Team:Kyoto/Notebook test

8/14

Transformation

Li, Uchino, Kaneko

Sample Name	Sample Volume (µl)	Competent Cells/(µl)	Medium
RFP 0.5ng/µl	2	DH5α /20	LB(CP)
RFP 5ng/µl	2	DH5α /20	LB(CP)
RFP 10ng/µl	2	DH5α /20	LB(CP)
RFP 20ng/µl	2	DH5α /20	LB(CP)
RFP 50ng/µl	2	DH5α /20

8/14

Gel Extraction

Li, Uchino, Kaneko

Lane	Restriction Enzyme Digestion Product	Volume/(µl)
1	10/1 PQ(3)	about 23.5µl
2	1kb ladder	6
3	10/1 PQ(3)	about 23.5µl
4	10/1 PQ(3)	about 24µl

Total volume of samples of lane 1~3 was 71.5µl and it was divided into three.

8/14

Transformation

Li, Uchino, Kaneko

Sample Name	Sample Volume	Competent Cells/(µl)	Medium
3C5	10µl	DH5α 20µl	LB(CP)
P+	10µl	DH5α 20µl	LB(CP)
P-	10µl	DH5α 20µl	LB(CP)
RBS	10µl	DH5α 20µl

8/14

Liquid Culture

Li, Uchino, Iguchi, Kaneko

Name	medium
8/13 transformation RFP 20ng/µl colony1	LB(CP)
8/13 transformation part RFP colony1	LB(CP)
8/13 transformation part RFP colony2	LB(CP)
8/13 transformation part RFP colony3	LB(CP)
8/13 transformation part RFP colony4	LB(CP)
8/13 transformation part RFP colony5	LB(CP)
8/13 transformation part RFP colony6	LB(CP)
8/13 transformation part RFP colony7	LB(CP)
8/13 transformation part RFP colony8	LB(CP)
8/13 transformation part RFP colony9	LB(CP)
8/13 transformation part RFP colony10	LB(CP)
	LB(CP)

8/15

Miniprep

Li, Uchino, Notsu, Kaneko, Watanabe

DNA	Concentration/(µg/ml)	260/280	260/230
RFP1 /2µl	82.5	1.40	1.22
RFP2 /4µl	142.5	1.69	2.05
RFP3 /4µl	50	1.55	1.71
RFP4 /4µl	57.5	1.64	1.88
RFP6 /4µl	98.75	1.71	1.40
RFP(20) /4µl	76.25	1.67

8/15

Electrophoresis

Notsu, Kaneko

Lane	Restriction Enzyme Digestion Products
1	1kb ladder 3µl
2	RFP1 10µl
3	RFP2 10µl
4	RFP3 10µl
5	RFP4 10µl
6	RFP6 10µl
7	RFP(20) 10µl
8	PCR1 5µl
9	PCR2 5µl
10	PCR3 5µl
11	PCR4 5µl
12	PCR5 5 µl
13	1kb ladder 3µl

8/15

Restriction Enzyme Digestion

Uchino, Notsu, Kaneko

Sample	DNA/(µl)	EcoR1/(µl)	Pst1/(µl)	Buffer/(µl)	MilliQ/(µl)	Total/(µl)
8/15Miniprep PSB1C3-RFP1	24	1	1	5(H)	19	50
8/15Miniprep PSB1C3-RFP2	14	1	1	5(H)	29	50
8/15Miniprep PSB1C3-RFP3	40	1	1	5(H)	3	50
8/15Miniprep PSB1C3-RFP4	35	1	1	5(H)	8	50
8/15Miniprep PSB1C3-RFP6	20	1	1	5(H)	23	50

8/15

colony PCR

Uchino, Notsu, Kaneko, Watanabe

colony Primers
8/14 transformation pSB1C3-RFP1 VF+VR
8/14 transformation pSB1C3-RFP2 VF+VR
8/14 transformation pSB1C3-RFP3 VF+VR
8/14 transformation pSB1C3-RFP4 VF+VR
8/14 transformation pSB1C3-RFP1 VF+VR

8/15

PCR (Steps)

Uchino, Notsu, Kaneko, Watanabe

Steps	Temparature	Time	Cycle
Pre Denature	94℃	2min	1
Denature	94℃	30sec	30
Annealing	55℃	30sec	30
Extension	68℃	1min	30
Final elongation	4℃	7min
Final Holding	4℃	-	-

8/16

Gel Extraction

Li, Iguchi, Notsu, Kaneko, Watanabe

Lane	Restriction Enzyme Digestion Product	Volume/(µl)
1	1kb ladder	10
2	blank
3	8/15 digestion pSB1C3-RFP 1	55
4	8/15 digestion pSB1C3-RFP 2	55
5	8/15 digestion pSB1C3-RFP 3	36
6	8/15 digestion pSB1C3-RFP 4	36
7	8/15 digestion pSB1C3-RFP 6

8/16

Electrophoresis

Li, Iguchi, Notsu, Kaneko, Watanabe

Lane	sample/(µl)	Length(bp)
1	8/16 Gel Extraction RFP1 /1
2	8/16 Gel Extraction RFP2 /1
3	8/16 Gel Extraction RFP3 /1
4	8/16 Gel Extraction RFP4 /1
5	blank
6	8/16 pSB1C3-vector /1
7	8/16 pSB1C3-vector /1
8

8/16

Ligation

Li, Iguchi, Notsu, Kaneko, Watanabe

Vector DNA /(µl) insert DNA /(µl) ligase /(µl) 10×buffer /(µl) MilliQ /(µl) Total /(µl)
pSB1C3 EPcut /1 RFP /4 0.5 1 3 9.5
pSB1C3 EPcut /1 RFP /2 0.5 1 5 9.5
pSB1C3 EPcut /1 RFP /4 0.5 1 3 9.5
pSB1C3 EPcut /1 RFP /4 0.5 1 3 9.5
pSB1C3 EPcut /1 - 0.5 1 7 9.5

8/17

Liquid Culture

Li

Name	medium
8/16 transformation pSB3C5 RFP1	LB(CP) /6
8/16 transformation pSB3C5 RFP2	LB(CP) /6
8/16 transformation pSB3C5 RFP1	LB(CP) /6

8/17

Transformation

Li

Sample Name	Sample Volume (µl)	Competent Cells/(µl)	Medium
pSB1C3 P+	2	DH5α /20	LB
pSB1C3 P-	2	DH5α /20	LB
pSB1C3 RBS	2	DH5α /20	LB
pSB3C5 RFP	2	DH5α /20	LB
8/16 ligation pSB1C3 RFP1	2	DH5α	LB

8/17

Electrophoresis

Iguchi

Lane	sample/(µl)	Length(bp)
1	1kbp ladder /3
2	blank
3	pSB1C3-RFP(ligate)1 /2
4	pSB1C3-RFP(ligate)2 /2
5	pSB1C3-RFP(ligate)3 /2
6	pSB1C3-RFP(ligate)4 /3
7	Vector (Ctrl) /5

8/18

Liquid Culture

Li, Iguchi, Notsu

Name	medium
INP [1] BBa_K811005	LB(CP) / 6ml
INP [2] BBa_K811005	LB(CP) / 6ml
INP [3] BBa_K811005	LB(CP) / 6ml
scfv04 [1] BBa_K875004	LB(CP) / 6ml
scfv04 [2] BBa_K875004	LB(CP) / 6ml
scfv04 [3] BBa_K875004	LB(CP) / 6ml
scfv06 [1] BBa__K875006	LB(CP) / 6ml
scfv06 [2] BBa_K875006	LB(CP) / 6ml
scfv06 [3] BBa_K875006	LB(CP) / 6ml

8/18

Miniprep

Li, Iguchi, Notsu

DNA	Concentration/(µg/ml)	260/280	260/230
8/17 Liquid Culture 3C5 [1] / 5µl	77	1.69	1.45
8/17 Liquid Culture 3C5 [2] / 2µl	40	1.67	1.17
8/17 Liquid Culture 3C5 [3] / 2µl	15	3.75	3.70

8/18

Electrophoresis

Li

Lane	sample/(µl)	Length(bp)
1	1kb ladder /1	-
2	[1] psb3c5(8/18 miniprep) /4.2	3807
3	[2] psb3c5(8/18 miniprep) /4.2	3807
4	[1'] psb3c5(8/18 miniprep) /4.2	3807
5	[2'] psb3c5(8/18 miniprep) /4.2	3807
6	[3'] psb3c5(8/18 miniprep) /4.2	3807

https://static.igem.org/mediawiki/2016/c/c5/T--Kyoto--8_18_No18_Electrophoresis_Kaneko_05_20_04_12_53.jpg

[img18]

8/18

Restriction Enzyme Digestion

Li, Iguchi, Notsu

Sample	DNA/(µl)	EcoR1/(µl)	Xba1/(µl)	Spe1/(µl)	Pst1/(µl)	Buffer/(µl)	BSA/(µl)	MilliQ/(µl)	Total/(µl)
	psb3c5(8/18miniprep) [1'], [2'], [3'] mixed / 50	1.0			1.0	5.6			56.6

8/19

Transformation

Li, Iguchi, Notsu

Sample Name	Sample Volume (µl)	Competent Cells/(µl)	Medium
BBa_ J61002 p+	2	DH5a /20	LB(Amp)
BBa_J61002 p-	2	DH5a /20	LB(Amp)
BBa_1A2 RBS	2	DH5a /20	LB(Amp)

Failed due to deactivation of Amp

8/19

MutationPCR

Templates /µl	Primer /µl	Primer STAR Max Premix /µl	MilliQ /µl	Total(µl)
scfv plasmid /0.1	m.scfv FW /1, m.scfvRV/1	25	22.9	50
scfv plasmid (nega) /0.1	0	5	4.9	10
(nega) /0	m.scfv FW/0.2, m.scfvRV/0.2	5	4.6	10

---30cycle---
98℃ 10''
55℃ 15''
72℃ 20''
----------------
4℃ holding

8/19

Electrophoresis

Notsu

Lane	sample/(µl)	Length(bp)
1	1kb ladder/2	-
2	scfv plasmid +primer(m.scfv FW, m.scfv RV) mutation PCR product /5	3600
3	scfv plasmid mutation PCR product /5	-
4	primer(m.scfv FW, m.scfv RV) mutation PCR product /5	-
5	psb3c5 EPcut product /5	2600(backborn), 1200(inseert)

[img22]

8/19

Concentration

Li, Notsu

Sample	OD600	Bacterial cells /ml
INP [1] K811005	0.643	5.14x10^8
scfv06 K875006	0.634	5.07x10^8
scfv04 K875004	0.618	4.94x10^8

1 OD 600 equals to 8x10^8 cells /ml

8/19

Miniprep

Li, Notsu

DNA	Concentration/(µg/ml)	260/280	260/230
scfv 04[1] K875004 / 3µl	56	1.63	1.45
scfv 04[2] K875004 / 3µl
scfv 04[3] K875004 / 3µl
scfv 06[1] K875006 /3µl
scfv 06[2] K875006 /3µl
scfv 06[3] K875006 /3µl
INP [1] K811005 /3µl
INP [2] K811005 /3µl
INP [3] K811005 /3µl

There was a problem with an instrument

8/20

Transformation

Li, Uchino

Sample Name	Sample Volume (µl)	Competent Cells/(µl)	Medium
8/19 mutation PCR scfv plasmid (nega)	1	DH5a/ 20	LB(CP)

8/19 mutation PCR:
1µl extraction of scfv plasmid (nega)/10µl was folded 300 times.

8/20

Transformation

Li, Uchino

Sample Name	Sample Volume (µl)	Competent Cells/(µl)	Medium
8/19 mutation PCR scfv plasmid	1 [note:1]	DH5a/20	LB(AMP)
8/19 mutation PCR scfv plasmid (nega)	1 [note:2]	DH5a/20	LB(AMP)
BBa_J61002 p+	1	DH5a/20	LB(AMP)
BBa_J61002 p-	1	DH5a/20	LB(AMP)
BBa_1A2 RBS	1	DH5a/20	LB(AMP)

[note:1]
8/19 mutation PCR:
1µl extraction of scfv plasmid (nega)/50µl was folded 300 times.
[note:2]
8/19 mutation PCR:
1µl extraction of scfv plasmid (nega)/10µl was folded 300 times.

8/20

Electrophoresis

Li

Lane	sample/(µl)	Length(bp)
1	1kb ladder /1.5
2	9/19 miniprep scfv04 [1]
3	9/19 miniprep scfv04 [2]
4	9/19 miniprep scfv04 [3]
5	9/19 miniprep scfv06 [1]
6	9/19 miniprep scfv06 [2]
7	9/19 miniprep scfv06 [3]
8	9/19 miniprep INP [1]
9	9/19 miniprep INP [2]
10	9/19 miniprep INP [3]

[img26]

8/21

Colony PCR

Uchino

Colony	Primers
8/20 transformation RBS	VF+VR
8/20 transformation p+	VF+VR
8/20 transformation p-	VF+VR
8/20 dilution INP	VF+VR

8/21

PCR (Steps)

Uchino

Steps	Temparature	Time	Cycle
Pre	94℃	2min	1
De	94℃	30 sec	30
An	55℃	30 sec	30
Ex	68℃	1min	30
Final E	4℃	7min	1
Final Hold	4℃	-	-

Autoclaved distrilled water /23µl
2xQuick taq /25µl
10 pmol/µl primer (VF) /1µl
10 pmol/µl primer (VR) /1µl
Template DNA /0µl
Total 50µl

8/21

Electrophoresis

Li

Lane	sample/(µl)	Length(bp)
1	1kb ladder /2
2	8/21 colony PCR RBS /all
3	8/21 colony PCR p+ /all
4	8/21 colony PCR p- /all
5	8/21 colony PCR INP /all
6	8/21 colony PCR Nega /all

[img29-1]
weak band except INP -> retry
colony PCR and Electrophoresis were conducted under the same condition.
[img29-2]
RBS and INP success.
P+ and P- failed -> retry.

8/21

PCR (Steps)

Li

Steps	Temparature	Time	Cycle
Predenature	95℃	3min	1
Denature	98℃	20sec	25
Annealing	70℃	15sec	25
Extension	72℃	30sec	25
Final Elongation	72℃	5min	1
Final Holding	4℃	-	1

8/21

PCR (Target)

Li

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	Buffer/(µl)	MilliQ/(µl)	Any Other/(µl)	Total/(µl)
scfv 8/20 transformation	cscfv.f /0.75	cscfv.r /0.75	2x KAPA Hifi /12.5	11		25
- (nega)	cscfv.f /0.3	cscfv.f /0.3	2x KAPA Hifi /5	4.4		10

8/21

Colony PCR

Li

Colony	Primers
8/20 transformation p+ [1]	VF+VR
8/20 transformation p+ [2]	VF+VR
8/20 transformation p+ [3]	VF+VR
8/20 transformation p- [1]	VF+VR
8/20 transformation p- [2]	VF+VR
8/20 transformation p- [3]	VF+VR
8/18 parts p+	VF+VR
8/18 parts p-	VF+VR

8/21

PCR (Steps)

Li

Steps	Temparature	Time	Cycle
pre	94℃	2min	1
de	94℃	30sec	30
an	55℃	30sec	30
ex	68℃	1min	30
F.elong	4℃	7min	1
F.hold	4℃	-	1

8/21

Liquid Culture

Li

Name	medium
strain	Medium/ml
8/20 transformation RBS	PG(AMP) /6
8/20 dilution INP7	PG(CP) /6
8/17 transformation 3c5, RFP	PG(CP) /6
8/20 transformation p+ [1]	PG(AMP) /6
8/20 transformation p- [1]	PG(AMP) /6

8/21

Electrophoresis

Li

Lane	sample/(µl)	Length(bp)
1	1kb ladder /1.5
2	8/21 cloning PCR scfv[1] /2
3	8/21 cloning PCR scfv[2] /2
4	nega /2
5	8/21 colony PCR p+ [1] /2
6	8/21 colony PCR p+ [2] /2
7	8/21 colony PCR p+ [3] /2
8	8/21 colony PCR p- [1] /2
9	8/21 colony PCR p- [2] /2
10	8/21 colony PCR p- [3] /2
11	8/21 colony PCR parts p+ /2
12	8/21 colony PCR parts p- /2

[img35]
2, 3, 4 cloning PCR failed.
1 forgot to put ladder
5, 7, 8, 9 strong band
6 weak
10 failed
P+[1] and p-[1] -> liquid culture

8/24

Ligation

Li

sample/µl	MilliQ (µl)	Buffer /µl	Ligase(µl)	Total(µl)
8/19 mutation PCR scfv /5	3.5	1	0.5	10

8/24

Liquid Culture

Li

Name	medium
8/20 transformation m.scfv colonyA /6	PG(AMP)

8/24

Miniprep

Li

DNA	Concentration/(µg/ml)	260/280	260/230
8/20 Liquid culture RBS	124.5	1.65	1.02
8/20 Liquid culture INP	147.8	1.66	0.98
8/20 Liquid culture 3c5.RFP	234.8	1.79	1.62
8/20 Liquid culture P+[1]	40.3	1.78	1.60
8/20 Liquid culture P-[1]	56.7	1.80	2.05

8/25

Miniprep

Li, Uchino, Notsu, Kaneko

DNA	Concentration/(µg/ml)	260/280	260/230
8/24 LIquid Culture m.scfv colonyA	-	-	-
8/24 LIquid Culture m.scfv colonyB	-	-

8/25

Electrophoresis

Lane	sample/(µl)	Length(bp)
1	1kb ladder /1.5
2	8/24 miniprep p+(J61002) /2
3	8/24 miniprep p-(J61002) /2
4	8/24 miniprep RFP (3c5) /2
5	8/24 miniprep INP (1c3) /2
6	8/24 miniprep RBS (1A2) /2

[img38]

8/25

Restriction Enzyme Digestion

Uchino

DNA/(µl)	EcoR1/(µl)	Xba1/(µl)	Spe1/(µl)	Pst1/(µl)	Buffer/(µl)	MilliQ/(µl)	Total/(µl)
p+/24.8			1	1	10xH /4	9.2	40
IINP /6.8		1		1	10xM /4	27.2	40
p- /17.6			1	1	10xH /4	16.4	40
RBS /8.0			1	1	10xH /4	26	40
RFP /4.3	1			1	10xH /4	29.7	40

8/25

Gel Extraction

Uchino, Notsu

Lane	Restriction Enzyme Digestion Product	Volume/(µl)
1	1kb ladder	6
2, 3	8/25 digestion p+	24 each
4, 5	8/25 digestion p-	24 each
6, 7	8/25 digestion RFP	24 each
8, 9	8/25 digestion INP	24 each
10, 11	8/25 digestion RBS	24 each

[img40]

8/25

Actvation check of Xba and Pst

Uchino

INP(µl)	Xba(µl)	Pst(µl)	10xM(µl)	BSA(µl)	MilliQ(µl)	Total(µl)
0.68	0.1	0.1	1	0.2	7.92	10
0.68	0.1	-	1	0.2	8.02	10
0.68	-	0.1	1	0.2	8.02	10
0.68	-	-	1	0.2	8.12	10

8/25

Electrophoresis

Notsu

Lane	sample/(µl)	Length(bp)
1	1kb ladder /3
2	[1] (inp xp cut) /10
3	[2] (inp x cut) /10
4	[3] (inp p cut) /10
5	[4] (inp no cut) /10

[img42]

8/26

Restriction Enzyme Digestion

Uchino, Notsu

DNA/(µl)	EcoR1/(µl)	Xba1/(µl)	BSA/(µl)	MilliQ/(µl)	Total/(µl)
8/24 miniprep INP 1c3 /0.68	-	1.0	0.2	7.12	10
8/15 miniprep RFP psb1c3 /1.3	-	1.0	0.2	6.5	10
8/24 miniprep INP 1c3 /0.68	0.1	1.0	0.2	7.02	10
8/15 miniprep RFP psb1c3 /1.3	0.1	1.0	0.2	6.4	10
8/24 miniprep INP 1c3/ 0.68	0.1	-	0.2	8.02	10
8/15 miniprep RFP psb1c3 /1.3	0.1	-	0.2	7.4	10
8/24 miniprep INP 1c3 /0.68	-	-	0.2	8.12	10
8/15 miniprep RFP psb1c3 /1.3	-	-	0.2	7.5	10

8/26

PCR (Target)

Li

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	Buffer/(µl)	MilliQ/(µl)	Any Other/(µl)	Total/(µl)
scfv from MK	c.scfv fw/0.75	c.scfv rv/0.75	2x KAPA Hifi /12.5	11		25
none	c.scfv fw/0.75	c.scfv rv/0.75	2x KAPA Hifi /5	4.4		10

8/26

Activation Check of Xba

Uchino

Lane	sample/(µl)	Length(bp)
1	1kb ladder/2
2	[8] RFP/10
3	[6] RFP EcoRI/10
4	[2] RFP XbaI/10
5	[4] RFP EcoRI, XbaI /10
6	[7] INP /10
7	[5] INP EcoRI /10
8	[1] INP XbaI /10
9	[3] INP EcoRI, XbaI/10

[img45-1]
[img45-2]

8/27

Electrophoresis

Li

Lane	sample/(µl)	Length(bp)
2	1kb ladder	2
3	8/25 PCR scfv	5
4	8/25 PCR none	5

8/27

PCR (Steps)

Li

Steps	Temparature	Time	Cycle
Predenature	95	3min	1
Denature	98	20sec	25
Annealing	65	15sec	25
Extension	72	30sec	25
Final Elogation	72	5min	1
Final Holding	4	-

8/27

PCR (Target)

Li

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	MilliQ/(µl)	Any Other/(µl)	Total/(µl)
s.BAN	BAN.Fw/ 0.75	BAN.Rv/ 0.75	11	2xKAPA HiFi/ 12.5	25
s.BAN	BAN.Fw/ 0.75	BAN.Rv/ 0.75	11	2xKAPA HiFi/ 12.5	25
s.BAN	BAN.Fw/ 0.75	BAN.Histag/ 0.75	11	2xKAPA HiFi/ 12.5	25
scfv	scfv.Fw/ 0.75	scfv.Rv/ 0.75	11	2xKAPA HiFi/ 12.5	25
8/24 miniprep INP	INP.Fw/ 0.75	INP.Rv/ 0.75	11	2xKAPA HiFi/ 12.5	25
8/24 miniprep INP	INP.Fw/ 0.75	INP.Rv.Histag/ 0.75	11	2xKAPA HiFi/ 12.5

9/8

Restriction Enzyme Digestion

Notsu

DNA/(µl)	EcoR1/(µl)	Spe1/(µl)	Pst1/(µl)	Buffer/(µl)	MilliQ/(µl)	Total/(µl)
[1] 8/15 miniprep 1c3 RFP /14	0.5	0.5	-	10xH /3	12	30
[2] 8/24 miniprep 3c5 RFP /8.5	0.5	-	0.5	10xH /3	17.5	30
[3] 8/24 miniprep 3c5 RFP /8.5	0.5	0.5	-	10xH /3	17.5	30
[4] 8/24 miniprep BBa_J61002 p- /24	0.5	-	0.5	10xH /3	2	30

9/9

Electrophoresis

Notsu

Lane	Restriction Enzyme Digestion Products
1	100bp ladder 9µL
2	8/24 miniprep p- EPcut 30µL

