	15 min	Dephosphorylation
2	65℃	5 min	Inactivation
Store	4℃	Hold	Store

PCR (3 STEP)

Mitsumoto

BBa_I0500, BBa_B0015, BBa_B0032, BBa_B0033, BBa_B0034 on pSB1C3

Reagent	Volume
BBa_I0500, BBa_B0015, BBa_B0032, BBa_B0033, BBa_B0034 on pSB1C3	1 μL
100UP - EX -F 10 μM	1 μL
200DN - PS - R 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	5 μL
2 mM dNTPs	5 μL
25 mM MgSO₄	3 μL
DW	33 μL
Total	50 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	35 cycle
Cycle 2	62.6℃	30 sec	Annealing	35 cycle
Cycle 3	68℃	elongation time	Elongation	35 cycle
Store	4℃	Hold	Store

2015/08/30

Electrophoresis

Mimata

BBa_R0010 - BBa_B0034 on pSB1C3, Thanatin - BBa_K759012 on pSB1C3

Gel Concentration	Voltage	Time	Buffer
1%	100 V	60 min	1/2 x TBE

Digestion

Mimata, Toyooka

XbaⅠ - Thanatin - SpeⅠ

Reagent	Volume
XbaⅠ - Thanatin - SpeⅠ	20 µL
SpeⅠ	1 µL
XbaⅠ	1 µL
10 × M Buffer	3 µL
DW	5 µL
Total	30 µL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	70℃	15 min	Inactivation
Store	4℃	Hold	Store

Digestion

Mimata, Toyooka

BBa_R0040 on pSB1A2, BBa_E0040 on pSB1A2

Reagent	Volume
BBa_R0040 on pSB1A2, BBa_E0040 on pSB1A2	20 µL
SpeⅠ	1 µL
CutSmart Buffer	3 µL
DW	6 µL
Total	30 µL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	70℃	15 min	Inactivation
Store	4℃	Hold	Store

Digestion

Mimata, Toyooka

BBa_B0030 on pSB1C3

Reagent	Volume
BBa_B0030 on pSB1C3	20 µL
SpeⅠ	1 µL
XbaⅠ	1 µL
10 × M Buffer	3 µL
DW	5 µL
Total	30 µL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	70℃	15 min	Inactivation
Store	4℃	Hold	Store

2015/08/31

PCR

Nishimura, Ono, Toyooka, Fujita

XbaⅠ - Thanatin - SpeⅠ

Reagent	Volume
XbaⅠ - Thanatin - SpeⅠ	1 µL
XbaⅠ - Thanatin - F 10 µM	1 µL
SpeⅠ - Thanatin - R 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

2 Step Cycle (Tm value ≥ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	30 cycle
Cycle 2	68℃	30 sec	Annealing / Elongation	30 cycle
Store	4℃	Hold	Store

PCR

Nishimura, Ono, Toyooka, Fujita

BamHⅠ- Thanatin - BglⅡ

Reagent	Volume
BamHⅠ- Thanatin - BglⅡ	1 µL
BamHⅠ- Thanatin - F 10 µM	1 µL
BglⅡ - D - Thanatin - R 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

2 Step Cycle (Tm value ≥ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	30 cycle
Cycle 2	68℃	30 sec	Annealing / Elongation	30 cycle
Store	4℃	Hold	Store

Electrophoresis

Ono, Nishimura, Toyooka, Fujita

BamHⅠ- Thanatin - BglⅡ, XbaⅠ - Thanatin - SpeⅠ

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

Ethanol Precipitation

Ono, Nishimura, Toyooka, Fujita

BamHⅠ- Thanatin - BglⅡ, XbaⅠ - Thanatin - SpeⅠ

Added 5 µL of NaOAc, 1.5 µL of glycogen and 150 µL of 100% ethanol.
Left it at -80℃ for 1 hr.
Centrifuged at 15,000 rpm for 15 min at 4℃.
Removed supernatant and added 100 µL of 70% ethanol.
Centrifuged at 15,000 rpm for 10 min at 4℃.
Removed supernatant and air-dried at room temperature with light shield.
Suspended with 10 µL of TE.

Digestion

Ono, Fujita, Toyooka, Nishimura

Thanatin fragment (Ethanol Precipitation product)

Reagent	Volume
Thanatin fragment (Ethanol Precipitation product)	20 µL
SpeⅠ	2 µL
XbaⅠ	1 µL
CutSmart Buffer	3 µL
DW	4 µL
Total	30 µL

Digestion

Step	Temp.	Time	Process
1	37℃	60 min	Digestion
Store	4℃	Hold	Store

Digestion

Ono, Fujita, Toyooka, Nishimura

Thanatin fragment (Ethanol Precipitation product)

Reagent	Volume
Thanatin fragment (Ethanol Precipitation product)	20 µL
BamHⅠ	2 µL
BglⅡ	6 µL
10 × K Buffer	10 µL
DW	60 µL
Total	100 µL

Digestion

Step	Temp.	Time	Process
1	37℃	60 min	Digestion
Store	4℃	Hold	Store

Ethanol Precipitation

Ono, Nishimura, Toyooka, Fujita

BamHⅠ- Thanatin - BglⅡ, XbaⅠ - Thanatin - SpeⅠ (Digestion product)

Added 5 µL of NaOAc, 1.5 µL of glycogen and 280 µL of 100% ethanol.
Left it at -80℃ for 1 hr.
Centrifuged at 15,000 rpm for 15 min at 4℃.
Removed supernatant and added 100 µL of 70% ethanol.
Centrifuged at 15,000 rpm for 10 min at 4℃.
Removed supernatant and air-dried at room temperature with light shield.
Suspended with 10 µL of TE.

Electrophoresis

Ono, Nishimura, Toyooka, Fujita, Mimata

BamHⅠ- Thanatin - BglⅡ, XbaⅠ - Thanatin - SpeⅠ (Ethanol Presipitation product)

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

Ligation

Ono

BBa_K759012 on pSB1C3 / BamHⅠ- Thanatin - BglⅡ

Reagent	Volume
BBa_K759012 on pSB1C3	95 µL
BamHⅠ- Thanatin - BglⅡ	0.5 µL
Mighty Mix	10 µL
Total	20 µL

Ligation

Step	Temp.	Time	Process
1	16℃	30 min	Ligation
Store	4℃	Hold	Store

Electrophoresis

Ono, Mimata

BBa_K759012 on pSB1C3, BBa_R0010 on pSB1C3

Gel Concentration	Voltage	Time	Buffer
1%	100 V	60 min	1/2 x TBE

Digestion

Onoda,

BBa_E1010 on pSB1C3

Reagent	Volume
BBa_E1010 on pSB1C3	10 µL
EcoRⅠ	1 µL
XbaⅠ	1 µL
10 × M Buffer	2 µL
DW	6 µL
Total	20 µL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	60℃	15 min	Inactivation
Store	4℃	Hold	Store

Digestion

Onoda

BBa_E1010 on pSB1C3

Reagent	Volume
BBa_E1010 on pSB1C3	10 µL
XbaⅠ	1 µL
CutSmart Buffer	2 µL
DW	7 µL
Total	20 µL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	60℃	15 min	Inactivation
Store	4℃	Hold	Store

Electrophoresis

Toyooka

BBa_E1010 on pSB1C3 (Digestion product)

Gel Concentration	Voltage	Time	Buffer
1%	100 V	40 min	1/2 x TBE

Gel Extract

Toyooka

BBa_E1010 on pSB1C3 (Digestion product)
FastGene^TM Gel Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel

Digestion

Ito, Sakai

HLA family

Reagent	Volume
HLA, HLZ, BLA, BLZ	20 µL
XbaⅠ	1 µL
SpeⅠ	1 µL
10 × M Buffer	3 µL
DW	5 µL
Total	30 µL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	80℃	20 min	Inactivation
Store	4℃	Hold	Store

Digestion

Ito, Sakai

HLA, HLZ, BLA, BLZ

Reagent	Volume
HLA, HLZ, BLA, BLZ	10 µL
XbaⅠ	1 µL
SpeⅠ	1 µL
10 x M buffer	2 µL
DW	6 µL
Total	20 µL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	80℃	20 min	Inactivation
Store	4℃	Hold	Store

Electrophoresis

Ito, Sakai

HLA family XbaⅠ & SpeⅠ (Digestion product)

Gel Concentration	Voltage	Time	Buffer
1%	100 V	40 min	1/2 x TBE

Gel Extract

Ito, Sakai

HLA family XbaⅠ & SpeⅠ (Digestion product)
FastGene^TM Gel Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel

September

2015/09/01

Gel Extract

Nishimura

BBa_B0032, BBa_B0033, BBa_B0034, BBa_B0015, BBa_I0500
FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel

Mini-prep

Nishimura

BBa_R0010 on pSB1C3, BBa_I0500 on pSB1C3
FastGene^TM Plasmid mini kit (Nippon Genetics Co.,Ltd)
standard protocol

Electrophoresis

Nishimura

BBa_R0010 - BBa_B0034 on pSB1C3 (Dephosphorylated product), BBa_R0010 - BBa_B0034 on pSB1C3 (Gel extract product)

Gel Concentration	Voltage	Time	Buffer
2%	100 V	60 min	1/2 x TBE

Ligation

Fujita, Nishimura

BBa_R0010 - BBa_B0034 on pSB1C3 / XbaⅠ - Thanatin - SpeⅠ

Reagent	Volume
BBa_R0010 - BBa_B0034 on pSB1C3	9.5 µL
XbaⅠ - Thanatin - SpeⅠ	0.5 µL
Mighty Mix	10 µL
Total	20 µL

Ligation

Step	Temp.	Time	Process
1	16℃	30 min	Ligation
Store	4℃	Hold	Store

Ligation

Fujita

Ag43 on pSB1C3 / BamHⅠ - Thanatin - BglⅡ

Reagent	Volume
BBa_R0010 - BBa_B0034 on pSB1C3	9.5 µL
BamHⅠ - Thanatin - BglⅡ	0.5 µL
Mighty Mix	10 µL
Total	20 µL

Ligation

Step	Temp.	Time	Process
1	16℃	30 min	Ligation
Store	4℃	Hold	Store

Ethanol Precipitation

Fujita

Thanatin - Ag43 on pSB1C3

Added 2 µL of NaOAc, 1.5 µL of glycogen and 60 µL of 100% ethanol.
Left it at -80℃ for 1 hr.
Centrifuged at 15,000 rpm for 15 min at 4℃.
Removed supernatant and added 100 µL of 70% ethanol.
Centrifuged at 15,000 rpm for 10 min at 4℃.
Removed supernatant and air-dried at room temperature with light shield.
Suspended with 10 µL of TE.

Transformation

Nishimura, Toyooka

BBa_I0500 - BBa_B0033 on pSB1C3,

Added 5 µL of BBa_B0033 on pSB1C3, BBa_R0040, BBa_I0500 BBa_B0032 on pSB1C3, BBa_I0500 BBa_B0033 on pSB1C3 to 50 µL of thawed competent cells (DH5α) on ice.
Incubated on ice for 30 min.
Heat-shocked for 30 sec at 42℃.
Added 200 µL of LB.
Incubated the cells for 2 hrs at 37℃.
Spread 300 µL of the culture onto plate with LBC.
Incubated the plate at 37℃ for 16 hours.

