Team:HokkaidoU Japan/Notebook/lcasei

Notebook

main1

L. casei

August

2015/08/11

Transformation

Mitsumoto, Onoda

pHIL253

  1. Added 5 µL of pHIL253 to µL of thawed competent cells (DH5α;) on ice.
  2. Incubated on ice for 30 min.
  3. Heat-shocked for 60 sec at 42℃.
  4. Spread 300 µL of the culture onto plate with LBA.
  5. Incubated the plate at 37℃ for 16 hrs.

2015/08/12

Liquid Culture

Mitsumoto

pHIL253

ReagentVolume
Single Colony-
LB2000 µL
Ampicillin2 µL

Cultured for 20 hrs.

2015/08/13

Mini-prep

Mitsumoto

pHIL253
FastGeneTM Plasmid mini kit (Nippon Genetics Co., Ltd)
standard protocol

Preparation of Bacteria

Mitsumoto

AHU1910

  1. Added 5 mL of L. casei to MRS medium.
  2. Cultured overnight.
  3. Measured OD600 and diluted with MRS medium to set OD600 to 0.2.
  4. Stand cultured for 1.5 ~ 2 hrs until OD600 is 0.4 ~ 0.5.
  5. Incubated the cells on ice for 10 min.
  6. Centrifuged at 6,000 g for 15 min at 4℃.
  7. Removed supernatant and added 30 mL of cooled PEB.
  8. Centrifuged at 6,000 g for 15 min at 4℃.
  9. Removed supernatant and added 30 mL of cooled PEB.
  10. Centrifuged at 6,000 g for 15 min at 4℃.
  11. Removed supernatant and added 1 mL of cooled PEB.

Electroporation

Mitsumoto

AHU1910

  1. Prepared the pHIL253 to 300 ng/10 µL (TE pH 8).
  2. Mixed 10 µL of plasmid and 200 µL of bacteria and incubated on ice for 10 min.
  3. Electoroporated at 2 kV with Gene Pulser (Bio-Rad) and Gene Pulser®/MicroPulserTM Electroporation Cuvettes (0.2 cm gap #1652086).
  4. Added 800 µL of cooled MRS medium and stand cultured for 3 hrs in 30℃.
  5. Spread on MRS plate (Em 5 µg/mL), (Amp 5 µg/mL).

2015/08/19

Electrophoresis

Mitsumoto

pHIL253

Gel ConcentrationVoltageTimeBuffer
2%100V30 min1/2 x TBE

PCR

Mitsumoto

XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - SpeⅠ, proCrp4

ReagentVolume
pHIL2531 µL
XbaⅠ - oripAMβ1 - repE - F - primer 10 µM1 µL
SpeⅠ - Sec signal sequence - 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 secDenaturation35 cycle
Cycle 268℃120 secAnnealing / Elongation35 cycle
Store4℃HoldStore

2015/08/24

Ligation

Onoda

BBa_E0040 on pSB1A2 / XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - SpeⅠ

ReagentVolume
BBa_E0040 on pSB1A21 µL
XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - SpeⅠ2 µL
Mighty Mix3 µL
DW4 µL
Total10 µL

Ligation

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

Ligation

Onoda

BBa_R0040 on pSB1C3 / XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - SpeⅠ

ReagentVolume
BBa_R0040 on pSB1C31 µL
XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - SpeⅠ2 µL
Mighty Mix3 µL
DW4 µL
Total10 µL

Ligation

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

Ligation

Onoda

pSB1C3 XbaⅠ & SpeⅠ (Digestion product) / XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - SpeⅠ

ReagentVolume
pSB1C3 XbaⅠ & SpeⅠ (Digestion product)1 µL
XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - SpeⅠ2 µL
Mighty Mix3 µL
DW4 µL
Total10 µL

Ligation

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

Transformation

Onoda

pHIL253, oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - BBa_R0040 on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - BBa_E0040 on pSB1A2

  1. Added 1 µL of pHIL253, oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - BBa_R0040 on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - BBa_E0040 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. 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 hrs.

2015/08/26

Preparation of Bacteria

Mimata, Sakai

AHU1910

  1. Added 5 mL of L. casei to MRS medium.
  2. Cultured overnight.
  3. Measured OD600 and diluted with MRS medium to set OD600 to 0.2.
  4. Stand cultured for 1.5 ~ 2 hrs until OD600 is 0.4 ~ 0.5.
  5. Incubated the cells on ice for 10 min.
  6. Centrifuged at 6,000 g for 15 min at 4℃.
  7. Removed supernatant and added 30 mL of cooled PEB.
  8. Centrifuged at 6,000 g for 15 min at 4℃.
  9. Removed supernatant and added 30 mL of cooled PEB.
  10. Centrifuged at 6,000 g for 15 min at 4℃.
  11. Removed supernatant and added 1 mL of cooled PEB.

