Team:HokkaidoU Japan/Notebook/ecoli

Notebook

main1

E. coli

January

2015/01/21

Transformation

Sakurai

BBa_K1524100

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

2015/01/22

Colony PCR

Sakurai

BBa_K1524100

ReagentVolume
Single Colony-
100UP - EX - F 10 µM0.8 µL
XhoⅠ - RBS - NcoⅠ 10 µM0.8 µL
KAPA Taq10 µL
DW8.4 µL
Total20 µL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation35 cycle
Cycle 268℃60 secAnnealing / Elongation35 cycle
Finish68℃60 secFinal Elongation
Store4℃HoldStore

Electrophoresis

Sakurai

BBa_K1524100

Gel ConcentrationVoltageTimeBuffer
1%100 V30 min1/2 x TBE

Liquid Culture

Sakurai

BBa_K1524100

ReagentVolume
Single Colony-
LB2 µL

Cultured for 16 hrs.

2015/01/26

Colony PCR

Sakurai

BBa_K1524100

ReagentVolume
Single Colony-
100UP - EX - F 10 µM0.4 µL
XhoⅠ - RBS - NcoⅠ 10 µM0.4 µL
KAPA Taq5 µL
DW4.2 µL
Total10 µL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation35 cycle
Cycle 268℃60 secAnnealing / Elongation35 cycle
Finish68℃60 secFinal Elongation
Store4℃HoldStore

Electrophoresis

Sakurai

BBa_K1524100

Gel ConcentrationVoltageTimeBuffer
1%100 V30 min1/2 x TBE

Colony PCR

Sakurai

BBa_K1524100

ReagentVolume
Single Colony-
100UP - EX - F 10 µM0.4 µL
XhoⅠ - RBS - NcoⅠ 10 µM0.4 µL
KAPA Taq5 µL
DW4.2 µL
Total10 µL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation35 cycle
Cycle 268℃60 secAnnealing / Elongation35 cycle
Finish68℃60 secFinal Elongation
Store4℃HoldStore

Colony PCR

Sakurai

BBa_K1524100

ReagentVolume
Single Colony-
100UP - EX - F 10 µM0.4 µL
200DN - PS - R 10 µM0.4 µL
KAPA Taq5 µL
DW4.2 µL
Total10 µL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation35 cycle
Cycle 268℃60 secAnnealing / Elongation35 cycle
Finish68℃60 secFinal Elongation
Store4℃HoldStore

March

2015/03/10

Competent Cells

Tanaka,Sakurai

BL21 (DE3) pLysS

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

2015/03/11

PCR

Sakurai

BBa_R0011

ReagentVolume
BBa_R00111 µL
100UP - EX - F 10 µM1.5 µL
200DN - PS - R 10 µM1.5 µL
KOD - Plus - Neo1 µL
10 x PCR Buffer for KOD - Plus - Neo 5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW32 µL
Total50 µL

BBa_0030 - BBa_E1010

ReagentVolume
BBa_0030 - BBa_E10101 µL
100UP - EX - F 10 µM1.5 µL
200DN - PS - R 10 µM1.5 µL
KOD - Plus - Neo1 µL
10 x PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW32 µL
Total50 µL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation30 cycle
Cycle 262.6℃30 secAnnealing30 cycle
Cycle 368℃1 minElongation30 cycle
Store4℃HoldStore

PCR Purification

Sakurai

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

Digestion

Sakurai

BBa_R0011

ReagentVolume
BBa_R001144 µL
XbaⅠ1 µL
CutSmart Buffer5 µL
Total50 µL

BBa_0030 - BBa_E1010

ReagentVolume
BBa_0030 - BBa_E101044 µL
XbaⅠ1 µL
CutSmart Buffer5 µL
Total50 µL

Digestion

StepTemp.TimeProcess
137℃300 minDigestion
260℃15 minInactivation
Store4℃HoldStore

Electrophoresis

Sakurai

BBa_R0011, BBa_0030 - BBa_E1010

Gel ConcentrationVoltageTimeBuffer
1%100 V30 min1/2 x TBE

Gel Extract

Sakurai

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

Electrophoresis

Sakurai

BBa_R0011, BBa_0030 - BBa_E1010

Gel ConcentrationVoltageTimeBuffer
1%100 V30 min1/2 x TBE

May

2015/05/13

Transformation

Onoda

pET15b

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

Transformation

Onoda, Sakurai

pET16b

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

Competent Cells

Onoda

Rosetta

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

2015/05/27

Transformation

Mimata, Onoda, Nishimura

BBa_E0040

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

Transformation

Mimata, Onoda, Ono, Nishimura

mBBa_R0040

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

2015/05/29

Mini-prep

Mimata, Onoda, Ono, Nishimura

BBa_E0040, mBBa_R0040
FastGeneTM Plasmid mini kit (Nippon Genetics Co.,Ltd)
standard protocol

PCR

Onoda, Ono

BBa_E0040

ReagentVolume
BBa_E00401 µL
100UP- EX - F 10 µM1 µL
200DN - PS - R 10 µM1 µL
KOD - FX - Neo1 µL
2 x PCR Buffer for KOD - FX - Neo 5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

mBBa_R0040

ReagentVolume
mBBa_R00401 µL
100UP - EX - F 10 µM1 µL
200DN - PS - R 10 µM1 µL
KOD - FX - Neo1 µL
2 x PCR Buffer for KOD - FX - Neo 5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation30 cycles
Cycle 268℃60 secAnnealing / Elongation30 cycles
Store4℃HoldStore

2015/05/30

Electrophoresis

Mimata, Onoda, Ono, Nishimura

BBa_E0040, mBBa_R0040

Gel ConcentrationVoltageTimeBuffer
2%100 V30 min1/2 x TBE

Gel Extract

Onoda, Ono, Nishimura

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

Digestion

Onoda, Ono, Nishimura

BBa_E0040

ReagentVolume
BBa_E004020 µL
DW5 µL
SpeⅠ1 µL
EcoRⅠ1 µL
CutSmart Buffer3 µL
Total30 µL

mBBa_R0040

ReagentVolume
mBBa_R004020 µL
DW5 µL
SpeⅠ1 µL
EcoRⅠ1 µL
CutSmart Buffer3 µL
Total30 µL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
260℃15 minInactivation
Store4℃HoldStore

Digestion

Onoda, Ono

pET15b

ReagentVolume
pET15b10 µL
DW6 µL
SpeⅠ1 µL
EcoRⅠ1 µL
CutSmart Buffer2 µL
Total20 µL

pET16b

ReagentVolume
pET16b10 µL
DW6 µL
SpeⅠ1 µL
EcoRⅠ1 µL
CutSmart Buffer2 µL
Total20 µL

pSB1A3

ReagentVolume
pSB1A310 µL
DW6 µL
SpeⅠ1 µL
EcoRⅠ1 µL
CutSmart Buffer2 µL
Total20 µL

pSB4C5

ReagentVolume
pSB4C52 µL
DW14 µL
SpeⅠ1 µL
EcoRⅠ1 µL
CutSmart Buffer2 µL
Total20 µL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
260℃15 minInactivation
Store4℃HoldStore

2015/05/31

Electrophoresis

Mimata, Onoda, Ono, Nishimura

BBa_E0040, BBa_R0040, pET15b, pET16b, pSB1A3, pSB4C5

Gel ConcentrationVoltageTimeBuffer
1%100 V30 min1/2 x TBE

Gel Extract

Mimata, Onoda, Ono, Nishimura

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

Electrophoresis

Mimata, Onoda, Ono, Nishimura

BBa_E0040, mBBa_R0040

Gel ConcentrationVoltageTimeBuffer
1%100 V30 min1/2 x TBE

PCR

Mimata, Onoda

BBa_E0040

ReagentVolume
BBa_E00401 µL
100UP - EX - F 10 µM1 µL
200DN - PS - R 10 µM1 µL
KOD - Plus - Neo1 µL
10 x Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

mBBa_R0040

ReagentVolume
mBBa_R00401 µL
100UP - EX - F 10 µM1 µL
200DN - PS - R 10 µM1 µL
KOD - Plus - Neo1 µL
10 x Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation30 cycle
Cycle 268℃60 secAnnealing / Elongation30 cycle
Store4℃HoldStore

June

2015/06/10

Digestion

Mimata, Onoda

BBa_E0040

ReagentVolume
BBa_E004016 µL
SpeⅠ1 µL
EcoRⅠ1 µL
CutSmart Buffer2 µL
Total20 µL

mBBa_R0040

ReagentVolume
mBBa_R004016 µL
SpeⅠ1 µL
EcoRⅠ1 µL
CutSmart Buffer2 µL
Total20 µL

Digestion

StepTemp.TimeProcess
137℃60 minDigestion
260℃15 minInactivation
Store4℃HoldStore

2015/06/16

PCR

Mimata

thanatin fragment for TA cloning

ReagentVolume
TA - F - primer1 µL
TA - R - primer1 µL
KAPA Taq25 µL
DW23 µL
Total50 µL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start95℃180 secInitialization
Cycle 195℃30 secDenaturation35 cycle
Cycle 263℃30 secAnnealing35 cycle
Cycle 372℃10 secElongation35 cycle
Finish72℃60 secFinal Elongation
Store4℃HoldStore

2015/06/17

Electrophoresis

Mimata

thanatin fragment for TA cloning

Gel ConcentrationVoltageTimeBuffer
2%100 V30 min1/2 x TBE

Annealing Oligos and Elongation

Ito

thanatin fragment for TA cloning

ReagentVolume
TA - F - primer 1 µM1 µL
TA - R - primer 1 µM1 µL
KAPA Taq25 µL
DW23 µL
Total50 µL

Annealing Oligos and Elongation

StepTemp.TimeProcessCycle
Start95℃1 minInitialization
Cycle195℃ to 23℃45 minAnnealing1 cycle
Cycle 272℃1 minElongation1 cycle
Store4℃HoldStore

2015/06/19

Electrophoresis

Ito

thanatin fragment for TA cloning

Gel ConcentrationVoltageTimeBuffer
2%100 V30min1/2 x TBE

Gel Extract

Ito

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

Ligation

Ito

pGEM - T vector / Thanatin fragment for TA cloning

ReagentVolume
pGEM - T vector1.7µL
Thanatin fragment for TA cloning0.15µL
Mighty Mix1.85µL
T4 Ligase0.18µL
DW6.12µL
Total10µL

Ligation

StepTemp.TimeProcess
116℃30 minLigation
265℃10 minInactivation
Store4℃HoldStore

2015/06/21

Transformation

Ito

pGEM - T vector

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

July

2015/07/25

Transformation

Onoda

BBa_B0015 on pSB1C3

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

Transformation

Onoda

BBa_R0010 - BBa_B0034 on pSB1C3

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

Transformation

Onoda

BBa_I0500 - BBa_B0034 on pSB1C3

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

Transformation

Onoda

BBa_B0034 on pSB1A2

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

PCR

Onoda

BBa_B0015 on pSB1C3

ReagentVolume
BBa_B0015 on pSB1C31 µL
100UP - EX - F 10 µM1 µL
200DN - PS - R 10 µM1 µL
KOD - Plus - Neo1 µL
10 x PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

BBa_R0010 - BBa_B0034 on pSB1C3

ReagentVolume
BBa_R0010 - BBa_B0034 on pSB1C31 µL
100UP - EX - F 10 µM1 µL
200DN - PS - R 10 µM1 µL
KOD - Plus - Neo1 µL
10 x PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

BBa_I0500 - BBa_B0034 on pSB1C3

ReagentVolume
BBa_I0500 - BBa_B0034 on pSB1C31 µL
100UP - EX - F 10 µM1 µL
200DN - PS - R 10 µM1 µL
KOD - Plus - Neo1 µL
10 x PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

BBa_B0034 on pSB1A2

ReagentVolume
BBa_B0034 on pSB1A21 µL
100UP - EX - F 10 µM1 µL
200DN - PS - R 10 µM1 µL
KOD - Plus - Neo1 µL
10 x PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation30 cycle
Cycle 262.6℃30 secAnnealing30 cycle
Cycle 368℃60 secElongation30 cycle
Store4℃HoldStore

Electrophoresis

Onoda

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

Gel ConcentrationVoltageTimeBuffer
2%100 V30 min1/2 x TBE

2015/07/26

Liquid Culture

Ono

BBa_I0500 - BBa_B0034 on pSB1A2

ReagentVolume
Single Colony-
LB2000 µL
Ampicillin2 µL

Cultured for 15 hours.

Mini-prep

Ito

BBa_R0010 - BBa_B0034 on pSB1C3, BBa_B0015 on pSB1C3, BBa_B0034 on pSB1A2
FastGeneTM 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 ConcentrationVoltageTimeBuffer
2%100 V30 min1/2 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
FastGeneTM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel

2015/07/27

Mini-prep

Ono

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

August

2015/08/04

Transformation

Ito

pGEM T vector

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

2015/08/05

Colony PCR

Ito

pGEM T vector

ReagentVolume
Single Colony-
NdeⅠ - F - primer 10 µM0.4 µL
BamHⅠ - R - primer 10 µM0.4 µL
KAPA Taq5.0 µL
DW4.2 µL
Total10 µL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃30 secDenaturation35 cycle
Cycle 268℃20 secAnnealing / Elongation35 cycle
Store4℃HoldStore

Electrophoresis

Ito

NdeⅠ - Thanatin - BamHⅠ

Gel ConcentrationVoltageTimeBuffer
2%100V60 min1/2 x TBE

Gel Extract

Ito

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

Ligation

Ito

pET vector / NdeⅠ - Thanatin - BamHⅠ

ReagentVolume
pET vector1 µL
NdeⅠ - Thanatin - BamHⅠ3 µL
10 × T4 DNA Ligase Buffer5 µL
T4 Ligase1 µL
Total10 µL

Ligation

StepTemp.TimeProcess
14℃30 minLigation
265℃10 minInactivation
Store4℃HoldStore

Transformation

Ito

BBa_E1010 - BBa_B0034 - BBa_E0040 - BBa_B0015 on pSB1C3

  1. Added 1 µL of Thanatin on pET vector to 50 µL of thawed competent cells (Rosetta) on ice.
  2. Incubated on ice for 30 min.
  3. Heat-shocked for 30 sec at 42℃.
  4. Spread 300 µL of the culture onto plate with LBA.
  5. 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

  1. Picked the colony with an inoculating loop from the agar plate.
  2. Draged the loop across on a new agar plate.
  3. 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
FastGeneTM 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

ReagentVolume
pSB1C320 µL
DW23 µL
Bgl Ⅱ2 µL
3.1 Buffer5 µL
Total50 µL

Digestion

StepTemp.TimeProcess
137℃60 minDigestion
270℃15 minInactivation
Store4℃HoldStore

Electrophoresis

Ito, Mimata, Onoda, Sakai, Nishimura, Kusumi

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

Gel ConcentrationVoltageTimeBuffer
1%100 V60 min1/2 x TBE

2015/08/12

Gel Extract

Nishimura, Sakai

BBa_I0500 - BBa_B0034 - BBa_K759012 (Signal - β domain) - BBa_B0015 on pSB1C3
FastGeneTM 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 ConcentrationVoltageTimeBuffer
1%100 V60 min1/2 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)

ReagentVolume
pSB1C31 µL
T7 promoter primer / SP6 promoter primer1.5 µL
Ready Reaction Premix1 µL
5 x Sequencing Buffer1.5 µL
DW5 µL
Total10 µL

Sequencing

StepTemp.TimeProcessCycle
Start96℃10 secDenaturation
Cycle 150℃5 sec-25 cycle
Cycle 260℃240 sec-25 cycle
Store4℃HoldStore

Ethanol Precipitation

Ito, Onoda, Nishimura, Fujita, Mimata

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

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

PCR

Ito, Onoda, Tanaka, Nishimura, Mimata

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

ReagentVolume
XbaⅠ - BBa_B0034 - XbaⅠ / SpeⅠ scar - Met - 10 × His tag - Thanatin - SpeⅠ1 µL
BamHⅠ - Thanatin forward Neo 10 µM1 µL
BglⅡ - Asp - Thanatin reverese Neo 10 µM1 µL
KOD - Plus - Neo1 µL
10 x PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation25 cycle
Cycle 268℃20 secAnnealing / Elongation25 cycle
Store4℃HoldStore

Digestion

Mimata

NdeⅠ - Thanatin - BamHⅠ on pET vector

ReagentVolume
NdeⅠ - Thanatin - BamHⅠ on pET vector10 µL
NdeⅠ1 µL
BamHⅠ1 µL
CutSmart Buffer5 µL
DW33 µL
Total50 µL

Digestion

StepTemp.TimeProcess
137℃60 minDigestion
270℃15 minInactivation
Store4℃HoldStore

2015/08/14

PCR

Fujita, Nishimura, Onoda

Thanatin (Mini-prep product)

ReagentVolume
Thanatin fragment1 µL
T7 - promoter primer 10 µM1 µL
SP6 - promoter primer 10 µM1 µL
KOD - Plus - Neo1 µL
10 × PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

3 Step Cycle (Tm value ≤ 66℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 194℃10 secDenaturation25 cycle
Cycle 25010 secAnnealing25 cycle
Cycle 368℃30 secElongation25 cycle
Store4℃HoldStore

Electrophoresis

Fujita, Nishimura, Onoda, Mimata

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

Gel ConcentrationVoltageTimeBuffer
2%100 V60 min1/2 x TBE

Annealing of Oligonucleotides

Onoda, Nishimura, Fujita

Thanatin fragment (derived from annealing TA primers)

ReagentVolume
TA - F - primer 10 µM1 µL
TA - R - primer 10 µM1 µL
KOD - Plus - Neo1 µL
10 x PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW34 µL
Total50 µL