Digestion

Nishimura, Onoda

HLA, HLZ, BLA, BLZ, BBa_B0030

Reagent	Volume
HLA, HLZ, BLA, BLZ, BBa_B0030	10 µL
SpeⅠ	1 µL
XbaⅠ	1 µL
10 × M Buffer	2 µL
DW	6 µL
Total	20 µL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	60℃	15 min	Inactivation
Store	4℃	Hold	Store

Electrophoresis

Nishimura

HLA, HLZ, BLA, BLZ

Gel Concentration	Voltage	Time	Buffer
2%	100 V	60 min	1/2 x TBE

Gel Extract

Nishimura, Sakai, Ito, Kusumi

HLA, HLZ, BLA, BLZ
FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel

Ligation

Fujita, Sakai, Nishimura

BBa_B0015 on pSB1C3 / HLA, BLA, BLZ

Reagent	Volume
BBa_B0015 on pSB1C3	10 µL
HLA , BLA, BLZ	30 µL
T4 Ligase	4.5 µL
10 × T4 DNA Ligase Buffer	5 µL
DW	0.5 µL
Total	50 µL

Ligation

Step	Temp.	Time	Process
1	16℃	30 min	Ligation
Store	4℃	Hold	Store

Ligation

Onoda, Nishimura, Fujita, Sakai

HLZ / BBa_B0015 on pSB1C3

Reagent	Volume
BBa_B0015 on pSB1C3	10 µL
HLZ	20 µL
T4 Ligase	3.5 µL
10 × T4 DNA Ligase Buffer	5 µL
DW	1.5 µL
Total	50 µL

Ligation

Step	Temp.	Time	Process
1	16℃	30 min	Ligation
Store	4℃	Hold	Store

Electrophoresis

Sakai

HLA, HLZ, BLA, BLZ

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

Ligation

Sakai

BBa_R0010 - BBa_B0034 on pSB1C3 / XbaⅠ - Thanatin - SpeⅠ (Digestion product)

Reagent	Volume
BBa_R0100 - BBa_B0034 on pSB1C3	15 µL
XbaⅠ - Thanatin - SpeⅠ	1.0 µL
T4 Ligase	1.6 µL
10 X T4 DNA Ligase Buffer	2.0 µL
DW	0.4 µL
Total	20 µL

Ligation

Step	Temp.	Time	Process
1	16℃	30 min	Ligation
Store	4℃	Hold	Store

Transformation

Sakai

BBa_R0010 - BBa_B0034 - XbaⅠ - Thanatin - SpeⅠ on pSB1C3

Added 1.0 µL of BBa_R0010 - BBa_B0034 - XbaⅠ - Thanatin - SpeⅠ on pSB1C3 to 50 µL of thawed competent cells (DH5α) on ice.
Incubated on ice for 30 min.
Heat-shocked for 30 sec at 42℃.
Added 200 µL of LB.
Incubated the cells for 2 hrs at 37℃.
Spread 300 µL of the culture onto plate with LBC.
Incubated the plate at 37℃ for 2 hours.

PCR

Ono

ABF-2, BLA, Thanatin, 10 × His tag - TEV - Thanatin, BLZ, HLZ

Reagent	Volume
ABF-2, BLA, Thanatin, 10 × His tag - TEV - Thanatin, BLZ, HLZ	1 µL
EX - F - Universal 10 µM	1 µL
PS - R 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO₄	3 µL
DW	33 µL
Total	50 µL

2 Step Cycle (Tm value ≥ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	30 cycle
Cycle 2	68℃	30 sec	Annealing / Elongation	30 cycle
Store	4℃	Hold	Store

2015/09/02

Gel Extract

Mimata

EcoRⅠ - BBa_B0032 - XbaⅠ, EcoRⅠ - BBa_B0033 - XbaⅠ, EcoRⅠ - BBa_B0034 - XbaⅠ, SpeⅠ - BBa_B0015 - PstⅠ, EcoRⅠ - BBa_I0500 - SpeⅠ
FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel

Transformation

Nishimura

HLA, BLA, HLZ, BLZ

Added 1 µL of HLA, BLA, HLZ, BLZ to 10 µL of thawed competent cells (DH5α) on ice.
Incubated on ice for 30 min.
Heat-shocked for 30 sec at 42℃.
Added 200 µL of LB.
Incubated the cells for 2 hrs at 37℃.
Spread 300 µL of the culture onto plate with LBC.
Incubated the plate at 37℃ for 16 hours.

Colony PCR

Nishimura, Ono, Onoda, Mimata

BBa_R0010 - BBa_B0034 - Thanatin

Reagent	Volume
Single Colony	-
100UP - EX - F 10 µM	0.4 µL
200DN - PS - R 10 µM	0.4 µL
KAPA Taq	5 µL
DW	4.2 µL
Total	10 µL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	40 cycle
Cycle 2	57℃	30 sec	Annealing	40 cycle
Cycle 3	72℃	40 sec	Elongation	40 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Colony PCR

Nishimura, Ono, Onoda, Mimata

BBa_R0010 - BBa_B0034 - Thanatin

Reagent	Volume
Single Colony	-
100UP - EX - F 10 µM	0.4 µL
SpeⅠ - Thanatin - Rv 10 µM	0.4 µL
KAPA Taq	5 µL
DW	4.2 µL
Total	10 µL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	40 cycle
Cycle 2	57℃	30 sec	Annealing	40 cycle
Cycle 3	72℃	40 sec	Elongation	40 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Colony PCR

Nishimura, Ono, Onoda, Mimata

Ag43 - Thanatin

Reagent	Volume
Single Colony	-
AGSP - BamHⅠ - Spindorin 10 µM	0.4 µL
BglⅡ - D - Thanatin - Rv 10 µM	0.4 µL
KAPA Taq	5 µL
DW	4.2 µL
Total	10 µL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	40 cycle
Cycle 2	65℃	30 sec	Annealing	40 cycle
Cycle 3	72℃	40 sec	Elongation	40 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Colony PCR

Nishimura, Ono, Onoda, Mimata

Ag43 - Thanatin

Reagent	Volume
Single Colony	-
AGSP - BamHⅠ - Spindorin 10 µM	0.4 µL
Ag43 - bunit - Rv 10 µM	0.4 µL
KAPA Taq	5 µL
DW	4.2 µL
Total	10 µL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	40 cycle
Cycle 2	65℃	30 sec	Annealing	40 cycle
Cycle 3	72℃	40 sec	Elongation	40 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Colony PCR

Nishimura, Ono, Onoda, Mimata

BBa_B0031 on pSB1A2

Reagent	Volume
Single Colony	-
100UP - EX - F 10 µM	0.4 µL
200DN - PS - R 10 µM	0.4 µL
KAPA Taq	5 µL
DW	4.2 µL
Total	10 µL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	40 cycle
Cycle 2	57℃	30 sec	Annealing	40 cycle
Cycle 3	72℃	40 sec	Elongation	40 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Electrophoresis

Nishimura, Ono, Onoda

BBa_R0010 - BBa_B0034 - Thanatin (colony PCR product), Ag43 Thanatin (colony PCR product), BBa_B0031 on pSB1C3(colony PCR product)

Gel Concentration	Voltage	Time	Buffer
1%	100 V	50 min	1/2 x TBE

Electrophoresis

Nishimura, Ono, Mimata

BBa_I0500

Gel Concentration	Voltage	Time	Buffer
1%	100 V	50 min	1/2 x TBE

Digestion

Nisimura, Ono, Mimata

BLZ, HLZ, ABF-2

Reagent	Volume
BLZ, HLZ, ABF-2	30 µL
SpeⅠ	1 µL
Total	31 µL

Digestion

Step	Temp.	Time	Process
1	37℃	60 min	Digestion
Store	4℃	Hold	Store

Electrophoresis

Nishimura, Ono, Mimata

BLZ, HLZ, ABF-2,

Gel Concentration	Voltage	Time	Buffer
2%	100 V	60 min	1/2 x TBE

Gel Extract

Mitsumoto

BLZ, HLZ, ABF-2
FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel

Transformation

Mimata

HLA on pSB1C3, HLZ on pSB1C3, BLA on pSB1C3, BLZ on pSB1C3, BBa_B0015 on pSB1C3

Added 1 µL of HLA on pSB1C3, HLZ on pSB1C3, BLA on pSB1C3, BLZ on pSB1C3, BBa_B0015 on pSB1C3 to 50 µL of thawed competent cells (DH5α) on ice.
Incubated on ice for 30 min.
Heat-shocked for 30 sec at 42℃.
Added 2000 µL of LB.
Incubated the cells for 2 hrs at 37℃.
Spread 300 µL of the culture onto plate with LBC.
Incubated the plate at 37℃ for 16 hours.

2015/09/03

Ligation

Nishimura

BBa_B0015 on pSB1C3 / HLZ

Reagent	Volume
BBa_B0015 on pSB1C3	6 µL
HLZ	8 µL
Mighty Mix	14 µL
Total	28 µL

Ligation

Step	Temp.	Time	Process
1	16℃	30 min	Ligation
Store	4℃	Hold	Store

Ligation

Nishimura

BBa_B0015 on pSB1C3 / HLA, BLA, BLZ

Reagent	Volume
BBa_B0015 on pSB1C3	6 µL
HLA, BLA, BLZ	14 µL
Mighty Mix	20 µL
Total	40 µL

Ligation

Step	Temp.	Time	Process
1	16℃	30 min	Ligation
Store	4℃	Hold	Store

Dephosphorylation

Nishimura

BBa_B0015 on pSB1C3

Reagent	Volume
BBa_B0015 on pSB1C3	40 µL
Antarctic Phosphatase	4 µL
Antarctic Phosphatase Buffer	8 µL
DW	28 µL
Total	80 µL

Dephosphorylation

Step	Temp.	Time	Process
1	37℃	30 min	Dephosphorylation
2	65℃	10 min	Inactivation
Store	4℃	Hold	Store

Colony PCR

Nishimura, Ono, Fujita

BBa_R0010 - BBa_B0034 - Thanatin

Reagent	Volume
Single Colony	-
100UP - EX - F 10 µM	0.4 µL
200DN - PS - R 10 µM	0.4 µL
KAPA Taq	5 µL
DW	4.2 µL
Total	10 µL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	40 cycle
Cycle 2	57℃	30 sec	Annealing	40 cycle
Cycle 3	72℃	40 sec	Elongation	40 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Colony PCR

Nishimura, Ono, Fujita

BBa_R0010 - BBa_B0034 - Thanatin

Reagent	Volume
Single Colony	-
100UP - EX - F 10 µM	0.4 µL
SpeⅠ - Thanatin - Rv 10 µM	0.4 µL
KAPA Taq	5 µL
DW	4.2 µL
Total	10 µL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	40 cycle
Cycle 2	57℃	30 sec	Annealing	40 cycle
Cycle 3	72℃	40 sec	Elongation	40 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Colony PCR

Nishimura, Ono, Fujita

Ag43 - Thanatin

Reagent	Volume
Single Colony	-
AGSP - BamHⅠ - Spindorin 10 µM	0.4 µL
BglⅡ - D - Thanatin - Rv 10 µM	0.4 µL
KAPA Taq	5 µL
DW	4.2 µL
Total	10 µL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	40 cycle
Cycle 2	65℃	30 sec	Annealing	40 cycle
Cycle 3	72℃	40 sec	Elongation	40 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Colony PCR

Nishimura, Ono, Fujita

Ag43 - Thanatin

Reagent	Volume
Single Colony	-
AGSP - BamHⅠ - Spindorin 10 µM	0.4 µL
Ag43 - bunit - Rv 10 µM	0.4 µL
KAPA Taq	5 µL
DW	4.2 µL
Total	10 µL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	40 cycle
Cycle 2	65℃	30 sec	Annealing	40 cycle
Cycle 3	72℃	40 sec	Elongation	40 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Colony PCR

Nishimura, Ono, Fujita

BBa_B0031 on pSB1A2

Reagent	Volume
Single Colony	-
100UP - EX - F 10 µM	0.4 µL
200DN - PS - R 10 µM	0.4 µL
KAPA Taq	5 µL
DW	4.2 µL
Total	10 µL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	40 cycle
Cycle 2	57℃	30 sec	Annealing	40 cycle
Cycle 3	72℃	40 sec	Elongation	40 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Electrophoresis

Nishimura, Ono, Fujita

BBa_R0010 - BBa_B0034 - Thanatin (colony PCR product), Ag43 Thanatin (colony PCR product), BBa_B0031 on pSB1C3(colony PCR product)

Gel Concentration	Voltage	Time	Buffer
1%	100 V	50 min	1/2 x TBE

Transformation

Nishimura, Fujita

HLA, BLA, HLZ, BLZ

Added 40 µL of HLA, BLA, HLZ, BLZ to 1 µL of thawed competent cells (DH5α) on ice.
Incubated on ice for 30 min.
Heat-shocked for 30 sec at 42℃.
Added 200 µL of LB.
Incubated the cells for 2 hrs at 37℃.
Spread 300 µL of the culture onto plate with LBC.
Incubated the plate at 37℃ for 16 hours.