9/9

Electrophoresis

Sukegawa, Notsu

Lane	Restriction Enzyme Digestion Products
1	1kbp ladder 9µL
2	8/15 miniprep 1C3RFP EScut 30µL
3	8/24 miniprep 3C5RFP EPcut 30µL
4	8/24 miniprep 3C5RFP EScut 30µL

9/14

PCR (Steps)

Li

Steps	Temparature	Time	Cycle
1	95	3min	１
2	98	70sec	25
3	68	15sec	25
4	72	30sec	25
5	72	5min	1
6	10	∞	1

9/14

PCR (Target)

Li

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	Buffer/(µl)	MilliQ/(µl)	Total/(µl)
8/24 miniprep INP	INPfw/0.75	INPRv/0.75	2×KAPAHiFi	11	25
8/24 miniprep INP	INPfw/0.75	INPHisRv/0.75	2×KAPAHiFi	11	25
BAN	BANFw/0/75	BANRv/0.75	2×KAPAHiFi	11	25
BAN	BANFw/0/75	BANHisRv/0.75	2×KAPAHiFi	11	25
BAN	BANFw/0/75	RBSRv/0.75	2×KAPAHiFi	11	25
scFv fromMk	scFvFw/0/75	scFvRv/0/75	2×KAPAHiFi	11	25
scFv fromMk	scFvHisFw/0.75		2×KAPAHiFi	11	25

9/14

Gel Extraction

Kaneko

Lane	Restriction Enzyme Digestion Products
2	ladder/6
4	9/14 PCR1/10
6	9/14 PCR2/10
8	9/14 PCR3/10
10	9/14 PCR4/10
12	9/14PCR5/10
14	9/14 PCR6/10
16	9/14 PCR7/10

9/14

PCR (Target)

Li

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	Buffer/(µl)	Total/(µl)
3sBAN/0.15+6scFvfromMK/0.15	BANFw/0/75	scFvRv/0.75	2×KAPAHiFi/12.5	25
4sBAN/0.15+7scFvfromMK/0.15	BANFw/0/75	scFvRv/0.75	2×KAPAHiFi/12.5	25
1INP8/24miniprep/0.15+5sBAN0.15+scFvfromMK/0.15	BANFw/0/75	scFvRv/0.75	2×KAPAHiFi/12.5	25
2INP8/24miniprep/0.15	BANFw/0/75	scFvRv/0.75	2×KAPAHiFi/12.5	25

9/15

Restriction Enzyme Digestion

Li, Uchino

Sample	DNA/(µl)	EcoR1/(µl)	Spe1/(µl)	Buffer/(µl)	Total/(µl)
3sBAN+6scFvfromMKPCRproduct	34	1	1	4	40
4sBAN+7scFvfromMK PCRproduct	34	1	1	4	40
1INP8/24miniprep+5sBAN+6scFvfromMKPCRproduct	16	1	1	2	20
2INP8/24miniprep+5sBAN+7scFvfromMk PCRproduct		1	1	4	40

9/15

Gel Extraction

Li, Uchino

Lane	Restriction Enzyme Digestion Product	Volume/(µl)
1	1kb ladder
2	3sBAN+6scFvfromMkPCR産物EScut	20
3	3sBAN+6scFvfromMkPCR産物EScut	20
4	4sBAN+7scFvfromMkPCR産物EScut	20
5	4sBAN+7scFvfromMkPCR産物EScut	20
6	1INP8/24miniprep+5sBAN+7scFvfromMkPCR産物EScut	20
7	2INP8/24miniprep+5sBAN+7scFvfromMkPCR産物EScut	20
8	2INP8/24miniprep+5sBAN+7scFvfromMkPCR産物EScut	20
9	RFP3C5PCR産物

9/16

Colony PCR (2Column)

Uchino, Michimori

Name I	Name II
9/15 transformation BANscFv 3C5 colony1~16	VF+VR
9/15 transformation INPscFv 3C5 colony1~16	VF+VR
9/15 transformation BANHisscFv 3C5 colony1~16	VF+VR
9/15 transformation INPHisscFv 3C5 colony1~16

9/16

PCR (Steps)

Uchino, Michimori

Steps	Temparature	Time	Cycle
1	94	2	1
2	94	0.5	25
3	55	0.5	25
4	68	1	25
5	4	7	1
6	4	∞

9/16

Gel Extraction

Li, Kaneko, Notsu

Lane	Restriction Enzyme Digestion Product	Volume/(µl)
1	1kb ladder	5
2	BANscFv3C5 colony 9	10
3	BANscFv3C5 colony 10	10
4	BANscFv3C5 colony 11	10
5	BANscFv3C5 colony 12	10
6	BANscFv3C5 colony 13	10
7	BANscFv3C5 colony 14	10
8	BANscFv3C5 colony 15	10
9	BANscFv3C5 colony 16	10
10	BANHisscFv3C5 colony 9	10
11	BANHisscFv3C5 colony 10	10
12	BANHisscFv3C5 colony 11	10
13	BANHisscFv3C5 colony 12	10
14	BANHisscFv3C5 colony 13	10
15	BANHisscFv3C5 colony 14	10
16	BANHisscFv3C5 colony 15	10
17	BANHisscFv3C5 colony 16	10
18	BANscFv3C5 colony 1	10

9/16

Gel Extraction

Li, Kaneko, Notsu

Lane	Restriction Enzyme Digestion Product	Volume/(µl)
1	1kb ladder	5
2	INPscFv3C5 colony 9	10
3	INPscFv3C5 colony 10	10
4	INPscFv3C5 colony 11	10
5	INPscFv3C5 colony 12	10
6	INPscFv3C5 colony 13	10
7	INPscFv3C5 colony 14	10
8	INPscFv3C5 colony 15	10
9	INPscFv3C5 colony 16	10
10	INPHisscFv3C5 colony 9	10
11	INPHisscFv3C5 colony 10	10
12	INPHisscFv3C5 colony 11	10
13	INPHisscFv3C5 colony 12	10
14	INPHisscFv3C5 colony 13	10
15	INPHisscFv3C5 colony 14	10
16	INPHisscFv3C5 colony 15	10
17	INPHisscFv3C5 colony 16	10
18	BANscFv3C5 colony 1	10

9/17

Electrophoresis

Michimori, Kaneko

Lane	Restriction Enzyme Digestion Products
1	buffer/5
2	1kb ladder/5
3	BAN 9 P/4.5
4	BAN 9 N/4.5
5	BAN14 P/4.5
6	BAN14 N/4.5
7	BANHis14 P/4.5
8	BANHis14/4.5

9/17

Electrophoresis

Kaneko

Lane	sample/(µl)	Length(bp)
1	1kbp ladder
2	none
3	BANHis16 p
4	BANHis16 n
5	INP 12 p
6	INP 14 p
7	INP 12 n
8

9/17

Electrophoresis

Kaneko

Lane	Restriction Enzyme Digestion Products
1	-
2	-
3	1kbladder/5
4	INPHis9 P/4.5
5	INPHis9N/4.5
6	INPHis12P/4.5
7	INPHis12N/4.5
8	INP14 N/4.5

9/17

Restriction Enzyme Digestion

Uchino, Michimori

Sample	DNA/(µl)	EcoR1/(µl)	Pst1/(µl)	Buffer/(µl)	MilliQ/(µl)	Total/(µl)
BAN9	2	0.5	0.5	10×H/1	6	10
BAN9	1	0	0	10×H/1	8	10
BAN14	2	0.5	0.5	10×H/1	6	10
BAN14	1	0	0	10×H/1	8	10
BANHis14	2	0.5	0.5	10×H/1	6	10
BANHis 14	1	0	0	10×H/1	8	10
BANHis 16	2	0.5	0.5	10×H/1	6	10
BANHis16	1	0	0	10×H/1	8	10
INP12	2	0.5	0.5	10×H/1	6	10
INP12	1	0	0	10×H/1	8	10
INP14	2	0.5	0.5	10×H/1	6	10
INP 14	1	0	0	10×H/1	8	10
INP 9	2	0.5	0.5	10×H/1	6	10
INP9	1	0	0	10×H/1	8	10
INPHis12	2	0.5	0.5	10×H/1	6	10
INPHis12	1	0	0	10×H/1	8	10

9/17

Miniprep

Notsu

DNA	Concentration/(µg/ml)	260/280	260/230
9/16 liquid culture BANscFv12
9/16 liquid culture BANscFv14
9/16 liquid culture BANHisscFv12
9/16 liquid culture BANHisscFv14
9/16 liquid culture INPHisscFv12
9/16 liquid culture INPHisscFv14
9/16 liquid culture INPHisscFv10
9/16 liquid culture BANscFv

2/4

PartsAwaking

Kaneko, Wan

Description	PartName	Backbone	Well	PlateYear	PlateNumber	MilliQ(µl)
RFP	E1010	pSB1C3	11N	2015	3

2/4

Transformation

Uchino, Iguchi, Notsu, Wan

Sample Name	Sample Volume (µl)	Competent Cells/(µl)	Medium
E1010	2	DH5α	LB(CP)

2/5

Colony PCR (typeA)

Uchino, Egashira, Yamamoto

Templates/(µl)	Primers/(µl)	MilliQ/(µl)	PreMix/(µl)	Total/(µl)
2/4 transformation E1010 colony1	VF 6.25µl VR 6.25µl	12.5	2×Quick Taq HS DyeMix 25µl	50

2/5

PCR (Steps)

Uchino, Egashira, Yamamoto

Steps	Temparature	Time	Cycle
Predenature	94℃	2min	1
Denature	94℃	30sec	25
Annealing	51℃	30sec	25
Extension	68℃	90sec	25
Final Elongation	4℃	-	-
Final Holding	4℃	-

2/5

Transformation

Uchino

Sample Name	Sample Volume	Competent Cells/(µl)
9/17 miniprep BAN9	2	DH5α/20µL
9/17　miniprep BANHis14	2	DH5α/20µL
9/17miniprep INPHis12	2	DH5α/20µL
9/17miniprep INPHis9	2

2/5

Liquid Culture

Uchino, Wan, Yamamoto

Name	medium
2/4 transformation E1010 colony1

2/5

Electrophoresis

Uchino

Lane	sample/(µl)	Length(bp)
1	1kb ladder /5
2	2/5 colony PCR E1010	1019
3	other sample	-
4	other sample	-
5	other sample	-
6	other sample	-
7	other sample	-
8	other sample

2/6

Miniprep

Notsu, Kaneko, Egashira, Wan, Yamamoto

DNA	Concentration/(µg/ml)	260/280	260/230
2/5 liquid culture E1010(RFP) colony1	48.0	1.93

2/6

Restriction Enzyme Digestion

Kaneko, Egashira, Wan, Yamamoto

Sample	DNA/(µl)	EcoR1/(µl)	Xba1/(µl)	Spe1/(µl)	Pst1/(µl)	Buffer/(µl)	BSA/(µl)	MilliQ/(µl)	Total/(µl)
2/6 miniprep E1010 colony1	34	0	1	0	1	1×M /4	0	0	40

2/7

Gel Extraction

Kaneko, Yamamoto

Lane	Restriction Enzyme Digestion Product	Volume/(µl)
1	1kb ladder	10
2	E1010 colony1 2/6 XPcut	40
3	other sample	-
4	other sample	-

2/7

Gel Extraction(Measurement)

Kaneko, Yamamoto

Sample/(µl)	Concentration(µg/µl)	260/280	260/230
E1010 colony1 2/6 XPcut	8.2	1.03	0.49

2/7

Liquid Culture

Uchino

Name	medium
2/5 transformation BAN9 colony1	LB(CP)
2/5 transformation BAN-HIS14 colony1	LB(CP)
2/5 transformation INP-HIS12 colony1	LB(CP)
2/5 transformation BAN9 colony1	LB(CP)

2/8

Liquid Culture

Uchino, Yamamoto

Name	medium
2/4 transformation E1010 colony2 1	LB(CP)
2/4 transformation E1010 colony2 2	LB(CP)

2/8

Miniprep

Uchino, Egashira, Yamamoto

DNA	Concentration/(µg/ml)	260/280	260/230
2/7 Liquid culture BAN9	123.6	1.87	2.07
2/7 Liquid culture BAN9	248.8	1.83	1.84
2/7 Liquid culture BAN-HIS14	268.9	1.86	2.15
2/7 Liquid culture BAN-HIS14	127.9	1.85	2.08
2/7 Liquid culture INP-HIS9	354.3	1.86	2.24
2/7 Liquid culture INP-HIS9	289.4	1.85	1.92
2/7 Liquid culture INP-HIS12	172.3	1.87	2.07
2/7 Liquid culture INP-HIS12	286.5	1.86

2/8

Electrophoresis

Uchino, Egashira, Yamamoto

Lane	sample/(µl)	Length(bp)
1	1kb ladder /5
2	2/8 miniprep BAN9 /5
3	2/8 miniprep BAN9 /5
4	2/8 miniprep BAN-HIS14 /5
5	2/8 miniprep BAN-HIS14 /5
6	2/8 miniprep INP-HIS9 /5
7	2/8 miniprep INP-HIS9 /5
8	2/8 miniprep INP-HIS14 /5
9	2/8 miniprep INP-HIS9 /5

2/9

Miniprep

Matsumoto, Notsu, Egashira, Yamamoto, Yamada

DNA	Concentration/(µg/ml)	260/280	260/230
2/8 liquid culture E1010 colony2 1and 2	58.5	1.89	2.16

2/9

Restriction Enzyme Digestion

Uchino, Egashira, Yamamoto, Yamada

Sample	DNA/(µl)	EcoR1/(µl)	Xba1/(µl)	Spe1/(µl)	Pst1/(µl)	Buffer/(µl)	BSA/(µl)	MilliQ/(µl)	Total/(µl)
2/8 liquid culture E1010 colony2	30	0	1	0	1	10×M /4	2	2	40

2/9

Restriction Enzyme Digestion

Uchino, Yamamoto, Yamada

Sample	DNA/(µl)	EcoR1/(µl)	Xba1/(µl)	Spe1/(µl)	Pst1/(µl)	Buffer/(µl)	BSA/(µl)	MilliQ/(µl)	Total/(µl)
2/8 liquid culture E1010 colony2	5	0	0	0	0	10×M /2	1	12	20

2/10

Electrophoresis

Uchino, Yamamoto, Yamada

Lane	sample/(µl)	Length(bp)
1	1kb ladder /5
2	2/9 Negative Control E1010 colony2 /10	2776
3	2/9 Digestion E1010 colony2 p+x /40	732
4	2/9 Negative Control E0040 colony2 /20	2799
5	2/9 Digestion E0040 colony2 p+x /40	746
6	2/9 Negative Control K592009 colony2 /20	2739
7	2/9 Digestion K592009 colony2 p+x /40	695
8	2/9 Negative Control E0022 colony2 /20	2832
9	2/9 Digestion E0022 colony2 p+x /40

2/10

Electrophoresis

Uchino, Yamamoto, Yamada

Lane	sample/(µl)	Length(bp)
1	1kb ladder /5
3	2/9 NC J23105 colony2 /20	2105
4	2/9 Digestion J23105 colony2 E+S /40	58
5	2/9 NC J23100 colony2 /20	2105
6	2/9 Digestion J23100 colony2 E+S /40	58
7	2/9 NC B0034 colony3 /20	2091
8	2/9 Digestion B0034 colony3 P+S /40

2/10

Miniprep

Michimori, Egashira, Yamamoto, Yamada

DNA	Concentration/(µg/ml)	260/280	260/230
E1010	9.4	2.13	0.61
K592009	5.2	1.74	0.29
E0040	15.0	1.94	0.38
E0022	8.5	1.04	0.86
B0034	21.2	1.84	1.06

Ideal MIniprep effficiency of E1010 would be 18.5ng/microL (presuming 100% Restriction Enzyme Digestion efficiency and 80 % Gel Extraction efficiency).

2/12

PartsAwaking

Uchino

Description	PartName	Backbone	Well	PlateYear	PlateNumber	MilliQ(µl)
sfGFP-CBDcex	K1321342	pSB1C3	18E	2015	5	10
sfGFP-CBDclos	K1321341	pSB1C3	18C	2015	5	10

2/12

Transformation

Uchino

Sample Name	Sample Volume (µl)	Competent Cells/(µl)	Medium
2/12 parts awakening sfGFP-CBDcex	1	DH5α /20	LB(CP)
2/12 parts awakening sfGFP-CBDclos	1	DH5α /20	LB(CP)

2/12

PCR (Target)

Uchino

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	Buffer/(µl)	MilliQ/(µl)	Any Other/(µl)	Total/(µl)
2/8 miniprep BAN-His14 4 /0.1	VF /0.75	m.scFv.Rv /0.75	-	10.9	2×KAPA HiFi /12.5	25
2/8 miniprep BAN-His14 4 /0.1	VR /0.75	m.scFv.Fw /0.75	-	10.9	2×KAPA HiFi /12.5	25

2/12

Electrophoresis

Uchino

Lane	sample/(µl)	Length(bp)
1	1kb ladder	5
2	2/12 PCR VF.m.scFv.Rv	25
3	2/12 PCR VR.m.Fw	25

2/12

PartsAwaking

Uchino

Description	PartName	Backbone	Well	PlateYear	PlateNumber	MilliQ(µl)
Promoter	J23114	J61002	19J	2015	4	10
Promoter	J23108	pSB1C3	4C	2015	4	10

2/12

Transformation

Uchino, Yamada

Sample Name	Sample Volume (µl)	Competent Cells/(µl)	Medium
2/12 Parts awakening J23114	1	DH5α /20	LB(AMP)
2/12 Parts awakening J23108	1	DH5α /20	LB(AMP)

2/12

Gel Extraction(Measurement)

Yamada

Sample/(µl)	Concentration(µg/µl)	260/280	260/230
2/12 PCR VF.m.scFv.Rv	18.8	1.95	1.52
2/12 PCR VR.m.scFv.Fw	28.6	2.08	0.07

2/15

Colony PCR (typeA)

Egashira

Templates/(µl)	Primers/(µl)	MilliQ/(µl)	PreMix/(µl)	Total/(µl)
2/12 transformation sfGFP-CBDcex colony1	VF /0.75 VR /0.75	11	2×KAPA HiFi /12.5	25
2/12 transformation sfGFP-CBDcex colony2	VF /0.75 VR /0.75	11	2×KAPA HiFi /12.5	25
2/12 transformation sfGFP-CBDclos colony1	VF /0.75 VR /0.75	11	2×KAPA HiFi /12.5	25
2/12 transformation sfGFP-CBDclos colony2	VF /0.75 VR /0.75	11	2×KAPA HiFi /12.5	25

2/15

Electrophoresis

Kaneko

Lane	sample/(µl)	Length(bp)
1	1kb ladder /5
2	2/12 transformation sfGFP-CBDcex colony1
3	2/12 transformation sfGFP-CBDcex colony2
4	2/12 transformation sfGFP-CBDclos colony1
5	2/12 transformation sfGFP-CBDclos colony2

2/15

Liquid Culture

Wan, Yamamoto

Name	medium
2/12 transformation sfGFP-CBDcex colony2	LB(CP)
2/12 transformation sfGFP-CBDclos colony2	LB(CP)

2/15

Colony PCR (typeA)

Egashira

Templates/(µl)	Primers/(µl)	MilliQ/(µl)	PreMix/(µl)	Total/(µl)
2/12 transformation J23114 colony1	VF 0.75 VR 0.75	11	2×KAPA HiFi /12.5	25
2/12 transformation J23114 colony2	VF 0.75 VR 0.75	11	2×KAPA HiFi /12.5	25
2/12 transformation J23108 colony1	VF 0.75 VR 0.75	11	2×KAPA HiFi /12.5	25
2/12 transformation J23108 colony2	VF 0.75 VR 0.75	11	2×KAPA HiFi /12.5	25

2/15

PCR (Steps)

Uchino

Steps	Temparature	Time	Cycle
Predenature	95℃	3min	1
Denature	98℃	20sec	35
Annealing	65℃	15sec	35
Extension	72℃	30sec	35
Final Elongation	72℃	5min	1
Final Holding	4℃	-	-

2/15

PCR (Target)

Uchino

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	Buffer/(µl)	MilliQ/(µl)	Any Other/(µl)	Total/(µl)
2/12 Gel extraction PCR VF/m.scFv.Rv /0.53 + 2/12 Gel extraction PCR VR/m.scFv.Fw /0.35	VF /0.75	VR /0.75	-	10.12	2×KAPA HiFi /12.5	25
2/12 Gel extraction PCR VF/m.scFv.Rv /0.53 + 2/12 Gel extraction PCR VR/m.scFv.Fw /0.35	VF /0.75	VR /0.75	-	10.12	2×KAPA HiFi /12.5	25

2/15

Electrophoresis

Kaneko

Lane	sample/(µl)	Length(bp)
1	1kb ladder /5
2	other sample	-
3	other sample	-
4	other sample	-
5	other sample	-
6	other sample	-
7	other sample	-
8	other sample	-
9	other sample	-
10	other sample	-
11	other sample	-
12	other sample	-
13	other sample	-
14	2/12 transformation J23114 colony1 /10	-
15	2/12 transformation J23114 colony2 /10
16	2/12 transformation J23108 colony1 /10
17	2/12 transformation J23108 colony2 /10

2/15

Gel Extraction

Yamamoto

Lane	Restriction Enzyme Digestion Product	Volume/(µl)
1	1ka ladder	5
2	2/15 OE-PCR VF/m.scFv.Rv+VR/m.scFv.Fw	25
3	2/15 OE-PCR VF/m.scFv.Rv+VR/m.scFv.Fw	25

2/15

Liquid Culture

Wan, Yamada

Name	medium
2/12 transformation J23114 colony2	LB(AMP)
2/12 transformation J23108 colony2	LB(AMP)

2/16

Miniprep

Wan, Yamamoto

DNA	Concentration/(µg/ml)	260/280	260/230
2/15 liquid culture sfGFP-CBDcex colony2	43.5	1.91	1.60
2/15 liquid culture sfGFP-CBDclos colony2	74.0	1.86	1.82

2/16

PCR (Steps)

Steps	Temparature	Time	Cycle
Predenature	95℃	5min	1
Denature	98℃	20sec	35
Annealing	65℃	15sec	35
Extension	72℃	30sec	35
Final Elongation	72℃	5min	1
Final Holding	4℃	-	-

2/16

PCR (Target)

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	Buffer/(µl)	MilliQ/(µl)	Any Other/(µl)	Total/(µl)
2/16 miniprep sfGFP-CBDcex colony2 /0.3	cex.Fw /0.75	VR /0.75	-	10.7	2×KAPA HiFi /12.5	25
2/16 miniprep sfGFP-CBDclos colony2 /0.3	cex.Fw /0.75	VR /0.75	-	10.7	2×KAPA HiFi /12.5	25

2/16

Gel Extraction(Measurement)

Yamamoto

Sample/(µl)	Concentration(µg/µl)	260/280	260/230
2/15 OE-PCR VF/m.scFv.Rv+VR/m.scFv.Fw	48.6	1.83	0.19

2/16

PCR (Target)

Yamamoto, Yamada

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	Buffer/(µl)	MilliQ/(µl)	Any Other/(µl)	Total/(µl)
2/15 Gel Extraction OE-PCR VF/m.scFv.Rv+VR/m.scFv.Fw /0.3	VF /0.75	VR /0.75	-	10.7	2×KAPA HiFi /12.5	25
2/15 Gel Extraction OE-PCR VF/m.scFv.Rv+VR/m.scFv.Fw /0.3	VF /0.75	VR /0.75	-	10.7	2×KAPA HiFi /12.5	25

