• Protocol

• E. coli
• January
• March
• May
• June
• July
• August
• September

• L. casei
• August
• September

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 hours.

2015/08/12

Liquid Culture

Mitsumoto

pHIL253

Reagent	Volume
Single Colony	-
LB	2000 μL
Amp	2 μL

Cultured for 20 hours.

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 hours 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 hours 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 - P casei - 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 - P casei - 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 - P casei - 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 hours.

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 hours 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 hours 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 hours 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 products), 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 hours 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 hours 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

2015/09/04

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 (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.

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

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