Mini-prep

Ono, Fujita

Ag43 - Thanatin on pSB1C3
FastGene^TM Plasmid mini kit (Nippon Genetics Co.,Ltd)
standard protocol

Digestion

Onoda

EcoRⅠ - BBa_R0010 - SpeⅠ

Reagent	Volume
EcoRⅠ - BBa_R0010 - SpeⅠ	28.5 µL
EcoRⅠ	1.5 µL
SpeⅠ	1.5 µL
10 × H Buffer	3.5 µL
Total	35 µL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
Store	4℃	Hold	Store

2015/09/04

Electrophoresis

Nishimura

ABF-2, BLA, Thanatin, 10 × His　tag - TEV - Thanatin, BLZ, HLZ, ABF-2

Gel Concentration	Voltage	Time	Buffer
2%	100 V	60 min	1/2 x TBE

Mini-prep

Nishimura, Ono

Ag43 - thanatin
FastGene^TM Plasmid mini kit (Nippon Genetics Co.,Ltd)
standard protocol

Sequencing

Nishimura

thanatin - Ag43

Reagent	Volume
Ag43 thanatin	1 µL
BBa_I0500 - Fw, Ag43 - Rv	1.5 µL
BigDye Terminator	1 µL
5 x Sequencing Buffer	1.5 µL
DW	5 µL
Total	10 µL

Sequencing

Step	Temp.	Time	Process	Cycle
Start	96℃	10 sec	Denaturation
Cycle 1	50℃	5 sec	-	25 cycle
Cycle 2	60℃	240 sec	-	25 cycle
Store	4℃	Hold	Store

Sequencing

Nishimura

BBa_I0500 - BBa_B0032, BBa_I0500 - BBa_B0033, BBa_I0500 - BBa_B0034

Reagent	Volume
100UP - EX - F	1 µL
200DN - PS - R	1.5 µL
Ready Reaction Premix	1 µL
5 x Sequencing Buffer	1.5 µL
DW	5 µL
Total	10 µL

Sequencing

Step	Temp.	Time	Process	Cycle
Start	96℃	10 sec	Denaturation
Cycle 1	50℃	5 sec	-	25 cycle
Cycle 2	60℃	240 sec	-	25 cycle
Store	4℃	Hold	Store

Ethanol Precipitation

Nishimura, Fujita, Mimata

Ag43 - thanatin, BBa_I0500 - BBa_B0032, BBa_I0500 - BBa_B0033, BBa_I0500 - BBa_B0034

Added 1 µL of NaOAc, 1.5 µL of glycogen and 30 µL of 100% ethanol.
Left it at -80℃ for 10 min.
Centrifuged at 15,000 rpm for 15 min at 4℃.
Removed supernatant and added 100 µL of 70% ethanol.
Centrifuged at 15,000 rpm for 10 min at 4℃.
Removed supernatant and air-dried at room temperature with light shield.
Suspended with 10 µL of HiDi.

Transformation

Fujita, Mimata

BLZ - BBa_B0015, HLZ - BBa_B0015, ABF-2 - BBa_B0015, BBa_R0010 - BBa_B0034 - 10 x His tag - TEV - Thanatin - BBa_B0015, BBa_R0010 - BBa_B0034 - Thanatin - BBa_B0015, BBa_R0010 - BBa_B0034 - ABF-2 - BBa_B0015, BBa_R0010 - BBa_B0034 - BLA - BBa_B0015

Added 5 µL of BLZ - BBa_B0015, HLZ - BBa_B0015, ABF-2 - BBa_B0015, BBa_R0010 - BBa_B0034 - 10 x His tag - TEV - Thanatin - BBa_B0015, BBa_R0010 - BBa_B0034 - Thanatin - BBa_B0015, BBa_R0010 - BBa_B0034 - ABF-2 - BBa_B0015, BBa_R0010 - BBa_B0034 - BLA - BBa_B0015 to 50 µL of thawed competent cells (DH5a) on ice.
Incubated on ice for 30 min.
Heat-shocked for 30 sec at 42℃.
Added 200 µL of LB.
Incubated the cells for 2 hrs at 37℃.
Spread 300 µL of the culture onto plate with LBC.
Incubated the plate at 37℃ for 18 hours.

Digestion

Fujita, Mimata

pSB1C3

Reagent	Volume
pSB1C3	5 µL
EcoRⅠ	1 µL
PstⅠ	1 µL
10 × H Buffer	5 µL
DW	38 µL
Total	50 µL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	80℃	20 min	Inactivation
Store	4℃	Hold	Store

Digestion

Fujita, Mimata

pSB1C3

Reagent	Volume
pSB1C3	5 µL
XbaⅠ	1 µL
SpeⅠ	1 µL
CutSmart Buffer	5 µL
DW	38 µL
Total	50 µL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	80℃	20 min	Inactivation
Store	4℃	Hold	Store

Digestion

Fujita, Mimata

ABF-2, Prefix - BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - Thanatin - BBa_B0015 - Suffix, 10 x His tag - TEV - Thanatin

Reagent	Volume
ABF-2, Prefix - BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - Thanatin - BBa_B0015 - Suffix, 10 x His tag - TEV - Thanatin	20 µL
EcoRⅠ	1 µL
PstⅠ	1 µL
10 x H Buffer	5 µL
DW	23 µL
Total	50 µL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	80℃	20 min	Inactivation
Store	4℃	Hold	Store

Digestion

Fujita, Mimata

BLA

Reagent	Volume
BLA	20 µL
XbaⅠ	1 µL
PstⅠ	1 µL
CutSmart Buffer	5 µL
DW	23 µL
Total	50 µL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	80℃	20 min	Inactivation
Store	4℃	Hold	Store

Digestion

Fujita, Mimata

BLZ, HLZ, ABF-2

Reagent	Volume
BLZ, HLZ, ABF-2	20 µL
EcoRⅠ	1 µL
SpeⅠ	1 µL
10 x H Buffer	5 µL
DW	23 µL
Total	50 µL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	80℃	20 min	Inactivation
Store	4℃	Hold	Store

Transformation

Mitsumoto

BLZ - BBa_B0015 on pSB1C3, HLZ - BBa_B0015 on pSB1C3, ABF-2 - BBa_B0015 on pSB1C3, BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - 10 x His tag - TEV - Thanatin - BBa_B0015 on pSB1C3, BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - Thanatin - BBa_B0015 on pSB1C3, BBa_R0010 - BBa_B0034 - ABF-2 - BBa_B0015 on pSB1C3, BBa_I0500 - BBa_B0034 - BLA - BBa_B0015 on pSB1C3

Added 5 μL of BLZ - BBa_B0015 on pSB1C3, HLZ - BBa_B0015 on pSB1C3, ABF-2 - BBa_B0015 on pSB1C3, BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - 10 x His tag - TEV - Thanatin - BBa_B0015 on pSB1C3, BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - Thanatin - BBa_B0015 on pSB1C3, BBa_R0010 - BBa_B0034 - ABF-2 - BBa_B0015 on pSB1C3, BBa_I0500 - BBa_B0034 - BLA - BBa_B0015 on pSB1C3 to 50 μL of thawed competent cells (DH5a) on ice.
Incubated on ice for 30 min.
Heat-shocked for 30 sec at 42℃.
Added 200 μL of LB.
Incubated the cells for 2 hrs at 37℃.
Spread 300 μL of the culture onto plate with LBCp.
Incubated the plate at 37℃ for 2 hours.

Colony PCR

Mitsumoto

BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - 10 x His tag - TEV - Thanatin - BBa_B0015 on pSB1C3, BBa_R0010 - BBa_B0034 - XbaⅠ/SpeⅠ scar - Thanatin - BBa_B0015 on pSB1C3

Reagent	Volume
Single Colony	-
XbaⅠ - Thanatin - F 10 μM	0.4 μL
200DN - PS - R 10 μM	0.4 μL
KAPA Taq	5 μL
DW	4.2μL
Total	10 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	35 cycle
Cycle 2	59.3℃	30 sec	Annealing	35 cycle
Cycle 3	68℃	90 sec	Elongation	35 cycle
Store	4℃	Hold	Store

Colony PCR

Mitsumoto

HLZ - BBa_B0015 on pSB1C3, nothing(other negative control)

Reagent	Volume
Single Colony	-
XbaⅠ - HLZ - F 10 μM	0.4 μL
200DN - PS - R 10 μM	0.4 μL
KAPA Taq	5 μL
DW	4.2μL
Total	10 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	35 cycle
Cycle 2	59.3℃	30 sec	Annealing	35 cycle
Cycle 3	68℃	90 sec	Elongation	35 cycle
Store	4℃	Hold	Store

Colony PCR

Mitsumoto

BLA - BBa_B0015 on pSB1C3, nothing(other negative control)

Reagent	Volume
Single Colony	-
XbaⅠ - BLA - F 10 μM	0.4 μL
200DN - PS - R 10 μM	0.4 μL
KAPA Taq	5 μL
DW	4.2μL
Total	10 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	35 cycle
Cycle 2	56.2℃	30 sec	Annealing	35 cycle
Cycle 3	68℃	90 sec	Elongation	35 cycle
Store	4℃	Hold	Store

Colony PCR

Mitsumoto

BBa_B0031 on pSB1A2

Reagent	Volume
Single Colony	-
100UP - EX - F 10 μM	0.4 μL
200DN - PS - R 10 μM	0.4 μL
KAPA Taq	5 μL
DW	4.2μL
Total	10 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	35 cycle
Cycle 2	56.2℃	30 sec	Annealing	35 cycle
Cycle 3	68℃	90 sec	Elongation	35 cycle
Store	4℃	Hold	Store

Colony PCR

Mitsumoto

BBa_R0010 - BBa_B0034 - Thanatin - BBa_B0015 on pSB1C3

Reagent	Volume
Single Colony	-
100UP - EX - F 10 μM	0.4 μL
200DN - PS - R 10 μM	0.4 μL
KAPA Taq	5 μL
DW	4.2μL
Total	10 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	35 cycle
Cycle 2	61.6℃	30 sec	Annealing	35 cycle
Cycle 3	68℃	90 sec	Elongation	35 cycle
Store	4℃	Hold	Store

Colony PCR

Mitsumoto

BBa_R0010 - BBa_B0034 - 10 x His tag - TEV - Thanatin - BBa_B0015 on pSB1C3, nothing(other negative control)

Reagent	Volume
Single Colony	-
100UP - EX - F 10 μM	0.4 μL
200DN - PS - R 10 μM	0.4 μL
KAPA Taq	5 μL
DW	4.2μL
Total	10 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	35 cycle
Cycle 2	61.6℃	30 sec	Annealing	35 cycle
Cycle 3	68℃	90 sec	Elongation	35 cycle
Store	4℃	Hold	Store

Electrophoresis

Mitsumoto

SpeⅠ - BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - ABF-2 - BBa_B0015 - PstⅠ, EcoRⅠ - BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - Thanatin - BBa_B0015 - PstⅠ, EcoRⅠ - BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - 10 x His tag - TEV - Thanatin - BBa_B0015 - PstⅠ, pSB1C3 EcoRⅠ & PstⅠ(Digestion product)

Gel Concentration	Voltage	Time	Buffer
1%	100 V	40 min	1/2 x TBE

Electrophoresis

Mitsumoto

XbaⅠ - BBa_B0034 - XbaⅠ / PstⅠ scar - BLA - BBa_B0015 - PstⅠ (Digestion product)

Gel Concentration	Voltage	Time	Buffer
1%	100 V	40 min	1/2 x TBE

Electrophoresis

Mitsumoto

pSB1C3 XbaⅠ & SpeⅠ(Digestion product)

Gel Concentration	Voltage	Time	Buffer
1%	100 V	40 min	1/2 x TBE

Electrophoresis

Mitsumoto

EcoRⅠ - standardized BLZ - SpeⅠ, EcoRⅠ - standardized HLZ - SpeⅠ, EcoRⅠ - codon optimized ABF-2 - SpeⅠ (Digestion product)