Annealing of Oligonucleotides

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 194℃10 secDenaturation10 cycle
Cycle 26010 secAnnealing10 cycle
Cycle 368℃30 secElongation10 cycle
Store4℃HoldStore

PCR

Nishimura, Onoda

Thanatin fragment (derived from annealing TA primers)

ReagentVolume
Thanatin fragment (derived from annealing TA primers)1 µL
BamHⅠ - Thanatin Neo 10 µM1 µL
BglⅡ - Asp - thanatin Neo 10 µM1 µL
KOD - Plus - Neo1 µL
10 x PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

2 Step Cycle (Tm value ≥ 66℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation35 cycle
Cycle 268℃60 secAnnealing / Elongation35 cycle
Store4℃HoldStore

PCR Purification

Nishimura, Onoda

Thanatin fragment from last 2 step PCR
FastGeneTM 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 ConcentrationVoltageTimeBuffer
2%100 V60 min1/2 x TBE

Transformation

Fujita, Mitsumoto

BBa_B0031 on pSB1A2, BBa_E0040 on pSB1A2

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

Transformation

Fujita, Mitsumoto

BBa_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0030

  1. 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.
  2. Incubated on ice for 30 min.
  3. Heat-shocked for 30 sec at 42℃.
  4. Added 200 µL of LB.
  5. Incubated the cells for 2 hrs at 37℃.
  6. Spread 300 µL of the culture onto plate with LBC.
  7. 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

ReagentVolume
Ba_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0031, BBa_B0030, BBa_E00401 µL
100UP - EX - F 10 µM1 µL
200DN - PS - R 10 µM1 µL
KOD - Plus - Neo1 µL
10 × PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation35 cycle
Cycle 268℃60 secAnnealing / Elongation35 cycle
Store4℃HoldStore

2015/08/15

Electrophoresis

Fujita, Nishimura

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

Gel ConcentrationVoltageTimeBuffer
2%100 V60 min1/2 x TBE

PCR

Fujita, Nishimura, Ono

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

ReagentVolume
BBa_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0031, BBa_B0030, BBa_E00401 µL
100UP - EX - F 1 µM1 µL
200DN - PS - R 1 µM1 µL
KOD - Plus - Neo1 µL
10 × PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation35 cycle
Cycle 268℃60 secAnnealing / Elongation35 cycle
Store4℃HoldStore

Electrophoresis

Fujita, Nishimura

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

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

PCR

Nishimura, Ono

Thanatin fragment (derived from annealing TA primers)

ReagentVolume
Thanatin fragment (derived from annealing TA primers)1 µL
NdeI - F - primer 10 µM1 µL
BamHI - R - primer 10 µM1 µL
KOD - Plus - Neo1 µL
10 × PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation25 cycle
Cycle 268℃30 secAnnealing / Elongation25 cycle
Store4℃HoldStore

PCR

Sakai, Ono

Thanatin fragment (PCR 2STEP product)

ReagentVolume
Thanatin fragment (PCR 2STEP product)1 µL
BamHI - Thanatin - F - Neo 10 µM1 µL
BglⅡ - Tanatin - R - Neo 10 µM1 µL
KOD - Plus - Neo1 µL
10 x Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation25 cycle
Cycle 268℃30 secAnnealing / Elongation25 cycle
Store4℃HoldStore

Annealing Oligos and Elongation

Onoda

Thanatin fragment (derived from annealing TA primers)

ReagentVolume
TA - F - primer 10 µM1 µL
TA - R - primer 10 µM1 µL
KOD - Plus - Neo1 µL
10 x PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW34 µL
Total50 µL

Annealing Oligos and Elongation

StepTemp.TimeProcessCycle
Start95℃1 minInitialization
Cycle195℃ to 23℃45 minAnnealing1 cycle
Cycle 268℃30 secElongation1 cycle
Store4℃HoldStore

Electrophoresis

Ono, Onoda

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

Gel ConcentrationVoltageTimeBuffer
2%100 V30 min1/2 x TBE

Gel Extract

Ono

BBa_B0031, BBa_B0030, BBa_E0040
FastGeneTM 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
FastGeneTM Plasmid mini kit (Nippon Genetics Co.,Ltd)
Standard protocol

2015/08/16

PCR

Nishimura, Ono

Thanatin fragment (derived from annealing TA primers)

ReagentVolume
Thanatin fragment (derived from annealing TA primers)1 µL
NdeI - F - primer 10 µM1 µL
BamHI - R - primer 10 µM1 µL
KOD - Plus - Neo1 µL
10 x PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation25 cycle
Cycle 268℃60 secAnnealing / Elongation25 cycle
Store4℃HoldStore

Electrophoresis

Nishimura, Ono

Thanatin fragment (PCR product)

Gel ConcentrationVoltageTimeBuffer
2%100 V60 min1/2 x TBE

Colony PCR

Onoda, Ono, Nishimura

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

ReagentVolume
Single Colony-
NdeI - F - primer 10 µM0.4 µL
BamHI - R - primer 10 µM0.4 µL
KAPA Taq5 µL
DW4.2 µL
Total10 µL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start95℃120 secInitialization
Cycle 195℃30 secDenaturation40 cycle
Cycle 262.930 secAnnealing40 cycle
Cycle 372℃40 secElongation40 cycle
Finish72℃120 secFinal Elongation
Store4℃HoldStore

Colony PCR

Onoda, Ono, Nishimura

BBa_B0031 on pSB1A2 into DH5α (as positive control)

ReagentVolume
Single Colony-
100UP - EX - F 10 µM0.4 µL
200DN - PS - R 10 µM0.4 µL
KAPA Taq5 µL
DW4.2 µL
Total10 µL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start95℃120 secInitialization
Cycle 195℃30 secDenaturation30 cycle
Cycle 257.230 secAnnealing30 cycle
Cycle 372℃30 secElongation30 cycle
Finish72℃120 secFinal Elongation
Store4℃HoldStore

Electrophoresis

Onoda, Ono

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

Gel ConcentrationVoltageTimeBuffer
2%100 V60 min1/2 x TBE

Electrophoresis

Ono, Mitsumoto, Fujita

BamHI - Thanatin - BglⅡ, NdeI - Thanatin - BamHI

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

PCR

Onoda

Thanatin fragment (Mini-prep product)

ReagentVolume
Thanatin fragment (Mini-prep product)1 µL
BamHI - Thanatin - F - Neo 10 µM1 µL
BglⅡ - Asp - Thanatin - R - Neo 10 µM1 µL
KOD - Plus - Neo1 µL
10 × PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation45 cycle
Cycle 265.130 secAnnealing45 cycle
Cycle 368℃30 secElongation45 cycle
Store4℃HoldStore

PCR

Onoda

Thanatin fragment (Mini-prep product)

ReagentVolume
Thanatin fragment (Mini-prep product)1 µL
NdeI - F - primer 10 µM1 µL
BamHI - R - primer 10 µM1 µL
KOD - Plus - Neo1 µL
10 × PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation45 cycle
Cycle 266.530 secAnnealing45 cycle
Cycle 368℃30 secElongation45 cycle
Store4℃HoldStore

Electrophoresis

Onoda

Thanatin fragment for TA cloning and last PCR prduct

Gel ConcentrationVoltageTimeBuffer
2%100 V60 min1/2 x TBE

PCR

Fujita, Mimata

Ba_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0031

ReagentVolume
Ba_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B00311 µL
100UP - EX - F 10 µM1 µL
200DN - PS - R 10 µM1 µL
KOD - Plus - Neo1 µL
10 x PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation35 cycle
Cycle 268℃60 secAnnealing / Elongation35 cycle
Store4℃HoldStore

Electrophoresis

Fujita, Mimata

Ba_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0031

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

Electrophoresis

Mitsumoto, Fujita

BamHI - Thanatin - BglⅡ, NdeI - Thanatin - BamHI

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

Colony PCR

Ono, Onoda

Thanatin fragment on pGEM - T vector into DH5α

ReagentVolume
Single Colony-
NdeI - F - primer 10 µM0.4 µL
BamHI - R - primer 10 µM0.4 µL
KAPA Taq5 µL
DW4.2 µL
Total10 µL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start95℃120 secInitialization
Cycle 195℃30 secDenaturation40 cycle
Cycle 262.930 secAnnealing40 cycle
Cycle 372℃40 secElongation40 cycle
Finish72℃120 secFinal Elongation
Store4℃HoldStore

Colony PCR

Ono, Onoda

Thanatin fragment on pGEM - T vector into DH5α

ReagentVolume
Single Colony-
T7 promoter primer 10 µM0.4 µL
SP6 promoter primer 10 µM0.4 µL
KAPA Taq5 µL
DW4.2 µL
Total10 µL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start95℃120 secInitialization
Cycle 195℃30 secDenaturation40 cycle
Cycle 25130 secAnnealing40 cycle
Cycle 372℃40 secElongation40 cycle
Finish72℃120 secFinal Elongation
Store4℃HoldStore

Colony PCR

Ono, Onoda

BBa_B0031 on pSB1A2 into DH5α (as positive control)

ReagentVolume
Single Colony-
100UP - EX - F 10 µM0.4 µL
200DN - PS - R 10 µM0.4 µL
KAPA Taq5 µL
DW4.2 µL
Total10 µL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start95℃120 secInitialization
Cycle 195℃30 secDenaturation40 cycle
Cycle 25730 secAnnealing40 cycle
Cycle 372℃40 secElongation40 cycle
Finish72℃120 secFinal Elongation
Store4℃HoldStore

Electrophoresis

Onoda

Thanatin fragment (Colony PCR product)

Gel ConcentrationVoltageTimeBuffer
1%100 V60 min1/2 x TBE

Annealing Oligos and Elongation

Mitsumoto

Thanatin fragment (derived from annealing TA primers)

ReagentVolume
TA - F - primer 10 µM1 µL
TA - R - primer 10 µM1 µL
KOD - Plus - Neo1 µL
10 x PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW34 µL
Total50 µL

Annealing Oligos and Elongation

StepTemp.TimeProcessCycle
Start95℃1 minInitialization
Cycle195℃ to 23℃60 secAnnealing45 cycle
Cycle 268℃30 secElongation45 cycle
Store4℃HoldStore

Annealing Oligos and Elongation

Mitsumoto

Thanatin fragment (derived from annealing TA primers)

ReagentVolume
TA - F - primer 10 µM1 µL
TA - R - primer 10 µM1 µL
KOD - Plus - Neo1 µL
10 x PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW34 µL
Total50 µL

Annealing Oligos and Elongation

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 194℃10 secDenaturation10 cycle
Cycle 26010 secAnnealing10 cycle
Cycle 368℃30 secElongation10 cycle
Store4℃HoldStore

Annealing Oligos and Elongation

Mitsumoto

Thanatin fragment (derived from annealing TA primers)

ReagentVolume
TA - F - primer 10 µM1 µL
TA - R - primer 10 µM1 µL
KOD - FX - Neo1 µL
10 × PCR Buffer for KOD - FX - Neo25 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW14 µL
Total50 µL

Annealing Oligos and Elongation

StepTemp.TimeProcessCycle
Start95℃1 minInitialization
Cycle195℃ to 23℃60 secAnnealing45 cycle
Cycle 268℃30 secElongation45 cycle
Store4℃HoldStore

Annealing Oligos and Elongation

Mitsumoto

Thanatin fragment (derived from annealing TA primers)

ReagentVolume
TA - F - primer 10 µM1 µL
TA - R - primer 10 µM1 µL
KOD - FX - NEO1 µL
2 × PCR Buffer for KOD - FX - Neo25 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW14 µL
Total50 µL

Annealing Oligos and Elongation

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 194℃10 secDenaturation10 cycle
Cycle 26010 secAnnealing10 cycle
Cycle 368℃30 secElongation10 cycle
Store4℃HoldStore

Annealing Oligos and Elongation

Mitsumoto

Thanatin fragment (derived from annealing TA primers)

ReagentVolume
Single Colony-
100UP - EX - F 10 µM1 µL
200DN - PS - R 10 µM1 µL
KAPA Taq25 µL
DW23 µL
Total50 µL

Annealing Oligos and Elongation

StepTemp.TimeProcessCycle
Start95℃1 minInitialization
Cycle 195℃ to 23℃60 secAnnealing45 cycle
Cycle 268℃30 secElongation45 cycle
Store4℃HoldStore

Annealing Oligos and Elongation

Mitsumoto

Thanatin fragment (derived from annealing TA primers)

ReagentVolume
Single Colony-
100UP - EX - F 10 µM1 µL
200DN - PS - R 10 µM1 µL
KAPA Taq25 µL
DW23 µL
Total50 µL

Annealing Oligos and Elongation

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 194℃10 secDenaturation10 cycle
Cycle 26010 secAnnealing10 cycle
Cycle 368℃30 secElongation10 cycle
Store4℃HoldStore

Electrophoresis

Mitsumoto

Thanatin fragment (derived from annealing TA primers)

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

2015/08/17

Annealing Oligos and Elongation

Nishimura, Onoda

Thanatin fragment (Mini-prep product)

ReagentVolume
TA - F - primer 10 µM1 µL
TA - R - primer 10 µM1 µL
KOD - Plus - Neo1 µL
10 x PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW34 µL
Total50 µL

Annealing Oligos and Elongation

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 194℃10 secDenaturation10 cycle
Cycle 26010 secAnnealing10 cycle
Cycle 368℃30 secElongation10 cycle
Store4℃HoldStore

Annealing Oligos and Elongation

Nishimura, Onoda

Thanatin fragment (Mini-prep product)

ReagentVolume
TA - F - primer 10 µM1 µL
TA - R - primer 10 µM1 µL
KAPA Taq25 µL
DW23 µL
Total50 µL

(Tm value ≤ -℃)

Annealing Oligos and Elongation

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 194℃10 secDenaturation10 cycle
Cycle 26010 secAnnealing10 cycle
Cycle 372℃30 secElongation10 cycle
Store4℃HoldStore

PCR

Nishimura, Ono, Onoda, Mimata

NdeⅠ - Thanatin - BamHⅠ

ReagentVolume
NdeⅠ - Thanatin - BamHⅠ1 µL
NdeⅠ - F - primer 10 µM1 µL
BamHⅠ - R - primer 10 µM1 µL
KOD - Plus - Neo1 µL
10 x PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation25 cycle
Cycle 268℃30 secAnnealing / Elongation25 cycle
Store4℃HoldStore

PCR

Nishimura, Ono, Onoda, Mimata

BamHI - Thanatin - BglⅡ

ReagentVolume
BamHI - Thanatin - BglⅡ1 µL
BamHI - Asp - Thanatin - R - Neo 10 µM1 µL
BglⅡ - D - Tanatin - R - Neo 10 µM1 µL
KOD - Plus - Neo1 µL
10 x PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation25 cycle
Cycle 268℃30 secAnnealing / Elongation25 cycle
Store4℃HoldStore

PCR Purification

Ono, MImata, Nishimura

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

Electrophoresis

Nishimura, Ono

Thanatin fragment (PCR 2STEP product)

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

PCR

Ito, Ono, Onoda

Thanatin fragment (derived from annealing TA cloning)

ReagentVolume
Thanatin fragment (derived from annealing TA cloning)1 µL
NdeⅠ - F - primer 10 µM1 µL
BamHⅠ - R - primer 10 µM1 µL
KOD - Plus - Neo1 µL
10 x PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation45 cycle
Cycle 265.130 secAnnealing45 cycle
Cycle 368℃30 secElongation45 cycle
Store4℃HoldStore

PCR

Ito, Ono, Onoda

BamHⅠ - Thanatin - BglⅡ

ReagentVolume
Thanatin fragment (derived from annealing TA cloning)1 µL
BamHⅠ - Asp - Thanatin - R - Neo 10 µM1 µL
BglⅡ - D - Tanatin - R - Neo 10 µM1 µL
KOD - Plus - Neo1 µL
10 x PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation45 cycle
Cycle 266.530 secAnnealing45 cycle
Cycle 368℃30 secElongation45 cycle
Store4℃HoldStore

Digestion

Ono, Onoda

NdeⅠ - Thanatin - BamHⅠ

ReagentVolume
NdeⅠ - Thanatin - BamHⅠ20 µL
NdeⅠ1 µL
BamHⅠ1 µL
10 × K Buffer2 µL
DW5 µL
Total30 µL

Digestion

StepTemp.TimeProcess
137℃60 minDigestion
270℃15 minInactivation
Store4℃HoldStore

Digestion

Ono, Onoda

BamHⅠ - Thanatin - BglⅡ

ReagentVolume
BamHⅠ - Thanatin - BglⅡ20 µL
BamHⅠ1 µL
BglⅡ1 µL
10 × K Buffer2 µL
DW5 µL
Total30 µL

Digestion

StepTemp.TimeProcess
137℃60 minDigestion
270℃15 minInactivation
Store4℃HoldStore

2015/08/18

Electrophoresis

Nishimura, Ono

Thanatin fragment (PCR 2STEP product)

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

PCR Purification

Ono, Mimata, Nishimura

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

Annealing Oligos and Elongation

Mimata

Thanatin fragment (derived from annealing TA cloning)

ReagentVolume
TA - F - primer 10 µM5 µL
TA - R - primer 10 µM5 µL
TE 0.8 M NaCl10 µL
Total50 µL

Annealing Oligos and Elongation

1
StepTemp.TimeProcessCycle
Start94℃2 minInitialization
Step195℃ to 25℃20 minAnnealing
Store4℃HoldStore

PCR

Mimata

Thanatin fragment (derived from annealing)

ReagentVolume
Thanatin fragment (derived from annealing)1 µL
NdeⅠ - F - primer 10 µM1 µL
BamHⅠ - R - primer 10 µM1 µL
KOD - Plus - Neo1 µL
10 x PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation25 cycle
Cycle 268℃30 secAnnealing / Elongation25 cycle
Store4℃HoldStore