2/16

Miniprep

Uchino, Yamada

DNA	Concentration/(µg/ml)	260/280	260/230
2/15 liquid culture J23114	178.8	1.91	2.20

2/16

PCR (Purification)

Yamada

Sample Name/(µl)	Concentration(ng/µl)	260/280	260/230
2/16 PCR m.scFv	152.6	1.86	2.10

2/16

Restriction Enzyme Digestion

Matsumoto, Notsu, Wan

Sample	DNA/(µl)	EcoR1/(µl)	Xba1/(µl)	Spe1/(µl)	Pst1/(µl)	Buffer/(µl)	BSA/(µl)	MilliQ/(µl)	Total/(µl)
2/16 PCR m.scFv	4	1	0	0	1	10×H /3	0	11	30
K592009	25	1	0	0	1	10×H /3	0	0	30

2/16

Gel Extraction

Yamamoto

Lane	Restriction Enzyme Digestion Product	Volume/(µl)
1	1kb ladder	5
2	m.scFv 2/16 EPcut	30
3	K592009 2/16EPcut	30

2/16

Liquid Culture

Uchino

Name	medium
2/12 transformation J23108 colony3	LB(AMP)

2/17

PCR (Steps)

Uchino, Kim

Steps	Temparature	Time	Cycle
Predenature	95℃	5min	1
Denature	98℃	20sec	25
Annealing	65℃	15sec	25
Extension	72℃	30sec	25
Final Elongation	72℃	5min	1
Final Holding	4℃	-	-

2/17

PCR (Target)

Uchino, Kim

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	Buffer/(µl)	MilliQ/(µl)	Any Other/(µl)	Total/(µl)
2/8 miniprep BAN-His14 /0.1	BAN-His-cex.Rv /0.75	VF /0.75	-	10.9	KAPA HiFi Ready Mix /12.5	25
2/8 miniprep BAN-His14 /0.1	BAN-His-clos.Rv /0.75	VF /0.75	-	10.9	KAPA HiFi Ready Mix /12.5	25
2/8 miniprep INP-His12 /0.1	INP-His-cex.Rv /0.75	VF /0.75	-	10.9	KAPA HiFi Ready Mix /12.5	25
2/8 miniprep INP-His12 /0.1	INP-His-clos.Rv /0.75	VF /0.75	-	10.9	KAPA HiFi Ready Mix /12.5	25

2/17

Gel Extraction

Michimori, Wan, Yamamoto, Kim

Lane	Restriction Enzyme Digestion Product	Volume/(µl)
1	blank
2	2/17PCR BAN-His-CBDcexRv+VF	25
3	2/17PCR BAN-His-CBDclosRv+VF	25
4	2/17PCR INP-His-CBDcexRv+VF	25
5	2/17PCR INP-His-CBDclosRv+VF	25
6	2/16PCR sf-GFP-CBDcex	50
7	2/16PCR sf-GFP-CBDclos	50
8	blank	-
9	1kb ladder	-

2/17

Gel Extraction(Measurement)

Michimori, Wan, Yamamoto, Kim

Sample/(µl)	Concentration(µg/µl)	260/280	260/230
2/17PCR BAN-His-CBDcexRv+VF	10.0	1.86	0.43
2/17PCR BAN-His-CBDclosRv+VF	34.6	1.91	1.81
2/17PCR INP-His-CBDcexRv+VF	100.2	1.90	1.24
2/17PCR INP-His-CBDclosRv+VF	116.5	1.90	2.08
2/16PCR sf-GFP-CBDcex	244.0	1.91	2.08
2/16PCR sf-GFP-CBDclos	208.9	1.90	2.01

2/17

PCR (Steps)

Yamamoto

Steps	Temparature	Time	Cycle
Predenature	95℃	3min	1
Denature	98℃	20sec	35
Annealing	65℃	15sec	35
Extension	72℃	30sec	35
Final Elongation	72℃	5min	1
Final Holding	4℃	-	-

2/17

PCR (Target)

Yamamoto

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	Buffer/(µl)	MilliQ/(µl)	Any Other/(µl)	Total/(µl)
2/17 Gel Extraction BAN-His-CBDcexRv+VF /8.5 + 3/17 Gel Extraction sf-GFP-CBDcex /0.5	VF /0.75	VR /0.75	-	2	2×KAPA HiFi /12.5	25
2/17 Gel Extraction BAN-His-CBDclosRv+VF /7 + 3/17 Gel Extraction 2/16PCR sf-GFP-CBDclos /2	VF /0.75	VR /0.75	-	2	2×KAPA HiFi /12.5	25
2/17 Gel Extraction INP-His-CBDcexRv+VF /6.5 + 3/17 Gel Extraction sf-GFP-CBDcex /1	VF /0.75	VR /0.75	-	3.5	2×KAPA HiFi /12.5	25
2/17 Gel Extraction INP-His-CBDclosRv+VF /9 + 3/17 Gel Extraction sf-GFP-CBDclos /2	VF /0.75	VR /0.75	-	0	2×KAPA HiFi /12.5	25

2/17

Miniprep

Matsumoto, Kim

DNA	Concentration/(µg/ml)	260/280	260/230
2/16 liquid culture J23108 colony3	20.8	1.90	1.28

2/17

Gel Extraction(Measurement)

Notsu, Wan

Sample/(µl)	Concentration(µg/µl)	260/280	260/230
2/16 Gel Extraction m.scFv EPcut	14.3	1.87	1.09
2/16 Gel Extraction K592009 EPcut	14.1	1.86	1.60

2/17

Ligation

Wan, Yamamoto

Vector/(µl)	Insert/(µl)	Ligation High/(µl)	Total/(µl)
2/16 Gel Extraction K592009 EPcut /2.5	2/16 Gel Extraction m.scFv EPcut /5.0	7.5	15

2/17

Transformation

Wan, Yamamoto

Sample Name	Sample Volume (µl)	Competent Cells/(µl)	Medium
2/17 ligation m.scFv EPcut+K592009 EPcut	2	DH5α /20	LB(CP)

2/18

Gel Extraction(Measurement)

Kim

Sample/(µl)	Concentration(µg/µl)	260/280	260/230
2/17 OE-PCR BAN-His-CBDcexRv+VF /8.5 + 3/17 Gel Extraction sf-GFP-CBDcex	72.7	1.87	1.93
2/17 OE-PCR BAN-His-CBDclosRv+VF /7 + 3/17 Gel Extraction 2/16PCR sf-GFP-CBDclos	112.1	1.92	1.34
2/17 OE-PCR INP-His-CBDcexRv+VF /6.5 + 3/17 Gel Extraction sf-GFP-CBDcex	102.4	1.89	1.96

2/18

Gel Extraction(Measurement)

Kim

Sample/(µl)	Concentration(µg/µl)	260/280	260/230
2/17 OE-PCR INP-His-CBDclosRv+VF /9 + 3/17 Gel Extraction sf-GFP-CBDclos	103.4	1.94	2.04

2/18

Gel Extraction

Kim

Lane	Restriction Enzyme Digestion Product	Volume/(µl)
1	1kb ladder	8
2	2/17 OE-PCR BAN-His-CBDcexRv+VF /8.5 + 3/17 Gel Extraction sf-GFP-CBDcex	25
3	2/17 OE-PCR BAN-His-CBDclosRv+VF /7 + 3/17 Gel Extraction 2/16PCR sf-GFP-CBDclos	25
4	2/17 OE-PCR INP-His-CBDcexRv+VF /6.5 + 3/17 Gel Extraction sf-GFP-CBDcex	25

2/18

Gel Extraction

Kim

Lane	Restriction Enzyme Digestion Product	Volume/(µl)
1	1kb ladder	8
2	2/17 OE-PCR INP-His-CBDclosRv+VF /9 + 3/17 Gel Extraction sf-GFP-CBDclos	25
3	blank	-
4	blank	-

2/18

Gel Extraction(Measurement)

Sample/(µl)	Concentration(µg/µl)	260/280	260/230
BAN-CBDcex	72.3	1.85	1.87
BAN-CBDclos	79.8	1.86	1.26
INP-CBDcex	83.6	1.80	1.57
INP-CBDclos	39.4	1.76	1.77

2/18

PCR (Target)

Kim

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	Buffer/(µl)	MilliQ/(µl)	Any Other/(µl)	Total/(µl)
BAN-CBDcex /0.2	VF /1.25	VR /1.25	-	9.8	KAPA Pre Mix /12.5	25
BAN-CBDclos /0.2	VF /1.25	VR /1.25	-	9.8	KAPA Pre Mix /12.5	25
INP-CBDcex /0.2	VF /1.25	VR /1.25	-	9.8	KAPA Pre Mix /12.5	25
INP-CBDclos /0.2	VF /1.25	VR /1.25	-	9.8	KAPA Pre Mix /12.5	25

2/18

Colony PCR (typeA)

Uchino

Templates/(µl)	Primers/(µl)	MilliQ/(µl)	PreMix/(µl)	Total/(µl)
2/17 transformation m.scFv EPcut+K592009 EPcut colony1	VF 1.25 VR 1.25	9.5	2×KAPA HiFi Fast Hotstart /12.5	25
2/17 transformation m.scFv EPcut+K592009 EPcut colony2	VF 1.25 VR 1.25	9.5	2×KAPA HiFi Fast Hotstart /12.5	25
2/17 transformation m.scFv EPcut+K592009 EPcut colony3	VF 1.25 VR 1.25	9.5	2×KAPA HiFi Fast Hotstart /12.5	25
2/17 transformation m.scFv EPcut+K592009 EPcut colony4	VF 1.25 VR 1.25	9.5	2×KAPA HiFi Fast Hotstart /12.5	25

2/18

PCR (Steps)

Uchino

Steps	Temparature	Time	Cycle
Predenature	95℃	2min	1
Denature	95℃	10sec	30
Annealing	55℃	15sec	30
Extension	72℃	30sec	30
Final Elongation	72℃	5min	1
Final Holding	4℃	-	-

2/18

PCR (Purification)

Michimori, Kim

Sample Name/(µl)	Concentration(ng/µl)	260/280	260/230
2/18 PCR m.scFv EPcut+K592009 EPcut colony1	49.8	1.92	0.08
2/18 PCR m.scFv EPcut+K592009 EPcut colony2	41.1	1.81	1.90
2/18 PCR m.scFv EPcut+K592009 EPcut colony3	47.2	1.87	1.99
2/18 PCR m.scFv EPcut+K592009 EPcut colony4	47.5	1.87	1.93

2/19

PCR (Purification)

Yamamoto

Sample Name/(µl)	Concentration(ng/µl)	260/280	260/230
BAN-cex/(25)	161.1	1.88	2.13
BAN-clos/(25)	217.9	1.86	2.10
INP-cex/(25)	158.6	1.86	1.98
INP-clos/(25)	163.6	1.85

2/19

Restriction Enzyme Digestion

Yamamoto

Sample	DNA/(µl)	EcoR1/(µl)	Pst1/(µl)	Buffer/(µl)	MilliQ/(µl)
BAN-cex	12.4	1	1	10×H/(3)	12.6
BAN-clos	9.2	1	1	10×H/(3)	15.8
INP-cex	12.6	1	1	10×H/(3)	12.4
INP-clos	12.2	1	1	10×H/(3)	12.8

2/16

Gel Extraction

Kosaka

Lane	Restriction Enzyme Digestion Product	Volume/(µl)
1	BAN-cex E, Pcut	30
2	BAN-clos E, Pcut	30
3	INP-cex E, Pcut	30
0	1kb ladder	5

2/19

Gel Extraction

Egashira, Wan, Yamamoto

Lane	Restriction Enzyme Digestion Product	Volume/(µl)
1	1kb ladder	5
2	2/19 Restriction Enzyme INP-clos EPcut	30
3	2/19 Restriction Enzyme E1010 EPcut	30
4	blank	-

2/16

PCR (Purification)

Wan

Sample Name/(µl)	Concentration(ng/µl)	260/280	260/230
2/19 Restriction Enzyme BAN-cex E, Pcut	9.6	1.85	0.84
〃BAN-clos E, Pcut	18.9	1.83	0.71
〃INP-cex E, Pcut	14.9	1.77	1.46
〃INP-clos E, Pcut	13.4	1.73	1.67
〃E1010 E, Pcut	19.6	1.86

2/19

Transformation

Wan, Egashira, Kim

Sample Name	Sample Volume (µl)	Competent Cells/(µl)	Medium
insertDNA/ul	VectoeDNA/ul	ligation high	total
BAN-cex/4	E1010cpsb1c3/2	6	12
BAN-clos/3	〃/3	6	12
INP-cex/4.5	〃/1.5	6	12
INP-clos/4.5	〃/1.5	6

2/19

Transformation

Wan

Sample Name	Sample Volume (µl)	Competent Cells/(µl)	Medium
2/19 Ligation BAN-cex+E1010	10	DH5a/(20)	LB(CP)
〃BAN-clos+E1010	10	DH5a/(20)	LB(CP)
〃INP-cex+E1010	10	DH5a/(20)	LB(CP)
〃INP-clos+E1010	10	DH5a/(20)

2/19

Restriction Enzyme Digestion

Michimori, Kim

Sample	DNA/(µl)	EcoR1/(µl)	Pst1/(µl)	Buffer/(µl)	Total/(µl)
2/18 purification m.scFv EPcut+K592009 EPcut colony1	12.5	0.5	0.5	10×H /1.5	15
2/18 purification m.scFv EPcut+K592009 EPcut colony2	12.5	0.5	0.5	10×H /1.5	15
2/18 purification m.scFv EPcut+K592009 EPcut colony3	12.5	0.5	0.5	10×H /1.5	15
2/18 purification m.scFv EPcut+K592009 EPcut colony4	12.5	0.5	0.5	10×H /1.5	15

2/19

Electrophoresis

Egashira

Lane	sample/(µl)	Length(bp)
1	1kb ladder /5
2	2/19 restricion enzyme digestion colony1 /10
3	2/19 restricion enzyme digestion colony2 /10
4	2/19 restricion enzyme digestion colony3 /10
5	2/19 restricion enzyme digestion colony4 /10

2/19

Liquid Culture

Egashira, Wan

Name	medium
2/17 transformaion m.scFv EPcut+K592009 EPcut colony2	LB(CP)
2/17 transformaion m.scFv EPcut+K592009 EPcut colony3	LB(CP)
2/17 transformaion m.scFv EPcut+K592009 EPcut colony4	LB(CP)

2/19

Colony PCR (typeA)

Yamada

Templates/(µl)	Primers/(µl)	MilliQ/(µl)	PreMix/(µl)	Total/(µl)
2/19 transformation BAN-His-CBDcex colony1	vf 1.25 vr 1.25	10	12.5	25
〃 colony2	〃	〃	〃	〃
〃 colony3	〃	〃	〃	〃
2/19 transformation BAN-His-CBDclos colony1	〃	〃	〃	〃
〃 colony2	〃	〃	〃	〃
〃 colony3	〃	〃	〃	〃
2/19 transformation INP-His-CBDcex colony1	〃	〃	〃	〃
〃 colony2	〃	〃	〃	〃
〃 colony3	〃	〃	〃	〃

2/22

Electrophoresis

Yamada

Lane	sample/(µl)	Length(bp)
1	ladder 1kb/(5)	/
2	2/22 PCR BAN-His-CBDcex colony1/(10)	515
3	〃colony2/(10)	515
4	〃colony3/(10)	515
5	2/22 PCR BAN-His-CBDclos colony3/(10)	487
6	2/22 PCR INP-His-CBDcex colony1/(10)	405
7	〃colony2/(10)	405
8
9

2/22

Liquid Culture

Yamada

Name	medium
2/22 PCR BAN-His-cex colony1	CP/5ml
〃colony2	〃
〃colony3	〃
2/22 PCR BAN-His-clos colony3

2/22

Miniprep

Egashira, Yamada

DNA	Concentration/(µg/ml)	260/280	260/230
2/19 liquid culture m.scFv EPcut+K592009 EPcut colony2	137.4	1.89	2.12
2/19 liquid culture m.scFv EPcut+K592009 EPcut colony3	164.0	1.89	2.09
2/19 liquid culture m.scFv EPcut+K592009 EPcut colony4	138.1	1.86	2.16

2/19

Ligation

Wan, Yamamoto

Sample Name	Sample Volume (µl)	Competent Cells/(µl)	Medium
insertDNA/ul	VectoeDNA/ul	ligation high	total
BAN-cex/4	E1010cpsb1c3/2	6	12
BAN-clos/3	〃/3	6	12
INP-cex/4.5	〃/1.5	6	12
INP-clos/4.5	〃/1.5	6

2/23

Transformation

Wan

Sample Name	Sample Volume (µl)	Competent Cells/(µl)	Medium
2/23 Ligation BAN-cex+E1010	2	DH5a/(20)	LB(CP)
〃clos〃	2	〃	〃
〃INP-cex〃	2	〃	〃
〃INP-clos〃	2	〃

2/23

Colony PCR (typeA)

Egashira

Templates/(µl)	Primers/(µl)	MilliQ/(µl)	PreMix/(µl)	Total/(µl)
2/23 transformation BAN-His-CBDclos colony1	vf1.25 vr1.25	10	12.5	25
〃colony2	〃	〃	〃	〃
〃colony3	〃	〃	〃	〃
2/23 transformation INP-His-CBDcex colony1	〃	〃	〃	〃
〃colony2	〃	〃	〃	〃
〃colony3	〃	〃	〃	〃
2/23 transformation INP-His-CBDclos colony1	〃	〃	〃

2/24

Miniprep

Yamada

DNA	Concentration/(µg/ml)	260/280	260/230
2/22 liqid culture BAN-Hi-cex colony1	45.3	1.88	2.08
〃colony2	47.1	1.89	1.98
〃colony3	43.8	1.96	2.08
2/22 BAN-His-clos colony3	53.7	1.82

2/24

Liquid Culture

Egashira

Name	medium
2/23 transformation BAN-clos colony1	LB(CP/5ml)
〃colony2	〃
〃colony3	〃
2/23 transformation INP-cex colony2	〃
〃colony3	〃
2/23 transformation INP-clos colony1

2/25

Miniprep

Egashira, Kim

DNA	Concentration/(µg/ml)	260/280	260/230
2/23 transformation BAN-His-CBDclos colony1	79.9	1.89	2.15
2/23 transformation BAN-His-CBDclos colony2	68.0	1.88	2.02
2/23 transformation BAN-His-CBDclos colony3	80.7	1.94	2.18
2/23 transformation INP-His-CBDcex colony2	106.0	1.91	2.17
2/23 transformation INP-His-CBDcex colony3	136.7	1.93	2.23
2/23 transformation INP-His-CBDclos colony1	183.7	1.91	2.21

2/25

Sequence

Michimori

Sample	DNA template/(µl)	Primer/(µl)	MilliQ(µl)	Total(µl)
Bcx 1-F	2/24 miniprep BAN-His-CBDcex colony1 /6.6	VF /0.2	3.2	10
Bcx 1-R	2/24 miniprep BAN-His-CBDcex colony1 /6.6	VR /0.2	3.2	10
Bcx 2-F	2/24 miniprep BAN-His-CBDcex colony2 /6.4	VF /0.2	3.4	10
Bcx 2-R	2/24 miniprep BAN-His-CBDcex colony2 /6.4	VR /0.2	3.4	10
Bcl1-F	2/25 miniprep BAN-His-CBDclos colony1 /3.8	VF /0.2	6.0	10
Bcl1-R	2/25 miniprep BAN-His-CBDclos colony1 /3.8	VR /0.2	6.0	10
Bcl2-F	2/25 miniprep BAN-His-CBDclos colony3 /3.7	VF /0.2	6.1	10
Bcl2-R	2/25 miniprep BAN-His-CBDclos colony3 /3.7	VR /0.2	6.1	10
Icx1-F	2/25 miniprep INP-His-CBDcex colony2 /2.8	VF /0.2	7.0	10
Icx1-R	2/25 miniprep INP-His-CBDcex colony2 /2.8	VR /0.2	7.0	10
Icx2-F	2/25 miniprep INP-His-CBDcex colony3 /2.2	VF /0.2	7.6	10
Icx2-R	2/25 miniprep INP-His-CBDcex colony3 /2.2	VR /0.2	7.6	10
Icl1-F	2/25 miniprep INP-His-CBDclos colony1 /1.6	VF /0.2	8.2	10
Icl1-R	2/25 miniprep INP-His-CBDclos colony1 /1.6	VR /0.2	8.2	10

2/25

Liquid Culture

Yamada

Name	medium
2/19 transformation BAN-His-cex colony1	LB(CP/5ml)
2/19 transformation BAN-His-cex colony2	LB(CP/5ml)
2/23 transformation BAN-His-clos colony1	LB(CP/5ml)
2/23 transformation BAN-His-clos colony3	LB(CP/5ml)
2/23 transformaation INP-His-cex colony2	LB(CP/5ml)
2/23 transformation INP-His-cex colony3	LB(CP/5ml)
INP-His-clos colony1

2/25

Sequence

Michimori

Sample	DNA template/(µl)	Primer/(µl)	MilliQ(µl)	Total(µl)
S2-F	2/22 miniprep scFv colony2 /2.2	VF /0.2	7.6	10
S2-R	2/22 miniprep scFv colony2 /2.2	VR /0.2	7.6	10
S3-F	2/22 miniprep scFv colony3 /1.8	VF /0.2	8.0	10
S3-R	2/22 miniprep scFv colony3 /1.8	VR /0.2	8.0	10
S4-F	2/22 miniprep scFv colony4 /2.2	VF /0.2	7.6	10
S4-R	2/22 miniprep scFv colony4 /2.2	VR /0.2	7.6	10

114C-R

2/25

Liquid Culture

Egashira, Kim, Yamada

Name	medium
2/17 transformaion m.scFv EPcut+K592009 EPcut colony2	LB(CP)
2/17 transformaion m.scFv EPcut+K592009 EPcut colony3	LB(CP)
2/17 transformaion m.scFv EPcut+K592009 EPcut colony4	LB(CP)

2/26

PCR (Target)

Li

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	MilliQ/(µl)	Any Other/(µl)	Total/(µl)
J23100	VF/ 0.75	J23100+RBSRv	11	PrimeSTARMax Premix/ 12.5	25
J23108	VF/ 0.75	J23108+RBSRv	11	PrimeSTARMax Premix/ 12.5	25
J23114	VF/ 0.75	J23114+RBSRv	11		25

2/26

PCR (Target)

Li

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	Buffer/(µl)	MilliQ/(µl)	Any Other/(µl)	Total/(µl)
J23100 iGEM Part	VF /0.75	J23100+RBS Rv /0.75	-	11	PrimeSTAR Max Premix /12.5	25
J23108 iGEM Part	VF /0.75	J23108+RBS Rv /0.75	-	11	PrimeSTAR Max Premix /12.5	25
J23114 iGEM Part	VF /0.75	J23114+RBS Rv /0.75	-	11	PrimeSTAR Max Premix /12.5	25

2/26

Electrophoresis

Egashira

Lane	sample/(µl)	Length(bp)
1	1kb ladder /5	-
2	2/26 PCR J23100 /5	193+α
3	2/26 PCR J23108 /5	193+α
4	2/26 PCR J23114 /5	193+α