Gel Concentration	Voltage	Time	Buffer
1%	100 V	40 min	1/2 x TBE

Electrophoresis

Mitsumoto

EcoRⅠ - BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - ABF-2 - BBa_B0015 - PstⅠ, EcoRⅠ - BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ - Thanatin - BBa_B0015 - SpeⅠ, EcoRⅠ - BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - 10 x His tag - TEV - Thanatin - BBa_B0015 - SpeⅠ, pSB1C3 (Digestion product)

Gel Concentration	Voltage	Time	Buffer
1%	100 V	40 min	1/2 x TBE

Electrophoresis

Mitsumoto

pSB1C3 XbaⅠ & SpeⅠ(Digestion product)

Gel Concentration	Voltage	Time	Buffer
1%	100 V	40 min	1/2 x TBE

Electrophoresis

Mitsumoto

XbaⅠ - BBa_B0034 - XbaⅠ / SpeⅠ scar - BLA - BBa_B0015 - PstⅠ XbaⅠ & PstⅠ (Digestion product)

Gel Concentration	Voltage	Time	Buffer
1%	100 V	40 min	1/2 x TBE

Electrophoresis

Mitsumoto

EcoRⅠ - BLZ - SpeⅠ, EcoRⅠ - HLZ - SpeⅠ, EcoRⅠ - ABF-2 - SpeⅠ (Digestion product)

Gel Concentration	Voltage	Time	Buffer
1%	100 V	40 min	1/2 x TBE

Digestion

Mitsumoto

BBa_I0500 - BBa_B0034 on pSB1C3, BBa_I0500 - B0033 on pSB1C3, BBa_I0500 - B0032 on pSB1C3

Reagent	Volume
BBa_I0500 - BBa_B0034 on pSB1C3, BBa_I0500 - B0033 on pSB1C3, BBa_I0500 - B0032 on pSB1C3	20 μL
SpeⅠ	2 μL
PstⅠ	2 μL
10 x H Buffer	3 μL
DW	3 μL
Total	30 μL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	60℃	15 min	Inactivation
Store	4℃	Hold	Store

Digestion

Mitsumoto

Ag43 - Thanatin (1mer)

Reagent	Volume
Ag43 - Thanatin (1mer)	20 μL
BglⅡ	2 μL
CutSmart Buffer	3 μL
DW	5 μL
Total	30 μL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	60℃	15 min	Inactivation
Store	4℃	Hold	Store

2015/09/05

Colony PCR

Fujita, Mimata

BLZ - BBa_B0015 on pSB1C3, HLZ - BBa_B0015 on pSB1C3, ABF-2 - BBa_B0015 on pSB1C3, BBa_R0010 - BBa_B0034 - 10 x His tag - TEV - Thanatin - BBa_B0015, BBa_R0010 - BBa_B0034 - Thanatin - BBa_B0015 on pSB1C3, BBa_R0010 - BBa_B0034 - ABF-2 - BBa_B0015 on pSB1C3, BBa_I0500 - BBa_B0034 - BLA - BBa_B0015 on pSB1C3

Reagent	Volume
Single Colony	-
100UP - EX - F 10 µM	0.4 µL
200DN - PS - R 10 µM	0.4 µL
KAPA Taq	5.0 µL
DW	4.2 µL
Total	10 µL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	35 cycle
Cycle 2	57.6℃	30 sec	Annealing	35 cycle
Cycle 3	72℃	210 sec	Elongation	35 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Electrophoresis

Fujita, Mimata

BLZ - BBa_B0015 on pSB1C3, HLZ - BBa_B0015 on pSB1C3, ABF-2 - BBa_B0015 on pSB1C3, BBa_R0010 - BBa_B0034 - 10 x His tag - TEV - Thanatin - BBa_B0015, BBa_R0010 - BBa_B0034 - Thanatin - BBa_B0015 on pSB1C3, BBa_R0010 - BBa_B0034 - ABF-2 - BBa_B0015 on pSB1C3, BBa_I0500 - BBa_B0034 - BLA - BBa_B0015 on pSB1C3

Gel Concentration	Voltage	Time	Buffer
1%	V	30 min	1/2 x TBE

Mini-prep

Mimata

BLZ - BBa_B0015 on pSB1C3, HLZ - BBa_B0015 on pSB1C3, ABF-2 - BBa_B0015 on pSB1C3, BBa_R0010 - BBa_B0034 - 10 x His tag - TEV - Thanatin - BBa_B0015, BBa_R0010 - BBa_B0034 - Thanatin - BBa_B0015 on pSB1C3, BBa_R0010 - BBa_B0034 - ABF-2 - BBa_B0015 on pSB1C3, BBa_I0500 - BBa_B0034 - BLA - BBa_B0015 on pSB1C3
FastGene^TM Plasmid mini kit (Nippon Genetics Co.,Ltd)
standard protocol

Ligation

Mimata

pSB1C3 / Prefix - BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - Thanatin - BBa_B0015 - Suffix

Reagent	Volume
pSB1C3	2.5 µL
Prefix - BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - Thanatin - BBa_B0015 - Suffix	40 µL
Mighty Mix	42.5 µL
Total	85 µL

Ligation

Step	Temp.	Time	Process
1	16℃	30 min	Ligation
2	70℃	10 min	Inactivation
Store	4℃	Hold	Store

Ligation

Mimata

pSB1C3 / Prefix - BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - 10 × His tag - TEV - Thanatin - BBa_B0015

Reagent	Volume
pSB1C3	2.5 µL
Prefix - BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - 10 × His tag - TEV - Thanatin - BBa_B0015	40 µL
Mighty Mix	42.5 µL
Total	85 µL

Ligation

Step	Temp.	Time	Process
1	16℃	30 min	Ligation
2	70℃	10 min	Inactivation
Store	4℃	Hold	Store

Electrophoresis

Mimata

Thanatin on pSB1C3, TEV - Thanatin on pSB1C3

Gel Concentration	Voltage	Time	Buffer
1%	100 V	60 min	1/2 x TBE

Digestion

Sakai

BBa_I0500 - BBa_B0034, BBa_I0500 - BBa_B0033, BBa_I0500 - BBa_B0032

Reagent	Volume
BBa_I0500 - BBa_B0034, BBa_I0500 - BBa_B0033, BBa_I0500 - BBa_B0032	20 µL
PstⅠ	1.5 µL
SpeⅠ	1.5 µL
10 x H Buffer	3 µL
DW	4 µL
Total	30 µL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	80℃	20 min	Inactivation
Store	4℃	Hold	Store

Digestion

Sakai

Ag43 - Thanatin

Reagent	Volume
Ag43 - Thanatin	20 µL
BglⅡ	2.0 µL
CutSmart Buffer	3 µL
DW	5 µL
Total	30 µL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	80℃	20 min	Inactivation
Store	4℃	Hold	Store

2015/09/06

Colony PCR

Fujita

BLZ - BBa_B0015 on pSB1C3, HLZ - BBa_B0015 on pSB1C3, ABF-2 - BBa_B0015 on pSB1C3, BBa_I0500 - BBa_B0034 - BLA - BBa_B0015 on pSB1C3

Reagent	Volume
Single Colony	-
100UP - EX - F 10 µM	0.4 µL
200DN - PS - R 10 µM	0.4 µL
KAPA Taq	5.0 µL
DW	4.2 µL
Total	10 µL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	35 cycle
Cycle 2	57.6℃	30 sec	Annealing	35 cycle
Cycle 3	72℃	210 sec	Elongation	35 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Electrophoresis

Fujita

BLZ - BBa_B0015 on pSB1C3, HLZ - BBa_B0015 on pSB1C3, ABF-2 - BBa_B0015 on pSB1C3, BBa_I0500 - BBa_B0034 - BLA - BBa_B0015 on pSB1C3 (Colony PCR product)

Gel Concentration	Voltage	Time	Buffer
1%	100 V	30 min	1/2 x TBE

2015/09/07

Electrophoresis

Nishimura

Ag43 - Thanatin -BglⅡ (Dephosphorylation and Digestion product, Mini-prep product)

Gel Concentration	Voltage	Time	Buffer
1%	100 V	60 min	1/2x TBE

Sequencing

Nishimura、Fujita

Ag43 Thanatin

Reagent	Volume
Ag43 Thanatin	1 μL
pBAD Fw / Ag43-Rv	1.5 μL
BigDye Terminator	1 μL
5x Sequencing Buffer	1.5 μL
DW	5 μL
Total	10 μL

Sequencing

Step	Temp.	Time	Process	Cycle
Start	96℃	10 sec	Denaturation
Cycle 1	50℃	5 sec	-	25 cycle
Cycle 2	60℃	240 sec	-	25 cycle
Store	4℃	Hold	Store

Sequencing

Nishimura, Fujita

pBAD_B0031

Reagent	Volume
pBAD_B0031	1 μL
100UP - EX - F / 200DN - PS - R	1.5 μL
BigDye Terminator	1 μL
5x Sequencing Buffer	1.5 μL
DW	5 μL
Total	10 μL

Sequencing

Step	Temp.	Time	Process	Cycle
Start	96℃	10 sec	Denaturation
Cycle 1	50℃	5 sec	-	25 cycle
Cycle 2	60℃	240 sec	-	25 cycle
Store	4℃	Hold	Store

Ethanol Precipitation

Nishimura

Ag43 thanatin on pSB1C3, pBAD_B0031

Added 2 μL of NaOAc, 1.5 μL of glycogen and 50 μL of 100% ethanol.
Left it at 24℃ for 15 min.
Centrifuged at 15,000 rpm for 15 min at 24℃.
Removed supernatant and added 100 μL of 70% ethanol.
Centrifuged at 15,000 rpm for 10 min at 24℃.
Removed supernatant and air-dried at room temperature with light shield.
Suspended with 11 μL of Hidi.

Liquid Culture

Mimata, Nishimura

Ag43 - Thanatin on pSB1C3, BBa_I0500 - BBa_B0032 on pSB1C3, BBa_I0500 - BBa_B0033 on pSB1C3, BBa_I0500 - BBa_B0034 on pSB1C3

Reagent	Volume
Single Colony	-
LBC	2000 μL
Chloramphenicol	2 μL

Cultured for 16 hours.

Digestion

Ono

Ag43 - Thanatin pSB1C3 (Mini-prep product)

Reagent	Volume
Ag43 - Thanatin pSB1C3 (Mini-prep product)	20 μL
BglⅡ	1 μL
DW	6 μL
10 × H Buffer	3 μL
Total	20 μL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	70℃	15 min	Inactivation
Store	4℃	Hold	Store

Electrophoresis

Nishimura

Ag43 - Thanatin -BglⅡ (Digestion product)

Gel Concentration	Voltage	Time	Buffer
1%	100 V	60 min	1/2x TBE

Colony PCR

Ono, Sakai

Ag43 - Thanatin on pSB1C3

Reagent	Volume
Single Colony	-
Agsp-BamH1-Spidroin 10 μM	0.4 μL
Ag43 - bunit - R 10 μM	0.4 μL
KAPA Taq	5 μL
DW	4.2 μL
Total	10 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	40 cycle
Cycle 2	65℃	30 sec	Annealing	40 cycle
Cycle 3	72℃	40 sec	Elongation	40 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Colony PCR

Ono, Sakai

Ag43 - Thanatin on pSB1C3

Reagent	Volume
Single Colony	-
Agsp-BamH1-Spidroin 10 μM	0.4 μL
BglⅡ - D - Thanatin - R 10 μM	0.4 μL
KAPA Taq	5 μL
DW	4.2 μL
Total	10 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	40 cycle
Cycle 2	65℃	30 sec	Annealing	40 cycle
Cycle 3	72℃	40 sec	Elongation	40 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Colony PCR

Ono, Sakai

BBa_B0031 on pSB1C3 (as a positive control)

Reagent	Volume
Single Colony	-
100UP - EX - F 10 μM	0.4 μL
200DN - PS - R 10 μM	0.4 μL
KAPA Taq	5 μL
DW	4.2 μL
Total	10 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	40 cycle
Cycle 2	57℃	30 sec	Annealing	40 cycle
Cycle 3	72℃	40 sec	Elongation	40 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Electrophoresis

Ono

Ag43 - Thanatin on pSB1C3 (Colony PCR 3STEP product), BBa_B0031 on pSB1C3 (as a positive control)

Gel Concentration	Voltage	Time	Buffer
1%	100 V	40 min	1/2x TBE

Transformation

Sakai

Ag43 - Thanatin on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - P_casei - RBS_casei - Sec signal sequence on pSB1C3

Added 1.0 μL of Ag43-Thanatin on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - P_casei - RBS_casei - Sec signal sequence on pSB1C3 to 50 μL of thawed competent cells (JM1O9) on ice.
Incubated on ice for 30 min.
Heat-shocked for 60 sec at 42℃.
Added 200 μL of LB.
Incubated the cells for 2 hrs at 37℃.
Spread 300 μL of the culture onto plate with LBChloramphenicol.
Incubated the plate at 37℃ for 2 hours.