PCR

Mimata

Thanatin fragment (derived from annealing)

ReagentVolume
Thanatin fragment (derived from annealing)1 µL
BamHⅠ - Asp - Thanatin - R - Neo 10 µM1 µL
BglⅡ - D - Tanatin - R - Neo 10 µM1 µL
KOD - Plus - Neo1 µL
10 x PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation25 cycle
Cycle 268℃30 secAnnealing / Elongation25 cycle
Store4℃HoldStore

PCR Purification

Ono, Mimata, Nishimura

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

Digestion

Ono, Onoda, Nishimura

NdeⅠ - Thanatin - BamHⅠ

ReagentVolume
NdeⅠ - Thanatin - BamHⅠ20 µL
NdeⅠ1 µL
BamHⅠ1 µL
10 × K Buffer2 µL
DW5 µL
Total30 µL

Digestion

StepTemp.TimeProcess
137℃60 minDigestion
270℃15 minInactivation
Store4℃HoldStore

Digestion

Ono, Onoda, Nishimura

BamHⅠ - Thanatin - BglⅡ

ReagentVolume
BamHⅠ - Thanatin - BglⅡ20 µL
BamHⅠ1 µL
BglⅡ1 µL
10 × K Buffer2 µL
DW5 µL
Total30 µL

Digestion

StepTemp.TimeProcess
137℃60 minDigestion
270℃15 minInactivation
Store4℃HoldStore

Ligation

Onoda, Mimata

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

ReagentVolume
pGEM T - vector1 µL
Thanatin fragment3 µL
2 × Ligation Buffer5 µL
T4 Ligase1 µL
Total10 µL

Ligation

StepTemp.TimeProcess
14℃6 hourLigation
265℃10 minInactivation
Store4℃HoldStore

2015/08/19

PCR

Ono

Thanatin fragment (derived from annealing)

ReagentVolume
Thanatin fragment (derived from annealing)1 µL
NdeⅠ - F - primer 10 µM1 µL
BamHⅠ - R - primer 10 µM1 µL
KOD - Plus - Neo1 µL
10 x PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 194℃10 secDenaturation40 cycle
Cycle 26030 secAnnealing40 cycle
Cycle 368℃30 secElongation40 cycle
Store4℃HoldStore

PCR

Ono

Thanatin fragment (derived from annealing)

ReagentVolume
Thanatin fragment (derived from annealing)1 µL
BamHⅠ - Thanatin - F 10 µM1 µL
BglⅡ - Tanatin - R 10 µM1 µL
KOD - Plus - Neo1 µL
10 x PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 194℃10 secDenaturation40 cycle
Cycle 26030 secAnnealing40 cycle
Cycle 368℃30 secElongation40 cycle
Store4℃HoldStore

Ethanol Precipitation

Mimata, Ono

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

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

Electrophoresis

Mimata

NdeⅠ - Thanatin - BamHⅠ, BamHⅠ - Thanatin - BglⅡ

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

PCR

Ono

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

ReagentVolume
BamHⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - BglⅡ1 µL
BamHⅠ - Thanatin - F 10 µM1 µL
BglⅡ - Thanatin - R 10 µM1 µL
KOD - Plus - Neo1 µL
10 x PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation40 cycle
Cycle 268℃30 secAnnealing / Elongation40 cycle
Store4℃HoldStore

PCR

Ono

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

ReagentVolume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ1 µL
XbaⅠ - Thanatin - F 10 µM1 µL
NdeⅠ - Thanatin - R 10 µM1 µL
KOD - Plus - Neo1 µL
10 x PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation40 cycle
Cycle 268℃30 secAnnealing / Elongation40 cycle
Store4℃HoldStore

Ethanol Precipitation

Ono

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

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

Digestion

Ono, Onoda, Mimata, Sakai

NdeⅠ - Thanatin - BamHⅠ

ReagentVolume
NdeⅠ - Thanatin - BamHⅠ20 µL
NdeⅠ1 µL
BamHⅠ1 µL
10 × K Buffer3 µL
DW5 µL
Total30 µL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
260℃15 minInactivation
Store4℃HoldStore

Digestion

Ono, Onoda, Mimata, Sakai

BamHⅠ - Thanatin - BglⅡ

ReagentVolume
BamHⅠ - Thanatin - BglⅡ20 µL
BamHⅠ1 µL
BglⅡ1 µL
10 × K Buffer3 µL
DW5 µL
Total30 µL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
260℃15 minInactivation
Store4℃HoldStore

Digestion

Ono, Onoda, Mimata, Sakai

pET - 15b vector, pET - 16b vector

ReagentVolume
pET - 15b vector, pET - 16b vector20 µL
NdeⅠ1 µL
BamHⅠ1 µL
10 × K Buffer3 µL
DW5 µL
Total30 µL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
260℃15 minInactivation
Store4℃HoldStore

Transformation

Onoda

Thanatin fragment on pGEM - T vector

  1. Added 1 µL of Thanatin fragment on pGEM - T vector to 50 µL of thawed competent cells (DH5α) on ice.
  2. Incubated on ice for 30 min.
  3. Heat-shocked for 30 sec at 42℃.
  4. Spread 300 µL of the culture onto plate with LBA.
  5. 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 ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

Electrophoresis

Ono, Nishimura, Mimata

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

Gel ConcentrationVoltageTimeBuffer
1%100 V60 min1/2 x TBE

Gel Extract

Nishimura, Ono

pET - 15b vector, pET - 16b vector
FastGeneTM 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)

ReagentVolume
BamHⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - BglⅡ (PCR 2STEP poduct)9 µL
BamHⅠ5 µL
BglⅡ5 µL
10 × K Buffer10 µL
DW71 µL
Total100 µL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
270℃15 minInactivation
Store4℃HoldStore

Digestion

Ono, Nisimura, Ito

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

ReagentVolume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ (PCR 2STEP poduct)9 µL
XbaⅠ5 µL
SpeⅠ5 µL
10 × K Buffer10 µL
DW71 µL
Total100 µL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
280℃20 minInactivation
Store4℃HoldStore

Digestion

Ono, Nishimura, Ito

BBa_B0015 on pSB1C3

ReagentVolume
BBa_B0015 on pSB1C320 µL
SpeⅠ1 µL
CutSmart Buffer3 µL
DW6 µL
Total30 µL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
280℃20 minInactivation
Store4℃HoldStore

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)

  1. Added 9 µL of NaOAc, 1.5 µL of glycogen and 270 µL of 100% ethanol.
  2. Left it at -80℃ for 1 hr.
  3. Centrifuged at 15,000 rpm for 15 min at 4℃.
  4. Removed supernatant and added 220 µL of 70% ethanol.
  5. Centrifuged at 15,000 rpm for 10 min at 4℃.
  6. Removed supernatant and air-dried at room temperature with light shield.
  7. 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 ConcentrationVoltageTimeBuffer
1%100 V60 min1/2 x TBE

2015/08/21

PCR

Ono, Nishimura, Onoda

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

ReagentVolume
BamHⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - BglⅡ1 µL
BamHⅠ - Thanatin - F 10 µM1 µL
BglⅡ - Thanatin - R 10 µM1 µL
KOD - Plus - Neo1 µL
10 x PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation40 cycle
Cycle 268℃30 secAnnealing / Elongation40 cycle
Store4℃HoldStore

PCR

Ono, Nishimura, Onoda

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

ReagentVolume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ1 µL
XbaⅠ - Thanatin - F 10 µM1 µL
SpeⅠ - Thanatin - R 10 µM1 µL
KOD - Plus - Neo1 µL
10 x PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation40 cycle
Cycle 268℃30 secAnnealing / Elongation40 cycle
Store4℃HoldStore

Electrophoresis

Ono, Onoda, Nishimura

XbaⅠ - Thanatin - SpeⅠ, BamHⅠ- Thanatin - BglⅡ

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 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

  1. Added 3 µL of NaOAc, 1 µL of glycogen and 90 µL of 100% ethanol.
  2. Left it at -80℃ for 10 min.
  3. Centrifuged at 15,000 rpm for 5 min at 4℃.
  4. Removed supernatant and added 100 µL of 70% ethanol.
  5. Centrifuged at 15,000 rpm for 5 min at 4℃.
  6. Removed supernatant and air-dried at room temperature.
  7. 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 ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

Ligation

Ono, Nishimura, Ito

BBa_K759012 on pSB1C3 / BamHⅠ - Thanatin - BglⅡ

ReagentVolume
BBa_K759012 on pSB1C35 µL
BamHⅠ - Thanatin - BglⅡ1 µL
10 × T4 DNA Ligase Buffer7 µL
T4 Ligase1 µL
Total14 µL

Ligation

StepTemp.TimeProcess
116℃30 minLigation
Store4℃HoldStore

Ligation

Ono, Nishimura, Ito

BBa_B0015 on pSB1C3 / XbaⅠ - Thanatin - SpeⅠ

ReagentVolume
BBa_B0015 on pSB1C35 µL
XbaⅠ - Thanatin - SpeⅠ1 µL
10 × T4 DNA Ligase Buffer7 µL
T4 DNA Ligase1 µL
Total14 µL

Ligation

StepTemp.TimeProcess
116℃30 minLigation
Store4℃HoldStore

Transformation

Onoda

Thanatin - BBa_K759012 on pSB1C3, Thanatin - BBa_B0015 on pSB1C3

  1. Added 5 µL of Thanatin - BBa_K759012 on pSB1C3, Thanatin - BBa_B0015 on pSB1C3 to 50 µL of thawed competent cells (DH5α) on ice.
  2. Incubated on ice for 30 min.
  3. Heat-shocked for 30 sec at 42℃.
  4. Added 200 µL of LB.
  5. Incubated the cells for 2 hrs at 37℃.
  6. Spread 300 µL of the culture onto plate with LBC.
  7. 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

ReagentVolume
BBa_B0033 on pSB1C3, XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ, XbaⅠ - BBa_K16800020 µL
XbaⅠ1 µL
SpeⅠ1 µL
10 × M Buffer3 µL
DW5 µL
Total30 µL

Digestion

StepTemp.TimeProcess
137℃60 minDigestion
280℃20 minInactivation
Store4℃HoldStore

Digestion

Onoda

BBa_E0040 on pSB1C3, BBa_R0040 on pSB1C3

ReagentVolume
BBa_E0040 on pSB1C3, BBa_R0040 on pSB1C320 µL
XbaⅠ1 µL
CutSmart Buffer3 µL
DW6 µL
Total30 µL

Digestion

StepTemp.TimeProcess
137℃60 minDigestion
260℃15 minInactivation
Store4℃HoldStore

2015/08/22

Electrophoresis

Ono, Nishimura

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

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

Digestion

Ono, Onoda

BBa_R0010 - BBa_B0034 on pSB1C3

ReagentVolume
BBa_R0010 - BBa_B0034 on pSB1C320 µL
SpeⅠ - HF1 µL
10 × M Buffer5 µL
DW24 µL
Total50 µL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
Store4℃HoldStore

Electrophoresis

Ono, Onoda

BBa_R0010 - BBa_B0034 on pSB1C3 (Digestion product)

Gel ConcentrationVoltageTimeBuffer
1%100 V45 min1/2 x TBE

Ethanol Precipitation

Ono

BBa_R0010 - BBa_B0034 on pSB1C3 (Digestion product)

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

Colony PCR

Ono, Onoda

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

ReagentVolume
Single Colony-
AGSP - BamHⅠ - Spidroin 10 µM0.4 µL
BglⅡ - D - Thanatin 10 µM0.4 µL
KAPA Taq5 µL
DW4.2 µL
Total10 µL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃30 secDenaturation40 cycle
Cycle 26540 secAnnealing40 cycle
Cycle 372℃40 secElongation40 cycle
Finish72℃120 secFinal Elongation
Store4℃HoldStore

Colony PCR

Ono, Onoda

BBa_B0031 on pSB1A2 (as Positive Control)

ReagentVolume
Single Colony-
100UP - EX - F 10 µM0.4 µL
200DN - PS - R 10 µM0.4 µL
KAPA Taq5 µL
DW4.2 µL
Total10 µL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start95℃120 secInitialization
Cycle 195℃30 secDenaturation40 cycle
Cycle 25730 secAnnealing40 cycle
Cycle 372℃40 secElongation40 cycle
Finish72℃120 secFinal Elongation
Store4℃HoldStore

Electrophoresis

Ono, Onoda

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

Gel ConcentrationVoltageTimeBuffer
1%100 V45 min1/2 x TBE

Ligation

Ono

BBa_R0010 - BBa_B0034 on pSB1C3 / XbaⅠ - Thanatin - SpeⅠ

ReagentVolume
BBa_R0010 - BBa_B0034 on pSB1C35 µL
XbaⅠ - Thanatin - SpeⅠ4 µL
Mighty Mix10 µL
DW1 µL
Total20 µL

Ligation

StepTemp.TimeProcess
116℃30 minLigation
Store4℃HoldStore

Transformation

Ono

BBa_R0010 - BBa_B0034 on pSB1C3

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

PCR

Onoda

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

ReagentVolume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ1 µL
XbaI - B0034 - XS scar - F 10 µM1 µL
SpeⅠ - Thanatin - R 10 µM1 µL
KOD - Plus - Neo1 µL
10 x PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation30 cycle
Cycle 268℃30 secAnnealing / Elongation30 cycle
Store4℃HoldStore

Electrophoresis

Onoda

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

Gel ConcentrationVoltageTimeBuffer
2%50 V50 min1/2 x TBE

PCR

Onoda

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

ReagentVolume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ1 µL
XbaI - B0034 - XS scar - F 10 µM1 µL
SpeⅠ - Thanatin - R 10 µM1 µL
KOD - Plus - Neo1 µL
10 x PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation30 cycle
Cycle 268℃30 secAnnealing / Elongation30 cycle
Store4℃HoldStore

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

  1. 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.
  2. Incubated on ice for 30 min.
  3. Heat-shocked for 30 sec at 42℃.
  4. Added 200 μL of LB.
  5. Incubated the cells for 2 hrs at 37℃.
  6. Spread 200 μL of the culture onto plate with LBC.
  7. Incubated the plate at 37℃ for 15 hours.

Transformation

Mitsumoto

BBa_B0034 - BBa_R0040 - BBa_B0015 on pSB1A2

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

ReagentVolume
Single Colony-
LBC2000 μL
Chloramphenicol2 μL

Cultured for 12 hours.

Liquid Culture

Mitsumoto

BBa_B0034 - BBa_R0040 - BBa_B0015 on pSB1A2

ReagentVolume
Single Colony-
LBA2000 μL
Ampicillin2 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 ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

Gel Extract

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

ReagentVolume
Single Colony-
100UP - EX - F 10 µM0.4 µL
SpeⅠ - Thanatin - R 10 µM0.4 µL
KAPA Taq5 µL
DW4.2 µL
Total10 µL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start95℃120 secInitialization
Cycle 195℃30 secDenaturation40 cycle
Cycle 25730 secAnnealing40 cycle
Cycle 372℃40 secElongation40 cycle
Finish72℃120 secFinal Elongation
Store4℃HoldStore

Colony PCR

Ito, Ono, Onoda

BBa_B0030 on pSB1A2 (as a positive control)

ReagentVolume
Single Colony-
100UP - EX - F 10 µM0.4 µL
200DN - PS - R 10 µM0.4 µL
KAPA Taq5 µL
DW4.2 µL
Total10 µL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start95℃120 secInitialization
Cycle 195℃30 secDenaturation40 cycle
Cycle 25730 secAnnealing40 cycle
Cycle 372℃40 secElongation40 cycle
Finish72℃120 secFinal Elongation
Store4℃HoldStore

2015/08/24

Mini-prep

Ono, Nishimura

Thanatin - BBa_K759012 on pSB1C3
FastGeneTM 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)

ReagentVolume
Single Colony-
100UP - EX - F 10 µM0.4 µL
SpeⅠ - Thanatin - R 10 µM0.4 µL
KAPA Taq5 µL
DW4.2 µL
Total10 µL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start95℃120 secInitialization
Cycle 195℃30 secDenaturation40 cycle
Cycle 25730 secAnnealing40 cycle
Cycle 372℃40 secElongation40 cycle
Finish72℃120 secFinal Elongation
Store4℃HoldStore

Colony PCR

Ono, Nishimura

Thanatin - BBa_K759012 on pSB1C3

ReagentVolume
Single Colony-
AGSP - BamHⅠ - Spidroin 10 µM0.4 µL
BBa_K759012 - R 10 µM0.4 µL
KAPA Taq5 µL
DW4.2 µL
Total10 µL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃30 secDenaturation40 cycle
Cycle 26540 secAnnealing40 cycle
Cycle 372℃40 secElongation40 cycle
Finish72℃120 secFinal Elongation
Store4℃HoldStore

Electrophoresis

Ono, Nishimura

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

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

Digestion

Ono, Nishimura

BBa_R0010 - BBa_B0034 - Thanatin on pSB1C3

ReagentVolume
BBa_R0010 - BBa_B0034 - Thanatin on pSB1C320 µL
SpeⅠ - HF1 µL
CutSmart Buffer3 µL
DW6 µL
Total30 µL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
280℃20 minInactivation
Store4℃HoldStore

Electrophoresis

Ono, Nishimura

BBa_R0010 - BBa_B0034 - Thanatin on pSB1C3 (digestion product)

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

Liquid Culture

Ono, Nishimura

Thanatin - BBa_K759012 on pSB1C3 into DH5α

ReagentVolume
Single Colony-
LB2000 µL
Chloramphenicol2 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

ReagentVolume
Single Colony-
LB2000 µL
Chloramphenicol2 µL

Cultured for 16 hours.