2/26

PCR (Target)

Egashira

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	Buffer/(µl)	MilliQ/(µl)	Any Other/(µl)	Total/(µl)
J23100 iGEM Part /0.1	VF 0.75	J23100+RBS Rv /0.75	-	10.9	KAPA HiFi /12.5	25
J23108 iGEM Part /0.1	VF 0.75	J23108+RBS Rv /0.75	-	10.9	KAPA HiFi /12.5	25
J23114 iGEM Part /0.1	VF 0.75	J23114+RBS Rv /0.75	-	10.9	KAPA HiFi /12.5	25

2/26

Gel Extraction

Egashira

Lane	Restriction Enzyme Digestion Product	Volume/(µl)
1	1kb ladder	5
2	2/26 PCR J23100	25
3	2/26 PCR J23108	25
4	2/26 PCR J23114

2/29

Gel Extraction(Measurement)

Egashira, Wan

Sample/(µl)	Concentration(µg/µl)	260/280	260/230
2/26 PCR J23100	1.5	2.71	0.09
2/26 PCR J23108	3.6	18.64	0.01
2/26 PCR J23114	1.7	3.05	0.13

2/29

PCR (Target)

Wan, Kim

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	Buffer/(µl)	MilliQ/(µl)	Any Other/(µl)	Total/(µl)
J23100 iGEM Part /0.2	VF /1.5	J23100+RBS Rv /1.5	-	21.8	KAPA HiFi /25	50
J23108 iGEM Part /0.2	VF /1.5	J23108+RBS Rv /1.5	-	21.8	KAPA HiFi /25	50
J23114 iGEM Part /0.2	VF /1.5	J23114+RBS Rv /1.5	-	21.8	KAPA HiFi /25	50

3/1

Electrophoresis

Li, Kim

Lane	sample/(µl)	Length(bp)
1	100bp ladder /6
2,3	J23114 /50	193+α
4,5	J23100 /50	193+α
6,7	J23108 /50	193+α

3/1

PCR (Purification)

Yamamoto, Kim

Sample Name/(µl)	Concentration(ng/µl)	260/280	260/230
2/29 PCR J23114	4.7	1.59	0.01
2/29 PCR J23100	2.4	2.60	0.01
2/29 PCR J23108	3.8	4.51	0.01

3/1

PCR (Target)

Yamada

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	Buffer/(µl)	MilliQ/(µl)	Any Other/(µl)	Total/(µl)
3/1 PCR purification J23100 /5	VF /0.75	J23100Rv /0.75	-	6	Prime Star Premix /12.5	25
3/1 PCR purification J23108 /5	VF /0.75	J23108Rv /0.75	-	6	Prime Star Premix /12.5	25
3/1 PCR purification J23114 /5	VF /0.75	J23114Rv /0.75	-	6	Prime Star Premix /12.5	25
2/22 miniprep m.scFv colony3 /0.5	BAN+scFv /0.75	VR /0.75	-	10.5	Prime Star Premix /12.5	25

3/1

Electrophoresis

Yamada

Lane	sample/(µl)	Length(bp)
1	1kb ladder /5	-
2	3/1 PCR J23100 /5	about 200
3	3/1 PCR J23108 /5	about 200
4	3/1 PCR J23114 /5	about 200
5	3/1 PCR m.scFv colony3 /5	949

3/1

Electrophoresis

Kim, Yamada

Lane	sample/(µl)	Length(bp)
1	100bp ladder /5	-
2	3/1 OE-PCR J23100 + m.scFv colony3 /2	about 1200
3	3/1 OE-PCR J23108 + m.scFv colony3 /2	about 1200
4	3/1 OE-PCR J23114 + m.scFv colony3 /2	about 1200

3/1

PCR (Purification)

Kim, Yamada

Sample Name/(µl)	Concentration(ng/µl)	260/280	260/230
3/1 OE-PCR J23100 + m.scFv colony3	180.5	1.89	1.83
3/1 OE-PCR J23108 + m.scFv colony3	183.3	1.87	1.96
3/1 OE-PCR J23114 + m.scFv colony3	178.8	1.87	2.08

3/1

PCR (Target)

Kim, Yamada

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	Buffer/(µl)	MilliQ/(µl)	Any Other/(µl)	Total/(µl)
3/1 PCR J23100 /1 + 3/1 PCR m.scFv colony3 /0.5	VF /0.5	VR /0.75	-	9.5	Prime Star Premix /12.5	25
3/1 PCR J23108 /1 + 3/1 PCR m.scFv colony3 /0.5	VF /0.5	VR /0.75	-	9.5	Prime Star Premix /12.5	25
3/1 PCR J23114 /2 + 3/1 PCR J23114 /0.5	VF /0.5	VR /0.75	-	8.5	Prime Star Premix /12.5	25

3/1

Gel Extraction

Kim, Yamada

Lane	Restriction Enzyme Digestion Product	Volume/(µl)
1	1kb ladder
2	3/1 EPcut J23114 + m.scFv	20
3	3/1 EPcut J23108 + m.scFv	20
4	3/1 EPcut J23100 + m.scFv	20

3/1

Restriction Enzyme Digestion

Li, Kim, Yamada

Sample	DNA/(µl)	EcoR1/(µl)	Pst1/(µl)	Buffer/(µl)	BSA/(µl)	MilliQ/(µl)	Total/(µl)
3/1 OE-PCR J23100 + m.scFv colony3	11	1	1	10×H /3	-	14	30
3/1 OE-PCR J23108 + m.scFv colony3	11	1	1	10×H /3	-	14	30
3/1 OE-PCR J23114 + m.scFv colony3	11	1	1	10×H /3	-	14	30

3/2

PartsAwaking

Michimori, Kim, Yamada

Description	PartName	Backbone	Well	PlateYear	PlateNumber	MilliQ(µl)
f1 origin	K314110	pSB1C3	1A	2016	1	10
T1 terminator from E. coli rrnB	K731722	pSB1C3	1B	2016	1	10
Promoter of the CaiF protein	K398326	pSB1C3	1C	2016	1	10

3/2

Transformation

Michimori

Sample Name	Sample Volume (µl)	Competent Cells/(µl)	Medium
3/2 Parts Awakening K314110	10	DH5α /20	LB(CP)
3/2 Parts Awakening K731722	10	DH5α /20	LB(CP)
3/2 Parts Awakening K398326	10	DH5α /20	LB(CP)

3/2

Gel Extraction(Measurement)

Yamada

Sample/(µl)	Concentration(µg/µl)	260/280	260/230
J23100+m.scFv 3/1 EPcut	17.7	1.90	1.68
J23108+m.scFv 3/1 EPcut	11.9	2.04	0.26
J23114+m.scFv 3/1 EPcut	16.8	1.97	1.79

3/2

Restriction Enzyme Digestion

Wan

Sample	DNA/(µl)	EcoR1/(µl)	Xba1/(µl)	Spe1/(µl)	Pst1/(µl)	Buffer/(µl)	BSA/(µl)	MilliQ/(µl)	Total/(µl)
E0020	40	2	0	0	2	10×H /6	-	10	60

3/2

Transformation

Yamada

Sample Name	Sample Volume (µl)	Competent Cells/(µl)	Medium
J23100+pSB1C3	2	DH5α /20	LB(CP)
J23108+pSB1C3	2	DH5α /20	LB(CP)
J23114+pSB1C3	2	DH5α /20	LB(CP)

3/3

PCR (Target)

Yamada

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	Buffer/(µl)	MilliQ/(µl)	Any Other/(µl)	Total/(µl)
2/9 miniprep E1010 /1	VF 0.75	VR 0.75	-	10	Prime Star premix 12.5	25
2/24 miniprep BAN-His-CBDcex colony2 /1	VF 0.75	VR 0.75	-	10	Prime Star premix 12.5	25
2/25 miniprep BAN-His-CBDclos /1	VF 0.75	VR 0.75	-	10	Prime Star premix 12.5	25
2/25 miniprep INP-His-CBDcex /1	VF 0.75	VR 0.75	-	10	Prime Star premix 12.5	25
2/25 miniprep INP-His-CBDclos /1	VF 0.75	VR 0.75	-	10	Prime Star premix 12.5	25

3/3

Gel Extraction(Measurement)

Egashira

Sample/(µl)	Concentration(µg/µl)	260/280	260/230
3/2 EPcut E0020	23.1	1.85	1.69

3/3

Gel Extraction

Egashira

Lane	Restriction Enzyme Digestion Product	Volume/(µl)
1	1kb ladder
2	3/2 EPcut E0020 (pSB1C3)	20

3/3

Ligation

Wan

Vector/(µl)	Insert/(µl)	Ligation High/(µl)	Total/(µl)
3/3 Gel Extraction E0020 EPcut /4	3/2 Gel Extraction J23100+m.scFv EPcut /12	16	32
3/3 Gel Extraction E0020 EPcut /4	3/2 Gel Extraction J23108+m.scFv EPcut /12	20	40
3/3 Gel Extraction E0020 EPcut /4	3/2 Gel Extraction J23114+m.scFv EPcut /12	16	32

3/3

Transformation

Wan, Kim

Sample Name	Sample Volume (µl)	Competent Cells/(µl)	Medium
J23100+PSB1C3	2	DH5alpha/ 20	LB(CP)
J23108+PSB1C3	2	DH5alpha/ 20	LB(CP)
J23114+PSB1C3	2	DH5alpha	LB(CP)

3/3

Liquid Culture

Wan, Kim

Name	medium
K314110 colony1	LB(CP)
K731722 colony1	LB(CP)
K398326 colony1	LB(CP)

3/4

Transformation

Li, Notsu

Sample Name	Sample Volume (µl)	Competent Cells/(µl)	Medium
J23100+pSB1C3	10	DH5α /20	LB(CP)
J23108+pSB1C3	10	DH5α /20	LB(CP)
J23114+pSB1C3	10	DH5α /20	LB(CP)

3/7

Miniprep

Yamada

DNA	Concentration/(µg/ml)	260/280	260/230
2/25 liquid culture BAN-His-CBDcex colony2	71.4	1.83	1.43
2/25 liquid culture BAN-His-CBDclos colony1	61.6	1.83	1.90
2/25 liquid culture INP-His-CBDcex colony3	83.4	1.94	1.92
2/25 liquid culture INP-His-CBDclos colony1	100.7	1.87	1.44

3/7

Transformation

Wan

Sample Name	Sample Volume (µl)	Competent Cells/(µl)	Medium
3/7 miniprep BAN-His-CBDcex	2	DH5α /20	LB(CP)
3/7 miniprep BAN-His-CBDclos	2	DH5α /20	LB(CP)
3/7 miniprep INP-His-CBDcex	2	DH5α /20	LB(CP)
3/7 miniprep INP-His-CBDclos	2	DH5α /20	LB(CP)

3/7

Transformation

Yamamoto

Sample Name	Competent Cells/(µl)	Medium
3/3 ligation J23100+pSB1C3	DH5α /20	LB(CP)
3/3 ligation J23108+pSB1C3	DH5α /20	LB(CP)
3/3 ligation J23114+pSB1C3	DH5α /20	LB(CP)

3/7

Colony PCR (typeA)

Yamamoto

Templates/(µl)	Primers/(µl)	MilliQ/(µl)	PreMix/(µl)	Total/(µl)
3/4 transformtion J23100 colony1	VF 1.25 VR 1.25	10	KAPA2G /12.5	25
3/4 transformtion J23100 colony2	VF 1.25 VR 1.25	10	KAPA2G /12.5	25
3/4 transformtion J23108 colony1	VF 1.25 VR 1.25	10	KAPA2G /12.5	25
3/4 transformtion J23108 colony2	VF 1.25 VR 1.25	10	KAPA2G /12.5	25
3/4 transformtion J23114 colony1	VF 1.25 VR 1.25	10	KAPA2G /12.5	25
3/4 transformtion J23114 colony2	VF 1.25 VR 1.25	10	KAPA2G /12.5	25

3/7

PCR (Steps)

Yamamoto

Steps	Temparature	Time	Cycle
Predenature	95℃	2min	1
Denature	95℃	15sec	35
Annealing	55℃	15sec	35
Extension	72℃	5sec	35
Final Elongation	72℃	10min	1
Final Holding	4℃	-	-

3/7

Electrophoresis

Wan, Yamamoto

Lane	sample/(µl)	Length(bp)
1	1kb ladder /5
2	3/7 colony PCR J23100 colony1 /10	1000+α
3	3/7 colony PCR J23100 colony2 /10	1000+α
4	3/7 colony PCR J23108 colony1 /10	1000+α
5	3/7 colony PCR J23108 colony2 /10	1000+α
6	3/7 colony PCR J23114 colony1 /10	1000+α
7	3/7 colony PCR J23114 colony2 /10	1000+α

3/7

PCR (Steps)

Yamada

Steps	Temparature	Time	Cycle
Predenature	95℃	5min	1
Denature	98℃	20sec	35
Annealing	60℃	15sec	35
Extension	72℃	5min	35
Final Elongation	4℃	-	-
Final Holding	4℃	-	-

3/7

PCR (Target)

Yamada

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	Buffer/(µl)	MilliQ/(µl)	Any Other/(µl)	Total/(µl)
3/1 purification J23100	VF /3	VR /3	-	44	KAPA HiFi /50	100
3/1 purification J23100	VF /3	VR /3	-	44	KAPA HiFi /50	100
3/1 purification J23108	VF /3	VR /3	-	44	KAPA HiFi /50	100
3/1 purification J23108	VF /3	VR /3	-	44	KAPA HiFi /50	100
3/1 purification J23114	VF /3	VR /3	-	44	KAPA HiFi /50	100
3/1 purification J23114	VF /3	VR /3	-	44	KAPA HiFi /50	100

3/7

Colony PCR (typeA)

Yamamoto

Templates/(µl)	Primers/(µl)	MilliQ/(µl)	PreMix/(µl)	Total/(µl)
3/7 transformation BAN-CBDcex colony1	VF 1.5 VR1.25	10	12.5	25
3/7 transformation BAN-cex colony2	VF 1.5 VR1.25	10	12.5	25
3/7 transformation BAN-clos colony1	VF 1.5 VR1.25	10	12.5	25
3/7 transformation BAN-closcolony1	VF 1.5 VR1.25	10	12.5
3/7 transformation INP-cex colony2	VF 1.5 VR1.25	10	12.5	25
3/7 transformation INP-cex colony2	VF 1.5 VR1.25	10	12.5	25
3/7 transformation INP-clos colony1	VF 1.5 VR1.25	10	12.5	25
3/7 transformation INP-clos colony 2	VF 1.5 VR1.25	10	12.5	25

3/8

PCR (Purification)

Uchino

Sample Name/(µl)	Concentration(ng/µl)	260/280	260/230
3/7 PCR J23100	423.3	1.84	2.20
3/7 PCR J23108	439.1	1.85	2.11
3/7 PCR J23114	451.2	1.81	1.91

3/8

Restriction Enzyme Digestion

Yamamoto

Sample	DNA/(µl)	EcoR1/(µl)	Pst1/(µl)	Buffer/(µl)	MilliQ/(µl)	Total/(µl)
3/8 purification J23100	5	1	1	10×H /3	20	30
3/8 purification J23108	5	1	1	10×H /3	20	30
3/8 purification J23114	5	1	1	10×H /3	20	30

3/8

Gel Extraction(Measurement)

Li, Kim, Yamada

Sample/(µl)	Concentration(µg/µl)	260/280	260/230
J23114 3/8 EPcut	13.7	3.62	0.03
J23108 3/8 EPcut	15.4	1.83	1.14
J23100 3/8 EPcut	14.5	1.79	1.03

3/9

Miniprep

Kim

DNA	Concentration/(µg/ml)	260/280	260/230
K731722	67.2	1.70	1.02
K314110	28.9	1.86	1.14
K398326	95.6	1.64	0.94

3/9

Restriction Enzyme Digestion

Yamamoto

Sample	DNA/(µl)	EcoR1/(µl)	Pst1/(µl)	Buffer/(µl)	Total/(µl)
3/1 miniprep K314110	25	1	1	10×H /3	30
3/1 miniprep K731722	25	1	1	10×H /3	30
3/1 miniprep K398326	25	1	1	10×H /3	30

3/9

Restriction Enzyme Digestion

Li

Sample	DNA/(µl)	EcoR1/(µl)	Xba1/(µl)	Spe1/(µl)	Pst1/(µl)	Buffer/(µl)	BSA/(µl)	MilliQ/(µl)	Total/(µl)
3/8 purification J23100	5	1	0	0	1	10×H /3	0	20	30
3/8 purification J23108	5	1	0	0	1	10×H /3	0	20	30
3/8 purification J23114	5	1	0	0	1	10×H /3	0	20	30
3/8 purification J23100	20	1	0	0	1	10×H /3	0	5	30
3/8 purification J23108	20	1	0	0	1	10×H /3	0	5	30
3/8 purification J23114	20	1	0	0	1	10×H /3	0	5	30
									30

3/11

Gel Extraction

Yamada

Lane	Restriction Enzyme Digestion Product	Volume/(µl)
1	1kb ladder	5
2	3/10 E.P. cut J23100+scFv	15
3	3/10 E.P. cut J23100+scFv	15
4	3/10 E.P.cut J23108+scFv	15
5	3/10 E.P.cut J23108+scFv	15
6	3/10 E.P.cut J23114+scFv	15
7	3/10 E.P.cut J23114+scFv	15
8	3/9 E.P. cut J23100+scFv	15
9	3/9 E.P. cut J23100+scFv	15
10	3/9 Ｅ.P. cut J23119 scFv	15
11	3/9 Ｅ.P. cut J23119 scFv	15
12	3/9 E.P.cut J23114+scFv

3/11

Gel Extraction

Yamada

Lane	Restriction Enzyme Digestion Product	Volume/(µl)
1	1kbp ladder	15
2	3/9 E.P. cut K314110	15
3	3/9 E.P. cut K314110	15
4	3/9 E.P cut K731722	15
5	3/9 E.P cut K731722	15
6	3/9 E.P cut K7398326	15
7	3/9 E.P cut K7398326	15
8	3/9 E.P> cut J23114+scFv

3/11

PCR (Purification)

Yoshimoto

Sample Name/(µl)	Concentration(ng/µl)	260/280	260/230
3/11 Gel Ex J23108+scFv	18.9	1.90	0.19
3/11 Gel Ex J23108+scFv	7.0	1.92	0.57
3/11 Gel Ex J23114+scFv	5.3	2.37	0.25
3/11 Gel Ex J23100+scFv	39.2	1.93	1.29
3/11 Gel Ex J23108+scFv	33.6	1.82	0.68
3/11 Gel Ex J23114+scFv	27.8	1.86	1.25
3/11 Gel Ex pSCIC3	29.0	1.86

3/11

Ligation

Kim

Vector/(µl)	Insert/(µl)	Ligation High/(µl)	Total/(µl)
3/11 Gel Extraction PSB1C3/ 5	3/11 Gel Extraction J23100+scfv/ 5.6	10.6	21.2
3/11 Gel Extraction PSB1C3/ 5	3/11 Gel Extraction J23108+scfv/ 5	11.5	23
3/11 Gel Extraction PSB1C3/ 5	3/11 Gel Extraction J23114+scfv/ 5	12.8

3/11

Transformation

Kim

Sample Name	Competent Cells/(µl)	Medium
3/11 Ligation PSB1C3+J23100+scFV	DH5α /20	LB
3/11 Ligation PSB1C3+J23108+scFv	DH5α /20	LB
3/11 Ligation PSB1C3+J23114+scFv	DH5α /20	LB

3/7

Electrophoresis

Yamada

Lane	sample/(µl)	Length(bp)
1	1kbp ladder
2	3/8 PCR BANcex colony1	515
3	3/8 PCR BAN-cex colony2	515
4	3/8 PCR BAN-clos colony1	487
5	3/8 PCR BAN-clos-colony2	487
6	3/8 PCR INP-cex colony 1	1405
7	3/8 PCR INP-cex colony 2	1405
8	3/8 PCR INP-clos colony 1	1378
9	3/8 PCR INP-clos colony 2	1378

3/14

PCR (Target)

Uchino, Egashira

Templates/(µl)	Primers/(µl)	MilliQ/(µl)	PreMix/(µl)	Total/(µl)
2/25 INPcex colony2	VF 0.75 CBDcex ctl. Rv0.75	11	KAPA HiFi 12.5	25
2/25 INPcex colony2	CBDcex ctl Fw 0.75 VR0.75	11	KAPA HiFi 12.5	25
2/18 INPclos Gel Extraction	VF 0.75 CBD clos ctl. Rv0.75	11	KAPA HiFi 12.5	25
2/18 INPclos Gel Extraction	CBDclos ctl Fw 0.75 VR0.75	11	KAPA HiFi 12.5	25

3/14

Gel Extraction

Uchino, Egashira

Lane	Restriction Enzyme Digestion Product	Volume/(µl)
4	3/14PCR INP cex (VF CBD cex ctl.Rv)25	5
5	3/14PCR INPcex (CBD cex ctl.Fw VR) 25	5
6	3/14PCR INP clos(VF CBDclos ctl.Rv)25	5
7	3/14PCR INPclos (CBDclos ctl.Fw VR) 25	5

3/15

Restriction Enzyme Digestion

Notsu

Sample	DNA/(µl)	Xba1/(µl)	Spe1/(µl)	Pst1/(µl)	Buffer/(µl)	MilliQ/(µl)	Total/(µl)
3/7 miniprep BAN-His-CBDcex colony2	30		1	1	4	4	40
3/7miniprep BAN-His-CBDclos colony1	30		1	1	4	4	40
3/7miniprep INP-His-CBDcex colony3	30		1	1	4	4	40
3/'miniprep INP-His-CBDcloscolony 1	40		1	1	5	3	50
2/9 miniprep J23105	61	1				0	70

3/14

OE-PCR (typeA)

Kim

Templates/(µl)	Primers/(µl)	MilliQ/(µl)	PreMix/(µl)	Total/(µl)
3/14 Gel Ex INP cex 0.5 VR ct Fw + 3/14 Gel Ex INP cex 0.5 VF ct Rv	VF1.5 VR1.5	21	25	50
3/14 Gel Ex INP clos 0.5 VR ct Fw + 3/14 Gel Ex INP clos 0.5 VF ct Rv	VF1.5 VR1.5	21	25	50

3/14

PCR (Steps)

Kim

Steps	Temparature	Time	Cycle
Pre Denature	95℃	5min	1
Denature	78℃	20sec	35
Annealing	60℃	15sec	35
Extension	72℃	45sec	35
Final Elongation	72℃	5min	1
Final Holding	4℃	-	-

3/14

Colony PCR (typeA)

Yoshimoto

Templates/(µl)	Primers/(µl)	MilliQ/(µl)	PreMix/(µl)	Total/(µl)
2/8 INP His9	VF 0.75 scFv ctl. Rv 0.75	11	12.5	25
2/8 INP His9	scFv ctl Fw 0.75 VR 0.75	11	12.5	25
2/25 INPcex colony2	VF 0.75 CBDcex ctl. Rv0.75	11	12.5	25
2/25 INPcex colony2	CBDcex ctl. Fw 0.75 VR 0.75	11	12.5	25
2/18 INPclos Gel Extraction	CBDclos ctl. Rv 0.75 VF 0.75	11	12.5	25
2/18 INPclos Gel Extraction	CBDclos ctl Fw 0.75	11	12.5