Ethanol Precipitation

Onoda

HLA, BLA, HLZ, BLZ(going through GP3 solution)

Added 25 μL of NaOAc, 1.5 μL of glycogen and 750 μL of 100% ethanol.
Left it at -80℃ for 1 hr.
Centrifuged at 15,000 rpm for 15 min at 4℃.
Removed supernatant and added 100 μL of 70% ethanol.
Centrifuged at 15,000 rpm for 10 min at 4℃.
Removed supernatant and air-dried at room temperature with light shield.
Suspended with 10 μL of TE.

Electrophoresis

Onoda

HLA, BLA, HLZ, BLZ (Ethanol Precipitation)

Gel Concentration	Voltage	Time	Buffer
1%	100 V	40 min	1/2x TBE

Colony PCR

Onoda

HLA, HLZ, BLZ

Reagent	Volume
Single Colony	-
XbalⅠ - HLA - F, XbalⅠ - HLZ - F, XbalⅠ - BLZ - F 10 μM	0.4 μL
200DN - PS - R 10 μM	0.4 μL
Kapa-Taq	5 μL
DW	4.2 μL
Total	10 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	40 cycle
Cycle 2	61.5℃	30 sec	Annealing	40 cycle
Cycle 3	72℃	60 sec	Elongation	40 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Colony PCR

Onoda

BLA, BBa_B0031 (as a positive control)

Reagent	Volume
Single Colony	-
BalⅠ - BLA - F, 100UP - EX - F 10 μM	0.4 μL
200DN - PS - R 10 μM	0.4 μL
Kapa-Taq	5 μL
DW	4.2 μL
Total	10 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	40 cycle
Cycle 2	57℃	30 sec	Annealing	40 cycle
Cycle 3	72℃	60 sec	Elongation	40 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Electrophoresis

Sakai

HLA - BBa_B0015, HLZ - BBa_B0015, BLA - BBa_B0015, BLZ - BBa_B0015

Gel Concentration	Voltage	Time	Buffer
1%	100 V	60 min	1/2x TBE

Ligation

Nishimura, Ono, Fujita

BBa_B0015 on pSB1C3 / BamHⅠ - Thanatin - BglⅡ

Reagent	Volume
BBa_B0015 on pSB1C3	10 μL
BamHⅠ - Thanatin - BglⅡ	1 μL
T4 Ligase	1 μL
10 × T4 Ligase	2 μL
DW	6 μL
Total	20 μL

Ligation

Step	Temp.	Time	Process
1	16℃	60 min	Ligation
Store	4℃	Hold	Store

2015/09/08

Colony PCR

Nishimura

HLA, HLZ, BLZ

Reagent	Volume
Single Colony	-
XbalⅠ - HLA - F, XbalⅠ - HLZ - F, XbalⅠ - BLZ - F 10 μM	0.4 μL
200DN - PS - R 10 μM	0.4 μL
Kapa-Taq	5 μL
DW	4.2 μL
Total	10 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	40 cycle
Cycle 2	61.5℃	30 sec	Annealing	40 cycle
Cycle 3	72℃	60 sec	Elongation	40 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Colony PCR

Nishimura

BLA, BBa_B0031 (as a positive control)

Reagent	Volume
Single Colony	-
BalⅠ - BLA - F, 100UP - EX - F 10 μM	0.4 μL
200DN - PS - R 10 μM	0.4 μL
Kapa-Taq	5 μL
DW	4.2 μL
Total	10 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	40 cycle
Cycle 2	57℃	30 sec	Annealing	40 cycle
Cycle 3	72℃	60 sec	Elongation	40 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Transformation

Nishimura

Ag43 - Thanatin on pSB1C3 (Ligation product)

Added 5 μL of Ag43 - Thanatin on pSB1C3 (Ligation product) to 50 μL of thawed competent cells (DH5α) on ice.
Incubated on ice for 30 min.
Heat-shocked for 30 sec at 42℃.
Added 200 μL of LB.
Incubated the cells for 2 hrs at 37℃.
Spread 300 μL of the culture onto plate with LBC.
Incubated the plate at 37℃ for 16 hours.

Electrophoresis

Nishimura

Gel Concentration	Voltage	Time	Buffer
1%	100 V	50 min	1/2x TBE

Mini-prep

Fujita

BLZ - BBa_B0015, HLZ - BBa_B0015, ABF-2 - BBa_B0015, BBa_R0010 - BBa_B0034 - ABF-2-BBa_B0015, BBa_I0500 - BBa_B0034 - BLA - BBa_B0015
FastGene^TM Plasmid mini kit (Nippon Genetics Co.,Ltd)
standard protocol

Liquid Culture

Nishimura

Ag43 - Thanatin on pSB1C3 into DH5α

Reagent	Volume
Single Colony	-
LB	2000 μL
Chloramphenicol	2 μL

Cultured for 16 hours.

Sequencing

Ito

Ag43 - Thanatin

Reagent	Volume
Ag43 - Thanatin	1 μL
BBa_I0500 - f2 / 200 - β domain - Ag43 - R	1.5 μL
BigDye Terminator	1 μL
5 x Sequencing Buffer	1.5 μL
DW	5 μL
Total	10 μL

Sequencing

Step	Temp.	Time	Process	Cycle
Start	96℃	10 sec	Denaturation
Cycle 1	50℃	5 sec	-	25 cycle
Cycle 2	60℃	240 sec	-	25 cycle
Store	4℃	Hold	Store

Sequencing

Ito

ABF-2, BLZ - BBa_B0015, BBa_I0500 - BLA, BBa_R0010 - ABF-2, HLZ, BBa_I0500 on pSB1A2, BBa_I0500 on pSB1C3

Reagent	Volume
ABF - 2	1 μL
100UP - EX - F / 200DN - PS - R	1.5 μL
Ready Reaction Premix	1 μL
5 x Sequencing Buffer	1.5 μL
DW	5 μL
Total	10 μL

Sequencing

Step	Temp.	Time	Process	Cycle
Start	96℃	10 sec	Denaturation
Cycle 1	50℃	5 sec	-	25 cycle
Cycle 2	60℃	240 sec	-	25 cycle
Store	4℃	Hold	Store

2015/09/09

Colony PCR

Mitsumoto

EcoRⅠ - BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - 10 x His tag - TEV - Thanatin - BBa_B0015 - PstⅠ, EcoRⅠ - BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - Thanatin - BBa_B0015 - PstⅠ

Reagent	Volume
Single Colony	-
100UP - EX - F 10 μM	0.4 μL
200DN - PS - R 10 μM	0.4 μL
KAPA Taq	5 μL
DW	4.2 μL
Total	10 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	35 cycle
Cycle 2	57.6℃	30 sec	Annealing	35 cycle
Cycle 3	68℃	150 sec	Elongation	35 cycle
Store	4℃	Hold	Store

Ethanol Precipitation

Nishimura

ABF-2,BLZ - BBa_B0015, BBa_I0500, BBa_R0010 - ABF-2, HLZ, BBa_I0500, BBa_I0500

Added 2 μL of NaOAc, 1.5 μL of glycogen and 50 μL of 100% ethanol.
Left it at 24℃ for 15min.
Centrifuged at 15,000 rpm for 15 min at 24℃.
Removed supernatant and added 100 μL of 70% ethanol.
Centrifuged at 15,000 rpm for 10 min at 24℃.
Removed supernatant and air-dried at room temperature with light shield.
Suspended with 11 μL of Hidi.

Mini-prep

Nishimura

Ag43 - Thanatin (Liquid culturing product)
FastGene^TM Plasmid mini kit (Nippon Genetics Co.,Ltd)
standard protocol

Digestion

Ono

Ag43 - Thanatin (Mini-prep product)

Reagent	Volume
Ag43 - Thanatin (Mini-prep product)	10 μL
BglⅡ	1 μL
SpeⅠ	1 μL
10 × H Buffer	2 μL
DW	6 μL
Total	20 μL

Digestion

Step	Temp.	Time	Process
1	37℃	60 min	Digestion
2	20 min	Inactivation
Store	4℃	Hold	Store

PCR

Nishimura

Ag43 - Thanatin (Mini-prep product)

Reagent	Volume
Ag43 - Thanatin (Mini-prep product)	1 μL
BamHⅠ - thanatin - F 10 μM	1 μL
200DN - PS - R 10 μM	1 μL
KOD Plus NEO	1 μL
KOD Plus NEO 10x Buffer	5 μL
2 mM dNTPS	5 μL
25 mM MgSO₄	3 μL
DW	33 μL
Total	50 μL

2 Step Cycle (Tm value ≥ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	35 cycle
Cycle 2	68℃	120 sec	Annealing / Elongation	35 cycle
Store	4℃	Hold	Store

Electrophoresis

Nishimura

Ag43 - Thanatin (Digestion product, PCR 2STEP Product)

Gel Concentration	Voltage	Time	Buffer
1%	100 V	50 min	1/2x TBE

Sequencing

Nishimura, Ono

Ag43 - Thanatin (Mini-prep product)

Reagent	Volume
Ag43 - Thanatin (Mini-prep product)	1 μL
pbad - F / 200DN Ag43 - R	1.5 μL
Ready Reaction Premix	1 μL
BigDye Terminator	1.5 μL
DW	5 μL
Total	10 μL

Sequencing

Step	Temp.	Time	Process	Cycle
Start	96℃	10 sec	Denaturation
Cycle 1	50℃	5 sec	-	25 cycle
Cycle 2	60℃	240 sec	-	25 cycle
Store	4℃	Hold	Store

PCR

Ono, Nishimura

Signal peptide - Thanatin - BglⅡ - β domain (Mini-prep product)

Reagent	Volume
Signal peptide - Thanatin - BglⅡ - β domain (Mini-prep product)	1 μL
Agsp - BamHⅠ - Spidroin 10 μM	1 μL
200DN - PS - R 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	5 μL
2 mM dNTPs	5 μL
25 mM MgSO₄	3 μL
DW	33 μL
Total	50 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	30 cycle
Cycle 2	65℃	30 sec	Annealing	30 cycle
Cycle 3	68℃	120 sec	Elongation	30 cycle
Store	4℃	Hold	Store

PCR

Ono, Nishimura

Signal peptide - Thanatin - BglⅡ - β domain (Mini-prep product)

Reagent	Volume
Signal peptide - Thanatin - BglⅡ - β domain (Mini-prep product)	1 μL
Ag43 - F4 10 μM	1 μL
200 β - domain Ag43 - R 10 μM	1 μL
KOD - FX - NEO	1 μL
10 × PCR Buffer for KOD -FX - NEO	25 μL
2 mM dNTPs	5 μL
25 mM MgSO₄	3 μL
DW	13 μL
Total	50 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	98℃	20 sec	Denaturation	30 cycle
Cycle 2	60℃	15 sec	Annealing	30 cycle
Cycle 3	68℃	180 sec	Elongation	30 cycle
Store	4℃	Hold	Store

PCR Purification

Nishimura

FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
Purification of PCR products

Electrophoresis

Nishimura

Thanatin - β domain - BBa_B0015, pSB1C3 - BBa_R0010 - Signal peptide - Thanatin (PCR 3STEP product, PCR purifying product)

