Team:HokkaidoU Japan/Notebook/lcasei

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Notebook

• Protocol

• E. coli
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• L. casei
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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

Reagent	Volume
Single Colony	-
LB	2000 µL
Ampicillin	2 µL

Cultured for 20 hrs.

2015/08/13

Mini-prep

Mitsumoto

pHIL253
FastGene^TM 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®/MicroPulser^TM 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 Concentration	Voltage	Time	Buffer
2%	100V	30 min	1/2 x TBE

PCR

Mitsumoto

XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - P_casei - RBS_casei - Sec signal sequence - SpeⅠ, proCrp4

Reagent	Volume
pHIL253	1 µL
XbaⅠ - oripAMβ1 - repE - F - primer 10 µM	1 µL
SpeⅠ - Sec signal sequence - R - primer 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 × PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO4	3 µL
DW	33 µL
Total	50 µL

2 Step Cycle (Tm value ≥ 63℃)

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

2015/08/24

Ligation

Onoda

BBa_E0040 on pSB1A2 / XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - P_casei - RBS_casei - Sec signal sequence - SpeⅠ

Reagent	Volume
BBa_E0040 on pSB1A2	1 µL
XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - SpeⅠ	2 µL
Mighty Mix	3 µL
DW	4 µL
Total	10 µL

Ligation

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

Ligation

Onoda

BBa_R0040 on pSB1C3 / XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - P_casei - RBS_casei - Sec signal sequence - SpeⅠ

Reagent	Volume
BBa_R0040 on pSB1C3	1 µL
XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - SpeⅠ	2 µL
Mighty Mix	3 µL
DW	4 µL
Total	10 µL

Ligation

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

Ligation

Onoda

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

Reagent	Volume
pSB1C3 XbaⅠ & SpeⅠ (Digestion product)	1 µL
XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - SpeⅠ	2 µL
Mighty Mix	3 µL
DW	4 µL
Total	10 µL

Ligation

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

Transformation

Onoda

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

1. Added 1 µL of pHIL253, oripAMβ1 - repE - Erythromycin resistant gene - P_casei - RBS_casei - Sec signal sequence - BBa_R0040 on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - P_casei - RBS_casei - 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®/MicroPulser^TM 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 - P_casei - RBS_casei - Sec signal sequence on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - P_casei - RBS_casei - Sec signal sequence - BBa_R0040 on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - P_casei - RBS_casei - Sec signal sequence BBa_E0040

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

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	10 sec	Denaturation	35 cycle
Cycle 2	57.6℃	30 sec	Annealing	35 cycle
Cycle 3	68℃	120 sec	Elongation	35 cycle
Store	4℃	Hold	Store

Colony PCR

Mitsumoto

oripAMβ1 - repE - Erythromycin resistant gene - P_casei - RBS_casei - Sec signal sequence on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - P_casei - RBS_casei - Sec signal sequence - BBa_R0040 on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - P_casei - RBS_casei - Sec signal sequence BBa_E0040

Reagent	Volume
Single Colony	-
100UP - EX - F 10 µM	0.4 μL
SpeⅠ - Sec signal sequence reverse 2 10 µM	0.4 µL
KAPA Taq	5.0 µL
DW	4.2 µL
Total	10 µL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	10 sec	Denaturation	35 cycle
Cycle 2	57.6℃	30 sec	Annealing	35 cycle
Cycle 3	68℃	120 sec	Elongation	35 cycle
Store	4℃	Hold	Store

2015/08/28

Electrophoresis

Mitsumoto

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

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

2015/08/31

Electrophoresis

Onoda

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

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

Dephosphorylation

Onoda

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

Reagent	Volume
vector	10 µL
Antarctic Phosphatase	1 µL
Antarctic Phosphatase Buffer	2 µL
DW	7 µL
Total	20 µL

Dephosphorylation

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

Electrophoresis

Onoda

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

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

Gel Extract

Onoda

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

Digestion

Onoda

oripAMβ1 - repE - Erythromycin resistant gene - P_casei - RBS_casei - Sec signal sequence

Reagent	Volume
oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence	20 µL
XbaⅠ	1 µL
SpeⅠ - HF	1 µL
10 × M buffer	3 µL
DW	5 µL
Total	30 µL

Digestion

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

Electrophoresis

Onoda

oripAMβ1 - repE - Erythromycin resistant gene - P_casei - RBS_casei - Sec signal sequence

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

Gel Extract

Onoda

oripAMβ1 - repE - Erythromycin resistant gene - P_casei - RBS_casei - Sec signal sequence
FastGene^TM 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 - P_casei - RBS_casei - Sec signal sequence

Reagent	Volume
BBa_E0040 on pSB1A2, BBa_R0040 on pSB1C3	3.5 µL
oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence	8 µL
Mighty Mix	11.5 µL
DW	7 µL
Total	30 µL

Ligation

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

Ligation

Onoda

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

Reagent	Volume
pSB1C3 XbaⅠ & SpeⅠ (Digestion product)	20 µL
oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence	3 µL
Mighty Mix	23 µL
DW	4 µL
Total	50 µL