3/14

Gel Extraction

Egashira

Lane	Restriction Enzyme Digestion Product	Volume/(µl)
1	1kb ladder	5
2	3/14 PCR INP His9 (VF scFv.ctl. Rv)	25
3	3/14 PCR INP His9 (scFv ctl.Fw VR)

3/15

Gel Extraction

Egashira

Lane	Restriction Enzyme Digestion Product	Volume/(µl)
1	1kb ladder 5
2	3/14 OE PCR CBDcex ctl 50	10
3	3/14 OEPCR CBDclos ctl 50

3/15

PCR (Target)

Egashira

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	Buffer/(µl)	MilliQ/(µl)	Any Other/(µl)	Total/(µl)
2/25 INPcex colony2	VF /1.5	CBDcex.ctl.RV /1.5	-	22	KAPA HiFi /25	50
2/25 INPcex colony2	CBDcex.ctl.Fw /1.5	VR /1.5	-	22	KAPA HiFi /25	50
2/25 INPclos colony1	VF /1.5	CBDclos.ctl.Rv /1.5	-	22	KAPA HiFi /25	50
2/25 INPclos colony1	CBDclos.ctl.Fw /1.5	VR /1.5	-	22	KAPA HiFi /25	50

3/15

Gel Extraction

Egashira

Lane	Restriction Enzyme Digestion Product	Volume/(µl)
4	3/15 PCR INPcex(VF 20, CBDcex ctl.Rv 20)	4
5	3/15 PCR INPcex(CBDcex ctl.Fw20, VR 20)	4
6	3/15 PCR INPclos(VF 20 CBDclos ctl.Rv 20)	4
7	3/15 PCR INPclos(CBDclos ctl.Fw20, VR 20)	4

3/15

Liquid Culture

Li, Uchino

Name	medium
3/11 scFv J23100 colony1,4 colony2,3(plate2)	LB(CP) 5×2
3/11 scFv J23108 colony 2(plate1) colony(plate2)	LB(CP) 5×2
3/14 scFv J23114 colony 1,2,4(plate1) colony1,2,(plate2)

3/16

PCR (Purification)

Kim, Yamada

Sample Name/(µl)	Concentration(ng/µl)	260/280	260/230
3/15 Gelex CBDcex ctl. Fw VR	55.3	1.95	0.11
3/15 Gelex CBDcex VF ctl.RV	8.2	1.74	1.04
3/15Gelex CBDclos ctl Fw VR	41.2	1.85	1.51
3/15Gelex CBDclos VF ctl RV	10.6	1.56	0.60
3/15Gelex BANcexSP	34.9	1.70	1.84
3/15Gelex BANclosSP	32.0	1.95	0.07
3/15 Gelex INPcex SP	40.3	1.76	1.76
3/15Gelex INPclosSP	106.8	1.83	0.19

3/16

Gel Extraction

Egashira

Lane	Restriction Enzyme Digestion Product	Volume/(µl)
3	3/15 PCR INPcex(VF 20, CBDcex ctl, RV 20)	4
4	3/15 PCR INPclos(VF 20, CBDclos ctl, RV 20)

3/16

Gel Extraction

Egashira

Lane	Restriction Enzyme Digestion Product	Volume/(µl)
4	CBDcex ctl
5	CBDclos ctl

3/16

PCR (Target)

Egashira

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	Buffer/(µl)	MilliQ/(µl)	Any Other/(µl)	Total/(µl)
3/15 Gel Ex INPcex 0.5(VF ctl.RV) 3/15 Gel Ex INPcex0.2(ctl.Fw VR)	VF1.25	VR1.25	KAPA taq 12.5	9.3		25
3/15 GEl EX INPclos 0.5(VF ctl RV) 3/15Gel Ex INPclos0.2(ctl.Fw VR)	VF1.25		KAPA taq 12.5	9.3		25

3/16

PCR (Target)

Kim

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	Buffer/(µl)	MilliQ/(µl)	Any Other/(µl)	Total/(µl)
3/14 Gel Extraction INP-His9 VR/ct/Rv /0.5 + 3/14 Gel Extraction INP-His9 /0.5	VF /1.5	VR /1.5	-	21	2×KAPA HiFi /25	50

3/16

PCR

Egashira

Templates/(µl)	Primers/(µl)	MilliQ/(µl)	KAPA HiFi/(µl)	Total/(µl)
2/8 INP HIs9	VF 1.5 scFvctl. RV1.5	22	25	50
2/8 INP His9	VR1.5 scFvctl. Fw 1.5 VR1.5	22	25

3/16

Miniprep

Egashira, Yamamoto

DNA	Concentration/(µg/ml)	260/280	260/230
J23100 scFv c1-p1	135.7	1.85	1.76
J23100 scFv C4-p1	135.1	1.86	1.85
J23100 scFv c3-p2	155.0	1.87	2.04
J23100 scFv c3-p2	205.9	1.76	1.32
J23108 scFv c2-p1	166.5	1.88	2.06
J23108 scFvc1-p2	95.7	1.84	1.83
J23114 scFv c1-p1	145.7	1.80	1.99
J23114 scFv c2-p1	111.7	1.90	2.16
J23114 scFv c4-p1	169.2	1.90	2.11
J23114 scFv c1-p2	127.4	1.87	2.00
J23114 scFv c2-p2	136.2	1.90	2.13

3/16

Sequence

Michimori

Sample	DNA template/(µl)	Primer/(µl)	MilliQ(µl)	Total(µl)
100A-F	3/15 miniprep J23100 scFv c1-p1 /2.2	VF /0.2	7.6	10
100A-R	3/15 miniprep J23100 scFv c1-p1 /2.2	VR /0.2	7.6	10
100B-F	3/15 miniprep J23100 scFv c4-p1 /2.2	VF /0.2	7.6	10
100b-R	3/15 miniprep J23100 scFv c4-p1 /2.2	VR /0.2	7.6	10
100C-F	3/15 miniprep J23100 scFv c2-p2 /1.9	VF /0.2	7.9	10
100C-R	3/15 miniprep J23100 scFv c2-p2 /1.9	VR /0.2	7.9	10
100D-F	3/15 miniprep J23100 scFv c3-p2 /1.5	VF /0.2	8.3	10
100D-R	3/15 miniprep J23100 scFv c3-p2 /1.5	VR /0.2	8.3	10
108A-F	3/15 miniprep J23108 scFv c2-p1 /1.8	VF /0.2	8.0	10
108A-R	3/15 miniprep J23108 scFv c2-p1 /1.8	VR /0.2	8.0	10
108B-F	3/15 miniprep J23108 scFv c1-p2 /3.1	VF /0.2	6.7	10
108B-R	3/15 miniprep J23108 scFv c1-p2 /3.1	VR /0.2	6.7	10
114A-F	3/15 miniprep J23114 scFv c1-p1 /2.1	VF /0.2	7.7	10
114A-R	3/15 miniprep J23114 scFv c1-p1 /2.1	VR /0.2	7.7	10
114B-F	3/15 miniprep J23114 scFv c2-p1 /2.7	VF /0.2	7.1	10
114B-R	3/15 miniprep J23114 scFv c2-p1 /2.7	VR /0.2	7.1	10

114C-R

3/16

Sequence

Michimori

Sample	DNA template/(µl)	Primer/(µl)	MilliQ(µl)	Total(µl)
114C-F	3/15 miniprep J23114 scFv c4-p1 /1.8	VF /0.2	8.0	10
114C-R	3/15 miniprep J23114 scFv c4-p1 /1.8	VR /0.2	8.0	10
114C-F	3/15 miniprep J23114 scFv c1-p2 /2.4	VF /0.2	7.4	10
114C-R	3/15 miniprep J23114 scFv c1-p2 /2.4	VR /0.2	7.4	10
114C-F	3/15 miniprep J23114 scFv c2-p2 /2.2	VF /0.2	7.6	10
114C-R	3/15 miniprep J23114 scFv c2-p2 /2.2	VR /0.2	7.6	10

114C-R

3/16

Gel Extraction

Egashira

Lane	Restriction Enzyme Digestion Product	Volume/(µl)
2	3/15 PCR INP His9(VF scFv CH. Rv)	20
3	3/15 PCR INP His9 (scFv ctl, Fw VR)

3/16

Gel Extraction

Egashira

Lane	Restriction Enzyme Digestion Product	Volume/(µl)
1	1kb ladder	5
2	3/15 PCR INP His9 (VF scFv ctl RV)

3/16

Gel Extraction

Egashira

Lane	Restriction Enzyme Digestion Product	Volume/(µl)
1	1kb ladder
2	scFv (VF ctl.Rv)
3

3/16

OEPCR (Target)

Egashira

Sample1/(µl)	Sample2/(µl)	Primer/(µl)	Buffer/(µl)	MilliQ/(µl)	Any Other/(µl)	Total/(µl)
3/15 Gel Extraction scfv (VF ctl.Rv)/ 0.5	3/15 Gel Extraction scfv (ctl.Fw VR)	VF/ 1.25, VR/1.25		9.3	KAPA Taq/ 12.5	25

3/16

PCR

Egashira

Templates/(µl)	Primers/(µl)	MilliQ/(µl)	PreMix/(µl)	Total/(µl)
scFv (VF ctl.RV) 0.5	VF 1.25 ctl.Rv) 1.25	9.5	12.5	25

3/16

PCR (Target)

Egashira

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	Buffer/(µl)	MilliQ/(µl)	Any Other/(µl)	Total/(µl)
3/16 Gel Extraction scFv(VF.ctl.Rv) /0.25+3/16 Gel Extraction scFv(ctl.Fw.VR) /0.25	VF /1.25	VR /1.25	-	9.5	2×KAPA HiFi /12.5	25

3/17

PCR (Target)

Egashira

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	Buffer/(µl)	MilliQ/(µl)	Any Other/(µl)	Total/(µl)
3/16 Gel Ex scFv(VF ctl.Rv)0.25, 3/15 Gel EX CBDcex(ctl.Fw VR) 0.25	VF 1.25	VR1.25	KAPA Taq 12.5	9.5		25
3/16 Gel Ex scFv(VF ctl.Rv) 0.25, Gel Ex CBDclos(ctl,Fw VR) 0.25	VF 1.25	VR1.25	KAPA Taq 12.5			25

3/17

Electrophoresis

Egashira

Lane	sample/(µl)	Length(bp)
3	3/16 OEPCR CBDcex ctl 25	500
4	3/16 OEPCR CBDclos ctl 25	500

3/17

Electrophoresis

Egashira

Lane	sample/(µl)	Length(bp)
2	CBDcex ctl 25
3	CBDclos ctl 25
4	1kb ladder 5

3/17

PCR (Target)

Egashira

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	Buffer/(µl)	MilliQ/(µl)	Any Other/(µl)	Total/(µl)
3/16 Gel Ex scFv(VF ctl,Rv)0.1 3/15 Gel Ex CBDcex(ctl,Fw VR) 0.1 3/15 Gel Ex CBDcex(ctl,Fw VR) 0.1	VF 1.25	VR1.25	KAPA 12.5	9.8		25
3/16 Gel Ex scFv(VF ctl,Rv) 0.1, 3/15 GelEx CBDclos(ctl,Fw VR) 0.1 ctl,Rv)0.1 3/15 Gel Ex CBDcex(ctl,Fw VR) 0.1	VF 1.25	VR1.25	KAPA 12.5	9.8		25

3/17

Miniprep

Yamamoto

DNA	Concentration/(µg/ml)	260/280	260/230
3/8 Liquid Culture BAN-His-cex	39.3	1.91	1.67
3/8 Liquid Culture BAN-His=clos	45.1	1.84	1.44

3/17

PCR (Steps)

Yamamoto

Steps	Temparature	Time	Cycle
Pre Denature	95℃	3min	1
Denature	95℃	15sec	40
Annealing	55℃	15sec	40
Elongation	72℃	1min	40
Final Elongation	72℃	1min	1
Final Holding	4℃	-	-

3/17

PCR (Target)

Yamamoto

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	Buffer/(µl)	MilliQ/(µl)	Total/(µl)
3/17 miniprep BAN-His-cex	CBDcexFw 1.25	VR1.25	12.5	9	25
3/17 miniprep BAN-His-clos	CBDclosFw 1.25	VR1.25	12.5	9	25
2/9 miniprep J23105_1	VF 1.25	RFP-cex RV 1.25	12.5	9	25
2/9 miniprep J23105_1	VF 1.25	RFP-clos Rv 1.25	12.5	9	25

3/17

Electrophoresis

Egashira

Lane	sample/(µl)	Length(bp)
1	1kb ladder 5
2

3/17

PCR (Target)

Egashira

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	Buffer/(µl)	MilliQ/(µl)	Any Other/(µl)	Total/(µl)
3/16 Gel Extraction scFv(VF.ctl.Rv) /0.1+3/15 Gel Extraction scFv(ctl.Fw.VR) /0.1	VF /1.25	VR /1.25	-	9.8	2×KAPA HiFi /12.5	25

3/18

Gel Extraction

Yamamoto

Lane	Restriction Enzyme Digestion Product	Volume/(µl)
1	1kb ladder	5
2	3/10 PCR BAN-cex CBDcexFW VR	25
3	3/10 PCR BAN-clos CBDclosFw VR	25
4	3/10 PCR J23105 VF RFPcexRv	25
5	3/10 PCRJ23105 VF RFPclos	25

3/18

Gel Extraction(Measurement)

Yamamoto

Sample/(µl)	Concentration(µg/µl)	260/280	260/230
3/17 PCR BAN-CBDcex CBDcex.Fw+Rv	127.7	1.90	1.57
3/17 PCR BAN-CBDclos CBDclos.Fw+VR	93.4	1.92	2.19
3/17 PCR J23105 VF+RFP-CBDcex.Rv	104.0	1.84	1.50
3/17 PCR J23105 VF+RFP-CBDclos.Rv	123.0	1.87	1.84

3/18

PCR (Steps)

Yamamoto

Steps	Temparature	Time	Cycle
Pre Denature	95℃	3min	1
Denature	95℃	15sec	40
Annealing	60℃	15sec	40
Elongation	72℃	1min	40
Final Elongation	72℃	1min	1
Final Holding	4℃	-	-

3/18

PCR (Target)

Yamamoto

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	Buffer/(µl)	MilliQ/(µl)	Any Other/(µl)	Total/(µl)
3/18 Gel Extraction BAN-His-cex 0.1 cexFw+VR , 3/18Gel Extraction J23105 VF+RFP-cexRv	VF 1.25	VR 1.25	KAPATaq 12.5	9.8		25
3/18 Gel Extraction BAN-His-clos 0.1 closFw+VR , 3/18Gel Extraction J23105 VF+RFP-closRv	VF 1.25	VR 1.25	KAPATaq 12.5	9.8		25

3/18

Gel Extraction

Yamamoto

Lane	Restriction Enzyme Digestion Product	Volume/(µl)
1	1kb ladder
2	3/18 OEPCR RFP-cex
3	3/18 OEPCR RFP-clos

3/18

PCR (Target)

Egashira

Templates/(µl)	Primers/(µl)	MilliQ/(µl)	PreMix/(µl)	Total/(µl)
2/25 INP cex colony2 0.1×2	VF 0.75×2 ctl RV 0.75	10.9×2	12.5×2	25×2
2/25 INP cex colony2 0.1×2	ctl Fw 0.75×2 VR 0.75×2	10.9×2	12.5×2	25×2
2/25 INP cex colony2 0.1×2	VF 0.75×2 VR 0.75×2	10.9×2	12.5×2	25×2
2/25 INP cex colony2 0.1×2	nothing	12.4	12.5×2	25×2
2/25 INP clos colony1 0.1	VF ctl.Rv 0.75×2	10.9	12.5×2	25×2
2/25 INP clos colony1 0.1	ctl.Rv 0.75×2	10.9	12.5×2	25×2
2/25 INP clos colony1 0.1	VF 0.75×2 VR 0.75×2	10.9	12.5×2	25×2
2/25 INP clos colony1 0.1	nothing	12.4	12.5×2	25×2

3/18

Colony PCR (typeA)

Notsu

Templates/(µl)	Primers/(µl)	MilliQ/(µl)	PreMix/(µl)	Total/(µl)
INP-His=CNDcex+RFP colony 1(3/16 ligation product)	VF 1.25, VR .125	10	12.5	25
INP-His-CBDcex+RFP colony (3/16 ligation product)	VF 1.25, VR .125	10	12.5	25
INP-His-CBDclos+RFP colony1(3/16 ligation product)	VF 1.25, VR .125	10	12.5	25
INP-His-CBDclos+RFP colony 2(3/16 ligation product)	VF 1.25, VR .125	10	12.5	25
INP-His-CBDcex+RFP colony1( 3/16 ligation product)	VF 1.25, VR .125	10	12.5	25
BAN-His-CBDcex+RFP colony2 (3/16 ligation product)	VF 1.25, VR .125	10	12.5	25
BAN-His-CBDclos+RFP colony1(3/16 ligation product)	VF 1.25, VR .125	10	12.5	25
BAN-His-CBDclos+RFP colony2(3/16 ligation product)	VF 1.25, VR .125	10	12.5	25

3/18

Electrophoresis

Kim, Yamada

Lane	sample/(µl)	Length(bp)
1	1kb ladder 5
2	1 kb ladder 5
3	2/25 INPcex colony2 10
4	2/25 INPcex colony2 10
5	2/25 INPcex colony2 10
6	2/25 INPcex colony210
7	2/25 INPclos colony1 10
8	2/25 INPclos colony1 10
9	2/25 INPclos colony1 10
10	2/25 INPclos colony1 10
11	100bp ladder 5
12	1kbp ladder 5

3/18

PCR

Egashira

Templates/(µl)	Primers/(µl)	MilliQ/(µl)	PreMix/(µl)	Total/(µl)
INP HIs9 0.1x2	VF 0.75x2 ctl. Rv 0.75x2	10.9x2	12.5x2	25x2
INP HIs9 0.1x2	ctl.Fw 1.75 VR 0.75	10.9x2	12.5x2	25x2
INP HIs9 0.1x2	VF 0.75 VR 0.75	10.9x2	12.5x2	25x2
INP HIs9 0.1x2	nothing	12.4	12.5x2

3/22

Electrophoresis

Notsu

Lane	sample/(µl)	Length(bp)
1	1kb ladder /7
2	INP-His-CBDcex+RFP colony1 (3/19 PCR) /20
3	INP-His-CBDcex+RFP colony2 (3/19 PCR) /20
4	blank
5	INP-His-CBDclos+RFP colony1 (3/19 PCR) /20
6	INP-His-CBDclos+RFP colony2 (3/19 PCR) /20
7	blank
8	BAN-His-CBDcex+RFP colony1 (3/19 PCR) /20
9	BAN-His-CBDcex+RFP colony2 (3/19 PCR) /20
10	blank
11	BAN-His-CBDclos+RFP colony1 (3/19 PCR) /20
12	BAN-His-CBDclos+RFP colony2 (3/19 PCR) /20
13	blank
14	100bp ladder /7
15	-
16	-
17	-

3/22

Electrophoresis

Egashira

Lane	sample/(µl)	Length(bp)
7	2/25 INPcexcolony2 (5) 10
8	2/25 INPcexcolony2 (6) 10
9	2/25 INPcexcolony2 (7) 10
10	2/25 INPcexcolony2 (8) 10
11	2/25 INPclos colony1(9) 10
12	2/25 INPclos colony1(10) 10
13	2/25 INPclos colony1(11) 10
14	2/25 INPclos colony1(12) 10
16	100b ladder 5
17	1kb ladder 5

3/22

Electrophoresis

Egashira

Lane	sample/(µl)	Length(bp)
5	3/22 PCR CBDcex(VF/ctl.Rv) 10
6	3/22 PCR CBDcex(ctl.Fw/VR) 10
7	3/22 PCR CBDclos(VP/ctl.RV) 10
8	3/22 PCR CBDclos(ctl.Fw/VR) 10

3/22

PCR (Target)

Templates/(µl)	Primers/(µl)	MilliQ/(µl)	PreMix/(µl)	Total/(µl)
2/25 INPcex colony2 0.1	VF 1.5 ctl, Rv 1.5	21.9	25	50
2/25 INPcex colony2 0.1	ctl.Fw 1.5 ,VR 1.5	21.9	25	50
2/25 INPclos colony1 0.1	VF 1.5, ctl,Rv 1.5	21.9	25	50
2/25 INPclos colony1 0.1	ctl,Fw 1.5 VR 1.5	21.9	25	50

3/22

Miniprep

Notsu, Yamada

DNA	Concentration/(µg/ml)	260/280	260/230
BANcex+RFP col.1	147.8	1.89	2.18
BANcex+RFP col.2	133.9	1.87	2.18
BANcex+RFP col.1	132.4	1.85	2.19
BANclos+RFP col.2	126.4	1.85	2.22
INPcex+RFP col.1	179.3	1.87	2.18
INPcex+RFP col.2	144.9	1.87	2.19
INPclos+RFPcol.1	152.2	1.87	2.23
INPclos+RFPcol.2	152.4	1.86

3/22

PCR (Target)

Yamada

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	Buffer/(µl)	MilliQ/(µl)	Any Other/(µl)	Total/(µl)
2/8 INP-His9 0.1	VF 1.5	ctl.Rv 1.5	25	21.9		50
2/8 INP-His9 0.1	scFv ctl.Fw 1.5	VR 1.5	25	21.9

3/22

Miniprep

Egashira, Kim

DNA	Concentration/(µg/ml)	260/280	260/230
3/22 PCR scFv (VF/ctl.RN)	105.4	1.81	1.54
3/22 PCR scFv (ctl. FV/VR)	127.4	1.87

3/22

OEPCR (Target)

Egashira

Sample1/(µl)	Sample2/(µl)	Primer/(µl)	Buffer/(µl)	MilliQ/(µl)	Any Other/(µl)	Total/(µl)
3/23 Gel Extraction scfv (VF ctl.Rv)/ 0.1	3/23 Gel Extraction scfv (ctl.Fw VR)/ 0.1	VF/ 1.25, VR/1.25		10.8	KAPA Taq/ 12.5	25

3/23

Electrophoresis

Egashira

Lane	sample/(µl)	Length(bp)
5	3/22 PCR CBDcex(VF/ctl.Rv) 40
6	3/22 PCR CBDcex(ctl.Fw/VR) 40
7	3/22 PCR CBDclos(VF/ctl.Rv) 40
8	3/22 PCR CBDclos(ctl.Fw/VR) 40

3/23

PCR (Steps)

Kim

Steps	Temparature	Time	Cycle
Pre Denature	95℃	2min	1
Denature	98℃	20sec	35
Annealing	60℃	15sec	35
Elongation	72℃	15sec	35
Final Elongation	72℃	5min	1
Final Holding	10℃	-	-

3/23

PCR (Target)

Kim

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	Buffer/(µl)	MilliQ/(µl)	Any Other/(µl)	Total/(µl)
3/18 Gelex BANcex Fw+VR 0.1, 3?18 Gelex J23105 VF+cexRv 0.1	VF1.25	VR 1.25	12.5	9.8		25
3/18 Gelex BANclos closFV VR 0.1, 3/18Gelex J23105 VF+closR 0.1	VF 1.25	VR 1.25	12.5	9.8		25