Gel Concentration	Voltage	Time	Buffer
1%	100 V	40 min	1/2 x TBE

Digestion

Ito

BBa_I0500 - B0034 - Thanatin - BglⅡ - Ag43 β domain - BBa_B0015 on pSB1C3

Reagent	Volume
BBa_I0500 - B0034 - Thanatin - BglⅡ - Ag43 β domain - BBa_B0015 on pSB1C3	10 μL
BglⅡ	1 μL
Spe1	1 μL
10 x H Buffer	2 μL
DW	6 μL
Total	20 μL

Digestion

Step	Temp.	Time	Process
1	37℃	60 min	Digestion
2	80℃	20 min	Inactivation
Store	4℃	Hold	Store

PCR

Ito

Ag43 - Thanatin on pSB1C3

Reagent	Volume
Ag43 - Thanatin on pSB1C3	1 μL
BamHⅠ - Thanatin - Fw 10 μM	1 μL
200DN - PS - R 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	5 μL
2 mM dNTPs	5 μL
25 mM MgSO₄	3 μL
DW	33 μL
Total	50 μL

2 Step Cycle (Tm value ≥ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	35 cycle
Cycle 2	68℃	120 sec	Annealing / Elongation	35 cycle
Store	4℃	Hold	Store

Sequencing

Ito

BBa_I0500 - BLA, ABF-2

Reagent	Volume
BBa_I0500 - BLA, ABF-2	1 μL
100UP - EX - F / 200DN - PS - R	1.5 μL
Ready Reaction Premix	1 μL
5 x Sequencing Buffer	1.5 μL
DW	5 μL
Total	10 μL

Sequencing

Step	Temp.	Time	Process	Cycle
Start	96℃	10 sec	Denaturation
Cycle 1	50℃	5 sec	-	25 cycle
Cycle 2	60℃	240 sec	-	25 cycle
Store	4℃	Hold	Store

Sequencing

Ito

BBa_I0500 - BLA

Reagent	Volume
BBa_I0500 - BLA	1 μL
pbad - f2 / 200 - β domain - Ag43 - Rv	1.5 μL
Ready Reaction Premix	1 μL
5 x Sequencing Buffer	1.5 μL
DW	5 μL
Total	10 μL

Sequencing

Step	Temp.	Time	Process	Cycle
Start	96℃	10 sec	Denaturation
Cycle 1	50℃	5 sec	-	25 cycle
Cycle 2	60℃	240 sec	-	25 cycle
Store	4℃	Hold	Store

2015/09/10

Mini-prep

Mitsumoto

EcoRⅠ - BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - 10 x His tag - TEV - Thanatin - BBa_B0015 - PstⅠ
FastGene^TM Plasmid mini kit (Nippon Genetics Co.,Ltd)
standard protocol

PCR

Nishimura

Signal peptide - Thanatin - BglⅡ - β domain(Mini-prep product)

Reagent	Volume
Signal peptide - Thanatin - BglⅡ - β domain (Mini-prep product)	1 μL
Agsp - BamHⅠ - Spidroin 10 μM	1 μL
200DN - PS - R 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	5 μL
2 mM dNTPs	5 μL
25 mM MgSO₄	3 μL
DW	33 μL
Total	50 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	30 cycle
Cycle 2	65℃	30 sec	Annealing	30 cycle
Cycle 3	68℃	120 sec	Elongation	30 cycle
Store	4℃	Hold	Store

PCR

Nishimura

Signal peptide - Thanatin - BglⅡ - β domain (Mini-prep product)

Reagent	Volume
Signal peptide - Thanatin - BglⅡ - β domain (Mini-prep product)	1 μL
Ag43 - F4 10 μM	1 μL
200 β - domain Ag43 - R 10 μM	1 μL
KOD - FX - NEO	1 μL
10 × PCR Buffer for KOD -FX - NEO	25 μL
2 mM dNTPs	5 μL
25 mM MgSO₄	3 μL
DW	13 μL
Total	50 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	98℃	20 sec	Denaturation	30 cycle
Cycle 2	60℃	15 sec	Annealing	30 cycle
Cycle 3	68℃	180 sec	Elongation	30 cycle
Store	4℃	Hold	Store

PCR Purification

Nishimura

Thanatin - β domain BBa_B0015, pSB1C3 - BBa_R0010 - Signal peptide - Thanatin (PCR 3STEP product)
FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
Purification of PCR products

Electrophoresis

Nishimjura

Thanatin - β domain BBa_B0015 (PCR 3STEP product, PCR purifying product)

Gel Concentration	Voltage	Time	Buffer
1%	100 V	40 min	1/2 x TBE

Electrophoresis

Nishimjura

pSB1C3 - BBa_R0010 - Signal peptide - Thanatin (PCR 3STEP product, PCR purifying product)

Gel Concentration	Voltage	Time	Buffer
1%	100 V	40 min	1/2 x TBE

PCR

Nishimura

Signal peptide - Thanatin - BglⅡ - β domain (Mini-prep product)

Reagent	Volume
Signal peptide - Thanatin - BglⅡ - β domain (Mini-prep product)	1 μL
Ag43 - F4 10 μM	1 μL
200 β - domain Ag43 - R 10 μM	1 μL
KOD - FX - NEO	1 μL
10 × PCR Buffer for KOD -FX - NEO	25 μL
2 mM dNTPs	5 μL
25 mM MgSO₄	3 μL
DW	13 μL
Total	50 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	98℃	20 sec	Denaturation	30 cycle
Cycle 2	60℃	15 sec	Annealing	30 cycle
Cycle 3	68℃	180 sec	Elongation	30 cycle
Store	4℃	Hold	Store

PCR Purification

Nishimura

pSB1C3 - BBa_R0010 - Signal peptide - Thanatin (PCR 3STEP product)
FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
Purification of PCR products

Electrophoresis

Nishimjura

pSB1C3 - BBa_R0010 - Signal peptide - Thanatin (PCR 3STEP product, PCR purifying product)

Gel Concentration	Voltage	Time	Buffer
1%	100 V	40 min	1/2 x TBE

Digestion

Nishimura, Fujita

Reagent	Volume
Thanatin - β domain BBa_B0015	25 μL
BamHⅠ	3 μL
SpeⅠ	0.5 μL
DW	16.5 μL
10 × K Buffer	5 μL
Total	50 μL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	70℃	20 min	Inactivation
Store	4℃	Hold	Store

Digestion

Nishimura, Fujita

Reagent	Volume
pSB1C3 - BBa_R0010 - Signal peptide - Thanatin	25 μL
BglⅡ	4 μL
SpeⅠ	0.5 μL
DW	15.5 μL
10 × H Buffer	5 μL
Total	50 μL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	70℃	20 min	Inactivation
Store	4℃	Hold	Store

Electrophoresis

Nishimura

Thanatin - β domain BBa_B0015, pSB1C3 - BBa_R0010 - Signal peptide - Thanatin (Digestion product)

Gel Concentration	Voltage	Time	Buffer
1%	100 V	40 min	1/2 x TBE

Gel Extract

Nishimura

Thanatin - β domain BBa_B0015, pSB1C3 - BBa_R0010 - Signal peptide - Thanatin (Digestion product)
FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel

Ligation

Nishimura

pSB1C3 - BBa_R0010 - Signal peptide - Thanatin / Thanatin - β domain BBa_B0015

Reagent	Volume
pSB1C3 - BBa_R0010 - Signal peptide - Thanatin	1.5 μL
Thanatin - β domain BBa_B0015	3.5 μL
Mighty Mix	5 μL
Total	10 μL

Ligation

Step	Temp.	Time	Process
1	60℃	60 min	Ligation
2	80℃	20 min	Inactivation
Store	4℃	Hold	Store

Transformation

Nishimura

BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar - Thanatin - β domain BBa_B0015 on pSB1C3

Added 5 μL of BBa_R0010 - Signal peptide - Thanatin - Bam / Bgl scar- Thanatin - β domain BBa_B0015 on pSB1C3 to 50 μL of thawed competent cells (HIT) on ice.
Incubated on ice for 30 min.
Heat-shocked for 30 sec at 42℃.
Added 500 μL of LB.
Incubated the cells for 2 hrs at 37℃.
Spread 500, 50 μL of the culture onto two plates with LBC.
Incubated the plate at 37℃ for hours.

Transformation

Nishimura

BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar - Thanatin - β domain BBa_B0015 on pSB1C3

Added 5 μL of BBa_R0010 - Signal peptide - Thanatin - Bam / Bgl scar- Thanatin - β domain BBa_B0015 on pSB1C3 to 50 μL of thawed competent cells (DH5α) on ice.
Incubated on ice for 30 min.
Heat-shocked for 30 sec at 42℃.
Added 500 μL of LB.
Incubated the cells for 2 hrs at 37℃.
Spread 500, 50 μL of the culture onto two plates with LBC.
Incubated the plate at 37℃ for hours.

Ethanol Precipitation

Ito

Thanatin ( 2mer )

Added 4 μL of NaOAc, 1.5 μL of glycogen and 120 μL of 100% ethanol.
Left it at -80℃ for 1 hr.
Centrifuged at 15,000 rpm for 15 min at 4℃.
Removed supernatant and added 220 μL of 70% ethanol.
Centrifuged at 15,000 rpm for 10 min at 4℃.
Removed supernatant and air-dried at room temperature with light shield.
Suspended with 10 μL of TE.

Sequencing

Ito

Prefix - BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - 10 × His tag - TEV - Thanatin - BBa_B0015

Reagent	Volume
Prefix - BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - 10 × His tag - TEV - Thanatin - BBa_B0015	1 μL
100UP - EX - F / 200DN - PS - R	1.5 μL
Ready Reaction Premix	1 μL
5 x Sequencing Buffer	1.5 μL
DW	5 μL
Total	10 μL

Sequencing

Step	Temp.	Time	Process	Cycle
Start	96℃	10 sec	Denaturation
Cycle 1	50℃	5 sec	-	25 cycle
Cycle 2	60℃	240 sec	-	25 cycle
Store	4℃	Hold	Store

Sequencing

Ito

Prefix - BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - 10 × His tag - TEV - Thanatin - BBa_B0015

Reagent	Volume
Prefix - BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - 10 × His tag - TEV - Thanatin - BBa_B0015	1 μL
BBa_I0500 - f2 / 200 - β domain - Ag43 - Rv	1.5 μL
Ready Reaction Premix	1 μL
5 x Sequencing Buffer	1.5 μL
DW	5 μL
Total	10 μL

Sequencing

Step	Temp.	Time	Process	Cycle
Start	96℃	10 sec	Denaturation
Cycle 1	50℃	5 sec	-	25 cycle
Cycle 2	60℃	240 sec	-	25 cycle
Store	4℃	Hold	Store

2015/09/11

Colony PCR

Mitsumoto

EcoRⅠ - BBa_R0010 - BBa_B0034 - XbaⅠ / PstⅠ scar - 10 x His tag - TEV - Thanatin - BBa_B0015 - PstⅠ, EcoRⅠ - BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - Thanatin - BBa_B0015 - PstⅠ

Reagent	Volume
Single Colony	-
100UP - EX - F 10 μM	0.4 μL
200DN - PS - R 10 μM	0.4 μL
KAPA Taq	5 μL
DW	4.2 μL
Total	10 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	35 cycle
Cycle 2	57.6℃	30 sec	Annealing	35 cycle
Cycle 3	72℃	90 sec	Elongation	35 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Sequencing

Nishimura

BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3

Reagent	Volume
BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3	1 μL
100UP - EX - F / 200DN - PS - R	1.5 μL
BigDye Terminator	1 μL
5 x Sequencing Buffer	1.5 μL
DW	5 μL
Total	10 μL

Sequencing

Step	Temp.	Time	Process	Cycle
Start	96℃	10 sec	Denaturation
Cycle 1	50℃	5 sec	-	25 cycle
Cycle 2	60℃	240 sec	-	25 cycle
Store	4℃	Hold	Store

Ethanol Precipitation

Nishimura

BLA

Added μL of NaOAc, 1.5 μL of glycogen and 150 μL of 100% ethanol.
Left it at -80℃ for 1 hr.
Centrifuged at 15,000 rpm for 15 min at 4℃.
Removed supernatant and added 220 μL of 70% ethanol.
Centrifuged at 15,000 rpm for 10 min at 4℃.
Removed supernatant and air-dried at room temperature with light shield.
Suspended with 10 μL of TE.