Ligation

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

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

2015/09/03

Electrophoresis

Mitsumoto

oripAMβ1 - repE - Erythromycin resistant gene - P_casei - RBS_casei - Sec signal sequence

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

Transformation

Mitsumoto

Erythromycin resistant gene - BBa_B0015 on pSB1C3, P_casei - B0034 - XbaⅠ / SpeⅠ scar - Sec signal sequence - Crp4 - B0034 - XbaⅠ / SpeⅠ scar - Sec signal sequence - BBa_E0040 - BBa_B0015 on pSB1C3, P_casei - 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, P_casei - B0034 - XbaⅠ / SpeⅠ scar - Sec signal sequence - Crp4 - B0034 - XbaⅠ / SpeⅠ scar - Sec signal sequence - BBa_E0040 - BBa_B0015 on pSB1C3, P_casei - 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 Concentration	Voltage	Time	Buffer
1%	100 V	40 min	1/2 x TBE

Electrophoresis

Mitsumoto

oripAMβ1 - repE - Erythromycin resistant gene - P_casei - RBS_casei - Sec signal sequence

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

Electrophoresis

Mitsumoto

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

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

PCR

Ono

Erythromycin resistant gene, pAMβ1, HD-5, Crp4

Reagent	Volume
Erythromycin resistant gene, oripAMβ1, HD-5, Crp4	1 µL
EX - F - Universal 10 µM	1 µL
PS - R 10 µM	1 µL
KOD - Plus - Neo	1 µL
10 x PCR Buffer for KOD - Plus - Neo	5 µL
2 mM dNTPs	5 µL
25 mM MgSO4	3 µL
DW	33 µL
Total	50 µL

2 Step Cycle (Tm value ≥ 63℃)

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

Electrophoresis

Nishimura, Ono, Mimata

Crp4, HD-5

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

Digestion

Nisimura, Ono, Mimata

Erythromycin resistant gene

Reagent	Volume
Erythromycin resistant gene	60 µL
SpeⅠ	2 µL
Total	62 µL

Digestion

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

Electrophoresis

Nishimura, Ono, Mimata

Erythromycin resistant gene

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

PCR Purification

Nishimura, Ono, Mimata

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

2015/09/04

Electrophoresis

Nishimura

oripAMβ1, HD-5, Crp-4

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

Electrophoresis

Nishimura

Erythromycin resistant gene, HD-5

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

Reagent	Volume
oripAMβ1, BBa_R0010 - BBa_B0034 - HD5-BBa_B0015, BBa_R0010 - BBa_B0034 - Crp4 - BBa_B0015	20 µL
EcoRⅠ	1 µL
PstⅠ	1 µL
10 x H Buffer	5 µL
DW	23 µL
Total	50 µL

Digestion

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

Digestion

Onoda

oripAMβ1 - repE - Erythromycin resistant gene - P_casei - RBS_casei - Sec signal sequence

Reagent	Volume
oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence	20 µL
XbaⅠ	1 µL
SpeⅠ	1 µL
10 x M Buffer	3 µL
DW	5 µL
Total	30 µL

Digestion

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

Electrophoresis

Onoda

oripAMβ1 - repE - Erythromycin resistant gene - P_casei - RBS_casei - Sec signal sequence (Digestion product)

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

2015/09/05

Colony PCR

Fujita, Mimata

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

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

3 Step Cycle (Tm value ≤ 63℃)

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

Electrophoresis

Fujita, Mimata

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

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

Mini-prep

Mimata

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

Ligation

Mimata, Onoda

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

Reagent	Volume
pSB1C3	2.5 µL
Pcasei - BBa_B0034 - Sec signal sequence - Crp4 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015	30 µL
Mighty Mix	32.5 µL
Total	65 µL

Ligation

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

Ligation

Mimata, Onoda

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

Reagent	Volume
pSB1C3	2.5 µL
EcoRⅠ - codon optimized - matured HD-5 - PstⅠ	30 µL
Mighty Mix	32.5 µL
Total	65 µL

Ligation

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

Electrophoresis

Mimata

pHIL253, HD-5 on pSB1C3, Crp4 on pSB1C3

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

Gel Extract

Mimata

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

Ligation

Mimata

pSB1C3 / oriAMβ1 - repE - Erythromycin resistant gene - P_casei - RBS_casei - Sec signal sequence

Reagent	Volume
pSB1C3	10 µL
oriAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence	20 µL
Mighty Mix	30 µL
Total	60 µL

Ligation

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

Digestion

Sakai

XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - SpeⅠ

Reagent	Volume
XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - SpeⅠ	30 µL
XbaⅠ	2 µL
SpeⅠ	2 µL
10 x M Buffer	4 µL
DW	2 µL
Total	40 µL

Digestion

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

2015/09/06

Colony PCR

Fujita

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

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

3 Step Cycle (Tm value ≤ 63℃)

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

Electrophoresis

Fujita

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

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

2015/09/07

Transformation

Sakai

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

1. Added 1.0 µL of oripAMβ1 - repE - Erythromycin resistant gene - P_casei - RBS_casei - 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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