2015/08/27

Preparation of Bacteria

Mimata, Sakai

AHU1910

  1. Added 5 mL of L. casei to MRS medium.
  2. Cultured overnight.
  3. Measured OD600 and diluted with MRS medium to set OD600 to 0.2.
  4. Stand cultured for 1.5 ~ 2 hrs until OD600 is 0.4 ~ 0.5.
  5. Incubated the cells on ice for 10 min.
  6. Centrifuged at 6,000 g for 15 min at 4℃.
  7. Removed supernatant and added 30 mL of cooled PEB.
  8. Centrifuged at 6,000 g for 15 min at 4℃.
  9. Removed supernatant and added 30 mL of cooled PEB.
  10. Centrifuged at 6,000 g for 15 min at 4℃.
  11. Removed supernatant and added 1 mL of cooled PEB.

Electroporation

Mimata, Sakai

AHU1910

  1. Prepared the plasmid to 300 ng/10 µL (TE pH 8).
  2. Mixed 10 µL of plasmid and 200 µL of bacteria and incubated on ice for 10 min.
  3. Electoroporated at 2 kV with Gene Pulser (Bio-Rad) and Gene Pulser®/MicroPulserTM Electroporation Cuvettes (0.2 cm gap #1652086).
  4. Added 800 µL of cooled MRS medium and stand cultured for 3 hrs in 30℃.
  5. Spread on MRS plate (Em 5 µg/mL).

Colony PCR

Mitsumoto

oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - BBa_R0040 on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence BBa_E0040

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℃10 secDenaturation35 cycle
Cycle 257.630 secAnnealing35 cycle
Cycle 368℃120 secElongation35 cycle
Store4℃HoldStore

Colony PCR

Mitsumoto

oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - BBa_R0040 on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence BBa_E0040

ReagentVolume
Single Colony-
100UP - EX - F 10 µM0.4 µL
SpeⅠ - Sec signal sequence reverse 2 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℃10 secDenaturation35 cycle
Cycle 257.630 secAnnealing35 cycle
Cycle 368℃120 secElongation35 cycle
Store4℃HoldStore

2015/08/28

Electrophoresis

Mitsumoto

colony PCR products of oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - BBa_R0040 on pSB1C3 (Colony PCR product), oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence BBa_E0040 (Colony PCR product)

Gel ConcentrationVoltageTimeBuffer
2%100V45 min1/2 x TBE

2015/08/31

Electrophoresis

Onoda

oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - BBa_0040 on pSB1A2(colony PCR product), oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - BBa_R0040 on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence on pSB1C3

Gel ConcentrationVoltageTimeBuffer
1%100 V40 min1/2 x TBE

Dephosphorylation

Onoda

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

ReagentVolume
vector10 µL
Antarctic Phosphatase1 µL
Antarctic Phosphatase Buffer2 µL
DW7 µL
Total20 µL

Dephosphorylation

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

Electrophoresis

Onoda

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

Gel ConcentrationVoltageTimeBuffer
1%100 V40 min1/2 x TBE

Gel Extract

Onoda

BBa_E0040 on pSB1A2, BBa_R0040 on pSB1C3, pSB1C3 XbaⅠ & SpeⅠ (Digestion product)
FastGeneTM Gel Extraction kit (Nippon Genetics Co., Ltd)
DNA extraction from gel

Digestion

Onoda

oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence

ReagentVolume
oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence20 µL
XbaⅠ1 µL
SpeⅠ - HF1 µL
10 × M buffer3 µL
DW5 µL
Total30 µL

Digestion

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

Electrophoresis

Onoda

oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence

Gel ConcentrationVoltageTimeBuffer
1%100 V40 min1/2 x TBE

Gel Extract

Onoda

oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence
FastGeneTM Gel Extraction kit (Nippon Genetics Co., Ltd)
DNA extraction from gel

Ligation

Onoda

BBa_E0040 on pSB1A2, BBa_R0040 on pSB1C3 / oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence

ReagentVolume
BBa_E0040 on pSB1A2, BBa_R0040 on pSB1C33.5 µL
oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence8 µL
Mighty Mix11.5 µL
DW7 µL
Total30 µL

Ligation

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

Ligation

Onoda

pSB1C3 XbaⅠ & SpeⅠ (Digestion product) / oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence

ReagentVolume
pSB1C3 XbaⅠ & SpeⅠ (Digestion product)20 µL
oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence3 µL
Mighty Mix23 µL
DW4 µL
Total50 µL

Ligation

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

September

2015/09/01

Preparation of Bacteria

Mimata

AHU1910

  1. Added 5 mL of L. casei to MRS medium.
  2. Cultured overnight.
  3. Measured OD600 and diluted with MRS medium to set OD600 to 0.2.
  4. Stand cultured for 1.5 ~ 2 hrs until OD600 is 0.4 ~ 0.5.
  5. Incubated the cells on ice for 10 min.
  6. Centrifuged at 6,000 g for 15 min at 4℃.
  7. Removed supernatant and added 30 mL of cooled PEB.
  8. Centrifuged at 6,000 g for 15 min at 4℃.
  9. Removed supernatant and added 30 mL of cooled PEB.
  10. Centrifuged at 6,000 g for 15 min at 4℃.
  11. Removed supernatant and added 1 mL of cooled PEB.