Colony PCR

Nishimura

BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3

Reagent	Volume
Single Colony	-
Agsp - BamHⅠ - Spidroin 10 μM	0.4 μL
Ag43 - bunit - R 10 μM	0.4 μL
KAPA Taq	5 μL
DW	4.2 μL
Total	10 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	30 sec	Denaturation	40 cycle
Cycle 2	65℃	30 sec	Annealing	40 cycle
Cycle 3	72℃	40 sec	Elongation	40 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Colony PCR

Nishimura

BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3

Reagent	Volume
Single Colony	-
Agsp - BamHⅠ - Spidroin 10 μM	0.4 μL
BglⅡ - D - Thanatin - R 10 μM	0.4 μL
KAPA Taq	5 μL
DW	4.2 μL
Total	10 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	30 sec	Denaturation	40 cycle
Cycle 2	65℃	30 sec	Annealing	40 cycle
Cycle 3	72℃	40 sec	Elongation	40 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Colony PCR

Nishimura

Prefix - BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - 10 × His tag - TEV - Thanatin - BBa_B0015, Prefix - BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - Thanatin - BBa_B0015 - Suffix, BBa_B0031 (as a positive control)

Reagent	Volume
Single Colony	-
100UP - EX - F 10 μM	0.4 μL
200DN - PS - R 10 μM	0.4 μL
KAPA Taq	5 μL
DW	4.2 μL
Total	10 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	30 sec	Denaturation	40 cycle
Cycle 2	65℃	30 sec	Annealing	40 cycle
Cycle 3	72℃	40 sec	Elongation	40 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Electrophoresis

Nishimura

BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3, Prefix - BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - 10 × His tag - TEV - Thanatin - BBa_B0015, Prefix - BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - Thanatin - BBa_B0015 - Suffix (Colony PCR procduct)

Gel Concentration	Voltage	Time	Buffer
1%	100 V	50 min	1/2 x TBE

2015/09/12

Dephosphorylation

Nishimura

BBa_R0010 - Signal peptide - Thanatin - BglⅡ on pSB1C3

Reagent	Volume
pSB1C3 - BBa_R0010 - Signal peptide - Thanatin - BglⅡ	20 μL
Antarctic Phosphatase	2 μL
Antarctic Phosphatase Buffer	4 μL
DW	14 μL
Total	40 μL

Dephosphorylation

Step	Temp.	Time	Process
1	37℃	30 min	Dephosphorylation
2	65℃	10 min	Inactivation
Store	4℃	Hold	Store

Ligation

Nishimura

pSB1C3 - BBa_R0010 - Signal peptide - Thanatin (Dephosphorylation product) / Thanatin - β domain - BBa_B0015

Reagent	Volume
pSB1C3 - BBa_R0010 - Signal peptide - Thanatin	1.5 μL
Thanatin - β domain - BBa_B0015	3.5 μL
Mighty Mix	5 μL
Total	10 μL

Ligation

Step	Temp.	Time	Process
1	16℃	60 min	Ligation
Store	4℃	Hold	Store

Ligation

Nishimura

BBa_R0010 - Signal peptide - Thanatin on pSB1C3 (Dephosphorylation product) / Thanatin - β domain - BBa_B0015

Reagent	Volume
BBa_R0010 - Signal peptide - Thanatin on pSB1C3	1.5 μL
Thanatin - β domain - BBa_B0015	3.5 μL
Mighty Mix	5 μL
Total	10 μL

Ligation

Step	Temp.	Time	Process
1	60℃	60 min	Ligation
2	80℃	20 min	Inactivation
Store	4℃	Hold	Store

Transformation

Nishimura

BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3 (Ligation product)

Added 5 μL of BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3 to 50 μL of thawed competent cells (DH5α) on ice.
Incubated on ice for 30 min.
Heat-shocked for 30 sec at 42℃.
Added 400 μL of LB.
Incubated the cells for 2 hrs at 37℃.
Spread 300 μL of the culture onto plate with LBC.
Incubated the plate at 37℃ for 16 hours.

Mini-prep

Nishimura

BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3
FastGene^TM Plasmid mini kit (Nippon Genetics Co.,Ltd)
standard protocol

Sequencing

Nishimura

BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3

Reagent	Volume
BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3	1 μL
pbad - f2 / 200 βdomein Ag43 Rv	1.5 μL
Ready Reaction Premix	1 μL
5 x Sequencing Buffer	1.5 μL
DW	5 μL
Total	10 μL

Sequencing

Step	Temp.	Time	Process	Cycle
Start	96℃	10 sec	Denaturation
Cycle 1	50℃	10 sec	-	25 cycle
Cycle 2	60℃	240 sec	-	25 cycle
Store	4℃	Hold	Store

Ethanol Precipitation

Nishimura

BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3

Added 7 μL of DW, 2 μL of NaOAc, 1 μL of glycogen and 50 μL of 100% ethanol.
Left it at 24℃ for 15 min.
Centrifuged at 15,000 rpm for 15 min at 24℃.
Removed supernatant and added 100 μL of 70% ethanol.
Centrifuged at 15,000 rpm for 10 min at 24℃.
Removed supernatant and air-dried at room temperature with light shield.
Suspended with 11 μL of HiDi.

Colony PCR

Nishimura

BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - 10 × His tag - TEV - Thanatin - BBa_B0015 on pSB1C3, BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - Thanatin - BBa_B0015 on pSB1C3

Reagent	Volume
Single Colony	-
100UP - EX - F 10 μM	0.4 μL
200DN - PS - R 10 μM	0.4 μL
KAPA Taq	5 μL
DW	4.2 μL
Total	10 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	40 cycle
Cycle 2	57℃	30 sec	Annealing	40 cycle
Cycle 3	72℃	40 sec	Elongation	40 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Liquid Culture

Nishimura

BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - 10 × His tag - TEV - Thanatin - BBa_B0015 on pSB1C3, BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - Thanatin - BBa_B0015 on pSB1C3

Reagent	Volume
Single Colony	-
LB	2000 μL
Chloramphenicol	2 μL

Cultured for 16 hours.

Electrophoresis

Nishimura

BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - 10 × His tag - TEV - Thanatin - BBa_B0015 on pSB1C3, BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - Thanatin - BBa_B0015 on pSB1C3

Gel Concentration	Voltage	Time	Buffer
1%	100 V	30 min	1/2 x TBE

Colony PCR

Mitsumoto

EcoRⅠ - BBa_R0010 - BBa_B0034 - XbaⅠ / PstⅠ scar - 10 x His tag - TEV - Thanatin - BBa_B0015 - PstⅠ, EcoRⅠ - BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - Thanatin - BBa_B0015 - PstⅠ

Reagent	Volume
Single Colony	-
100UP - EX - F 10 μM	0.4 μL
200DN - PS - R 10 μM	0.4 μL
KAPA Taq	5 μL
DW	4.2 μL
Total	10 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	35 cycle
Cycle 2	57.6℃	30 sec	Annealing	35 cycle
Cycle 3	72℃	90 sec	Elongation	35 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

MIC Test

Onoda, Sakai, Ito

50μM Thanatin

Cultured E. coli DH5α, JM109 in 2 ml of LB overnight.
Took 80 μl of it and cultured for 1 hr in 2 mL of LB culture until the OD₆₀₀ became 0.4.
100,000 fold dilutied it with PB culture.
Spread them on LB plate and counted the number of colony.
Made 20 μl of dilution series of supernatant and cultured 80 μL of diluted DH5α, JM109 at 30℃, 120,000 rpm for 18 hrs.
Incubated in refrigerator.
Observed the appearance of colony and decided the effective concentration of supernatant.

2015/09/13

PCR

Nishimura

BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3

Reagent	Volume
BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3	1 μL
Agsp - BamHⅠ - Spidroin 10 μM	1 μL
200DN - PS - R 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	5 μL
2 mM dNTPs	5 μL
25 mM MgSO₄	3 μL
DW	33 μL
Total	50 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	30 cycle
Cycle 2	65℃	15 sec	Annealing	30 cycle
Cycle 3	68℃	120 sec	Elongation	30 cycle
Store	4℃	Hold	Store

PCR

Nishimura

BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3

Reagent	Volume
BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3	1 μL
Ag43 - f4 10 μM	1 μL
Xba - RBS - scar 10 μM	1 μL
KOD - FX - Neo	1 μL
2x PCR Buffer for KOD - FX - Neo	25 μL
2 mM dNTPs	5 μL
25 mM MgSO₄	3 μL
DW	13 μL
Total	50 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	30 cycle
Cycle 2	60℃	15 sec	Annealing	30 cycle
Cycle 3	68℃	180 sec	Elongation	30 cycle
Store	4℃	Hold	Store

Electrophoresis

Nishimura

BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3

Gel Concentration	Voltage	Time	Buffer
1%	100 V	60 min	1/2 x TBE

PCR Purification

Nishimura

BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3
FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
Purification of PCR products

Digestion

Nishimura

BglⅡ - Thanatin - β domain BBa_B0015

Reagent	Volume
BglⅡ - Thanatin - β domain BBa_B0015	25 μL
BamHⅠ	3 μL
SpeⅠ	0.5 μL
10 × K Buffer	5 μL
DW	16.5 μL
Total	50 μL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	70℃	20 min	Inactivation
Store	4℃	Hold	Store

Digestion

Nishimura

BBa_R0010 - Signal peptide - Thanatin - BglⅡ on pSB1C3

Reagent	Volume
BBa_R0010 - Signal peptide - Thanatin - BglⅡ on pSB1C3	25 μL
BglⅡ	4 μL
SpeⅠ	0.5 μL
10 × H Buffer	5 μL
DW	15.5 μL
Total	50 μL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	70℃	20 min	Inactivation
Store	4℃	Hold	Store

Electrophoresis

Nishimura

BglⅡ - Thanatin - β domain BBa_B0015, BBa_R0010 - Signal peptide - Thanatin - BglⅡ on pSB1C3

Gel Concentration	Voltage	Time	Buffer
1%	100 V	60 min	1/2 x TBE

Gel Extract

Nishimura

BglⅡ - Thanatin - β domain BBa_B0015, BBa_R0010 - Signal peptide - Thanatin - BglⅡ on pSB1C3
FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel

Ligation

Nishimura

BBa_R0010 - Signal peptide - Thanatin - BglⅡ on pSB1C3 ( 2mer ) / Thanatin - β domain - BBa_B0015 ( 1mer )

Reagent	Volume
BBa_R0010 - Signal peptide - Thanatin - BglⅡ on pSB1C3 ( 2mer )	3.5 μL
Thanatin - β domain - BBa_B0015 ( 1mer )	1.5 μL
Mighty Mix	5 μL
Total	10 μL

Ligation

Step	Temp.	Time	Process
1	16℃	30 min	Ligation
2	65℃	10 min	Inactivation
Store	4℃	Hold	Store

Ligation

Nishimura

BBa_R0010 - Signal peptide - Thanatin - BglⅡ on pSB1C3 ( 2mer ) / Thanatin - β domain - BBa_B0015 ( 1mer )

Reagent	Volume
BBa_R0010 - Signal peptide - Thanatin - BglⅡ on pSB1C3 ( 2mer )	1.5 μL
Thanatin - β domain - BBa_B0015 ( 1mer )	3.5 μL
Mighty Mix	5 μL
Total	10 μL

Ligation

Step	Temp.	Time	Process
1	16℃	30 min	Ligation
2	65℃	10 min	Inactivation
Store	4℃	Hold	Store

Ligation

Nishimura

BBa_R0010 - Signal peptide - Thanatin - BglⅡ on pSB1C3 ( 2mer ) / Thanatin - β domain - BBa_B0015 ( 2mer )

Reagent	Volume
BBa_R0010 - Signal peptide - Thanatin - BglⅡ on pSB1C3 ( 2mer )	3.5 μL
Thanatin - β domain - BBa_B0015 ( 2mer )	1.5 μL
Mighty Mix	5 μL
Total	10 μL

Ligation

Step	Temp.	Time	Process
1	16℃	30 min	Ligation
2	65℃	10 min	Inactivation
Store	4℃	Hold	Store

Transformation

Nishimura

BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3, BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3

Added 5 μL of BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3, BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3 to 50 μL of thawed competent cells (DH5α) on ice.
Incubated on ice for 30 min.
Heat-shocked for 30 sec at 42℃.
Added 400 μL of LB.
Incubated the cells for 2 hrs at 37℃.
Spread 300 μL of the culture onto plate with LBC.
Incubated the plate at 37℃ for 16 hours.