3/23

Gel Extraction (Measurement)

Egashira

Sample Name/(µl)	Concentration(ng/µl)	260/280	260/230
3/22 PCR CBDcex(ctlFw/VR)	26.9	1.75	1.04
3/22PCR CBDclos(ctl Fw/VR)	100.6	1.90	1.65

3/23

Electrophoresis

Kim

Lane	sample/(µl)	Length(bp)
3	3/18 OEPCR BANcex cexFw+VR J23105 VF+cexRv 25
4	BANclos clos Fw+VR J23105 VF+clos 25
1	1kb ladder 10
2	100bp ladder 10

3/23

Electrophoresis

Egashira

Lane	sample/(µl)	Length(bp)
4	3/23 OEPCR CBDcex ctl 25
5	3/23 OEPCR CBDclos ctl 25

3/23

PCR (Target)

Egashira

Templates/(µl)	Primers/(µl)	MilliQ/(µl)	PreMix/(µl)	Total/(µl)
3/23 Gel Ex scFv(VF/ctl.Rv) 0.1, 3/23 Gel Ex CBDcex(ctl.Fw/VR) 0.1	VF 0.75 VR 0.75	10.8	12.5	25
3/23 Gel Ex scFv (VF/ctl.Rv), 3/23 Gel Ex clos(ctl.Fw/VR)	VF 0.75 VR 0.75	10.8	12.5	25

3/23

Electrophoresis

Yamada

Lane	sample/(µl)	Length(bp)
1	1kb ladder 5
2	100b ladder 7.5
3	3/22 PCR scFv(VP/ctl.Rv) 40
4	3/22 PCR scFv(ctl.Fw/VR)

3/24

PCR (Steps)

Kim

Steps	Temparature	Time	Cycle
Pre Denature	95℃	2min	1
Denature	98℃	20sec	30
Annealing	60℃	15sec	30
Elongation	72℃	15sec	30
Final Elongation	72℃	5min	1
Final Holding	10℃	-	-

3/24

PCR (Target)

Kim

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	Buffer/(µl)	MilliQ/(µl)	Any Other/(µl)	Total/(µl)
3/18 Gelcex BAN cex cexFv VR 0.2, 3/18 Gelex J23105 VF cexFv 0.2	VF1.25	VR 1.25	12.5	9.6		25
3/18 Gelcex BAN clos clos Fv VR 0.2, 3/18 Gelex J23105 VF clos Rv 0.2	VF 1.25	VR 1.25	12.5	9.6		25

3/24

PCR (Purification)

Egashira

Sample Name/(µl)	Concentration(ng/µl)	260/280	260/230
3/24 OEPCR CBDcex ctl	468.6	1.82	2.06
3/24 OEPCR CBDclos ctl	321.8	1.86	0.45

3/24

PCR (Purification)

Kim

DNA	Concentration/(µg/ml)	260/280	260/230
3/24 OEPCR BANcex ctl	77.2	1.81	1.40
3/24 OEPCR BAN clos ctl	23.8	1.75	1.56

3/24

PCR (Purification)

Kim

DNA	Concentration/(µg/ml)	260/280	260/230
3/24 OEPCR BANcex ctl	77.2	1.81	1.40
3/24 OEPCR BAN clos ctl	23.8	1.75	1.56

3/24

PCR (Purification)

Egashira

Sample Name/(µl)	Concentration(ng/µl)	260/280	260/230
3/24 OEPCR scFv ctl	394.2	1.83

3/24

Restriction Enzyme Digestion

Egashira

Sample	DNA/(µl)	EcoR1/(µl)	Xba1/(µl)	Spe1/(µl)	Pst1/(µl)	Buffer/(µl)	BSA/(µl)	MilliQ/(µl)	Total/(µl)
3/24 OE PCR scFv ctl	5.1	1			1	3		19.9	30

3/24

Transformation

Egashira

Sample Name	Sample Volume (µl)	Competent Cells/(µl)	Medium
PCBTC3+scFv+ctl.		DH5α

3/25

Restriction Enzyme Digestion

Egashira

Sample	DNA/(µl)	EcoR1/(µl)	Pst1/(µl)	Buffer/(µl)	MilliQ/(µl)	Total/(µl)
3/24 OEPCR CBDcex ctl	4.3	1	1	10×H /3	20.7	30
3/24 OEPCR CBDclos ctl	6.2	1	1	10×H /3	18.8	30
3/15 J23100+scFv C3-P2	9.7	1	1	10×H /3	15.3	30

3/25

Restriction Enzyme Digestion

Kim

Sample	DNA/(µl)	EcoR1/(µl)	Xba1/(µl)	Spe1/(µl)	Pst1/(µl)	Buffer/(µl)	BSA/(µl)	MilliQ/(µl)	Total/(µl)
3/24 OEPCR BAN-CBDcex ctl	10	1	0	0	1	10×H /3	0	15	30
3/24 OEPCR BAN-CBDclos ctl	10	1	0	0	1	10×H /3	0	15	30

3/25

Ligation

Yamada

Vector/(µl)	Insert/(µl)	Ligation High/(µl)	Total/(µl)
3/24 EPcut pSB1C3 /8	3/24 EPcut BAN-CBDcex.ctl /3.7	11.7	23.4
3/24 EPcut pSB1C3 /7	3/24 EPcut BAN-CBDclos.ctl /4.7	11.7	23.4

3/25

Transformation

Yamada

Sample Name	Sample Volume (µl)	Competent Cells/(µl)	Medium
pSB1C3+BAN-CBDcex.ctl 3/25 ligation	-	20	LB(CP)
pSB1C3+BAN-CBDclos.ctl 3/25 ligation	-	20	LB(CP)
pSB1C3+CBDcex 3/24 ligation	-	20	LB(CP)
pSB1C3+CBDclos 3/24 ligation	-	20	LB(CP)
pSB1C3+scFv.ctl 3/24 ligation	-	20	LB(CP)

3/28

Colony PCR (typeA)

Yamada

Templates/(µl)	Primers/(µl)	MilliQ/(µl)	PreMix/(µl)	Total/(µl)
BANcex ctl col.1	VF 1.25 VR 1.25	10	12.5	25
BAncex ctl col.2	VF 1.25 VR 1.25	10	12.5	25
BANclos ctl col.1	VF 1.25 VR 1.25	10	12.5	25
BANclos ctl col.2	VF 1.25 VR 1.25	10	12.5	25
CBDcex col.1	VF 1.25 VR 1.25	10	12.5	25
CBDcex col.2	VF 1.25 VR 1.25	10	12.5	25
CBDclos col,1	VF 1.25 VR 1.25	10	12.5	25
CBDclos col.2	VF 1.25 VR 1.25	10	12.5
scFv ctl col.1
scFv ctl col.2

3/28

Electrophoresis

Lane	sample/(µl)	Length(bp)
1	1kb ladder(10)
2	100bb ladder(10)
3	BANcex ctl col.1
4	BANcex ctl col.2
5	BANclos ctl col.1
6	BANclos ctl col.2
7	CBDcex col.1
8	CBDcex col.2
9	CBDclos col.1
10	CBDclos col.2
11	scFv ctl col.1
12	scFv ctl col.2

7/21

Liquid Culture

Yamamoto

Name	medium
BclA-His-scFv glycerol stock1	LB(CP)
BclA-His-scFv glycerol stock2	LB(CP)
BclA-His-CBDcex glycerol stock1	LB(CP)
BclA-His-CBDcex glycerol stock2	LB(CP)
BclA-His-CBDclos glycerol stock1	LB(CP)
BclA-His-CBDclos glycerol stock2	LB(CP)
INP-His-CBDcex glycerol stock1	LB(CP)
INP-His-CBDcex glycerol stock2	LB(CP)
INP-His-CBDclos glycerol stock1	LB(CP)
INP-His-CBDclos glycerol stock2	LB(CP)

7/21

Transformation

Yamamoto

Sample Name	Sample Volume (µl)	Competent Cells/(µl)	Medium
2/8 miniprep INP-His-scFv	1	DH5α /20	LB(CP)
2/8 miniprep BclA-His-scFv14	1	DH5α /20	LB(CP)
2/24 miniprep BclA-His-CBDcex	1	DH5α /20	LB(CP)
2/24 miniprep BclA-His-CBDclos	1	DH5α /20	LB(CP)
2/25 miniprep INP-His-CBDcex	1	DH5α /20	LB(CP)
2/25 miniprep INP-His-CBDclos	1	DH5α /20	LB(CP)

7/22

Transformation

Yamamoto

Sample Name	Sample Volume (µl)	Competent Cells/(µl)	Medium
2/4 parts awakening E0040	1	DH5α /20	LB(AMP)

7/22

Liquid Culture

Yamamoto

Name	medium
7/21 transformation BclA-His-scFv colony1	LB(CP)
7/21 transformation BclA-His-scFv colony2	LB(CP)
7/21 transformation INP-His-scFv colony1	LB(CP)
7/21 transformation INP-His-scFv colony2	LB(CP)
7/21 transformation BclA-His-CBDcex colony1	LB(CP)
7/21 transformation BclA-His-CBDcex colony2	LB(CP)
7/21 transformation BclA-His-CBDclos colony1	LB(CP)
7/21 transformation BclA-His-CBDclos colony2	LB(CP)
7/21 transformation INP-His-CBDcex colony1	LB(CP)
7/21 transformation INP-His-CBDcex colony2	LB(CP)
7/21 transformation INP-His-CBDclos colony1	LB(CP)
7/21 transformation INP-His-CBDclos colony 2	LB(CP)

7/25

Miniprep

Yamamoto

DNA	Concentration/(µg/ml)	260/280	260/230
7/22 liquid culture BclA-His-scFv1	183.5	1.85	2.05
7/22 liquid culture BclA-His-scFv2	379.0	1.86	2.20
7/22 liquid culture INP-His-scFv1	189.6	1.85	2.21
7/22 liquid culture INP-His-scFv2	375.3	1.85	2.21
7/22 liquid culture BclA-His-CBDcex1	91.9	1.87	1.90
7/22 liquid culture BclA-His-CBDcex2	187.1	1.87	1.98
7/22 liquid culture BclA-His-CBDclos1	41.2	1.88	2.21
7/22 liquid culture BclA-His-CBDclos2	109.4	1.94	2.24
7/22 liquid culture INP-His-CBDcex1	71.0	1.73	1.19
7/22 liquid culture INP-His-CBDcex2	55.5	1.89	2.23
7/22 liquid culture INP-His-CBDclos1	88.6	1.82	1.61
7/22 liquid culture INP-His-CBDclos2	75.8	1.86	2.17

7/25

Restriction Enzyme Digestion

Yamamoto

Sample	DNA/(µl)	Xba1/(µl)	Spe1/(µl)	Pst1/(µl)	Buffer/(µl)	BSA/(µl)	MilliQ/(µl)	Total/(µl)
2/25 miniprep BclA-His-scFv1	10.9	0	0	0	10×L /3	0	14.1	30
2/25 miniprep BclA-His-scFv2	5.3	1	0	1	10×H /3	3.0	16.7	30
2/25 miniprep INP-His-scFv1	10.5	0	0	0	10×L /3	0	14.5	30
2/25 miniprep BclA-His-CBDcex2	10.7	0	1	1	10×M /3	0	14.3	30
2/25 miniprep BclA-His-CBDclos2	18.3	0	1	1	10×M /3	0	6.7	30
2/25 miniprep INP-His-CBDcex1	25	0	1	1	10×M /3	0	0	30
2/25 miniprep INP-His-CBDclos1	22.6	0	1	1	10×M /3	0	2.4	30

Samples of "2/25 miniprep BclA-His-scFv1" and "2/25 miniprep INP-His-scFv1" include 2µl of DraⅡ(Eco0109Ⅰ) (DraⅡis restriction enzyme).

7/26

Gel Extraction

Yamamoto

Lane	Restriction Enzyme Digestion Product	Volume/(µl)
1	1kb ladder	5
2	7/25 restriction enzyme digestion BclA-His-scFv1 DraⅡcut	30
3	7/25 restriction enzyme digestion BclA-His-scFv2 XPcut	30
4	7/25 restriction enzyme digestion INP-His-scFv1 DraⅡcut	30
5	7/25 restriction enzyme digestion BclA-His-CBDcex2 SPcut	30
6	7/25 restriction enzyme digestion BclA-His-CBDclos2 SPcut	30
7	7/25 restriction enzyme digestion INP-His-CBDcex1 SPcut	30
8	7/25 restriction enzyme digestion INP-His-CBDclos1 SPcut	30

7/26

Gel Extraction(Measurement)

Yamamoto

Sample/(µl)	Concentration(µg/µl)	260/280	260/230
7/26 Gel Extraction BclA-His-scFv2 XPcut	3.8	5.14	0.01
7/26 Gel Extraction BclA-His-CBDcex2 SPcut	10.4	3.24	0.01
7/26 Gel Extraction BclA-His-CBDclos2 SPcut	6.0	2.67	0.01
7/26 Gel Extraction INP-His-CBDcex1 SPcut	8.1	2.03	0.02
7/26 Gel Extraction INP-His-CBDclos1 SPcut	14.6	2.57	0.02

7/26

Restriction Enzyme Digestion

Yamamoto

Sample	DNA/(µl)	EcoR1/(µl)	Xba1/(µl)	Spe1/(µl)	Pst1/(µl)	Buffer/(µl)	BSA/(µl)	MilliQ/(µl)	Total/(µl)
7/25 miniprep BclA-His-scFv1	10.9	0	0	0	0	10×L /3	0	14.1	30
7/25 miniprep BclA-His-scFv1	3.6	1	0	0	1	10×H /3	0	3.4	10
7/25 miniprep BclA-His-scFv1	3.6	0	0	0	0	0	0	6.4	10
7/25 miniprep BclA-His-scFv2	5.3	0	1	0	1	10×H /3	3.0	16.7	30
/25 miniprep INP-His-scFv1	10.5	0	0	0	0	10×L /3	0	14.5	30
/25 miniprep INP-His-scFv1	3.5	0	0	0	1	10×H /1	0	3.5	10
/25 miniprep INP-His-scFv1	3.5	0	0	0	0	0	0	6.5	10
7/25 miniprep BclA-His-CBDcex2	10.7	0	0	1	1	10×M /3	0	14.3	30
7/25 miniprep BclA-His-CBDclos2	18.3	0	0	1	1	10×M /3	0	6.7	30
7/25 miniprep INP-His-CBDcex2	25	0	0	1	1	10×M /3	0	0	30
7/25 miniprep INP-His-CBDclos2	25	0	0	1	1	10×M /3	0	0	30

Samples of "2/25 miniprep BclA-His-scFv(10.9µl)" and "2/25 miniprep INP-His-scFv1(10.5µl)" include 2µl of DraⅡ(Eco0109Ⅰ) (DraⅡis restriction enzyme).

7/26

Gel Extraction

Yamamoto

Lane	Restriction Enzyme Digestion Product	Volume/(µl)
1	1kb ladder	5
2	2/26Restriction Enzyme Digestion BclA-His-scFv1 DraⅡcut	30
3	2/26Restriction Enzyme Digestion BclA-His-scFv1 XPcut	10
4	2/26Restriction Enzyme Digestion BclA-His-scFv1	10
5	2/26Restriction Enzyme Digestion INP-His-scFv1 DraⅡcut	30
6	2/26Restriction Enzyme Digestion INP-His-scFv1EPcut	10
7	2/26Restriction Enzyme Digestion INP-His-scFv1	10
-	-	-
1'	1kb ladder	5
2'	2/26Restriction Enzyme Digestion BclA-His-scFv2 XPcut	30
3'	2/26Restriction Enzyme Digestion BclA-His-CBDcex2 SPcut	30
4'	2/26Restriction Enzyme Digestion BclA-His-CBDclos2 SPcut	30
5'	2/26Restriction Enzyme Digestion INP-His-CBDcex2 SPcut	30
6'	2/26Restriction Enzyme Digestion INP-His-CBDclos2 SPcut	30

7/27

Ligation

Yamamoto

Insert DNA(7/27purification) /µ?	Vector DNA(7/25purification) /µ?	Ligation High /µ?	Total
-	BclA-scFv1 DraⅡcut /7.5	7.5	15
-	INP-scFv1 DraⅡcut /7.5	7.5	15
BclAscFv2 XPcut /7.16	BclA-CBDcex2 SPcut /0.34	7.5	15
BclAscFv2 XPcut /3.75	BclA-CBDclos2 SPcut /3.75	7.5	15
BclAscFv2 XPcut /1.88	INP-CBDcex2 SPcut /5.62	7.5	15
BclAscFv2 XPcut /5.0	INP-CBDclos2 SPcut /2.5	7.5	15

7/27

Transformation

Yamamoto

Sample Name	Sample Volume (µl)	Competent Cells/(µl)	Medium
BclAscFv1 DraⅡcut	2	DH5α /20	LB(CP)
INPscFv1 DraⅡcut	2	DH5α /20	LB(CP)
BclAscFv+BclACBDcex	2	DH5α /20	LB(CP)
BclAscFv+BclACBDclos	2	DH5α /20	LB(CP)
BclAscFv+INPCBDcex	2	DH5α /20	LB(CP)
BclAscfv+INPCBDclos	2	DH5α /20	LB(CP)

7/27

Gel Extraction(Measurement)

Yamamoto

Sample/(µl)	Concentration(µg/µl)	260/280	260/230
7/27 Gel Extraction BclA-His-scFv1 DraⅡcut	4.8	-7.11	1.04
7/27 Gel Extraction INP-His-scFv1 DraⅡcut	40.4	1.32	1.00
7/27 Gel Extraction BclA-His-scFv2 XPcut	9.3	-4.24	0.02
7/27 Gel Extraction BclA-His-CBDcex2 SPcut	174.5	1.32	0.84
7/27 Gel Extraction BclA-His-CBDclos2 SPcut	9.7	5.84	0.19
7/27 Gel Extraction INP-His-CBDcex2 SPcut	3.7	-2.82	0.14
7/27 Gel Extraction INP-His-CBDclos2 SPcut	22.6	2.51	0.21

7/28

Restriction Enzyme Digestion

Yamamoto

DNA /µ?	Xba1 /µ?	Spe1 /µ?	Pst1 /µ?	DraⅡ /µ?	Buffer /µ?	BSA /µ?	MilliQ /µ?	Total /µ?
BclAscFv2 /5.3	0	0	0	1	10×L /3	0	20.7	30
BclAscFv2 /5.3	1	0	1	0	10×H /3	3	16.7	30
INPscFv2 /5.3	0	0	0	1	10×L /3	0	20.7	30
BclAcex2 /10.7	0	1	1	0	10×M /3	0	14.3	30
BclAclos1 /25	0	1	1	0	10×M /3	0	0	30
INPcex1,2 /25	0	1	1	0	10×M /3	0	0	30
INPclos1,2 /25	0	1	1	0	10×M /3	0	0	30

7/28

Gel Extraction

Kaneko

Lane	Restriction Enzyme Digestion Product	Volume/(µl)
	7/28 BclA-scFv2 XPcut

0.5µg/µl
260/280 2.36
260/230 1.15

7/28

Transformation

Kaneko

Sample Name	Sample Volume (µl)	Competent Cells/(µl)	Medium
2/22 scFv colony2	2	DH5α /20

7/28

Ligation

Yamamoto

Insert DNA /(µl) Vector DNA /(µl) Ligation high /(µl) Total /(µl)
- 7/28 Restriction Enzyme BclA-scFv DraⅡcut /7.5 7.5 15
- 7/28 Restriction Enzyme INP-scFv DraⅡcut /7.5 7.5 1.5

7/28

Transformation

Yamada

Sample Name	Sample Volume (µl)	Competent Cells/(µl)	Medium
7/28 ligation BclA-scFv control	2	DH5α /20	LB(CP)
7/28 ligation INP-scFv control	2	DH5α /20	LB(CP)
2/22 miniprep BAN-scFv		DH5α /20

7/28

Liquid Culture

Yamamoto

Name	medium
BclA-scFv glycerol stock	LB(CP)
7/21 transformation INP-scFv colony1	LB(CP)
7/21 transformation BclA-CBDcex colony1	LB(CP)
7/21 transformation BclA-CBDclos colony1	LB(CP)
7/21 transformation INP-CBDcex colony1	LB(CP)
7/21 transformation INP-CBDclos colony1	LB(CP)
7/21 transformation	LB(CP)

8/1

Colony PCR (typeA)

Yamamoto

Templates/(µl)	Primers/(µl)	MilliQ/(µl)	PreMix/(µl)	Total/(µl)
7/28 transformaton BclA control colony4	VF/1.25 VR/1.25	10	12.5	25
7/28 transformaton BclA control colony5	VF/1.25 VR/1.25	10	12.5	25
7/28 transformaton BclA control colony6	VF/1.25 VR/1.25	10	12.5	25
7/28 transformaton INP control colony4	VF/1.25 VR/1.25	10	12.5	25
7/28 transformaton INP control colony5	VF/1.25 VR/1.25	10	12.5	25
7/28 transformaton INP control colony6	VF/1.25 VR/1.25	10	12.5

8/1

PCR (Steps)

Yamamoto

Steps	Temparature	Time	Cycle
Predenature	95℃	1min	1
Denature	95℃	15sec	30
Annealing	60℃	15sec	30
Extension	72℃	10sec	30
Final Elogation	72℃	5min	1
Final Holding		-	1

8/1

Miniprep

Yamamoto

DNA	Concentration/(µg/ml)	260/280	260/230
7/28 liquid culture BclA-scFv	239.1	1.87	1.78
7/28 liquid culture INP-scFv colony1	263.4	1.86	1.99
7/28 liquid culture E0040 colony1	98.8	1.84	2.15
7/28 liquid culture BclA-CBDcex colony1	109.9	1.82	1.37
7/28 liquid culture BclA-CBDclos colony1	94.1	1.89	1.93
7/28 liquid culture INP-CBDcex colony1	111.4	1.82	1.39
7/28 liquid culture INP-CBDclos colony1	185.1	1.81

8/1

Electrophoresis

Iguchi

Lane	sample/(µl)	Length(bp)
1	8/1 colonyPCR BclA control colony4 /10	672
2	8/1 colonyPCR BclA control colony5 /10	672
3	8/1 colonyPCR BclA control colony6 /10	672
4	8/1 colonyPCR INP control colony4 /10	1563
5	8/1 colonyPCR INP control colony5 /10	1563
6	8/1 colonyPCR INP control colony6 /10
7	1kb ladder /5

8/1

Restriction Enzyme Digestion

Iguchi

Sample	DNA/(µl)	Xba1/(µl)	Spe1/(µl)	Pst1/(µl)	Buffer/(µl)	BSA/(µl)	MilliQ/(µl)	Total/(µl)
8/1 miniprep BclA-scFv	8.36	1	0	1	10×H 3	3	13.64	30
8/1 miniprep BclA-CBDcex colony1	18.2	0	1	1	10×H /3	0	6.8	30
8/1 miniprep BclA-CBDclos colony1	21.3	0	1	1	10×M /3	0	3.7	30
8/1 miniprep INP-CBDcex colony1	18.0	0	1	1	10×M /3	0	7.0	30
8/1 miniprep INP-CBDclos colony1	10.8	0	1	1	10×M /3	0	16.2