Sequencing

Ono, Nishimura

Thanatin - BBa_K759012 on pSB1C3

ReagentVolume
Thanatin - BBa_K759012 on pSB1C31 µL
pbad - F2 / 200 - βdomain BBa_K759012 - R1.5 µL
BigDye Terminator1 µL
5 x Sequencing Buffer1.5 µL
DW5 µL
Total10 µL

Sequencing

StepTemp.TimeProcessCycle
Start96℃10 secDenaturation
Cycle 150℃5 sec-30 cycle
Cycle 260℃240 sec-30 cycle
Store4℃HoldStore

Ethanol Precipitation

Ono, Nishimura

Thanatin - BBa_K759012 on pSB1C3

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

PCR

Ono, Nishimura

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

ReagentVolume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ1 µL
XbaⅠ - BBa_B0034 - XS scar - F 10 µM1 µL
SpeⅠ - Thanatin - R 10 µM1 µL
KOD - Plus - Neo1 µL
10 x PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation40 cycle
Cycle 25510 secAnnealing40 cycle
Cycle 368℃30 secElongation40 cycle
Store4℃HoldStore

Ethanol Precipitation

Fujita, Nishimura

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

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

Electrophoresis

Fujita

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

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

2015/08/26

PCR

Ono、Nishimura

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

ReagentVolume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ1 µL
XbaⅠ - BBa_B0034 - XS scar - F 10 µM1 µL
SpeⅠ - Thanatin - R 10 µM1 µL
KOD - Plus - Neo1 µL
10 x PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation40 cycle
Cycle 25510 secAnnealing40 cycle
Cycle 368℃30 secElongation40 cycle
Store4℃HoldStore

PCR

Ono, Nishimura

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

ReagentVolume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ1 µL
XbaⅠ - BBa_B0034 - XS scar - F 10 µM1 µL
SpeⅠ - Thanatin - R 10 µM1 µL
KOD - Plus - Neo1 µL
10 x PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation40 cycle
Cycle 25330 secAnnealing40 cycle
Cycle 368℃30 secElongation40 cycle
Store4℃HoldStore

PCR

Ono, Nishimura, Fujita

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

ReagentVolume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ1 µL
XbaⅠ - BBa_B0034 - XbaⅠ / SpeⅠ scar - F 10 µM1 µL
SpeⅠ - Thanatin - R 10 µM1 µL
KOD - Plus - Neo1 µL
10 x PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation30 cycle
Cycle 268℃30 secAnnealing / Elongation30 cycle
Store4℃HoldStore

Electrophoresis

Ono, Nishimura, Fujita

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

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

Ethanol Precipitation

Fujita, Nishimura, Ono

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

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

Mini-prep

Nishimura, Ono

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

Sequencing

Fujita, Nishimura, Ono

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

ReagentVolume
BBa_R0010 - BBa_B0034 - Thanatin on pSB1C3 (Mini-prep producrt)1 µL
100UP - EX - F / 200DN - PS - R1.5 µL
Ready Reaction Premix1 µL
5 x Sequencing Buffer1.5 µL
DW5 µL
Total10 µL

Sequencing

StepTemp.TimeProcessCycle
Start96℃10 secDenaturation
Cycle 150℃5 sec-30 cycle
Cycle 260℃240 sec-30 cycle
Store4℃HoldStore

Ethanol Precipitation

Fujita, Nishimura, Ono

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

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

2015/08/27

Ligation

Ono, Ito

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

ReagentVolume
BBa_K759012 on pSB1C35 µL
BamHⅠ - Thanatin - BglⅡ4 µL
Mighty Mix10 µL
DW1 µL
Total20 µL

Ligation

StepTemp.TimeProcess
116℃30 minLigation
Store4℃HoldStore

Ligation

Ono, Ito

BBa_K759012 on pSB1C3 (not phosphorylated)

ReagentVolume
BBa_K759012 on pSB1C32 µL
Mighty Mix2 µL
DW6 µL
Total10 µL

Ligation

StepTemp.TimeProcess
116℃30 minLigation
Store4℃HoldStore

Ligation

Ono, Ito

BBa_K759012 on pSB1C3 (phosphorylated)

ReagentVolume
BBa_K759012 on pSB1C32 µL
Mighty Mix2 µL
DW6 µL
Total10 µL

Ligation

StepTemp.TimeProcess
116℃30 minLigation
Store4℃HoldStore

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)

  1. 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.
  2. Incubated on ice for 30 min.
  3. Heat-shocked for 30 sec at 42℃.
  4. Added 200 µL of LB.
  5. Incubated the cells for 2 hrs at 37℃.
  6. Spread 300 µL of the culture onto plate with LBC.
  7. Incubated the plate at 37℃ for 12 hours.

Colony PCR

Ito, Ono

Thanatin - BBa_K759012 on pSB1C3

ReagentVolume
Single Colony-
Agsp - BamHⅠ - Spidroin 10 µM0.4 µL
BBa_K759012 - bunit - R / BglⅡ - D - Thanatin - R 10 µM0.4 µL
KAPA Taq5 µL
DW4.2 µL
Total10 µL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start95℃120 secInitialization
Cycle 195℃30 secDenaturation40 cycle
Cycle 26530 secAnnealing40 cycle
Cycle 372℃60 secElongation40 cycle
Finish72℃120 secFinal Elongation
Store4℃HoldStore

2015/08/28

Electrophoresis

Fujita, Ono

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

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

Ethanol Precipitation

Fujita

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

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

Electrophoresis

Fujita, Ono

BamHⅠ - Thanatin - BglⅡ

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

Gel Extract

Fujita

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

PCR

Fujita, Ono

XbaⅠ - Thanatin - SpeⅠ

ReagentVolume
XbaⅠ - Thanatin - SpeⅠ1 µL
XbaⅠ - Thanatin - F 10 µM1 µL
SpeⅠ - Thanatin - R 10 µM1 µL
KOD - Plus - Neo1 µL
10 x PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation30 cycle
Cycle 268℃30 secAnnealing / Elongation30 cycle
Store4℃HoldStore

PCR

Fujita, Ono

XbaⅠ - Thanatin - SpeⅠ

ReagentVolume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ1 µL
XbaⅠ - Thanatin - F 10 µM1 µL
SpeⅠ - Thanatin - R 10 µM1 µL
KOD - Plus - Neo1 µL
10 x PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation30 cycle
Cycle 26730 secAnnealing30 cycle
Cycle 368℃30 secElongation30 cycle
Store4℃HoldStore

PCR

Fujita, Ono

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

ReagentVolume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ1 µL
XbaⅠ - Thanatin - F 10 µM1 µL
SpeⅠ - Thanatin - R 10 µM1 µL
KOD - Plus - Neo1 µL
10 x PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation30 cycle
Cycle 268℃30 secAnnealing / Elongation30 cycle
Store4℃HoldStore

PCR

Fujita, Ono

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

ReagentVolume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ1 µL
XbaⅠ - Thanatin - F 10 µM1 µL
SpeⅠ - Thanatin - R 10 µM1 µL
KOD - Plus - Neo1 µL
10 x PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation30 cycle
Cycle 26730 secAnnealing30 cycle
Cycle 368℃30 secElongation30 cycle
Store4℃HoldStore

PCR

Fujita, Ono

BamHⅠ - Thanatin - BglⅡ

ReagentVolume
BamHⅠ - Thanatin - BglⅡ1 µL
BamHⅠ - Thanatin - F 10 µM1 µL
BglⅡ - Thanatin - R 10 µM1 µL
KOD - Plus - Neo1 µL
10 x PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation30 cycle
Cycle 268℃30 secAnnealing / Elongation30 cycle
Store4℃HoldStore

PCR

Fujita, Ono

BamHⅠ - Thanatin - BglⅡ

ReagentVolume
BamHⅠ - Thanatin - BglⅡ1 µL
BamHⅠ - Thanatin - F 10 µM1 µL
BglⅡ - Thanatin - R 10 µM1 µL
KOD - Plus - Neo1 µL
10 x PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation30 cycle
Cycle 26730 secAnnealing30 cycle
Cycle 368℃30 secElongation30 cycle
Store4℃HoldStore

Electrophoresis

Fujita, Ono

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

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

Digestion

Ono, Fujita

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

ReagentVolume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ20 µL
SpeⅠ1 µL
XbaⅠ1 µL
10 × M Buffer3 µL
0.1%BSA3 µL
DW2 µL
Total30 µL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
Store4℃HoldStore

Digestion

Ono, Fujita

BamHⅠ - Thanatin - BglⅡ

ReagentVolume
BamHⅠ - Thanatin - BglⅡ20 µL
BamHⅠ1 µL
BglⅡ1 µL
10 × K Buffer3 µL
DW5 µL
Total30 µL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
Store4℃HoldStore

2015/08/29

Electrophoresis

Fujita, Ono

XbaⅠ - Thanatin - SpeⅠ

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

Gel Extract

Fujita, Ono

XbaⅠ - Thanatin - SpeⅠ


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

Ethanol Precipitation

Fujita, Ono

XbaⅠ - Thanatin - SpeⅠ

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

Electrophoresis

Fujita, Ono

XbaⅠ - Thanatin - SpeⅠ

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

PCR

Ono

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

ReagentVolume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ1 µL
XbaⅠ - Thanatin - F 10 µM1 µL
SpeⅠ - Thanatin - R 10 µM1 µL
KOD - Plus - Neo1 µL
10 x PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation40 cycle
Cycle 268℃30 secAnnealing / Elongation40 cycle
Store4℃HoldStore

Ethanol Precipitation

Ono

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

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

Electrophoresis

Fujita, Mimata

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

Gel ConcentrationVoltageTimeBuffer
1%100 V60 min1/2 x TBE

Electrophoresis

Fujita, Mimata

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

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

Digestion

Fujita

BBa_R0010 - BBa_B0034 on pSB1C3

ReagentVolume
BBa_R0010 - BBa_B0034 on pSB1C320 µL
SpeⅠ - HF1 µL
CutSmart Buffer3 µL
DW6 µL
Total30 µL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
280℃20 minInactivation
Store4℃HoldStore

Transformation

Mitsumoto

BBa_B0015 on pSB1C3

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

Transformation

Mitsumoto

BBa_I0500 on pSB2K3

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

PCR (2 STEP)

Mitsumoto

HLA on pCOLA

ReagentVolume
HLA on pCOLA1 μL
XbaI - HLA - F - primer 10 μM1 μL
SpeI - HLA - R - primer 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation35 cycle
Cycle 268℃elongation timeAnnealing / Elongation35 cycle
Store4℃HoldStore

PCR (3 STEP)

Mitsumoto

HLZ on pCOLA

ReagentVolume
HLZ on pCOLA1 μL
XbaI - HLZ - F - primer 10 μM1 μL
SpeI - HLZ - R - primer 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation35 cycle
Cycle 262.7℃30 secAnnealing35 cycle
Cycle 368℃elongation timeElongation35 cycle
Store4℃HoldStore

PCR (3 STEP)

Mitsumoto

BLA on pCOLA

ReagentVolume
BLA on pCOLA1 μL
XbaI - BLA - F - primer 10 μM1 μL
SpeI - BLA - R - primer 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation35 cycle
Cycle 260.9℃30 secAnnealing35 cycle
Cycle 368℃elongation timeElongation35 cycle
Store4℃HoldStore

PCR (3 STEP)

Mitsumoto

BBa_B0015, BBa_B0032, BBa_B0033, BBa_B0034 on pSB1C3

ReagentVolume
BBa_B0015, BBa_B0032, BBa_B0033, BBa_B0034 on pSB1C31 μL
100UP - EX - F 10 μM1 μL
200DN - PS - R 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation35 cycle
Cycle 262.6℃30 secAnnealing35 cycle
Cycle 368℃elongation timeElongation35 cycle
Store4℃HoldStore

Electrophoresis

Mitsumoto

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

Gel ConcentrationVoltageTimeBuffer
2%100 V30 min1/2 x TBE

Dephosphorylation

Mitsumoto

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

ReagentVolume
BBa_R0040 on pSB1C3, BBa_E0040 on pSB1A2 XbaI (Digestion product)10 μL
Antarctic Phosphatase1 μL
Antarctic Phosphatase Buffer2 μL
DW7 μL
Total20 μL

Dephosphorylation

StepTemp.TimeProcess
137℃15 minDephosphorylation
265℃5 minInactivation
Store4℃HoldStore

PCR (3 STEP)

Mitsumoto

BBa_I0500, BBa_B0015, BBa_B0032, BBa_B0033, BBa_B0034 on pSB1C3

ReagentVolume
BBa_I0500, BBa_B0015, BBa_B0032, BBa_B0033, BBa_B0034 on pSB1C31 μL
100UP - EX -F 10 μM1 μL
200DN - PS - R 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation35 cycle
Cycle 262.6℃30 secAnnealing35 cycle
Cycle 368℃elongation timeElongation35 cycle
Store4℃HoldStore

2015/08/30

Electrophoresis

Mimata

BBa_R0010 - BBa_B0034 on pSB1C3, Thanatin - BBa_K759012 on pSB1C3

Gel ConcentrationVoltageTimeBuffer
1%100 V60 min1/2 x TBE

Digestion

Mimata, Toyooka

XbaⅠ - Thanatin - SpeⅠ

ReagentVolume
XbaⅠ - Thanatin - SpeⅠ20 µL
SpeⅠ1 µL
XbaⅠ1 µL
10 × M Buffer 3 µL
DW5 µL
Total30 µL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
270℃15 minInactivation
Store4℃HoldStore

Digestion

Mimata, Toyooka

BBa_R0040 on pSB1A2, BBa_E0040 on pSB1A2

ReagentVolume
BBa_R0040 on pSB1A2, BBa_E0040 on pSB1A220 µL
SpeⅠ1 µL
CutSmart Buffer3 µL
DW6 µL
Total30 µL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
270℃15 minInactivation
Store4℃HoldStore

Digestion

Mimata, Toyooka

BBa_B0030 on pSB1C3

ReagentVolume
BBa_B0030 on pSB1C320 µL
SpeⅠ1 µL
XbaⅠ1 µL
10 × M Buffer 3 µL
DW5 µL
Total30 µL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
270℃15 minInactivation
Store4℃HoldStore

2015/08/31

PCR

Nishimura, Ono, Toyooka, Fujita

XbaⅠ - Thanatin - SpeⅠ

ReagentVolume
XbaⅠ - Thanatin - SpeⅠ1 µL
XbaⅠ - Thanatin - F 10 µM1 µL
SpeⅠ - Thanatin - R 10 µM1 µL
KOD - Plus - Neo1 µL
10 x PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation30 cycle
Cycle 268℃30 secAnnealing / Elongation30 cycle
Store4℃HoldStore

PCR

Nishimura, Ono, Toyooka, Fujita

BamHⅠ- Thanatin - BglⅡ

ReagentVolume
BamHⅠ- Thanatin - BglⅡ1 µL
BamHⅠ- Thanatin - F 10 µM1 µL
BglⅡ - D - Thanatin - R 10 µM1 µL
KOD - Plus - Neo1 µL
10 x PCR Buffer for KOD - Plus - Neo5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation30 cycle
Cycle 268℃30 secAnnealing / Elongation30 cycle
Store4℃HoldStore

Electrophoresis

Ono, Nishimura, Toyooka, Fujita

BamHⅠ- Thanatin - BglⅡ, XbaⅠ - Thanatin - SpeⅠ

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

Ethanol Precipitation

Ono, Nishimura, Toyooka, Fujita

BamHⅠ- Thanatin - BglⅡ, XbaⅠ - Thanatin - SpeⅠ

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

Digestion

Ono, Fujita, Toyooka, Nishimura

Thanatin fragment (Ethanol Precipitation product)

ReagentVolume
Thanatin fragment (Ethanol Precipitation product)20 µL
SpeⅠ2 µL
XbaⅠ1 µL
CutSmart Buffer3 µL
DW4 µL
Total30 µL

Digestion

StepTemp.TimeProcess
137℃60 minDigestion
Store4℃HoldStore

Digestion

Ono, Fujita, Toyooka, Nishimura

Thanatin fragment (Ethanol Precipitation product)

ReagentVolume
Thanatin fragment (Ethanol Precipitation product)20 µL
BamHⅠ2 µL
BglⅡ6 µL
10 × K Buffer10 µL
DW60 µL
Total100 µL

Digestion

StepTemp.TimeProcess
137℃60 minDigestion
Store4℃HoldStore

Ethanol Precipitation

Ono, Nishimura, Toyooka, Fujita

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

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

Electrophoresis

Ono, Nishimura, Toyooka, Fujita, Mimata

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

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

Ligation

Ono

BBa_K759012 on pSB1C3 / BamHⅠ- Thanatin - BglⅡ

ReagentVolume
BBa_K759012 on pSB1C395 µL
BamHⅠ- Thanatin - BglⅡ0.5 µL
Mighty Mix10 µL
Total20 µL

Ligation

StepTemp.TimeProcess
116℃30 minLigation
Store4℃HoldStore

Electrophoresis

Ono, Mimata

BBa_K759012 on pSB1C3, BBa_R0010 on pSB1C3

Gel ConcentrationVoltageTimeBuffer
1%100 V60 min1/2 x TBE

Digestion

Onoda,

BBa_E1010 on pSB1C3

ReagentVolume
BBa_E1010 on pSB1C310 µL
EcoRⅠ1 µL
XbaⅠ1 µL
10 × M Buffer 2 µL
DW6 µL
Total20 µL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
260℃15 minInactivation
Store4℃HoldStore

Digestion

Onoda

BBa_E1010 on pSB1C3

ReagentVolume
BBa_E1010 on pSB1C310 µL
XbaⅠ1 µL
CutSmart Buffer2 µL
DW7 µL
Total20 µL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
260℃15 minInactivation
Store4℃HoldStore

Electrophoresis

Toyooka

BBa_E1010 on pSB1C3 (Digestion product)