Electroporation

Mimata, Sakai

AHU1910

  1. Prepared the plasmid to 300 ng/10 µL (TE pH 8).
  2. Mixed 10 µL of plasmid and 200 µL of bacteria and incubated on ice for 10 min.
  3. Electoroporated at 2 kV with Gene Pulser (Bio-Rad) and Gene Pulser®/MicroPulserTM Electroporation Cuvettes(0.2 cm gap #1652086).
  4. Added 800 µL of cooled MRS medium and stand cultured for 3 hrs in 30℃.
  5. Spread on MRS plate (Em 5 µg/mL).

2015/09/02

Gel Extract

Mitsumoto

Erythromycin resistant gene EcoRⅠ & SpeⅠ(Digestion product)
FastGeneTM Gel/PCR Extraction kit (Nippon Genetics Co., Ltd)
DNA extraction from gel

2015/09/03

Electrophoresis

Mitsumoto

oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence

Gel ConcentrationVoltageTimeBuffer
1%100 V40 min1/2 x TBE

Transformation

Mitsumoto

Erythromycin resistant gene - BBa_B0015 on pSB1C3, Pcasei - B0034 - XbaⅠ / SpeⅠ scar - Sec signal sequence - Crp4 - B0034 - XbaⅠ / SpeⅠ scar - Sec signal sequence - BBa_E0040 - BBa_B0015 on pSB1C3, Pcasei - B0034 - XbaⅠ / SpeⅠ scar - Sec signal sequence - HD-5 - B0034 - XbaⅠ / SpeⅠ scar - Sec signal sequence - BBa_E0040 - BBa_B0015 on pSB1C3,

  1. Added 5 µL of Erythromycin resistant gene - BBa_B0015 on pSB1C3, Pcasei - B0034 - XbaⅠ / SpeⅠ scar - Sec signal sequence - Crp4 - B0034 - XbaⅠ / SpeⅠ scar - Sec signal sequence - BBa_E0040 - BBa_B0015 on pSB1C3, Pcasei - B0034 - XbaⅠ / SpeⅠ scar - Sec signal sequence - HD-5 - B0034 - XbaⅠ / SpeⅠ scar - Sec signal sequence - BBa_E0040 - 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 LBCp.
  7. Incubated the plate at 37℃ for 2 hrs.

Electrophoresis

Mitsumoto

EcoRⅠ - codon optimized - matured - HD-5 - PstⅠ

Gel ConcentrationVoltageTimeBuffer
1%100 V40 min1/2 x TBE

Electrophoresis

Mitsumoto

oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence

Gel ConcentrationVoltageTimeBuffer
1%100 V40 min1/2 x TBE

Electrophoresis

Mitsumoto

EcoRⅠ - oripAMB1 - repE - PstⅠ, EcoRⅠ - codon optimized - matured - HD-5 - PstⅠ

Gel ConcentrationVoltageTimeBuffer
1%100 V40 min1/2 x TBE

PCR

Ono

Erythromycin resistant gene, pAMβ1, HD-5, Crp4

ReagentVolume
Erythromycin resistant gene, oripAMβ1, HD-5, Crp41 µL
EX - F - Universal 10 µM1 µL
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 secDenaturation30 cycle
Cycle 268℃30 secAnnealing / Elongation30 cycle
Store4℃HoldStore

Electrophoresis

Nishimura, Ono, Mimata

Crp4, HD-5

Gel ConcentrationVoltageTimeBuffer
1%100 V50 min1/2 x TBE

Digestion

Nisimura, Ono, Mimata

Erythromycin resistant gene

ReagentVolume
Erythromycin resistant gene60 µL
SpeⅠ2 µL
Total62 µL

Digestion

StepTemp.TimeProcess
137℃60 minDigestion
Store4℃HoldStore

Electrophoresis

Nishimura, Ono, Mimata

Erythromycin resistant gene

Gel ConcentrationVoltageTimeBuffer
2%100 V60 min1/2 x TBE

PCR Purification

Nishimura, Ono, Mimata

repE, Erythromycin resistant gene, Crp4, HD-5
FastGeneTM Gel/PCR Extraction kit (Nippon Genetics Co., Ltd)
Purification of PCR products