8/2

Gel Extraction

Yamamoto

Lane	Restriction Enzyme Digestion Product	Volume/(µl)
1	BclA-scFv XPcut	30
2	BclA-CBDcex SPcut	30
3	BclA-CBDclos SPcut	30
4	INP-CBDcex SPcut	30
5	INP-CBDclos SPcut	30
6	1kb ladder	30

8/2

Gel Extraction

Yamamoto

Sample Concentration ng/µl 260/280 260/230
8/2 Electrophoresis BclA-scFv XPcut 1.0 23.20 0.12
8/2 Electrophoresis BclA-CBDcex SPcut 10.8 1.89 1.35
8/2 Electrophoresis BclA-CBDclos SPcut 18.0 1.92 1.88
8/2 Electrophoresis INP-CBDcex SPcut 25.4 1.93 1.81
8/2 Electrophoresis INP-CBDclos SPcut 60.9

8/2

Ligation

Yamamoto

Vector/(µl)	Insert/(µl)	Ligation High/(µl)	Total/(µl)
8/2 purification BclA-CBDcex SPcut /3.5	8/2 purification BclA-scFv XPcut /4	7.5	15
8/2 purification BclA-CBDclos SPcut /3.5	8/2 purification BclA-scFv XPcut /4	7.5	15
8/2 purification INP-CBDcex SPcut /3.5	8/2 purification BclA-scFv XPcut /4	7.5	15
8/2 purification INP-CBDclos SPcut /3.5	8/2 purification BclA-scFv XPcut /4	7.5

8/2

Transformation

Yamamoto, Yamada

Sample Name	Sample Volume (µl)	Competent Cells/(µl)	Medium
8/2 ligation BclA-scFv+BclA-CBDcex	2	DH5α /20	LB(CP)
8/2 ligation BclA-scFv+BclA-CBDclos	2	DH5α /20	LB(CP)
8/2 ligation BclA-scFv+INP-CBDcex	2	DH5α /20	LB(CP)
8/2 ligation BclA-scFv+INP-CBDclos	2	DH5α /20

8/2

Liquid Culture

Yamada

Name	medium
BclA-CBDcex glycerol stock	LB(CP)
BclA-CBDclos glycerol stock	LB(CP)
INP-CBDcex glycerol stock	LB(CP)
INP-CBDclos glycerol stock

8/3

Liquid Culture

Yamada

Name	medium
7/28 transformation BclA control colony5	LB(CP)
7/28 transformation BclA control colony6	LB(CP)
7/28 transformation INP control colony4	LB(CP)
7/28 transformation INP control colony5
7/28 transformation INP control colony6

8/3

Restriction Enzyme Digestion

Yamamoto

Sample	DNA/(µl)	Xba1/(µl)	Spe1/(µl)	Pst1/(µl)	Buffer/(µl)	BSA/(µl)	MilliQ/(µl)	Total/(µl)
8/1 miniprep BclA-scFv	8.36	1	0	1	10×H /3	3	13.64	30
8/1 miniprep BclA-CBDcex	18.2	0	1	1	10×H /3	0	6.8	30
8/1 miniprep BclA-CBDclos	21.3	0	1	1	10×M /3	0	3.7	30
8/1 miniprep INP-CBDcex	18.0	0	1	1	10×M /3	0	7.0	30
8/1 miniprep INP-CBDclos	10.8	0	1	1	10×M /3	0	16.2

8/3

Ligation

Yamamoto

Vector/(µl)	Insert/(µl)	Ligation High/(µl)	Total/(µl)
8/1 Restriction Enzyme Digestion BclA-CBDcex /3.5	8/1 Restriction Enzyme Digestion BclA-scFv /4	7.5	15
8/1 Restriction Enzyme Digestion BclA-CBDclos /3.5	8/1 Restriction Enzyme Digestion BclA-scFv /4	7.5	15
8/1 Restriction Enzyme Digestion INP-CBDcex /3.5	8/1 Restriction Enzyme Digestion BclA-scFv /4	7.5	15
8/1 Restriction Enzyme Digestion INP-CBDclos /3.5	8/1 Restriction Enzyme Digestion BclA-scFv /4	7.5	15

8/3

Transformation

Yamada

Sample Name	Sample Volume (µl)	Competent Cells/(µl)	Medium
8/3 ligation BclA-scFv+BclA-CBDcex	15	DH5α /20	LB(CP)
8/3 ligation BclA-scFv+BclA-CBDclos	15	DH5α /20	LB(CP)
8/3 ligation BclA-scFv+INP-CBDcex	15	DH5α /20	LB(CP)
8/3 ligation BclA-scFv+INP-CBDclos	15	DH5α /20

8/4

Colony PCR (typeA)

Yamamoto

Templates/(µl)	Primers/(µl)	MilliQ/(µl)	PreMix/(µl)	Total/(µl)
8/3 transformation BclA-scFv+BclA-CBDcex colony1	VF 1.25 VR1.25	10	12.5	25
8/3 transformation BclA-scFv+BclA-CBDcex colony2	VF 1.25 VR1.25	10	12.5	25
8/3 transformation BclA-scFv+BclA-CBDcex colony3	VF 1.25 VR1.25	10	12.5	25
8/3 transformation BclA-scFv+BclA-CBDcex colony4	VF 1.25 VR1.25	10	12.5	25
8/3 transformation BclA-scFv+BclA-CBDclos colony1	VF 1.25 VR1.25	10	12.5	25
8/3 transformation BclA-scFv+BclA-CBDclos colony2	VF 1.25 VR1.25	10	12.5	25
8/3 transformation BclA-scFv+BclA-CBDclos colony3	VF 1.25 VR1.25	10	12.5	25
8/3 transformation BclA-scFv+BclA-CBDclos colony4	VF 1.25 VR1.25	10	12.5	25
8/3 transformation BclA-scFv+INP-CBDcex colony1	VF 1.25 VR1.25	10	12.5	25
8/3 transformation BclA-scFv+INP-CBDcex colony2	VF 1.25 VR1.25	10	12.5	25
8/3 transformation BclA-scFv+INP-CBDcex colony3	VF 1.25 VR1.25	10	12.5	25
8/3 transformation BclA-scFv+INP-CBDcex colony4	VF 1.25 VR1.25	10	12.5	25
8/3 transformation BclA-scFv+INP-CBDclos colony1	VF 1.25 VR1.25	10	12.5	25
8/3 transformation BclA-scFv+INP-CBDclos colony2	VF 1.25 VR1.25	10	12.5	25
8/3 transformation BclA-scFv+INP-CBDclos colony3	VF 1.25 VR1.25	10	12.5	25
8/3 transformation BclA-scFv+INP-CBDclos colony4	VF 1.25 VR1.25	10	12.5	25

8/4

PCR (Steps)

Yamamoto

Steps	Temparature	Time	Cycle
Predenature	95℃	1min	1
Denature	95℃	15sec	30
Annealing	60℃	15sec	30
Extension	72℃	10sec	30
Final Elongation	72℃	5min	1
Final Holding	4℃	-	-

8/4

Miniprep

Yamada

DNA	Concentration/(µg/ml)	260/280	260/230
8/3 liquid culture BclA control colony5	137.2	1.77	1.03
8/3 liquid culture BclA control colony6	117.6	1.87	1.54
8/3 liquid culture INP control4	165.2	1.87	1.68
8/3 liquid culture INP control5	150.6	1.90	1.81
8/3 liquid culture INP control6	147.4	1.79

8/4

Sequence

Yamamoto, Yamada

No primer(10µM) MilliQ Total
1 / 2 BclA_5_VF(2.18µl) / BclA_5_VR(2.18µl) 2µl / 2µl 5.82µl / 5.82µl 10µl
3 / 4 BclA_6_VF(2.55µl) / BclA_6_VR(2.55µl) 2µl / 2µl 5.45µl / 5.45µl 10µl
5 / 6 INP_4_VF(1.81µl) / INP_4_VR(1.81µl) 2µl / 2µl 6.19µl / 6.19µl 10µl
7 / 8 INP_5_VF(1.99µl) / INP_5_VR(1.99µl) 2µl / 2µl 6.01µl / 6.01µl 10µl

8/4

Electrophoresis

Yamada

Lane	sample/(µl)	Length(bp)
1	8/4 colony PCR BclA-scFv+BclA-CBDcex colony1 /10	1716
2	8/4 colony PCR BclA-scFv+BclA-CBDcex colony2 /10	1716
3	8/4 colony PCR BclA-scFv+BclA-CBDcex colony3 /10	1716
4	8/4 colony PCR BclA-scFv+BclA-CBDcex colony4 /10	1716
5	8/4 colony PCR BclA-scFv+BclA-CBDclos colony1 /10	1716
6	8/4 colony PCR BclA-scFv+BclA-CBDclos colony2 /10	1716
7	8/4 colony PCR BclA-scFv+BclA-CBDclos colony3 /10	1716
8	8/4 colony PCR BclA-scFv+BclA-CBDclos colony4 /10	1716
9	100bp ladder /5	-
10	1kb ladder /5	-
11	-	-
12	-	-

8/4

Electrophoresis

Yamada

Lane	sample/(µl)	Length(bp)
1	8/4 colony PCR BclA-scFv+INP-CBDcex colony1 /10	2606
2	8/4 colony PCR BclA-scFv+INP-CBDcex colony2 /10	2606
3	8/4 colony PCR BclA-scFv+INP-CBDcex colony3 /10	2606
4	8/4 colony PCR BclA-scFv+INP-CBDcex colony4 /10	2606
5	8/4 colony PCR BclA-scFv+INP-CBDclos colony1 /10	2606
6	8/4 colony PCR BclA-scFv+INP-CBDclos colony2 /10	2606
7	8/4 colony PCR BclA-scFv+INP-CBDclos colony3 /10	2606
8	8/4 colony PCR BclA-scFv+INP-CBDclos colony4 /10	2606
9	100bp ladder /5	-
10	1kb ladder /5
11	-	-
12	-	-

8/5

PartsAwaking

Yamamoto

Description	PartName	Backbone	Well	PlateYear	PlateNumber	MilliQ(µl)
RFP	J23105	J61002	17N	2016	4

8/5

Transformation

Yamamoto

Sample Name	Sample Volume (µl)	Competent Cells/(µl)	Medium
8/5 Parts awakening J23105	1µl	DH5α /20

8/9

Restriction Enzyme Digestion

Iguchi, Yamada

Sample	DNA/(µl)	Xba1/(µl)	Spe1/(µl)	Pst1/(µl)	Buffer/(µl)	BSA/(µl)	MilliQ/(µl)	Total/(µl)
8/1 miniprep BclA-scFv	8.36	1	0	1	10×H /3	3	13.69	30
8/1 and 7/25 miniprep BclA-CBDcex	18.	0	1	1	10×H /3	0	6.8	30
2/25 miniprep BclA-CBDclos	21.3	0	1	1	10×M /3	0	3.7	30
2/25 and 7/25 minniprep INP-CBDcex	18.0	0	1	1	10×M /3	0	7.0	30
8/1 miniprep INP-CBDclos	10.8	0	1	1	10×M /3	0	16.2	30

8/9

Liquid Culture

Yamada

Name	medium
8/1 transformation J23105

8/9

Miniprep

Yamada

DNA	Concentration/(µg/ml)	260/280	260/230
8/3 liquid culture BclA-CBDccex	225.8	1.74	1.63
8/3 liquid culture BclA-CBDclos	102.3	1.80	1.66
8/3 liquid culture INP-CBDcex	112.4	1.86	1.77
8/3 liquid culture INP-CBDclos	42.8	1.76	0.78

8/9

Electrophoresis

Yamada

Lane	sample/(µl)	Length(bp)

8/10

Gel Extraction

Yamada

Lane	Restriction Enzyme Digestion Product	Volume/(µl)
A1	1kb ladder	10
A2	BclA-scFv 8/9XPcut	30
A3	BclA-CBDcex 8/9 SPcut	30
A4	BclA-CBDclos 8/9 SPcu	30
-	-	-
B1	1kb ladder	10
B2	INP-CBDcex 8/9 SPcut	30
B3	INP-CBDclos 8/9 SPcut

8/10

Gel Extraction(Measurement)

Yamada

Sample/(µl)	Concentration(µg/µl)	260/280	260/230
BclA-scFv 8/9XPcut	2.6	1.13	0.58
BclA-CBDcex 8/9 SPcut	6.3	1.54	0.97
BclA-CBDclos 8/9 SPcut	4.7	1.49	0.48
INP-CBDcex 8/9 SPcut	4.4	1.39	0.67
INP-CBDclos 8/9 SPcut	3.5	1.29

8/10

Miniprep

Notsu

DNA	Concentration/(µg/ml)	260/280	260/230
8/9 liquid culture J23105 (AMP) RFP colony3	178.8	1.98	1.92

8/12

Liquid Culture

Iguchi

Name	medium
8/2 liquid culture INP-CBDclos	LB(CP)
BclA-scFv glycerol stock 1	LB(CP)
BclA-scFv glycerol stock 2	LB(CP)
BclA-scFv glycerol stock 3	LB(CP)
BclA-scFv glycerol stock 4	LB(CP)

8/18

Miniprep

Notsu

DNA	Concentration/(µg/ml)	260/280	260/230
8/12 liquid culture INP-CBDclos	45.3	1.82	1.31
8/12 liquid culture BclA-scFv glycerol stock 1	101.6	1.59	0.71
8/12 liquid culture BclA-scFv glycerol stock 2	100.7	1.67	0.78
8/12 liquid culture BclA-scFv glycerol stock 3	77.8	1.60	0.70
8/12 liquid culture BclA-scFv glycerol stock 4	41.0	1.87	1.27

8/20

Liquid Culture

Ohara, Isoda

Name	medium
BclA-CBDcex	LB(CP)
BclA-CBDclos	LB(CP)
INP-CBDcex	LB(CP)
INP-CBDclos

8/21

Liquid Culture

Ohara

Name	medium
BclA-scFv [1](glisto）	LB(CP)
BclA-scFv [2]	LB(CP)
BclA-scFv [3]	LB(CP)
BclA-scFv [4]

8/22

Electrophoresis

Sassa

Lane	sample/(µl)	Length(bp)
8/22 Restraction Ensyme Digestion 1.1kb ladder	5
8/22 Restraction Ensyme Digestion Bcla-cex	25	2520
8/22 Restraction Ensyme Digestion Bcla-clos	25	2520
8/22 Restraction Ensyme Digestion INP-cex(s,pcut)	25	3500
8/22 Restraction Ensyme Digestion INP-clos	25	3500
8/22 Restraction Ensyme Digestion INP-cex	25	2541
8/22 Restraction Ensyme Digestion J23105 (AMP) RFP 8/21 old	25	950
8/22 Restraction Ensyme Digestion Liquid culture Bcla-scfv glisto 4	25	932
8/22 Restraction Ensyme Digestion Bcla-scfv glisto 2	25	932
100ladder	5

8/22

Restriction Enzyme Digestion

Ohara

Sample	DNA/(µl)	EcoR1/(µl)	Xba1/(µl)	Spe1/(µl)	Pst1/(µl)	Buffer/(µl)	BSA/(µl)	MilliQ/(µl)	Total/(µl)
8/12 Liquid cultureBclA-scFv glisto [4]	38	0	1	0	1	5	5		50
8/12 Liquid cultureBclA-scFv glisto [2]	38	0	1	0	1	5	5	0	50

8/22

Liquid Culture

Yoshimoto, Ohara, Isoda

Name	medium
BclA control	LB(CP)
INP control

8/22

Transformation

Ohara

Sample Name	Sample Volume (µl)	Competent Cells/(µl)	Medium
2/9 J23105 RFP		DH5α /20

8/22

CBD 1st

Yoshimoto

sample	before	after	wash
BclAclos+	0.208/0.206/0.206	0.131/0.135/0.14	0.111/0.112/0.109
INPclos+	0.183/0.186/0.184	0.239/0.23/0.22	0.158/0.155/0.169

8/22

CBD 2nd

Yoshimoto

sample	before	after	wash
BclAclos+	0.208/0.205/0.204	0.078/0.074/0.075	0.056/0.056/0.039
INPclos+	0.183/0.181/0.187	0.093/0.097/0.099	0.067/0.073/0.069

8/23

CBD 3rd

Yamada

sample	before	after	wash
BclAcex+ (15min)	0.247/0.247/0.248	0.221/0.224/0.218	0.085/0.075/0.073
BclAcex+ (30min)	0.247/0.247/0.248	0.243/0.229/0.228	0.034/0.048/0.038
BclAcex+ (60min)	0.247/0.247/0.248	0.224/0.221/0.217	0.065/0.064/0.065
BclAcex- (15min)	0.247/0.247/0.248	0.257/0.253/0.258
BclAcex- (30min)	0.247/0.247/0.248	0.236/0.230/0.235
BclAcex- (60min)	0.247/0.247/0.248	0.249/0.248/0.247
cellulose only (60min)		0.021/0.021/0.023

8/23

CBD 4th

Yamada

sample	before	after	wash
BclAcex+ (2ml tube)	0.227/0.226/0.225	0.108/0.108/0.108	0.076/0.074/0.081
BclActl+ (2ml tube)	0.186/0.203/0.189	0.082/0.081/0.082	0.055/0.046/0.044
BclAcex- (2ml tube)	0.235/0.237/0.233	0.261/0.262/0.254
BclAcex+ (1.5ml tube)	0.237/0.226/0.236	0.159/0.157/0.158	0.066/0.063/0.065
BclActl+ (1.5ml tube)	0.177/0.173/0.179	0.112/0.109/0.113	0.080/0.076/0.095
BclAcex- (1.5ml tube)	0.240/0.235/0.238	0.255/0.254/0.253

8/24

Liquid Culture

Sassa

Name	medium
8/5 transormation J2305

8/24

Liquid Culture

Ohara

Name	medium
8/24 BclA-scFv 1	LB(CP)
8/24 BclA-scFv 2	LB(CP)
8/24 BclA-scFv 3	LB(CP)
8/24 BclA-scFv 4

8/29

Miniprep

Sassa

DNA	Concentration/(µg/ml)	260/280	260/230
1. 8/23Liquidculture Bcla scFv	111.6	1.98	1.82
2. 8/23Liquidculture Bcla scFv	143.1	2.00	2.03
2'. 8/23Liquidculture Bcla scFv	141.6	2.04	2.10
3. 8/23Liquidculture Bcla scFv	145.2	2.07	2.01
8/23Liquidculture J23105	63.1	2.01	1.69
8/23Liquidculture J23105	68.1	2.00

8/24

CBD 5th

Isoda

sample	before	after	wash
INPcex+	0.195/0.191/0.192	0.103/0.102/0.109	0.059/0.051/0.055
INPcex-	0.190/0.191/0.205	0.186/0.187/0.186
INPclos+	0.187/0.187/0.188	0.121/0.122/0.126	0.051/0.058/0.059
INPclos-	0.196/0.195/0.191	0.206/0.204/0.202
INPctl+	0.172/0.163/0.162	0.116/0.177/0.124	0.066/0.063/0.062
INPctl-	0.171/0.175/0.165	0.167/0.182/0.171

8/24

CBD 6th

Isoda

sample	before	after	wash
INPcex+	0.184/0.184/0.189	0.104/0.106/0.106	0.044/0.044/0.056
INPcex-	0.186/0.190/0.184	0.182/0.181/0.184
INPclos+	0.184/0.189/0.185	0.097/0.098/0.099	0.037/0.037/0.038
INPclos-	0.187/0.192/0.190	0.185/0.184/0.189
INPctl+	0.173/0.184/0.180	0.095/0.099/0.095	0.045/0.046/0.046
INPctl-	0.185/0.184/0.183	0.175/0.175/0.175
BclAcex+	0.170/0.168/0.174	0.099/0.094/0.092	0.037/0.032/0.036
BclAcex-	0.186/0.189/0.171	0.164/0.167/0.164
cellulose only	0.006/0.005/0.006	0.013/0.010/0.006

8/25

CBD 7th

Yamada

sample	before	after	wash
BclAcex+	0.181/0.181/0.181	0.192/0.196/0.195	0/0/0
BclAcex-	0.149/0.150/0.151	0.221/0.213/0.211
BclActl+	0.169/0.165/0.167	0.154/0.156/0.157	0.012/0.013/0.011
BclActl-	0.151/0.153/0.154	0.187/0.178/0.173

8/26

PCR (Target)

Sassa

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	Buffer/(µl)	MilliQ/(µl)	Any Other/(µl)	Total/(µl)
8/10 miniprep J23105 /3	VF /1.25	VR /1.25	-	7	KAPA2GFAST /12.5	25
8/24 OD0.206 INP-CBDclos /3	VF /1.25	VR /1.25	-	7	KAPA2GFAST /12.5	25
8/24 OD0.202 INP-CBDcex	VF /1.25	VR /1.25	-	7	KAPA2GFAST /12.5	25

8/26

Electrophoresis

Yamamoto, Sassa

Lane	sample/(µl)	Length(bp)
1	1kb ladder /5
2	8/26 PCR INP-CBDcex VFVR /15
3	8/26 PCR INP-CBDclos VFVR /15

8/26

Miniprep

Sassa

DNA	Concentration/(µg/ml)	260/280	260/230
8/26 Gel Extraction Bcla cex	8.3	1.52	0.25
8/26 Gel Extraction Bcla clos	10.2	1.70	0.26
8/26 Gel Extraction INP cex	11.4	1.76	0.15
8/26 Gel Extraction INP clos	7.7	1.62	0.09
8/26 Gel Extraction Bcla scFv glisto	7.0	1.59	0.20
8/26 Gel Extraction Bcla scFv glisto	14.1	6.33

8/26

Miniprep

Yamamoto, Sassa

DNA	Concentration/(µg/ml)	260/280	260/230
8/26 Gel Extraction Bcla cex	8.3	1.52	0.25
8/26 Gel Extraction Bcla clos	10.2	1.70	0.26
8/26 Gel Extraction INP cex	11.4	1.76	0.15
8/26 Gel Extraction INP clos	7.7	1.62	0.09
8/26 Gel Extraction Bcla scFv glisto	7.0	1.59	0.20
8/26 Gel Extraction Bcla scFv glisto	14.1	6.33

8/26

CBD 8th

Yoshimoto

sample	before	after	wash
BclAcex+	0.183/0.185/0.188	0.116/0.115/0.114	0.086/0.086/0.089
BclActl+	0.116/0.115/0.114	0.096/0.092/0.087	0.09/0.079/0.084
cellulose only		0.037/0.035/0.037

8/26

CBD 9th

Ohara

sample	before	after	wash
BclAcex+	0.186/0.186/0.184	0.170/0.169/0.170	0.021/0.024/0.020
BclAcex-	0.154/0.149/0.149	0.142/0.143/0.141
BclActl+	0.194/0.189/0.189	0.167/0.164/0.159	0.010/0.009/0.008
BclActl-	0.162/0.168/0.169	0.159/0.162/0.165

8/29

Ligation

Sassa

Insert DNA	vector DNA	Ligation High	Total
8/26 Gel Extraction BclAscFv /3.4	8/26 Gel Extraction Bcla CBDcex /4.1	7.5	15
8/26 Gel Extraction BclAscFv /3.1	8/26 Gel Extraction Bcla CBD clos /4.4	7.5	15
8/26 Gel Extraction BclAscFvBclAscFv /3.8	8/26 Gel Extraction Bcla CBD cex /4.7	7.5	15
8/26 Gel Extraction BclAscFv /2.6	8/26 Gel Extraction Bcla CBD clos /4.9	7.5