Gel ConcentrationVoltageTimeBuffer
1%100 V40 min1/2 x TBE

Gel Extract

Toyooka

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

Digestion

Ito, Sakai

HLA family

ReagentVolume
HLA, HLZ, BLA, BLZ20 µL
XbaⅠ1 µL
SpeⅠ1 µL
10 × M Buffer 3 µL
DW5 µL
Total30 µL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
280℃20 minInactivation
Store4℃HoldStore

Digestion

Ito, Sakai

HLA, HLZ, BLA, BLZ

ReagentVolume
HLA, HLZ, BLA, BLZ10 µL
XbaⅠ1 µL
SpeⅠ1 µL
10 x M buffer 2 µL
DW6 µL
Total20 µL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
280℃20 minInactivation
Store4℃HoldStore

Electrophoresis

Ito, Sakai

HLA family XbaⅠ & SpeⅠ (Digestion product)

Gel ConcentrationVoltageTimeBuffer
1%100 V40 min1/2 x TBE

Gel Extract

Ito, Sakai

HLA family XbaⅠ & SpeⅠ (Digestion product)
FastGeneTM 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
FastGeneTM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel

Mini-prep

Nishimura

BBa_R0010 on pSB1C3, BBa_I0500 on pSB1C3
FastGeneTM 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 ConcentrationVoltageTimeBuffer
2%100 V60 min1/2 x TBE

Ligation

Fujita, Nishimura

BBa_R0010 - BBa_B0034 on pSB1C3 / XbaⅠ - Thanatin - SpeⅠ

ReagentVolume
BBa_R0010 - BBa_B0034 on pSB1C39.5 µL
XbaⅠ - Thanatin - SpeⅠ0.5 µL
Mighty Mix10 µL
Total20 µL

Ligation

StepTemp.TimeProcess
116℃30 minLigation
Store4℃HoldStore

Ligation

Fujita

Ag43 on pSB1C3 / BamHⅠ - Thanatin - BglⅡ

ReagentVolume
BBa_R0010 - BBa_B0034 on pSB1C39.5 µL
BamHⅠ - Thanatin - BglⅡ0.5 µL
Mighty Mix10 µL
Total20 µL

Ligation

StepTemp.TimeProcess
116℃30 minLigation
Store4℃HoldStore

Ethanol Precipitation

Fujita

Thanatin - Ag43 on pSB1C3

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

Transformation

Nishimura, Toyooka

BBa_I0500 - BBa_B0033 on pSB1C3,

  1. 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.
  2. Incubated on ice for 30 min.
  3. Heat-shocked for 30 sec at 42℃.
  4. Added 200 µL of LB.
  5. Incubated the cells for 2 hrs at 37℃.
  6. Spread 300 µL of the culture onto plate with LBC.
  7. Incubated the plate at 37℃ for 16 hours.

Digestion

Nishimura, Onoda

HLA, HLZ, BLA, BLZ, BBa_B0030

ReagentVolume
HLA, HLZ, BLA, BLZ, BBa_B003010 µL
SpeⅠ1 µL
XbaⅠ1 µL
10 × M Buffer2 µL
DW6 µL
Total20 µL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
260℃15 minInactivation
Store4℃HoldStore

Electrophoresis

Nishimura

HLA, HLZ, BLA, BLZ

Gel ConcentrationVoltageTimeBuffer
2%100 V60 min1/2 x TBE

Gel Extract

Nishimura, Sakai, Ito, Kusumi

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

Ligation

Fujita, Sakai, Nishimura

BBa_B0015 on pSB1C3 / HLA, BLA, BLZ

ReagentVolume
BBa_B0015 on pSB1C310 µL
HLA , BLA, BLZ30 µL
T4 Ligase4.5 µL
10 × T4 DNA Ligase Buffer5 µL
DW0.5 µL
Total50 µL

Ligation

StepTemp.TimeProcess
116℃30 minLigation
Store4℃HoldStore

Ligation

Onoda, Nishimura, Fujita, Sakai

HLZ / BBa_B0015 on pSB1C3

ReagentVolume
BBa_B0015 on pSB1C310 µL
HLZ20 µL
T4 Ligase3.5 µL
10 × T4 DNA Ligase Buffer5 µL
DW1.5 µL
Total50 µL

Ligation

StepTemp.TimeProcess
116℃30 minLigation
Store4℃HoldStore

Electrophoresis

Sakai

HLA, HLZ, BLA, BLZ

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

Ligation

Sakai

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

ReagentVolume
BBa_R0100 - BBa_B0034 on pSB1C315 µL
XbaⅠ - Thanatin - SpeⅠ1.0 µL
T4 Ligase1.6 µL
10 X T4 DNA Ligase Buffer2.0 µL
DW0.4 µL
Total20 µL

Ligation

StepTemp.TimeProcess
116℃30 minLigation
Store4℃HoldStore

Transformation

Sakai

BBa_R0010 - BBa_B0034 - XbaⅠ - Thanatin - SpeⅠ on pSB1C3

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

PCR

Ono

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

ReagentVolume
ABF-2, BLA, Thanatin, 10 × His tag - TEV - Thanatin, BLZ, HLZ1 µL
EX - F - Universal 10 µM1 µL
PS - R 10 µM1 µL
KOD - Plus - Neo1 µL
10 x PCR Buffer for KOD - Plus - Neo 5 µL
2 mM dNTPs5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation30 cycle
Cycle 268℃30 secAnnealing / Elongation30 cycle
Store4℃HoldStore

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

Transformation

Nishimura

HLA, BLA, HLZ, BLZ

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

Colony PCR

Nishimura, Ono, Onoda, Mimata

BBa_R0010 - BBa_B0034 - Thanatin

ReagentVolume
Single Colony-
100UP - EX - F 10 µM0.4 µL
200DN - PS - R 10 µM0.4 µL
KAPA Taq5 µL
DW4.2 µL
Total10 µL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start95℃120 secInitialization
Cycle 195℃30 secDenaturation40 cycle
Cycle 25730 secAnnealing40 cycle
Cycle 372℃40 secElongation40 cycle
Finish72℃120 secFinal Elongation
Store4℃HoldStore

Colony PCR

Nishimura, Ono, Onoda, Mimata

BBa_R0010 - BBa_B0034 - Thanatin

ReagentVolume
Single Colony-
100UP - EX - F 10 µM0.4 µL
SpeⅠ - Thanatin - Rv 10 µM0.4 µL
KAPA Taq5 µL
DW4.2 µL
Total10 µL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start95℃120 secInitialization
Cycle 195℃30 secDenaturation40 cycle
Cycle 25730 secAnnealing40 cycle
Cycle 372℃40 secElongation40 cycle
Finish72℃120 secFinal Elongation
Store4℃HoldStore

Colony PCR

Nishimura, Ono, Onoda, Mimata

Ag43 - Thanatin

ReagentVolume
Single Colony-
AGSP - BamHⅠ - Spindorin 10 µM0.4 µL
BglⅡ - D - Thanatin - Rv 10 µM0.4 µL
KAPA Taq5 µL
DW4.2 µL
Total10 µL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start95℃120 secInitialization
Cycle 195℃30 secDenaturation40 cycle
Cycle 26530 secAnnealing40 cycle
Cycle 372℃40 secElongation40 cycle
Finish72℃120 secFinal Elongation
Store4℃HoldStore

Colony PCR

Nishimura, Ono, Onoda, Mimata

Ag43 - Thanatin

ReagentVolume
Single Colony-
AGSP - BamHⅠ - Spindorin 10 µM0.4 µL
Ag43 - bunit - Rv 10 µM0.4 µL
KAPA Taq5 µL
DW4.2 µL
Total10 µL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start95℃120 secInitialization
Cycle 195℃30 secDenaturation40 cycle
Cycle 26530 secAnnealing40 cycle
Cycle 372℃40 secElongation40 cycle
Finish72℃120 secFinal Elongation
Store4℃HoldStore

Colony PCR

Nishimura, Ono, Onoda, Mimata

BBa_B0031 on pSB1A2

ReagentVolume
Single Colony-
100UP - EX - F 10 µM0.4 µL
200DN - PS - R 10 µM0.4 µL
KAPA Taq5 µL
DW4.2 µL
Total10 µL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start95℃120 secInitialization
Cycle 195℃30 secDenaturation40 cycle
Cycle 25730 secAnnealing40 cycle
Cycle 372℃40 secElongation40 cycle
Finish72℃120 secFinal Elongation
Store4℃HoldStore

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 ConcentrationVoltageTimeBuffer
1%100 V50 min1/2 x TBE

Electrophoresis

Nishimura, Ono, Mimata

BBa_I0500

Gel ConcentrationVoltageTimeBuffer
1%100 V50 min1/2 x TBE

Digestion

Nisimura, Ono, Mimata

BLZ, HLZ, ABF-2

ReagentVolume
BLZ, HLZ, ABF-230 µL
SpeⅠ1 µL
Total31 µL

Digestion

StepTemp.TimeProcess
137℃60 minDigestion
Store4℃HoldStore

Electrophoresis

Nishimura, Ono, Mimata

BLZ, HLZ, ABF-2,

Gel ConcentrationVoltageTimeBuffer
2%100 V60 min1/2 x TBE

Gel Extract

Mitsumoto

BLZ, HLZ, ABF-2
FastGeneTM 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

  1. 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.
  2. Incubated on ice for 30 min.
  3. Heat-shocked for 30 sec at 42℃.
  4. Added 2000 µL of LB.
  5. Incubated the cells for 2 hrs at 37℃.
  6. Spread 300 µL of the culture onto plate with LBC.
  7. Incubated the plate at 37℃ for 16 hours.

2015/09/03

Ligation

Nishimura

BBa_B0015 on pSB1C3 / HLZ

ReagentVolume
BBa_B0015 on pSB1C36 µL
HLZ8 µL
Mighty Mix14 µL
Total28 µL

Ligation

StepTemp.TimeProcess
116℃30 minLigation
Store4℃HoldStore

Ligation

Nishimura

BBa_B0015 on pSB1C3 / HLA, BLA, BLZ

ReagentVolume
BBa_B0015 on pSB1C36 µL
HLA, BLA, BLZ14 µL
Mighty Mix20 µL
Total40 µL

Ligation

StepTemp.TimeProcess
116℃30 minLigation
Store4℃HoldStore

Dephosphorylation

Nishimura

BBa_B0015 on pSB1C3

ReagentVolume
BBa_B0015 on pSB1C340 µL
Antarctic Phosphatase4 µL
Antarctic Phosphatase Buffer8 µL
DW28 µL
Total80 µL

Dephosphorylation

StepTemp.TimeProcess
137℃30 minDephosphorylation
265℃10 minInactivation
Store4℃HoldStore

Colony PCR

Nishimura, Ono, Fujita

BBa_R0010 - BBa_B0034 - Thanatin

ReagentVolume
Single Colony-
100UP - EX - F 10 µM0.4 µL
200DN - PS - R 10 µM0.4 µL
KAPA Taq5 µL
DW4.2 µL
Total10 µL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start95℃120 secInitialization
Cycle 195℃30 secDenaturation40 cycle
Cycle 25730 secAnnealing40 cycle
Cycle 372℃40 secElongation40 cycle
Finish72℃120 secFinal Elongation
Store4℃HoldStore

Colony PCR

Nishimura, Ono, Fujita

BBa_R0010 - BBa_B0034 - Thanatin

ReagentVolume
Single Colony-
100UP - EX - F 10 µM0.4 µL
SpeⅠ - Thanatin - Rv 10 µM0.4 µL
KAPA Taq5 µL
DW4.2 µL
Total10 µL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start95℃120 secInitialization
Cycle 195℃30 secDenaturation40 cycle
Cycle 25730 secAnnealing40 cycle
Cycle 372℃40 secElongation40 cycle
Finish72℃120 secFinal Elongation
Store4℃HoldStore

Colony PCR

Nishimura, Ono, Fujita

Ag43 - Thanatin

ReagentVolume
Single Colony-
AGSP - BamHⅠ - Spindorin 10 µM0.4 µL
BglⅡ - D - Thanatin - Rv 10 µM0.4 µL
KAPA Taq5 µL
DW4.2 µL
Total10 µL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start95℃120 secInitialization
Cycle 195℃30 secDenaturation40 cycle
Cycle 26530 secAnnealing40 cycle
Cycle 372℃40 secElongation40 cycle
Finish72℃120 secFinal Elongation
Store4℃HoldStore

Colony PCR

Nishimura, Ono, Fujita

Ag43 - Thanatin

ReagentVolume
Single Colony-
AGSP - BamHⅠ - Spindorin 10 µM0.4 µL
Ag43 - bunit - Rv 10 µM0.4 µL
KAPA Taq5 µL
DW4.2 µL
Total10 µL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start95℃120 secInitialization
Cycle 195℃30 secDenaturation40 cycle
Cycle 26530 secAnnealing40 cycle
Cycle 372℃40 secElongation40 cycle
Finish72℃120 secFinal Elongation
Store4℃HoldStore

Colony PCR

Nishimura, Ono, Fujita

BBa_B0031 on pSB1A2

ReagentVolume
Single Colony-
100UP - EX - F 10 µM0.4 µL
200DN - PS - R 10 µM0.4 µL
KAPA Taq5 µL
DW4.2 µL
Total10 µL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start95℃120 secInitialization
Cycle 195℃30 secDenaturation40 cycle
Cycle 25730 secAnnealing40 cycle
Cycle 372℃40 secElongation40 cycle
Finish72℃120 secFinal Elongation
Store4℃HoldStore

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 ConcentrationVoltageTimeBuffer
1%100 V50 min1/2 x TBE

Transformation

Nishimura, Fujita

HLA, BLA, HLZ, BLZ

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

Mini-prep

Ono, Fujita

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

Digestion

Onoda

EcoRⅠ - BBa_R0010 - SpeⅠ

ReagentVolume
EcoRⅠ - BBa_R0010 - SpeⅠ28.5 µL
EcoRⅠ1.5 µL
SpeⅠ1.5 µL
10 × H Buffer3.5 µL
Total35 µL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
Store4℃HoldStore

2015/09/04

Electrophoresis

Nishimura

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

Gel ConcentrationVoltageTimeBuffer
2%100 V60 min1/2 x TBE

Mini-prep

Nishimura, Ono

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

Sequencing

Nishimura

thanatin - Ag43

ReagentVolume
Ag43 thanatin1 µL
BBa_I0500 - Fw, Ag43 - Rv1.5 µL
BigDye Terminator1 µL
5 x Sequencing Buffer1.5 µL
DW5 µL
Total10 µL

Sequencing

StepTemp.TimeProcessCycle
Start96℃10 secDenaturation
Cycle 150℃5 sec-25 cycle
Cycle 260℃240 sec-25 cycle
Store4℃HoldStore

Sequencing

Nishimura

BBa_I0500 - BBa_B0032, BBa_I0500 - BBa_B0033, BBa_I0500 - BBa_B0034

ReagentVolume
100UP - EX - F1 µL
200DN - PS - R1.5 µL
Ready Reaction Premix1 µL
5 x Sequencing Buffer1.5 µL
DW5 µL
Total10 µL

Sequencing

StepTemp.TimeProcessCycle
Start96℃10 secDenaturation
Cycle 150℃5 sec-25 cycle
Cycle 260℃240 sec-25 cycle
Store4℃HoldStore

Ethanol Precipitation

Nishimura, Fujita, Mimata

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

  1. Added 1 µL of NaOAc, 1.5 µL of glycogen and 30 µL of 100% ethanol.
  2. Left it at -80℃ for 10 min.
  3. Centrifuged at 15,000 rpm for 15 min at 4℃.
  4. Removed supernatant and added 100 µL of 70% ethanol.
  5. Centrifuged at 15,000 rpm for 10 min at 4℃.
  6. Removed supernatant and air-dried at room temperature with light shield.
  7. Suspended with 10 µL of HiDi.
  8. 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

    1. 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.
    2. Incubated on ice for 30 min.
    3. Heat-shocked for 30 sec at 42℃.
    4. Added 200 µL of LB.
    5. Incubated the cells for 2 hrs at 37℃.
    6. Spread 300 µL of the culture onto plate with LBC.
    7. Incubated the plate at 37℃ for 18 hours.