2015/09/04

Electrophoresis

Nishimura

oripAMβ1, HD-5, Crp-4

Gel ConcentrationVoltageTimeBuffer
1%100 V60 min1/2 x TBE

Electrophoresis

Nishimura

Erythromycin resistant gene, HD-5

Gel ConcentrationVoltageTimeBuffer
2%100 V60 min1/2 x TBE

Transformation

Fujita, Mimata

Erythromycin resistant gene - BBa_B0015, BBa_R0010 - BBa_B0034 - Crp4 - BBa_B0015, BBa_R0010 - BBa_B0034 - HD5 - BBa_B0015

  1. Added 5 µL of Erythromycin resistant gene - BBa_B0015, BBa_R0010 - BBa_B0034 - Crp4 - BBa_B0015, BBa_R0010 - BBa_B0034 - HD5 - BBa_B0015 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 18 hrs.

Digestion

Fujita, Mimata

oripAMβ1, BBa_R0010 - BBa_B0034 - HD5-BBa_B0015, BBa_R0010 - BBa_B0034 - Crp4 - BBa_B0015

ReagentVolume
oripAMβ1, BBa_R0010 - BBa_B0034 - HD5-BBa_B0015, BBa_R0010 - BBa_B0034 - Crp4 - BBa_B001520 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

Onoda

oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence

ReagentVolume
oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence20 µL
XbaⅠ1 µL
SpeⅠ1 µL
10 x M Buffer3 µL
DW5 µL
Total30 µL

Digestion

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

Electrophoresis

Onoda

oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence (Digestion product)

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

2015/09/05

Colony PCR

Fujita, Mimata

Erythromycin resistant gene - BBa_B0015 on pSB1C3, Pcasei - BBa_B0034 - Sec signal sequence - Crp4 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015 on pSB1C3, Pcasei - BBa_B0034 - Sec signal sequence - HD5 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015

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

Erythromycin resistant gene - BBa_B0015 on pSB1C3, Pcasei - BBa_B0034 - Sec signal sequence - Crp4 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015 on pSB1C3, Pcasei - BBa_B0034 - Sec signal sequence - HD5 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015

Gel ConcentrationVoltageTimeBuffer
1% V30 min1/2 x TBE

Mini-prep

Mimata

Erythromycin resistant gene - BBa_B0015 on pSB1C3, Pcasei - BBa_B0034 - Sec signal sequence - Crp4 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015 on pSB1C3, Pcasei - BBa_B0034 - Sec signal sequence - HD5 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015
FastGeneTM Plasmid mini kit (Nippon Genetics Co., Ltd)
standard protocol

Ligation

Mimata, Onoda

pSB1C3 / Pcasei - BBa_B0034 - Sec signal sequence - Crp4 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015

ReagentVolume
pSB1C32.5 µL
Pcasei - BBa_B0034 - Sec signal sequence - Crp4 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B001530 µL
Mighty Mix32.5 µL
Total65 µL

Ligation

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

Ligation

Mimata, Onoda

pSB1C3 / EcoRⅠ - codon optimized - matured HD-5 - PstⅠ

ReagentVolume
pSB1C32.5 µL
EcoRⅠ - codon optimized - matured HD-5 - PstⅠ30 µL
Mighty Mix32.5 µL
Total65 µL

Ligation

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

Electrophoresis

Mimata

pHIL253, HD-5 on pSB1C3, Crp4 on pSB1C3

Gel ConcentrationVoltageTimeBuffer
1%100 V60 min1/2 x TBE

Gel Extract

Mimata

pHIL253
FastGeneTM Gel/PCR Extraction kit (Nippon Genetics Co., Ltd)
DNA extraction from gel

Ligation

Mimata

pSB1C3 / oriAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence

ReagentVolume
pSB1C310 µL
oriAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence20 µL
Mighty Mix30 µL
Total60 µL

Ligation

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

Digestion

Sakai

XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - SpeⅠ

ReagentVolume
XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - SpeⅠ30 µL
XbaⅠ2 µL
SpeⅠ2 µL
10 x M Buffer4 µL
DW2 µL
Total40 µL

Digestion

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

2015/09/06

Colony PCR

Fujita

Erythromycin resistant gene - BBa_B0015 on pSB1C3, Pcasei - BBa_B0034 - Sec signal sequence - Crp4 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015 on pSB1C3, Pcasei - BBa_B0034 - Sec signal sequence - HD5 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015

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

Erythromycin resistant gene - BBa_B0015 on pSB1C3, Pcasei - BBa_B0034 - Sec signal sequence - Crp4 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015 on pSB1C3, Pcasei - BBa_B0034 - Sec signal sequence - HD5 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015 (Colony PCR product)

Gel ConcentrationVoltageTimeBuffer
1%100 V30 min1/2 x TBE

2015/09/07

Transformation

Sakai

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

  1. Added 1.0 µL of 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 LBC.
  7. Incubated the plate at 37℃ for 12 hrs.

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