2015/09/14

Colony PCR

Nishimura

BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3, BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3

Reagent	Volume
Single Colony	-
Agsp - BamHⅠ - Spindroin 10 μM	0.4 μL
200 - β domein - Ag43 - Rv 10 μM	0.4 μL
KAPA Taq	5 μL
DW	4.2 μL
Total	10 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	30 sec	Denaturation	40 cycle
Cycle 2	65℃	30 sec	Annealing	40 cycle
Cycle 3	72℃	40 sec	Elongation	40 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Electrophoresis

Nishimura

BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3, BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3

Gel Concentration	Voltage	Time	Buffer
1%	100 V	30 min	1/2 x TBE

2015/09/15

Sequencing

Nishimura

BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3, BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3

Reagent	Volume
BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3, BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3	1 μL
pbad - f2	1.5 μL
Ready Reaction Premix	1 μL
5 x Sequencing Buffer	1.5 μL
DW	5 μL
Total	10 μL

Sequencing

Step	Temp.	Time	Process	Cycle
Start	96℃	10 sec	Denaturation
Cycle 1	50℃	5 sec	-	25 cycle
Cycle 2	60℃	240 sec	-	25 cycle
Store	4℃	Hold	Store

Sequencing

Nishimura

BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3, BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3

Reagent	Volume
BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3, BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3	1 μL
200 - β domein - Ag43 - Rv	1.5 μL
Ready Reaction Premix	1 μL
5 x Sequencing Buffer	1.5 μL
DW	5 μL
Total	10 μL

Sequencing

Step	Temp.	Time	Process	Cycle
Start	96℃	10 sec	Denaturation
Cycle 1	50℃	5 sec	-	25 cycle
Cycle 2	60℃	240 sec	-	25 cycle
Store	4℃	Hold	Store

Mini-prep

Nishimura

BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3, BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3
FastGene^TM Plasmid mini kit (Nippon Genetics Co.,Ltd)
standard protocol

Liquid Culture

Nishimura

BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3, BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3

Reagent	Volume
Single Colony	-
LB	2000 μL
Chloramphenicol	2 μL

Cultured for 16 hours.

Mini-prep

Nishimura

BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3, BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3
FastGene^TM Plasmid mini kit (Nippon Genetics Co.,Ltd)
standard protocol

Mini-prep

Nishimura

BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - 10 × His tag - TEV - Thanatin - BBa_B0015 on pSB1C3, BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - Thanatin - BBa_B0015 on pSB1C3
FastGene^TM Plasmid mini kit (Nippon Genetics Co.,Ltd)
Standard Protocol

Liquid Culture

Nishimura

BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - 10 × His tag - TEV - Thanatin - BBa_B0015 on pSB1C3, BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - Thanatin - BBa_B0015 on pSB1C3

Reagent	Volume
Single Colony	-
LB	15000 μL
Chloramphenicol	1.5 μL

Cultured for 16 hours.

Sequencing

Nishimura

BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - 10 × His tag - TEV - Thanatin - BBa_B0015 on pSB1C3, BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - Thanatin - BBa_B0015 on pSB1C3

Reagent	Volume
BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - 10 × His tag - TEV - Thanatin - BBa_B0015 on pSB1C3, BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - Thanatin - BBa_B0015 on pSB1C3	1 μL
100UP - EX - F	1.5 μL
Ready Reaction Premix	1 μL
5 x Sequencing Buffer	1.5 μL
DW	5 μL
Total	10 μL

Sequencing

Step	Temp.	Time	Process	Cycle
Start	96℃	10 sec	Denaturation
Cycle 1	50℃	5 sec	-	25 cycle
Cycle 2	60℃	240 sec	-	25 cycle
Store	4℃	Hold	Store

Sequencing

Nishimura

BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - 10 × His tag - TEV - Thanatin - BBa_B0015 on pSB1C3, BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - Thanatin - BBa_B0015 on pSB1C3

Reagent	Volume
BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - 10 × His tag - TEV - Thanatin - BBa_B0015 on pSB1C3, BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - Thanatin - BBa_B0015 on pSB1C3	1 μL
200DN - PS - R	1.5 μL
Ready Reaction Premix	1 μL
5 x Sequencing Buffer	1.5 μL
DW	5 μL
Total	10 μL

Sequencing

Step	Temp.	Time	Process	Cycle
Start	96℃	10 sec	Denaturation
Cycle 1	50℃	5 sec	-	25 cycle
Cycle 2	60℃	240 sec	-	25 cycle
Store	4℃	Hold	Store

Sequencing

Nishimura

BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - 10 × His tag - TEV - Thanatin - BBa_B0015 on pSB1C3, BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - Thanatin - BBa_B0015 on pSB1C3

Reagent	Volume
BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - 10 × His tag - TEV - Thanatin - BBa_B0015 on pSB1C3, BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - Thanatin - BBa_B0015 on pSB1C3	1 μL
100UP - EX - F	1.5 μL
Ready Reaction Premix	1 μL
5 x Sequencing Buffer	1.5 μL
DW	5 μL
Total	10 μL

Sequencing

Step	Temp.	Time	Process	Cycle
Start	96℃	10 sec	Denaturation
Cycle 1	50℃	5 sec	-	25 cycle
Cycle 2	60℃	240 sec	-	25 cycle
Store	4℃	Hold	Store

Sequencing

Nishimura

BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - 10 × His tag - TEV - Thanatin - BBa_B0015 on pSB1C3, BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - Thanatin - BBa_B0015 on pSB1C3

Reagent	Volume
BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - 10 × His tag - TEV - Thanatin - BBa_B0015 on pSB1C3, BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - Thanatin - BBa_B0015 on pSB1C3	1 μL
200DN - PS - R	1.5 μL
Ready Reaction Premix	1 μL
5 x Sequencing Buffer	1.5 μL
DW	5 μL
Total	10 μL

Sequencing

Step	Temp.	Time	Process	Cycle
Start	96℃	10 sec	Denaturation
Cycle 1	50℃	5 sec	-	25 cycle
Cycle 2	60℃	240 sec	-	25 cycle
Store	4℃	Hold	Store

2015/09/16

PCR

Nishimura

BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3, BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3

Reagent	Volume
BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3, BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3	1 μL
Agsp - BamHⅠ - Spidroin 10 μM	1 μL
200DN - PS - R 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	5 μL
2 mM dNTPs	5 μL
25 mM MgSO₄	3 μL
DW	33 μL
Total	50 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	30 cycle
Cycle 2	65℃	15 sec	Annealing	30 cycle
Cycle 3	68℃	120 sec	Elongation	30 cycle
Store	4℃	Hold	Store

PCR

Nishimura

BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3, BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3

Reagent	Volume
BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3, BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3	1 μL
Ag43 - f4 10 μM	1 μL
XbaⅠ - RBS - scar 10 μM	1 μL
KOD - FX - Neo	1 μL
2x PCR Buffer for KOD - FX - Neo	25 μL
2 mM dNTPs	5 μL
25 mM MgSO₄	3 μL
DW	13 μL
Total	50 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	30 cycle
Cycle 2	60℃	15 sec	Annealing	30 cycle
Cycle 3	68℃	180 sec	Elongation	30 cycle
Store	4℃	Hold	Store

Electrophoresis

Nishimura

BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3, BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3

Gel Concentration	Voltage	Time	Buffer
1%	100 V	60 min	1/2 x TBE

PCR Purification

Nishimura

BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3, BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C3
FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
Purification of PCR products

Ethanol Precipitation

Nishimura

BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - 10 × His tag - TEV - Thanatin - BBa_B0015 on pSB1C3, BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - Thanatin - BBa_B0015 on pSB1C3

Added 7 μL of DW, 2 μL of NaOAc, 1 μL of glycogen and 50 μL of 100% ethanol.
Left it at 24℃ for 15 min.
Centrifuged at 15,000 rpm for 15 min at 24℃.
Removed supernatant and added 100 μL of 70% ethanol.
Centrifuged at 15,000 rpm for 10 min at 24℃.
Removed supernatant and air-dried at room temperature with light shield.
Suspended with 10 μL of HiDi.

2015/09/17

Measurement of Optical Density

Mimata

Ag43 - Thanatin (1mer) into DH5α

Cultured the colony for 10 hours.
Diluted with midium to set OD₆₀₀ to 0.1.
Measured OD₆₀₀ 12 times with an hour interval each.

Measurement of Optical Density

Mimata

Ag43 - Thanatin (2mer) into DH5α

Cultured the colony for 10 hours.
Diluted with midium to set OD₆₀₀ to 0.1.
Measured OD₆₀₀ 12 times with an hour interval each.

Measurement of Optical Density

Mimata

Ag43 - Thanatin (3mer) into DH5α

Cultured the colony for 10 hours.
Diluted with midium to set OD₆₀₀ to 0.1.
Measured OD₆₀₀ 12 times with an hour interval each.

Measurement of Optical Density

Mimata

Ag43 - Thanatin (4mer) into DH5α

Cultured the colony for 10 hours.
Diluted with midium to set OD₆₀₀ to 0.1.
Measured OD₆₀₀ 12 times with an hour interval each.

Measurement of Optical Density

Mimata

Ag43 (without Thanatin) into DH5α

Cultured the colony for 10 hours.
Diluted with midium to set OD₆₀₀ to 0.1.
Measured OD₆₀₀ 12 times with an hour interval each.

MIC Test

Ito, Ono

Thanatin (1mer, 2mer, 3mer, 4mer, nothing (as a negative control))

Cultured E. coli DH5α in 2 ml of LB overnight.
Took 80 μl of it and cultured for 1 hr in 2 mL of LB culture until the OD₆₀₀ became 0.4.
100,000 fold dilutied it with PB culture.
Spread them on LB plate and counted the number of colony.
Made 20 μl of dilution series of supernatant and cultured 80 μL of diluted JM109 at 30℃, 120,000 rpm for 18 hrs.
Incubated in refrigerator.
Observed the appearance of colony and decided the effective concentration of supernatant.

2015/09/18

MIC Test

Ito, Onoda

Thanatin (4mer)

Cultured E. coli DH5α in 2 ml of LB overnight.
Took 80 μl of it and cultured for 1 hr in 2 mL of LB culture until the OD₆₀₀ became 0.4.
100,000 fold dilutied it with PB culture.
Spread them on LB plate and counted the number of colony.
Made 20 μl of dilution series of supernatant and cultured 80 μL of diluted DH5α at 30℃, 120,000 rpm for 18 hrs.
Incubated in refrigerator.
Observed the appearance of colony and decided the effective concentration of supernatant.

←Notebook Top

Team:HokkaidoU Japan/Notebook/ecoli

E. coli

January

2015/01/21

2015/01/22

2015/01/26

March

2015/03/10

Competent Cells

2015/03/11

PCR

PCR Purification

Digestion

Electrophoresis

Gel Extract

Electrophoresis

May

2015/05/13

Transformation

Transformation

Competent Cells

2015/05/27

Transformation

Transformation

2015/05/29

Mini-prep

PCR

2015/05/30

Electrophoresis

Gel Extract

Digestion

Digestion

2015/05/31

Electrophoresis

Gel Extract

Electrophoresis

PCR

June

2015/06/10

Digestion

2015/06/16

PCR

2015/06/17

Electrophoresis

Annealing Oligos and Elongation

2015/06/19

Electrophoresis

Gel Extract

Ligation

2015/06/21

Transformation

July

2015/07/25

Transformation

Transformation

Transformation

Transformation

PCR

Electrophoresis

2015/07/26

Liquid Culture

Mini-prep

Electrophoresis

Gel Extract

2015/07/27

Mini-prep

August

September