    Digestion

    Fujita, Mimata

    pSB1C3

    ReagentVolume
    pSB1C35 µL
    EcoRⅠ1 µL
    PstⅠ1 µL
    10 × H Buffer5 µL
    DW38 µL
    Total50 µL

    Digestion

    StepTemp.TimeProcess
    137℃120 minDigestion
    280℃20 minInactivation
    Store4℃HoldStore

    Digestion

    Fujita, Mimata

    pSB1C3

    ReagentVolume
    pSB1C35 µL
    XbaⅠ1 µL
    SpeⅠ1 µL
    CutSmart Buffer5 µL
    DW38 µL
    Total50 µL

    Digestion

    StepTemp.TimeProcess
    137℃120 minDigestion
    280℃20 minInactivation
    Store4℃HoldStore

    Digestion

    Fujita, Mimata

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

    ReagentVolume
    ABF-2, Prefix - BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - Thanatin - BBa_B0015 - Suffix, 10 x His tag - TEV - Thanatin20 µL
    EcoRⅠ1 µL
    PstⅠ1 µL
    10 x H Buffer5 µL
    DW23 µL
    Total50 µL

    Digestion

    StepTemp.TimeProcess
    137℃120 minDigestion
    280℃20 minInactivation
    Store4℃HoldStore

    Digestion

    Fujita, Mimata

    BLA

    ReagentVolume
    BLA20 µL
    XbaⅠ1 µL
    PstⅠ1 µL
    CutSmart Buffer5 µL
    DW23 µL
    Total50 µL

    Digestion

    StepTemp.TimeProcess
    137℃120 minDigestion
    280℃20 minInactivation
    Store4℃HoldStore

    Digestion

    Fujita, Mimata

    BLZ, HLZ, ABF-2

    ReagentVolume
    BLZ, HLZ, ABF-220 µL
    EcoRⅠ1 µL
    SpeⅠ1 µL
    10 x H Buffer5 µL
    DW23 µL
    Total50 µL

    Digestion

    StepTemp.TimeProcess
    137℃120 minDigestion
    280℃20 minInactivation
    Store4℃HoldStore

    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

    1. 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.
    2. Incubated on ice for 30 min.
    3. Heat-shocked for 30 sec at 42℃.
    4. Added 200 μL of LB.
    5. Incubated the cells for 2 hrs at 37℃.
    6. Spread 300 μL of the culture onto plate with LBCp.
    7. 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

    ReagentVolume
    Single Colony-
    XbaⅠ - Thanatin - F 10 μM0.4 μL
    200DN - PS - R 10 μM0.4 μL
    KAPA Taq5 μL
    DW4.2μL
    Total10 μL

    3 Step Cycle (Tm value ≤ 63℃)

    StepTemp.TimeProcessCycle
    Start95℃120 secInitialization
    Cycle 195℃30 secDenaturation35 cycle
    Cycle 259.330 secAnnealing35 cycle
    Cycle 368℃90 secElongation35 cycle
    Store4℃HoldStore

    Colony PCR

    Mitsumoto

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

    ReagentVolume
    Single Colony-
    XbaⅠ - HLZ - F 10 μM0.4 μL
    200DN - PS - R 10 μM0.4 μL
    KAPA Taq5 μL
    DW4.2μL
    Total10 μL

    3 Step Cycle (Tm value ≤ 63℃)

    StepTemp.TimeProcessCycle
    Start95℃120 secInitialization
    Cycle 195℃30 secDenaturation35 cycle
    Cycle 259.330 secAnnealing35 cycle
    Cycle 368℃90 secElongation35 cycle
    Store4℃HoldStore

    Colony PCR

    Mitsumoto

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

    ReagentVolume
    Single Colony-
    XbaⅠ - BLA - F 10 μM0.4 μL
    200DN - PS - R 10 μM0.4 μL
    KAPA Taq5 μL
    DW4.2μL
    Total10 μL

    3 Step Cycle (Tm value ≤ 63℃)

    StepTemp.TimeProcessCycle
    Start95℃120 secInitialization
    Cycle 195℃30 secDenaturation35 cycle
    Cycle 256.230 secAnnealing35 cycle
    Cycle 368℃90 secElongation35 cycle
    Store4℃HoldStore

    Colony PCR

    Mitsumoto

    BBa_B0031 on pSB1A2

    ReagentVolume
    Single Colony-
    100UP - EX - F 10 μM0.4 μL
    200DN - PS - R 10 μM0.4 μL
    KAPA Taq5 μL
    DW4.2μL
    Total10 μL

    3 Step Cycle (Tm value ≤ 63℃)

    StepTemp.TimeProcessCycle
    Start95℃120 secInitialization
    Cycle 195℃30 secDenaturation35 cycle
    Cycle 256.230 secAnnealing35 cycle
    Cycle 368℃90 secElongation35 cycle
    Store4℃HoldStore

    Colony PCR

    Mitsumoto

    BBa_R0010 - BBa_B0034 - Thanatin - BBa_B0015 on pSB1C3

    ReagentVolume
    Single Colony-
    100UP - EX - F 10 μM0.4 μL
    200DN - PS - R 10 μM0.4 μL
    KAPA Taq5 μL
    DW4.2μL
    Total10 μL

    3 Step Cycle (Tm value ≤ 63℃)

    StepTemp.TimeProcessCycle
    Start95℃120 secInitialization
    Cycle 195℃30 secDenaturation35 cycle
    Cycle 261.630 secAnnealing35 cycle
    Cycle 368℃90 secElongation35 cycle
    Store4℃HoldStore

    Colony PCR

    Mitsumoto

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

    ReagentVolume
    Single Colony-
    100UP - EX - F 10 μM0.4 μL
    200DN - PS - R 10 μM0.4 μL
    KAPA Taq5 μL
    DW4.2μL
    Total10 μL

    3 Step Cycle (Tm value ≤ 63℃)

    StepTemp.TimeProcessCycle
    Start95℃120 secInitialization
    Cycle 195℃30 secDenaturation35 cycle
    Cycle 261.630 secAnnealing35 cycle
    Cycle 368℃90 secElongation35 cycle
    Store4℃HoldStore

    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 ConcentrationVoltageTimeBuffer
    1%100 V40 min1/2 x TBE

    Electrophoresis

    Mitsumoto

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

    Gel ConcentrationVoltageTimeBuffer
    1%100 V40 min1/2 x TBE

    Electrophoresis

    Mitsumoto

    pSB1C3 XbaⅠ & SpeⅠ(Digestion product)

    Gel ConcentrationVoltageTimeBuffer
    1%100 V40 min1/2 x TBE

    Electrophoresis

    Mitsumoto

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

    Gel ConcentrationVoltageTimeBuffer
    1%100 V40 min1/2 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 ConcentrationVoltageTimeBuffer
    1%100 V40 min1/2 x TBE

    Electrophoresis

    Mitsumoto

    pSB1C3 XbaⅠ & SpeⅠ(Digestion product)

    Gel ConcentrationVoltageTimeBuffer
    1%100 V40 min1/2 x TBE

    Electrophoresis

    Mitsumoto

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

    Gel ConcentrationVoltageTimeBuffer
    1%100 V40 min1/2 x TBE

    Electrophoresis

    Mitsumoto

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

    Gel ConcentrationVoltageTimeBuffer
    1%100 V40 min1/2 x TBE

    Digestion

    Mitsumoto

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

    ReagentVolume
    BBa_I0500 - BBa_B0034 on pSB1C3, BBa_I0500 - B0033 on pSB1C3, BBa_I0500 - B0032 on pSB1C320 μL
    SpeⅠ2 μL
    PstⅠ2 μL
    10 x H Buffer3 μL
    DW3 μL
    Total30 μL

    Digestion

    StepTemp.TimeProcess
    137℃120 minDigestion
    260℃15 minInactivation
    Store4℃HoldStore

    Digestion

    Mitsumoto

    Ag43 - Thanatin (1mer)

    ReagentVolume
    Ag43 - Thanatin (1mer)20 μL
    BglⅡ2 μL
    CutSmart Buffer3 μL
    DW5 μL
    Total30 μL

    Digestion

    StepTemp.TimeProcess
    137℃120 minDigestion
    260℃15 minInactivation
    Store4℃HoldStore

    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

    ReagentVolume
    Single Colony-
    100UP - EX - F 10 µM0.4 µL
    200DN - PS - R 10 µM0.4 µL
    KAPA Taq5.0 µL
    DW4.2 µL
    Total10 µL

    3 Step Cycle (Tm value ≤ 63℃)

    StepTemp.TimeProcessCycle
    Start95℃120 secInitialization
    Cycle 195℃30 secDenaturation35 cycle
    Cycle 257.630 secAnnealing35 cycle
    Cycle 372℃210 secElongation35 cycle
    Finish72℃120 secFinal Elongation
    Store4℃HoldStore

    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 ConcentrationVoltageTimeBuffer
    1% V30 min1/2 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
    FastGeneTM Plasmid mini kit (Nippon Genetics Co.,Ltd)
    standard protocol

    Ligation

    Mimata

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

    ReagentVolume
    pSB1C32.5 µL
    Prefix - BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - Thanatin - BBa_B0015 - Suffix40 µL
    Mighty Mix42.5 µL
    Total85 µL

    Ligation

    StepTemp.TimeProcess
    116℃30 minLigation
    270℃10 minInactivation
    Store4℃HoldStore

    Ligation

    Mimata

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

    ReagentVolume
    pSB1C32.5 µL
    Prefix - BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - 10 × His tag - TEV - Thanatin - BBa_B001540 µL
    Mighty Mix42.5 µL
    Total85 µL

    Ligation

    StepTemp.TimeProcess
    116℃30 minLigation
    270℃10 minInactivation
    Store4℃HoldStore

    Electrophoresis

    Mimata

    Thanatin on pSB1C3, TEV - Thanatin on pSB1C3

    Gel ConcentrationVoltageTimeBuffer
    1%100 V60 min1/2 x TBE

    Digestion

    Sakai

    BBa_I0500 - BBa_B0034, BBa_I0500 - BBa_B0033, BBa_I0500 - BBa_B0032

    ReagentVolume
    BBa_I0500 - BBa_B0034, BBa_I0500 - BBa_B0033, BBa_I0500 - BBa_B003220 µL
    PstⅠ1.5 µL
    SpeⅠ1.5 µL
    10 x H Buffer3 µL
    DW4 µL
    Total30 µL

    Digestion

    StepTemp.TimeProcess
    137℃120 minDigestion
    280℃20 minInactivation
    Store4℃HoldStore

    Digestion

    Sakai

    Ag43 - Thanatin

    ReagentVolume
    Ag43 - Thanatin20 µL
    BglⅡ2.0 µL
    CutSmart Buffer3 µL
    DW5 µL
    Total30 µL

    Digestion

    StepTemp.TimeProcess
    137℃120 minDigestion
    280℃20 minInactivation
    Store4℃HoldStore

    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

    ReagentVolume
    Single Colony-
    100UP - EX - F 10 µM0.4 µL
    200DN - PS - R 10 µM0.4 µL
    KAPA Taq5.0 µL
    DW4.2 µL
    Total10 µL

    3 Step Cycle (Tm value ≤ 63℃)

    StepTemp.TimeProcessCycle
    Start95℃120 secInitialization
    Cycle 195℃30 secDenaturation35 cycle
    Cycle 257.630 secAnnealing35 cycle
    Cycle 372℃210 secElongation35 cycle
    Finish72℃120 secFinal Elongation
    Store4℃HoldStore

    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 ConcentrationVoltageTimeBuffer
    1%100 V30 min1/2 x TBE

    2015/09/07

    Electrophoresis

    Nishimura

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

    Gel ConcentrationVoltageTimeBuffer
    1%100 V60 min1/2x TBE

    Sequencing

    Nishimura、Fujita

    Ag43 Thanatin

    ReagentVolume
    Ag43 Thanatin1 μL
    pBAD Fw / Ag43-Rv1.5 μL
    BigDye Terminator1 μL
    5x Sequencing Buffer1.5 μL
    DW5 μL
    Total10 μL

    Sequencing

    StepTemp.TimeProcessCycle
    Start96℃10 secDenaturation
    Cycle 150℃5 sec-25 cycle
    Cycle 260℃240 sec-25 cycle
    Store4℃HoldStore

    Sequencing

    Nishimura, Fujita

    pBAD_B0031

    ReagentVolume
    pBAD_B00311 μL
    100UP - EX - F / 200DN - PS - R1.5 μL
    BigDye Terminator1 μL
    5x Sequencing Buffer1.5 μL
    DW5 μL
    Total10 μL

    Sequencing

    StepTemp.TimeProcessCycle
    Start96℃10 secDenaturation
    Cycle 150℃5 sec-25 cycle
    Cycle 260℃240 sec-25 cycle
    Store4℃HoldStore

    Ethanol Precipitation

    Nishimura

    Ag43 thanatin on pSB1C3, pBAD_B0031

    1. Added 2 μL of NaOAc, 1.5 μL of glycogen and 50 μL of 100% ethanol.
    2. Left it at 24℃ for 15 min.
    3. Centrifuged at 15,000 rpm for 15 min at 24℃.
    4. Removed supernatant and added 100 μL of 70% ethanol.
    5. Centrifuged at 15,000 rpm for 10 min at 24℃.
    6. Removed supernatant and air-dried at room temperature with light shield.
    7. 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

    ReagentVolume
    Single Colony-
    LBC2000 μL
    Chloramphenicol2 μL

    Cultured for 16 hours.

    Digestion

    Ono

    Ag43 - Thanatin pSB1C3 (Mini-prep product)

    ReagentVolume
    Ag43 - Thanatin pSB1C3 (Mini-prep product)20 μL
    BglⅡ1 μL
    DW6 μL
    10 × H Buffer3 μL
    Total20 μL

    Digestion

     
    StepTemp.TimeProcess
    137℃120 minDigestion
    270℃15 minInactivation
    Store4℃HoldStore

    Electrophoresis

    Nishimura

    Ag43 - Thanatin -BglⅡ (Digestion product)

    Gel ConcentrationVoltageTimeBuffer
    1%100 V60 min1/2x TBE

    Colony PCR

    Ono, Sakai

    Ag43 - Thanatin on pSB1C3

    ReagentVolume
    Single Colony-
    Agsp-BamH1-Spidroin 10 μM0.4 μL
    Ag43 - bunit - R 10 μM0.4 μL
    KAPA Taq5 μL
    DW4.2 μL
    Total10 μL

    3 Step Cycle (Tm value ≤ 63℃)

    StepTemp.TimeProcessCycle
    Start95℃120 secInitialization
    Cycle 195℃30 secDenaturation40 cycle
    Cycle 26530 secAnnealing40 cycle
    Cycle 372℃40 secElongation40 cycle
    Finish72℃120 secFinal Elongation
    Store4℃HoldStore

    Colony PCR

    Ono, Sakai

    Ag43 - Thanatin on pSB1C3

    ReagentVolume
    Single Colony-
    Agsp-BamH1-Spidroin 10 μM0.4 μL
    BglⅡ - D - Thanatin - R 10 μM0.4 μL
    KAPA Taq5 μL
    DW4.2 μL
    Total10 μL

    3 Step Cycle (Tm value ≤ 63℃)

    StepTemp.TimeProcessCycle
    Start95℃120 secInitialization
    Cycle 195℃30 secDenaturation40 cycle
    Cycle 26530 secAnnealing40 cycle
    Cycle 372℃40 secElongation40 cycle
    Finish72℃120 secFinal Elongation
    Store4℃HoldStore

    Colony PCR

    Ono, Sakai

    BBa_B0031 on pSB1C3 (as a positive control)

    ReagentVolume
    Single Colony-
    100UP - EX - F 10 μM0.4 μL
    200DN - PS - R 10 μM0.4 μL
    KAPA Taq5 μL
    DW4.2 μL
    Total10 μL

    3 Step Cycle (Tm value ≤ 63℃)

    StepTemp.TimeProcessCycle
    Start95℃120 secInitialization
    Cycle 195℃30 secDenaturation40 cycle
    Cycle 25730 secAnnealing40 cycle
    Cycle 372℃40 secElongation40 cycle
    Finish72℃120 secFinal Elongation
    Store4℃HoldStore

    Electrophoresis

    Ono

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

    Gel ConcentrationVoltageTimeBuffer
    1%100 V40 min1/2x TBE

    Transformation

    Sakai

    Ag43 - Thanatin on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence on pSB1C3

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

    Ethanol Precipitation

    Onoda

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

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

    Electrophoresis

    Onoda

    HLA, BLA, HLZ, BLZ (Ethanol Precipitation)

    Gel ConcentrationVoltageTimeBuffer
    1%100 V40 min1/2x TBE

    Colony PCR

    Onoda

    HLA, HLZ, BLZ

    ReagentVolume
    Single Colony-
    XbalⅠ - HLA - F, XbalⅠ - HLZ - F, XbalⅠ - BLZ - F 10 μM0.4 μL
    200DN - PS - R 10 μM0.4 μL
    Kapa-Taq5 μL
    DW4.2 μL
    Total10 μL

    3 Step Cycle (Tm value ≤ 63℃)

    StepTemp.TimeProcessCycle
    Start95℃120 secInitialization
    Cycle 195℃30 secDenaturation40 cycle
    Cycle 261.530 secAnnealing40 cycle
    Cycle 372℃60 secElongation40 cycle
    Finish72℃120 secFinal Elongation
    Store4℃HoldStore

    Colony PCR

    Onoda

    BLA, BBa_B0031 (as a positive control)

    ReagentVolume
    Single Colony-
    BalⅠ - BLA - F, 100UP - EX - F 10 μM0.4 μL
    200DN - PS - R 10 μM0.4 μL
    Kapa-Taq5 μL
    DW4.2 μL
    Total10 μL

    3 Step Cycle (Tm value ≤ 63℃)

    StepTemp.TimeProcessCycle
    Start95℃120 secInitialization
    Cycle 195℃30 secDenaturation40 cycle
    Cycle 25730 secAnnealing40 cycle
    Cycle 372℃60 secElongation40 cycle
    Finish72℃120 secFinal Elongation
    Store4℃HoldStore

    Electrophoresis

    Sakai

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

    Gel ConcentrationVoltageTimeBuffer
    1%100 V60 min1/2x TBE

    Ligation

    Nishimura, Ono, Fujita

    BBa_B0015 on pSB1C3 / BamHⅠ - Thanatin - BglⅡ

    ReagentVolume
    BBa_B0015 on pSB1C310 μL
    BamHⅠ - Thanatin - BglⅡ1 μL
    T4 Ligase1 μL
    10 × T4 Ligase2 μL
    DW6 μL
    Total20 μL

    Ligation

    StepTemp.TimeProcess
    116℃60 minLigation
    Store4℃HoldStore

    2015/09/08

    Colony PCR

    Nishimura

    HLA, HLZ, BLZ

    ReagentVolume
    Single Colony-
    XbalⅠ - HLA - F, XbalⅠ - HLZ - F, XbalⅠ - BLZ - F 10 μM0.4 μL
    200DN - PS - R 10 μM0.4 μL
    Kapa-Taq5 μL
    DW4.2 μL
    Total10 μL

    3 Step Cycle (Tm value ≤ 63℃)

    StepTemp.TimeProcessCycle
    Start95℃120 secInitialization
    Cycle 195℃30 secDenaturation40 cycle
    Cycle 261.530 secAnnealing40 cycle
    Cycle 372℃60 secElongation40 cycle
    Finish72℃120 secFinal Elongation
    Store4℃HoldStore

    Colony PCR

    Nishimura

    BLA, BBa_B0031 (as a positive control)

    ReagentVolume
    Single Colony-
    BalⅠ - BLA - F, 100UP - EX - F 10 μM0.4 μL
    200DN - PS - R 10 μM0.4 μL
    Kapa-Taq5 μL
    DW4.2 μL
    Total10 μL

    3 Step Cycle (Tm value ≤ 63℃)

    StepTemp.TimeProcessCycle
    Start95℃120 secInitialization
    Cycle 195℃30 secDenaturation40 cycle
    Cycle 25730 secAnnealing40 cycle
    Cycle 372℃60 secElongation40 cycle
    Finish72℃120 secFinal Elongation
    Store4℃HoldStore

    Transformation

    Nishimura

    Ag43 - Thanatin on pSB1C3 (Ligation product)

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

    Electrophoresis

    Nishimura

    Gel ConcentrationVoltageTimeBuffer
    1%100 V50 min1/2x TBE
    →failed

    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
    FastGeneTM Plasmid mini kit (Nippon Genetics Co.,Ltd)
    standard protocol

    Liquid Culture

    Nishimura

    Ag43 - Thanatin on pSB1C3 into DH5α

    ReagentVolume
    Single Colony-
    LB2000 μL
    Chloramphenicol2 μL

    Cultured for 16 hours.

    Sequencing

    Ito

    Ag43 - Thanatin

    ReagentVolume
    Ag43 - Thanatin1 μL
    BBa_I0500 - f2 / 200 - β domain - Ag43 - R1.5 μL
    BigDye Terminator1 μL
    5 x Sequencing Buffer1.5 μL
    DW5 μL
    Total10 μL

    Sequencing

    StepTemp.TimeProcessCycle
    Start96℃10 secDenaturation
    Cycle 150℃5 sec-25 cycle
    Cycle 260℃240 sec-25 cycle
    Store4℃HoldStore

    Sequencing

    Ito

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

    ReagentVolume
    ABF - 21 μL
    100UP - EX - F / 200DN - PS - R1.5 μL
    Ready Reaction Premix1 μL
    5 x Sequencing Buffer1.5 μL
    DW5 μL
    Total10 μL

    Sequencing

    StepTemp.TimeProcessCycle
    Start96℃10 secDenaturation
    Cycle 150℃5 sec-25 cycle
    Cycle 260℃240 sec-25 cycle
    Store4℃HoldStore

    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

    ReagentVolume
    Single Colony-
    100UP - EX - F 10 μM0.4 μL
    200DN - PS - R 10 μM0.4 μL
    KAPA Taq5 μL
    DW4.2 μL
    Total10 μL

    3 Step Cycle (Tm value ≤ 63℃)

    StepTemp.TimeProcessCycle
    Start95℃120 secInitialization
    Cycle 195℃30 secDenaturation35 cycle
    Cycle 257.630 secAnnealing35 cycle
    Cycle 368℃150 secElongation35 cycle
    Store4℃HoldStore

    Ethanol Precipitation

    Nishimura

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

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

    Mini-prep

    Nishimura

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

    Digestion

    Ono

    Ag43 - Thanatin (Mini-prep product)

    ReagentVolume
    Ag43 - Thanatin (Mini-prep product)10 μL
    BglⅡ1 μL
    SpeⅠ1 μL
    10 × H Buffer2 μL
    DW6 μL
    Total20 μL

    Digestion

    StepTemp.TimeProcess
    137℃60 minDigestion
    220 minInactivation
    Store4℃HoldStore

    PCR

    Nishimura

    Ag43 - Thanatin (Mini-prep product)

    ReagentVolume
    Ag43 - Thanatin (Mini-prep product) 1 μL
    BamHⅠ - thanatin - F 10 μM1 μL
    200DN - PS - R 10 μM1 μL
    KOD Plus NEO1 μL
    KOD Plus NEO 10x Buffer5 μL
    2 mM dNTPS5 μL
    25 mM MgSO43 μL
    DW33 μL
    Total50 μL

    2 Step Cycle (Tm value ≥ 63℃)

    StepTemp.TimeProcessCycle
    Start94℃120 secInitialization
    Cycle 198℃10 secDenaturation35 cycle
    Cycle 268℃120 secAnnealing / Elongation35 cycle
    Store4℃HoldStore

    Electrophoresis

    Nishimura

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

    Gel ConcentrationVoltageTimeBuffer
    1%100 V50 min1/2x TBE

    Sequencing

    Nishimura, Ono

    Ag43 - Thanatin (Mini-prep product)

    ReagentVolume
    Ag43 - Thanatin (Mini-prep product)1 μL
    pbad - F / 200DN Ag43 - R1.5 μL
    Ready Reaction Premix1 μL
    BigDye Terminator1.5 μL
    DW5 μL
    Total10 μL

    Sequencing

    StepTemp.TimeProcessCycle
    Start96℃10 secDenaturation
    Cycle 150℃5 sec-25 cycle
    Cycle 260℃240 sec-25 cycle
    Store4℃HoldStore

    PCR

    Ono, Nishimura

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

    ReagentVolume
    Signal peptide - Thanatin - BglⅡ - β domain (Mini-prep product)1 μL
    Agsp - BamHⅠ - Spidroin 10 μM1 μL
    200DN - PS - R 10 μM1 μL
    KOD - Plus - Neo1 μL
    10 x PCR Buffer for KOD - Plus - Neo5 μL
    2 mM dNTPs5 μL
    25 mM MgSO43 μL
    DW33 μL
    Total50 μL

    3 Step Cycle (Tm value ≤ 63℃)

    StepTemp.TimeProcessCycle
    Start95℃120 secInitialization
    Cycle 198℃10 secDenaturation30 cycle
    Cycle 26530 secAnnealing30 cycle
    Cycle 368℃120 secElongation30 cycle
    Store4℃HoldStore

    PCR

    Ono, Nishimura

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

    ReagentVolume
    Signal peptide - Thanatin - BglⅡ - β domain (Mini-prep product)1 μL
    Ag43 - F4 10 μM1 μL
    200 β - domain Ag43 - R 10 μM1 μL
    KOD - FX - NEO1 μL
    10 × PCR Buffer for KOD -FX - NEO 25 μL
    2 mM dNTPs5 μL
    25 mM MgSO43 μL
    DW13 μL
    Total50 μL

    3 Step Cycle (Tm value ≤ 63℃)

    StepTemp.TimeProcessCycle
    Start95℃120 secInitialization
    Cycle 198℃20 secDenaturation30 cycle
    Cycle 26015 secAnnealing30 cycle
    Cycle 368℃180 secElongation30 cycle
    Store4℃HoldStore

    PCR Purification

    Nishimura


    FastGeneTM 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 ConcentrationVoltageTimeBuffer
    1%100 V40 min1/2 x TBE

    Digestion

    Ito

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

    ReagentVolume
    BBa_I0500 - B0034 - Thanatin - BglⅡ - Ag43 β domain - BBa_B0015 on pSB1C310 μL
    BglⅡ1 μL
    Spe11 μL
    10 x H Buffer2 μL
    DW6 μL
    Total20 μL

    Digestion

    StepTemp.TimeProcess
    137℃60 minDigestion
    280℃20 minInactivation
    Store4℃HoldStore

    PCR

    Ito

    Ag43 - Thanatin on pSB1C3

    ReagentVolume
    Ag43 - Thanatin on pSB1C31 μL
    BamHⅠ - Thanatin - Fw 10 μM1 μL
    200DN - PS - R 10 μM1 μL
    KOD - Plus - Neo1 μL
    10 x PCR Buffer for KOD - Plus - Neo5 μL
    2 mM dNTPs5 μL
    25 mM MgSO43 μL
    DW33 μL
    Total50 μL

    2 Step Cycle (Tm value ≥ 63℃)

    StepTemp.TimeProcessCycle
    Start94℃120 secInitialization
    Cycle 198℃10 secDenaturation35 cycle
    Cycle 268℃120 secAnnealing / Elongation35 cycle
    Store4℃HoldStore

    Sequencing

    Ito

    BBa_I0500 - BLA, ABF-2

    ReagentVolume
    BBa_I0500 - BLA, ABF-21 μL
    100UP - EX - F / 200DN - PS - R1.5 μL
    Ready Reaction Premix1 μL
    5 x Sequencing Buffer1.5 μL
    DW5 μL
    Total10 μL

    Sequencing

    StepTemp.TimeProcessCycle
    Start96℃10 secDenaturation
    Cycle 150℃5 sec-25 cycle
    Cycle 260℃240 sec-25 cycle
    Store4℃HoldStore

    Sequencing

    Ito

    BBa_I0500 - BLA

    ReagentVolume
    BBa_I0500 - BLA1 μL
    pbad - f2 / 200 - β domain - Ag43 - Rv1.5 μL
    Ready Reaction Premix1 μL
    5 x Sequencing Buffer1.5 μL
    DW5 μL
    Total10 μL

    Sequencing

    StepTemp.TimeProcessCycle
    Start96℃10 secDenaturation
    Cycle 150℃5 sec-25 cycle
    Cycle 260℃240 sec-25 cycle
    Store4℃HoldStore

    2015/09/10

    Mini-prep

    Mitsumoto

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

    PCR

    Nishimura

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

    ReagentVolume
    Signal peptide - Thanatin - BglⅡ - β domain (Mini-prep product)1 μL
    Agsp - BamHⅠ - Spidroin 10 μM1 μL
    200DN - PS - R 10 μM1 μL
    KOD - Plus - Neo1 μL
    10 x PCR Buffer for KOD - Plus - Neo5 μL
    2 mM dNTPs5 μL
    25 mM MgSO43 μL
    DW33 μL
    Total50 μL

    3 Step Cycle (Tm value ≤ 63℃)

    StepTemp.TimeProcessCycle
    Start95℃120 secInitialization
    Cycle 198℃10 secDenaturation30 cycle
    Cycle 26530 secAnnealing30 cycle
    Cycle 368℃120 secElongation30 cycle
    Store4℃HoldStore

    PCR

    Nishimura

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

    ReagentVolume
    Signal peptide - Thanatin - BglⅡ - β domain (Mini-prep product)1 μL
    Ag43 - F4 10 μM1 μL
    200 β - domain Ag43 - R 10 μM1 μL
    KOD - FX - NEO1 μL
    10 × PCR Buffer for KOD -FX - NEO 25 μL
    2 mM dNTPs5 μL
    25 mM MgSO43 μL
    DW13 μL
    Total50 μL

    3 Step Cycle (Tm value ≤ 63℃)

    StepTemp.TimeProcessCycle
    Start95℃120 secInitialization
    Cycle 198℃20 secDenaturation30 cycle
    Cycle 26015 secAnnealing30 cycle
    Cycle 368℃180 secElongation30 cycle
    Store4℃HoldStore

    PCR Purification

    Nishimura

    Thanatin - β domain BBa_B0015, pSB1C3 - BBa_R0010 - Signal peptide - Thanatin (PCR 3STEP product)
    FastGeneTM 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 ConcentrationVoltageTimeBuffer
    1%100 V40 min1/2 x TBE

    Electrophoresis

    Nishimjura

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

    Gel ConcentrationVoltageTimeBuffer
    1%100 V40 min1/2 x TBE
    →failed

    PCR

    Nishimura

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

    ReagentVolume
    Signal peptide - Thanatin - BglⅡ - β domain (Mini-prep product)1 μL
    Ag43 - F4 10 μM1 μL
    200 β - domain Ag43 - R 10 μM1 μL
    KOD - FX - NEO1 μL
    10 × PCR Buffer for KOD -FX - NEO 25 μL
    2 mM dNTPs5 μL
    25 mM MgSO43 μL
    DW13 μL
    Total50 μL

    3 Step Cycle (Tm value ≤ 63℃)

    StepTemp.TimeProcessCycle
    Start95℃120 secInitialization
    Cycle 198℃20 secDenaturation30 cycle
    Cycle 26015 secAnnealing30 cycle
    Cycle 368℃180 secElongation30 cycle
    Store4℃HoldStore

    PCR Purification

    Nishimura

    pSB1C3 - BBa_R0010 - Signal peptide - Thanatin (PCR 3STEP product)
    FastGeneTM 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 ConcentrationVoltageTimeBuffer
    1%100 V40 min1/2 x TBE

    Digestion

    Nishimura, Fujita

    ReagentVolume
    Thanatin - β domain BBa_B001525 μL
    BamHⅠ3 μL
    SpeⅠ0.5 μL
    DW16.5 μL
    10 × K Buffer5 μL
    Total50 μL

    Digestion

    StepTemp.TimeProcess
    137℃120 minDigestion
    270℃20 minInactivation
    Store4℃HoldStore

    Digestion

    Nishimura, Fujita

    ReagentVolume
    pSB1C3 - BBa_R0010 - Signal peptide - Thanatin25 μL
    BglⅡ4 μL
    SpeⅠ0.5 μL
    DW15.5 μL
    10 × H Buffer5 μL
    Total50 μL

    Digestion

    StepTemp.TimeProcess
    137℃120 minDigestion
    270℃20 minInactivation
    Store4℃HoldStore

    Electrophoresis

    Nishimura

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

    Gel ConcentrationVoltageTimeBuffer
    1%100 V40 min1/2 x TBE

    Gel Extract

    Nishimura

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

    ReagentVolume
    pSB1C3 - BBa_R0010 - Signal peptide - Thanatin1.5 μL
    Thanatin - β domain BBa_B00153.5 μL
    Mighty Mix5 μL
    Total10 μL

    Ligation

    StepTemp.TimeProcess
    160℃60 minLigation
    280℃20 minInactivation
    Store4℃HoldStore

    Transformation

    Nishimura

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

    1. 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.
    2. Incubated on ice for 30 min.
    3. Heat-shocked for 30 sec at 42℃.
    4. Added 500 μL of LB.
    5. Incubated the cells for 2 hrs at 37℃.
    6. Spread 500, 50 μL of the culture onto two plates with LBC.
    7. Incubated the plate at 37℃ for hours.

    Transformation

    Nishimura

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

    1. 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.
    2. Incubated on ice for 30 min.
    3. Heat-shocked for 30 sec at 42℃.
    4. Added 500 μL of LB.
    5. Incubated the cells for 2 hrs at 37℃.
    6. Spread 500, 50 μL of the culture onto two plates with LBC.
    7. Incubated the plate at 37℃ for hours.

    Ethanol Precipitation

    Ito

    Thanatin ( 2mer )

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

    Sequencing

    Ito

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

    ReagentVolume
    Prefix - BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - 10 × His tag - TEV - Thanatin - BBa_B00151 μL
    100UP - EX - F / 200DN - PS - R1.5 μL
    Ready Reaction Premix1 μL
    5 x Sequencing Buffer1.5 μL
    DW5 μL
    Total10 μL

    Sequencing

    StepTemp.TimeProcessCycle
    Start96℃10 secDenaturation
    Cycle 150℃5 sec-25 cycle
    Cycle 260℃240 sec-25 cycle
    Store4℃HoldStore

    Sequencing

    Ito

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

    ReagentVolume
    Prefix - BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - 10 × His tag - TEV - Thanatin - BBa_B00151 μL
    BBa_I0500 - f2 / 200 - β domain - Ag43 - Rv1.5 μL
    Ready Reaction Premix1 μL
    5 x Sequencing Buffer1.5 μL
    DW5 μL
    Total10 μL

    Sequencing

    StepTemp.TimeProcessCycle
    Start96℃10 secDenaturation
    Cycle 150℃5 sec-25 cycle
    Cycle 260℃240 sec-25 cycle
    Store4℃HoldStore

    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

    ReagentVolume
    Single Colony-
    100UP - EX - F 10 μM0.4 μL
    200DN - PS - R 10 μM0.4 μL
    KAPA Taq5 μL
    DW4.2 μL
    Total10 μL

    3 Step Cycle (Tm value ≤ 63℃)

    StepTemp.TimeProcessCycle
    Start95℃120 secInitialization
    Cycle 195℃30 secDenaturation35 cycle
    Cycle 257.630 secAnnealing35 cycle
    Cycle 372℃90 secElongation35 cycle
    Finish72℃120 secFinal Elongation
    Store4℃HoldStore

    Sequencing

    Nishimura

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

    ReagentVolume
    BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C31 μL
    100UP - EX - F / 200DN - PS - R1.5 μL
    BigDye Terminator1 μL
    5 x Sequencing Buffer1.5 μL
    DW5 μL
    Total10 μL

    Sequencing

    StepTemp.TimeProcessCycle
    Start96℃10 secDenaturation
    Cycle 150℃5 sec-25 cycle
    Cycle 260℃240 sec-25 cycle
    Store4℃HoldStore

    Ethanol Precipitation

    Nishimura

    BLA

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

    Colony PCR

    Nishimura

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

    ReagentVolume
    Single Colony-
    Agsp - BamHⅠ - Spidroin 10 μM0.4 μL
    Ag43 - bunit - R 10 μM0.4 μL
    KAPA Taq5 μL
    DW4.2 μL
    Total10 μL

    3 Step Cycle (Tm value ≤ 63℃)

    StepTemp.TimeProcessCycle
    Start94℃120 secInitialization
    Cycle 198℃30 secDenaturation40 cycle
    Cycle 26530 secAnnealing40 cycle
    Cycle 372℃40 secElongation40 cycle
    Finish72℃120 secFinal Elongation
    Store4℃HoldStore

    Colony PCR

    Nishimura

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

    ReagentVolume
    Single Colony-
    Agsp - BamHⅠ - Spidroin 10 μM0.4 μL
    BglⅡ - D - Thanatin - R 10 μM0.4 μL
    KAPA Taq5 μL
    DW4.2 μL
    Total10 μL

    3 Step Cycle (Tm value ≤ 63℃)

    StepTemp.TimeProcessCycle
    Start94℃120 secInitialization
    Cycle 198℃30 secDenaturation40 cycle
    Cycle 26530 secAnnealing40 cycle
    Cycle 372℃40 secElongation40 cycle
    Finish72℃120 secFinal Elongation
    Store4℃HoldStore

    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)

    ReagentVolume
    Single Colony-
    100UP - EX - F 10 μM0.4 μL
    200DN - PS - R 10 μM0.4 μL
    KAPA Taq5 μL
    DW4.2 μL
    Total10 μL

    3 Step Cycle (Tm value ≤ 63℃)

    StepTemp.TimeProcessCycle
    Start94℃120 secInitialization
    Cycle 198℃30 secDenaturation40 cycle
    Cycle 26530 secAnnealing40 cycle
    Cycle 372℃40 secElongation40 cycle
    Finish72℃120 secFinal Elongation
    Store4℃HoldStore

    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 ConcentrationVoltageTimeBuffer
    1%100 V50 min1/2 x TBE

    2015/09/12

    Dephosphorylation

    Nishimura

    BBa_R0010 - Signal peptide - Thanatin - BglⅡ on pSB1C3

    ReagentVolume
    pSB1C3 - BBa_R0010 - Signal peptide - Thanatin - BglⅡ20 μL
    Antarctic Phosphatase2 μL
    Antarctic Phosphatase Buffer4 μL
    DW14 μL
    Total40 μL

    Dephosphorylation

    StepTemp.TimeProcess
    137℃30 minDephosphorylation
    265℃10 minInactivation
    Store4℃HoldStore

    Ligation

    Nishimura

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

    ReagentVolume
    pSB1C3 - BBa_R0010 - Signal peptide - Thanatin1.5 μL
    Thanatin - β domain - BBa_B00153.5 μL
    Mighty Mix5 μL
    Total10 μL

    Ligation

    StepTemp.TimeProcess
    116℃60 minLigation
    Store4℃HoldStore

    Ligation

    Nishimura

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

    ReagentVolume
    BBa_R0010 - Signal peptide - Thanatin on pSB1C31.5 μL
    Thanatin - β domain - BBa_B00153.5 μL
    Mighty Mix5 μL
    Total10 μL

    Ligation

    StepTemp.TimeProcess
    160℃60 minLigation
    280℃20 minInactivation
    Store4℃HoldStore

    Transformation

    Nishimura

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

    1. 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.
    2. Incubated on ice for 30 min.
    3. Heat-shocked for 30 sec at 42℃.
    4. Added 400 μL of LB.
    5. Incubated the cells for 2 hrs at 37℃.
    6. Spread 300 μL of the culture onto plate with LBC.
    7. 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
    FastGeneTM 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

    ReagentVolume
    BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C31 μL
    pbad - f2 / 200 βdomein Ag43 Rv1.5 μL
    Ready Reaction Premix1 μL
    5 x Sequencing Buffer1.5 μL
    DW5 μL
    Total10 μL

    Sequencing

    StepTemp.TimeProcessCycle
    Start96℃10 secDenaturation
    Cycle 150℃10 sec-25 cycle
    Cycle 260℃240 sec-25 cycle
    Store4℃HoldStore

    Ethanol Precipitation

    Nishimura

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

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

    ReagentVolume
    Single Colony-
    100UP - EX - F 10 μM0.4 μL
    200DN - PS - R 10 μM0.4 μL
    KAPA Taq5 μL
    DW4.2 μL
    Total10 μL

    3 Step Cycle (Tm value ≤ 63℃)

    StepTemp.TimeProcessCycle
    Start95℃120 secInitialization
    Cycle 195℃30 secDenaturation40 cycle
    Cycle 25730 secAnnealing40 cycle
    Cycle 372℃40 secElongation40 cycle
    Finish72℃120 secFinal Elongation
    Store4℃HoldStore

    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

    ReagentVolume
    Single Colony-
    LB2000 μL
    Chloramphenicol2 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 ConcentrationVoltageTimeBuffer
    1%100 V30 min1/2 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

    ReagentVolume
    Single Colony-
    100UP - EX - F 10 μM0.4 μL
    200DN - PS - R 10 μM0.4 μL
    KAPA Taq5 μL
    DW4.2 μL
    Total10 μL

    3 Step Cycle (Tm value ≤ 63℃)

    StepTemp.TimeProcessCycle
    Start95℃120 secInitialization
    Cycle 195℃30 secDenaturation35 cycle
    Cycle 257.630 secAnnealing35 cycle
    Cycle 372℃90 secElongation35 cycle
    Finish72℃120 secFinal Elongation
    Store4℃HoldStore

    MIC Test

    Onoda, Sakai, Ito

    50μM Thanatin

    1. Cultured E. coli DH5α, JM109 in 2 ml of LB overnight.
    2. Took 80 μl of it and cultured for 1 hr in 2 mL of LB culture until the OD600 became 0.4.
    3. 100,000 fold dilutied it with PB culture.
    4. Spread them on LB plate and counted the number of colony.
    5. Made 20 μl of dilution series of supernatant and cultured 80 μL of diluted DH5α, JM109 at 30℃, 120,000 rpm for 18 hrs.
    6. Incubated in refrigerator.
    7. 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

    ReagentVolume
    BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C31 μL
    Agsp - BamHⅠ - Spidroin 10 μM1 μL
    200DN - PS - R 10 μM1 μL
    KOD - Plus - Neo1 μL
    10 x PCR Buffer for KOD - Plus - Neo5 μL
    2 mM dNTPs5 μL
    25 mM MgSO43 μL
    DW33 μL
    Total50 μL

    3 Step Cycle (Tm value ≤ 63℃)

    StepTemp.TimeProcessCycle
    Start95℃120 secInitialization
    Cycle 198℃10 secDenaturation30 cycle
    Cycle 26515 secAnnealing30 cycle
    Cycle 368℃120 secElongation30 cycle
    Store4℃HoldStore

    PCR

    Nishimura

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

    ReagentVolume
    BBa_R0010 - Signal peptide - Thanatin - BamHⅠ / BglⅡ scar- Thanatin - β domain BBa_B0015 on pSB1C31 μL
    Ag43 - f4 10 μM1 μL
    Xba - RBS - scar 10 μM1 μL
    KOD - FX - Neo1 μL
    2x PCR Buffer for KOD - FX - Neo25 μL
    2 mM dNTPs5 μL
    25 mM MgSO43 μL
    DW13 μL
    Total50 μL

    3 Step Cycle (Tm value ≤ 63℃)

    StepTemp.TimeProcessCycle
    Start95℃120 secInitialization
    Cycle 198℃10 secDenaturation30 cycle
    Cycle 26015 secAnnealing30 cycle
    Cycle 368℃180 secElongation30 cycle
    Store4℃HoldStore

    Electrophoresis

    Nishimura

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

    Gel ConcentrationVoltageTimeBuffer
    1%100 V60 min1/2 x TBE

    PCR Purification

    Nishimura

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

    Digestion

    Nishimura

    BglⅡ - Thanatin - β domain BBa_B0015

    ReagentVolume
    BglⅡ - Thanatin - β domain BBa_B001525 μL
    BamHⅠ3 μL
    SpeⅠ0.5 μL
    10 × K Buffer5 μL
    DW16.5 μL
    Total50 μL

    Digestion

    StepTemp.TimeProcess
    137℃120 minDigestion
    270℃20 minInactivation
    Store4℃HoldStore

    Digestion

    Nishimura

    BBa_R0010 - Signal peptide - Thanatin - BglⅡ on pSB1C3

    ReagentVolume
    BBa_R0010 - Signal peptide - Thanatin - BglⅡ on pSB1C325 μL
    BglⅡ4 μL
    SpeⅠ0.5 μL
    10 × H Buffer5 μL
    DW15.5 μL
    Total50 μL

    Digestion

    StepTemp.TimeProcess
    137℃120 minDigestion
    270℃20 minInactivation
    Store4℃HoldStore

    Electrophoresis

    Nishimura

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

    Gel ConcentrationVoltageTimeBuffer
    1%100 V60 min1/2 x TBE

    Gel Extract

    Nishimura

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

    ReagentVolume
    BBa_R0010 - Signal peptide - Thanatin - BglⅡ on pSB1C3 ( 2mer )3.5 μL
    Thanatin - β domain - BBa_B0015 ( 1mer )1.5 μL
    Mighty Mix5 μL
    Total10 μL

    Ligation

    StepTemp.TimeProcess
    116℃30 minLigation
    265℃10 minInactivation
    Store4℃HoldStore

    Ligation

    Nishimura

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

    ReagentVolume
    BBa_R0010 - Signal peptide - Thanatin - BglⅡ on pSB1C3 ( 2mer )1.5 μL
    Thanatin - β domain - BBa_B0015 ( 1mer )3.5 μL
    Mighty Mix5 μL
    Total10 μL

    Ligation

    StepTemp.TimeProcess
    116℃30 minLigation
    265℃10 minInactivation
    Store4℃HoldStore

    Ligation

    Nishimura

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

    ReagentVolume
    BBa_R0010 - Signal peptide - Thanatin - BglⅡ on pSB1C3 ( 2mer )3.5 μL
    Thanatin - β domain - BBa_B0015 ( 2mer )1.5 μL
    Mighty Mix5 μL
    Total10 μL

    Ligation

    StepTemp.TimeProcess
    116℃30 minLigation
    265℃10 minInactivation
    Store4℃HoldStore

    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

    1. 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.
    2. Incubated on ice for 30 min.
    3. Heat-shocked for 30 sec at 42℃.
    4. Added 400 μL of LB.
    5. Incubated the cells for 2 hrs at 37℃.
    6. Spread 300 μL of the culture onto plate with LBC.
    7. 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

    ReagentVolume
    Single Colony-
    Agsp - BamHⅠ - Spindroin 10 μM0.4 μL
    200 - β domein - Ag43 - Rv 10 μM0.4 μL
    KAPA Taq5 μL
    DW4.2 μL
    Total10 μL

    3 Step Cycle (Tm value ≤ 63℃)

    StepTemp.TimeProcessCycle
    Start94℃120 secInitialization
    Cycle 198℃30 secDenaturation40 cycle
    Cycle 26530 secAnnealing40 cycle
    Cycle 372℃40 secElongation40 cycle
    Finish72℃120 secFinal Elongation
    Store4℃HoldStore

    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 ConcentrationVoltageTimeBuffer
    1%100 V30 min1/2 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

    ReagentVolume
    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 pSB1C31 μL
    pbad - f21.5 μL
    Ready Reaction Premix1 μL
    5 x Sequencing Buffer1.5 μL
    DW5 μL
    Total10 μL

    Sequencing

    StepTemp.TimeProcessCycle
    Start96℃10 secDenaturation
    Cycle 150℃5 sec-25 cycle
    Cycle 260℃240 sec-25 cycle
    Store4℃HoldStore

    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

    ReagentVolume
    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 pSB1C31 μL
    200 - β domein - Ag43 - Rv1.5 μL
    Ready Reaction Premix1 μL
    5 x Sequencing Buffer1.5 μL
    DW5 μL
    Total10 μL

    Sequencing

    StepTemp.TimeProcessCycle
    Start96℃10 secDenaturation
    Cycle 150℃5 sec-25 cycle
    Cycle 260℃240 sec-25 cycle
    Store4℃HoldStore

    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
    FastGeneTM 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

    ReagentVolume
    Single Colony-
    LB2000 μL
    Chloramphenicol2 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
    FastGeneTM 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
    FastGeneTM 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

    ReagentVolume
    Single Colony-
    LB15000 μL
    Chloramphenicol1.5 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

    ReagentVolume
    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 pSB1C31 μL
    100UP - EX - F1.5 μL
    Ready Reaction Premix1 μL
    5 x Sequencing Buffer1.5 μL
    DW5 μL
    Total10 μL

    Sequencing

    StepTemp.TimeProcessCycle
    Start96℃10 secDenaturation
    Cycle 150℃5 sec-25 cycle
    Cycle 260℃240 sec-25 cycle
    Store4℃HoldStore

    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

    ReagentVolume
    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 pSB1C31 μL
    200DN - PS - R1.5 μL
    Ready Reaction Premix1 μL
    5 x Sequencing Buffer1.5 μL
    DW5 μL
    Total10 μL

    Sequencing

    StepTemp.TimeProcessCycle
    Start96℃10 secDenaturation
    Cycle 150℃5 sec-25 cycle
    Cycle 260℃240 sec-25 cycle
    Store4℃HoldStore

    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

    ReagentVolume
    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 pSB1C31 μL
    100UP - EX - F1.5 μL
    Ready Reaction Premix1 μL
    5 x Sequencing Buffer1.5 μL
    DW5 μL
    Total10 μL

    Sequencing

    StepTemp.TimeProcessCycle
    Start96℃10 secDenaturation
    Cycle 150℃5 sec-25 cycle
    Cycle 260℃240 sec-25 cycle
    Store4℃HoldStore

    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

    ReagentVolume
    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 pSB1C31 μL
    200DN - PS - R1.5 μL
    Ready Reaction Premix1 μL
    5 x Sequencing Buffer1.5 μL
    DW5 μL
    Total10 μL

    Sequencing

    StepTemp.TimeProcessCycle
    Start96℃10 secDenaturation
    Cycle 150℃5 sec-25 cycle
    Cycle 260℃240 sec-25 cycle
    Store4℃HoldStore

    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

    ReagentVolume
    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 pSB1C31 μL
    Agsp - BamHⅠ - Spidroin 10 μM1 μL
    200DN - PS - R 10 μM1 μL
    KOD - Plus - Neo1 μL
    10 x PCR Buffer for KOD - Plus - Neo5 μL
    2 mM dNTPs5 μL
    25 mM MgSO43 μL
    DW33 μL
    Total50 μL

    3 Step Cycle (Tm value ≤ 63℃)

    StepTemp.TimeProcessCycle
    Start95℃120 secInitialization
    Cycle 198℃10 secDenaturation30 cycle
    Cycle 26515 secAnnealing30 cycle
    Cycle 368℃120 secElongation30 cycle
    Store4℃HoldStore

    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

    ReagentVolume
    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 pSB1C31 μL
    Ag43 - f4 10 μM1 μL
    XbaⅠ - RBS - scar 10 μM1 μL
    KOD - FX - Neo1 μL
    2x PCR Buffer for KOD - FX - Neo25 μL
    2 mM dNTPs5 μL
    25 mM MgSO43 μL
    DW13 μL
    Total50 μL

    3 Step Cycle (Tm value ≤ 63℃)

    StepTemp.TimeProcessCycle
    Start95℃120 secInitialization
    Cycle 198℃10 secDenaturation30 cycle
    Cycle 26015 secAnnealing30 cycle
    Cycle 368℃180 secElongation30 cycle
    Store4℃HoldStore

    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 ConcentrationVoltageTimeBuffer
    1%100 V60 min1/2 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
    FastGeneTM 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

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

    2015/09/17

    Measurement of Optical Density

    Mimata

    Ag43 - Thanatin (1mer) into DH5α

    1. Cultured the colony for 10 hours.
    2. Diluted with midium to set OD600 to 0.1.
    3. Measured OD600 12 times with an hour interval each.

    Measurement of Optical Density

    Mimata

    Ag43 - Thanatin (2mer) into DH5α

    1. Cultured the colony for 10 hours.
    2. Diluted with midium to set OD600 to 0.1.
    3. Measured OD600 12 times with an hour interval each.

    Measurement of Optical Density

    Mimata

    Ag43 - Thanatin (3mer) into DH5α

    1. Cultured the colony for 10 hours.
    2. Diluted with midium to set OD600 to 0.1.
    3. Measured OD600 12 times with an hour interval each.

    Measurement of Optical Density

    Mimata

    Ag43 - Thanatin (4mer) into DH5α

    1. Cultured the colony for 10 hours.
    2. Diluted with midium to set OD600 to 0.1.
    3. Measured OD600 12 times with an hour interval each.

    Measurement of Optical Density

    Mimata

    Ag43 (without Thanatin) into DH5α

    1. Cultured the colony for 10 hours.
    2. Diluted with midium to set OD600 to 0.1.
    3. Measured OD600 12 times with an hour interval each.

    MIC Test

    Ito, Ono

    Thanatin (1mer, 2mer, 3mer, 4mer, nothing (as a negative control))

    1. Cultured E. coli DH5α in 2 ml of LB overnight.
    2. Took 80 μl of it and cultured for 1 hr in 2 mL of LB culture until the OD600 became 0.4.
    3. 100,000 fold dilutied it with PB culture.
    4. Spread them on LB plate and counted the number of colony.
    5. Made 20 μl of dilution series of supernatant and cultured 80 μL of diluted JM109 at 30℃, 120,000 rpm for 18 hrs.
    6. Incubated in refrigerator.
    7. Observed the appearance of colony and decided the effective concentration of supernatant.

    2015/09/18

    MIC Test

    Ito, Onoda

    Thanatin (4mer)

    1. Cultured E. coli DH5α in 2 ml of LB overnight.
    2. Took 80 μl of it and cultured for 1 hr in 2 mL of LB culture until the OD600 became 0.4.
    3. 100,000 fold dilutied it with PB culture.
    4. Spread them on LB plate and counted the number of colony.
    5. Made 20 μl of dilution series of supernatant and cultured 80 μL of diluted DH5α at 30℃, 120,000 rpm for 18 hrs.
    6. Incubated in refrigerator.
    7. Observed the appearance of colony and decided the effective concentration of supernatant.

    ←Notebook Top

Link to iGEM HokkaidoU website main1 Link to iGEM HokkaidoU twitter
Link to iGEM HokkaidoU facebook page main5