8/29

Western Blotting

Ohara, Sassa

lane	sample
1	Marker
2	Sano VLP 0.5µl
3	Sano VLP 0.2µl
4	Sano VLP 0.1µl
5	Sano VLP 0.05µl
6	Sano VLP 0.02µl
7	Sano VLP 0.05µl

blotting CC
1'antibody time/buffer 1:3000 6ml 1h
2'antibody time/buffer 1:10000 10ml 1h
Blocking 1day
transfer 100mA 1h

8/29

PCR (Target)

Miyazaki

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	Buffer/(µl)	MilliQ/(µl)	Any Other/(µl)	Total/(µl)
BclA-CBDcex	VF /1.25	VR /1.25	-	10	KAPA2GFAST /12.5	25
BclA-CBDclos	VF /1.25	VR /1.25	-	10	KAPA2GFAST /12.5	25
INP-CBDcex	VF /1.25	VR /1.25	-	10	KAPA2GFAST /12.5	25
INP-CBDclos	VF /1.25	VR /1.25	-	10	KAPA2GFAST /12.5	25

8/29

ligation

Miyazaki

Insert DNA	Vector DNA	Ligation High	Total
BclAscFv XPcut 6.3	INPCBDcex SPcut 1.2	7.5	15
BclAscFv XPcut 6.4	INPCBDclos SPcut 1.1	7.5	15

8/29

Miniprep

Sassa, Miyazaki

DNA	Concentration/(µg/ml)	260/280	260/230
BclA scFv 1	8.4	1.63	0.11
BclA scFv 2	5.6	1.91	0.04
INP CBDcex	44.8	1.90	0.70
INP CBDclos	33.1	1.82	0.68
BclA cex	26.3	1.93	0.62
BclA clos	24.4	1.93	0.66
J23105 VF VR	33.2	1.88	0.24

9/5

Restriction Enzyme Digestion

Miyazaki

Sample	DNA/(µl)	EcoR1/(µl)	Xba1/(µl)	Spe1/(µl)	Pst1/(µl)	Buffer/(µl)	BSA/(µl)	MilliQ/(µl)	Total/(µl)
8/10 J23105 RFP miniprep	20		1	0	1	10×H 3	3	2

9/5

Transformation

Yamamoto

Sample Name	Sample Volume (µl)	Competent Cells/(µl)	Medium
8/29 Ligation Bclascfv-BclaCBDcex	2	CH5a/20	LB(CP)
〃Bclascfv-BclaCBDclos	2	〃	〃
〃Bclascfv-INPCBDcex	2	〃	〃
〃Bclascfv-INPCBDclos	2	〃

9/5

Electrophoresis

Yamamoto

Lane	sample/(µl)	Length(bp)
1	8/20 SPcut Bcla-CBDcex/25	2520
2	〃Bcla-CBDclos/25	〃
3	〃INP-CBDcex/25	〃
4	〃INP-CBDclos/25

9/5

Electrophoresis

Miyazaki

Lane	sample/(µl)	Length(bp)
1	100bp ladder/5	6×loading Dye -
2	8/10 miniprep 9/5 xpcut J23105RFP/30

9/5

Liquid Culture

Isoda

Name	medium
BclA-CBDcex /20ml	LB(CP)
BclA-control /20ml	LB(CP)

9/5

Gel Extraction

Sassa, Miyazaki

Lane	Restriction Enzyme Digestion Product	Volume/(µl)
9/5 electrophoresis 2	J23105 RFP XPcut backbone
9/5 electrophoresis 2	J23105 RFP XPcut insert
9/5 electrophoresis 1	BclAcex SPcut
9/5 electrophoresis 2	BclAclos SPcut
9/5 electrophoresis 3	INPcex SPcut
9/5 electrophoresis 4	INPclos SPcut

9/6

Colony PCR (typeA)

Yamamoto

Templates/(µl)	Primers/(µl)	MilliQ/(µl)	PreMix/(µl)	Total/(µl)
Bclascfv+BclaCBDcex colony1	vf1.25 vr1.25	10	KAPA2GFAST 12.5	25
〃colony2	〃	〃	〃	〃
〃colony3	〃	〃	〃	〃
〃colony4	〃	〃	〃	〃
Bclascfv+BclaCBDclos colony1	〃	〃	〃	〃
〃colony2	〃	〃	〃	〃
〃colony3	〃	〃	〃	〃
〃colony4	〃	〃	〃	〃
Bclascfv+INPCBDcex colony1	〃	〃	〃	〃
〃colony2	〃	〃	〃	〃
〃colony3	〃	〃	〃	〃
〃colony4	〃	〃	〃	〃
Bclascfv+INPCBDclos colony1	〃	〃	〃	〃
〃colony2	〃	〃	〃	〃
〃colony3	〃	〃	〃	onaji
〃colony4	〃	〃	〃	〃

9/6

Ligation

Yamamoto

Insert DNA(µl)	Vector DNA/(µl)	Ligation High/(µl)	Total/(µl)
9/5 Gel Extraction BclA cex SPcut/7	9/5 Gel Extraction J23105 xpcut 36/0.5	7.5	15
9/5 Gel Extraction BclA clos SPcut/7	9/5 Gel Extraction J23105 xpcut 36/0.5	7.5	15
9/5 Gel Extraction INP cex SPcut/7	9/5 Gel Extraction J23105 xpcut 36/0.5	7.5	15
9/5 Gel Extraction INP clos SPcut/7	9/5 Gel Extraction J23105 xpcut 36/0.5	7.5	15

9/6

Electrophoresis

Lane	sample/(µl)	Length(bp)
1	1kb ladder/5	0
2	9/6 PCR scfv+Bclacex colony1/15	0
3	〃2/15	0
4	〃3/15	0
5	〃4/15	0
6	9/6 PCR scfv+Bclaclos colony1	0
7	〃2	0
8	〃3	0
9	〃4	0
10	9/6 PCR scfv+INPcex colony1	0
11	〃2	0
12	〃3	0
13	〃4	0
14	9/6 PCR scfv+INPclos colony1	0
15	〃2	0
16	〃3	0
17	〃4

9/6

Western Blotting

Ohara, Sassa

lane	sample
1	Marker
2	Sano VLP 0.5µl
3	Sano VLP 0.2µl
4	Sano VLP 0.1µl
5	Sano VLP 0.05µl
6	Sano VLP 0.02µl
7	Sano VLP 0.05µl

blotting CC
1'antibody time/buffer 1:3000 6ml 1h
2'antibody time/buffer 1:10000 10ml 1h
Blocking 1day
transfer 100mA 1h

9/6

CBD 10th

Isoda

sample	before	after
BclAcex+	0.239	0.222
BclActl+	0.212	0.190
BclAcex-	0.239	0.233
BclActl-	0.221	0.210

9/6

CBD 11th

Isoda

sample	before	after
BclAcex+ (NaCl)	0.202	0.176
BclAcex- (NaCl)	0.202	0.206
BclAcex- (NaCl)	0.202	0.264
BclAcex+ (PBS buffer)	0.212	0.179
BclAcex- (PBS buffer)	0.212	0.209
BclActl+ (PBS buffer)	0.211	0.172

9/7

Colony PCR (typeA)

Yamamoto

Templates/(µl)	Primers/(µl)	MilliQ/(µl)	PreMix/(µl)	Total/(µl)
Bclascfv-BclaCBDcex colony5	vf12.5 vr12.5	10	KAPA2GFASTHSRM 12.5	25
〃6	=	=	=	=
〃7	=	=	=	=
〃8	=	=	=	=
〃9	=	=	=	=
〃10	=	=	=	=
9/5 Bclascfv-BclaCBDclos 5	=	=	=	=
〃6	=	=	=	=
9/5 Bclascfv-INPCBDcex1	=	=	=	=
〃2	=	=	=	=
〃3	=	=	=	=
〃4	=	=	=	=
Bclascfv-INPCBDclos1	=	=	=	=
〃2	=	=	=	=
〃3	=	=	=	=
〃4	=	=	=

9/7

Miniprep

Yamamoto

DNA	Concentration/(µg/ml)	260/280	260/230
Bcla-CBDcex	99.7	1.94	2.08
Bcla-CBDclos	73.5	1.96	2.15
INP-CBDcex	45.3	1.99	2.14
INP-CBDclos	49.6	1.89

9/7

Restriction Enzyme Digestion

Yamamoto

Sample	DNA/(µl)	Xba1/(µl)	Spe1/(µl)	Pst1/(µl)	Buffer/(µl)	BSA/(µl)	Total/(µl)
8/5 miniprep INP-CBDcex	25	0	1	1	10×M/3	0	30
8/5 miniprep INP-CBDclos	25	0	1	1	10×M/3	0	30
9/13 miniprep BclA-scFv1	22	1	0	1	10×H/3	3	30

9/7

Liquid Culture

Yamamoto

Name	medium
9/5 Transformation BclascFv-BclaCBDcex colony5	LB(CP)/5ml
〃BclascFv-BclaCBDclos colony4

9/7

SEM

Ohara

number	sample	OD600	VLP(µl)
1	BclAscFv 3c5	0.990	1
2	BclAscFv 1c5	1.001	1
3	INPscFv 3c5	1.062	1
4	BclA CTL	1.053	1
5	INP CTL	0.952	1

9/7

Liquid Culture

Isoda

Name	medium
Bcla-CBDcex	LB(CP)/30ml
Bcla-control

9/7

CBD 12th

Isoda

sample	before	after
BclAcex+ (PBS buffer)	0.195/0.192	0.188/0.189
BclActl+ (PBS buffer)	0.198/0.195	0.192/0.189
BclAcex+ (CB buffer)	0.186/0.184	0.176/0.173
BclActl+ (CB buffer)	0.172/0.169	0.151/0.155

9/7

CBD 13th

Isoda

sample	before	after
BclAcex+ (PBS buffer) (Bemliese)	0.201/0.201	0.201/0.201
BclActl+ (PBS buffer) (Bemliese)	0.224/0.223	0.218/0.215
BclAcex+ (CB buffer) (Bemliese)	0.266/0.257	0.243/0.242
BclActl+ (CB buffer) (Bemliese)	0.178/0.177	0.157/0.149
BclAcex+ (PBS buffer) (Kimwipe)	0.203/0.201	0.200/0.196
BclActl+ (PBS buffer) (Kimwipe)	0.221/0.220	0.224/0.229
BclAcex+ (CB buffer) (Kimwipe)	0.273/0.266	0.244/0.242
BclActl+ (CB buffer) (Kimwipe)	0.172/0.172	0.154/0.154

9/8

Gel Extraction

Yamamoto

Lane	Restriction Enzyme Digestion Product	Volume/(µl)
1	1kb ladder	5
2	9/7 restriction enzyme design BclA scFv X/Pcut	30
3	9/7 restriction enzyme design INP CBDcex S/Pcut	30
4	9/7 restriction enzyme design INP CBDclos S/Pcut	30
5	100bp ladder	5

9/8

Colony PCR (typeA)

Yamamoto

Templates/(µl)	Primers/(µl)	MilliQ/(µl)	PreMix/(µl)	Total/(µl)
9/5 TransformationINPcex-scFv colony5	vf 1.25 vr1.25	10	KAPA2GFAST/12.5	25
〃colony6	=	=	=	=
〃colony7	=	=	=	=
〃colony8	=	=	=	=
9/5 Transformation INPclos-BclascFv colony1	=	=	=	=
〃colony2	=	=	=	=
〃colony5	=	=	=	=
〃colony6	=	=	=

9/8

Western Blotting

Ohara, Sassa

lane	sample
1	Marker
2	Sano VLP 0.5µl
3	Sano VLP 0.2µl
4	Sano VLP 0.1µl
5	Sano VLP 0.05µl
6	Sano VLP 0.02µl
7	Sano VLP 0.05µl

1'antibody time/buffer 1:3000 6ml 1h
2'antibody time/buffer 1:10000 10ml 1h
Blocking 1day
transfer 100mA 1h

9/8

Ligation

Yamamoto

Insert/µl	Vector/µl	Ligation High/µl	Total
9/8 Gel Extraction BclA scFv X/Pcut /6	9/8 Gel Extraction INP CBDcex S/Pcut /1.5	7.5	15
9/8 Gel Extraction BclA scFv X/Pcut /6	9/8 Gel Extraction INP CBDcex S/Pcut /1.5	7.5	15

9/8

Transformation

Yamamoto, Sassa

Sample Name	Sample Volume (µl)	Competent Cells/(µl)	Medium
9/8 Ligation Bclascfv-INPCBDcex	2	DH5a/20	LB(CP)
〃BclascFv-INPCBDclos	2	〃	〃
9/7Miniprep BclaCBDcex+8/24 Miniprep J23105	1	〃	LB(CP+AMP)
〃Bcla-CBDclos+8/24 J23105	1	〃	〃
9/7 Miniprep INP-CBDcex+8/24 Miniprep J23105	1	〃	〃
9/7 Miniprep INP-CBDclos+8/24 Miniprep J23105	1	〃

9/8

Liquid Culture

Isoda

Name	medium
Bcla-CBDcex	LB(CP)/30ml
Bcla-CBDcontrol

9/8

CBD 14th

Isoda

sample	before	after
CBDcex+ (CB buffer)	0.241/0.236	0.215/0.212
CBDctl+ (CB buffer)	0.182/0.180	0.159/0.159
BclAcex+ (CB buffer + Na2CO3)	0.235/0.235	0.177/0.177
BclActl+ (CB buffer+Na2CO3)	0.227/0.218	0.155/0.156

9/9

Glycerol Stock

Yamamoto

sample /ml	glycerol /ml
BclA control /0.8	0.2
BclA control /0.8	0.2

9/9

Glycerol Stock

Yamamoto

sample /ml	glycerol /ml
INP control /0.8	0.2
INP control /0.8	0.2

9/9

Colony PCR (typeA)

Iguchi

Templates/(µl)	Primers/(µl)	MilliQ/(µl)	PreMix/(µl)	Total/(µl)
INPcex-scFv colony9	vf1.25 vr1.25	8.5	KAPA2GFAST 12.5	25
〃colony10	〃	〃	〃	〃
〃colony11	〃	〃	〃	〃
9/5 transformation INPclos-scFv colony1	〃	〃	〃	〃
〃colony5	〃	〃	〃

9/9

Liquid Culture

Miyazaki

Name	medium
scFv lc3	LB()

9/9

Western Blotting

Ohara, Sassa

lane	sample
1	Marker
2	Sano VLP 0.5µl
3	Sano VLP 1µl
4	Sano VLP 0.2µl
5	Sano VLP 0.1µl
6	Sano VLP 0.05µl
7	Sano VLP 0.02µl
8	Sano VLP 0.5µl 95℃ without boiling
9	only SDS lysis buffer

1'antibody time/buffer 1:3000 9ml 1h
2'antibody time/buffer 1:10000 10ml 1h
Blocking 1h
transfer 100mA 1h

9/9

Miniprep

Miyazaki

DNA	Concentration/(µg/ml)	260/280	260/230
scFv Bcla/1ul	271.8	2.09	2.15
VLP/1ul	1.67	1.98

9/9

Colony PCR (typeA)

Yamamoto

Templates/(µl)	Primers/(µl)	MilliQ/(µl)	PreMix/(µl)	Total/(µl)
BclaCBDcexJ23105	vf1.25 vr1.25	10	KAPA2GFAST HSRM withDye/12.5	25
9/6 Transformation BclaCBDclos J23105	〃	〃	〃	〃
〃INPCBDcex J23105	〃	〃	〃	〃
〃INPCBDcexJ23105	〃	〃	〃	〃

9/9

Transformation

Sample Name	Sample Volume (µl)	Competent Cells/(µl)	Medium
2/24 miniprep Bcla-his-cex colony2	0.1	DH5a/20ul

9/9

Liquid Culture

Iguchi

Name	medium
9/5 transformation INPcex-BclascFv colony9	LB(CP)/5ml
〃INPclos-BclascFv colony1

9/9

Colony PCR (typeA)

Yamamoto

Templates/(µl)	Primers/(µl)	MilliQ/(µl)	PreMix/(µl)	Total/(µl)
9/5 transformation Bcla CBDcex J23105	vf1.0 vr1.0	23	Quick Taq/25	50
〃BclaCBDclos J23105	〃	〃	〃	〃
〃INPCBDcex J23105	〃	〃	〃	〃
〃INPCBDclos J23105	〃	〃	〃

9/12

Restriction Enzyme Digestion

Miyazaki

Sample	DNA/(µl)	EcoR1/(µl)	Xba1/(µl)	Spe1/(µl)	Pst1/(µl)	Buffer/(µl)	BSA/(µl)	MilliQ/(µl)	Total/(µl)
9/9miniprep scFv Bcla lc3	29ul		1	0	1	10×H/5	5	9

9/12

Electrophoresis

Miyazaki

Lane	sample/(µl)	Length(bp)
1	1kb ladder/5	-
2	Bcla CBDcex j23105/15[1]	789 1140
3	〃[2]	〃
4	Bcla CBDclos J23105[1]	757 1140
5	〃[2]	〃
6	INP CBDcex J23105[1]	1664 1140
7	〃[2]	〃
8	INP CBDclos J23105[1]	1796 1140
9	〃[2]	〃
10	100bp ladder

9/12

Colony PCR (typeA)

Miyazaki

Templates/(µl)	Primers/(µl)	MilliQ/(µl)	PreMix/(µl)	Total/(µl)
Bcla CBDcex J23105[1]	vf0.5 vr0.5	11.5	Quick Taq/12.5	25
〃[2]	〃	〃	〃	〃
〃[3]	〃	〃	〃	〃
〃[4]	〃	〃	〃	〃
Bcla CBDclos J23105[1]	〃	〃	〃	〃
〃[2]	〃	〃	〃	〃
〃[3]	〃	〃	〃	〃
〃[4]	〃	〃	〃

9/12

Colony PCR (typeA)

Miyazaki

Templates/(µl)	Primers/(µl)	MilliQ/(µl)	PreMix/(µl)	Total/(µl)
INP CBDcex J23105	vf0.5 vr0.5	11.5	Quick Taq12.5	25
INP〃[2]	=	=	=	=
〃[3]	=	=	=	=
〃[4]	=	=	=	=
〃clos[1]	=	=	=	=
〃[2]	=	=	=	=
〃[3]	=	=	=	=
〃[4]	=	=	=

9/13

Western Blotting

Ohara, Sassa

lane	sample
1	Marker
2	Sano VLP 1µl
3	Sano VLP 1µl
4	Sano VLP 0.2µl
5	Sano VLP 0.1µl
6	Sano VLP 0.05µl
7	Sano VLP 0.02µl
8	Sano VLP 0.5µl 95℃ without boiling
9	only SDS lysis buffer

1'antibody time/buffer 1:3000 9ml 1h
2'antibody time/buffer 1:10000 10ml 1h
Blocking 1h
transfer 100mA 1h
30ml TBS-T/blocking reagent 1.5g

9/14

Liquid Culture

Isoda

Name	medium
9/9 transformation BclA-CBDcex	LB(CP)
8/24 transformation BclA-CBDclos	LB(CP)
8/24 transformation INP-CBDcex	LB(CP)
8/20 transformation	LB(CP)

9/14

Western Blotting

Ohara, Sassa

lane	sample
1	Marker
2	Sano VLP 0.5µl＋INP-scFv 100µl
3	Sano VLP 0.5µl＋BAN-scFv 100µl
4	Sano VLP 0.2µl ＋BAN-scFv 100µl
5	Sano VLP 0.2µl ＋BAN-scFv 70µl ＋BANcontrol30µl
6	Sano VLP 0.2µl ＋BAN-scFv 20µl ＋BANcontrol80µl
7	Sano VLP 0.2µl ＋BANcontrol100µl
8	Sano VLP 0.2µl 95℃ without boiling
9	Sano VLP 0.5µl 95℃ without boiling
10	Marker

1'antibody time/buffer 1:1000 10ml / Blocking buffer　overnight
2'antibody time/buffer 1:2500 10ml / Blocking buffer 1h
Blocking 1h
transfer 100mA 1h
25ml TBS-T/blocking reagent 1.3g

9/15

Western Blotting

Ohara, Sassa

lane	sample
1	Marker
2	Sano VLP 0.5µl＋INP-scFv 100µl
3	Sano VLP 0.5µl＋BAN-scFv 100µl
4	Sano VLP 0.2µl ＋BAN-scFv 100µl
5	Sano VLP 0.2µl ＋BAN-scFv 70µl ＋BANcontrol30µl
6	Sano VLP 0.2µl ＋BAN-scFv 20µl ＋BANcontrol80µl
7	Sano VLP 0.2µl ＋BANcontrol100µl
8	Sano VLP 0.2µl 95℃ without boiling
9	Sano VLP 0.5µl 95℃ without boiling
10	Marker

1'antibody time/buffer 1:1000 10ml / Blocking buffer　overnight
2'antibody time/buffer 1:2500 10ml / Blocking buffer 1h
Blocking 1h
transfer 100mA 1h
25ml TBS-T/blocking reagent 1.3g

9/15

Liquid Culture

Sassa

Name	medium
9/7 BclA scFv 3C5 9/7 2	LB(CP) /20mL
9/14 BclA cex (9/9 transformation)	LB(CP) /20mL
9/7 INP scFv 3C5	LB(CP) /20mL
9/9 BclA scFv 1c3	LB(CP) /20mL
9/7 INP CTL	LB(CP) /20mL
9/7 BclA CTL 2	LB(CP) /20mL

9/15

Liquid Culture

Ohara, Isoda

Name	medium
BclA CBDclos glycerol stock	LB(CP)
INP-CBDcex glycerol stock	LB(CP)
INP-CBDclos glycerol stock	LB(CP)

9/16

Transformation

Uchino, Sassa

Sample Name	Sample Volume (µl)	Competent Cells/(µl)	Medium
8/20 J23105 RFP 1/100	20	DH5α /20	LB(CP)

9/19

Transformation

Sassa, Takimoto

Sample Name	Sample Volume (µl)	Competent Cells/(µl)	Medium
2/24 BAN-cex	1	DH5α /20	LB(CP)
8/5 INP-cex	1	DH5α /20	LB(CP)
2/8 INP HIsR	1	DH5α /20	LB(CP)
2/19 BAN-clos	1	DH5α /20	LB(CP)
8/5 INP-clos	1	DH5α /20	LB(CP)
2/4 E1010	1	DH5α /20
8/18 BclA-scFv	1	DH5α /20	LB(CP)

9/19

Liquid Culture

Sassa, Takimoto

Name	medium
9/15 Liquid culture BclA scFv 3C5[2]	LB(CP) /20
9/19 Liquid culture　Bcla CTL	LB(CP) /20
9/15 Liquid culture INP-scFv 3C5	LB(CP) /20
9/15 Liquid culture INP CTL

9/20

Liquid Culture

Sassa

Name	medium
9/19 liquid culture BclA-scFv 3C5-2	LB(CP)
9/19 liquid culture BclA-ctl	LB(CP)
9/19 liquid culture INP-scFv 3C5	LB(CP)
9/19 liquid culture INP-ctl	LB(CP)

9/20

Western Blotting

Ohara, Sassa

lane	sample
Marker	M.Marker
1	INPNC-6His-scFv 300 µl＋VLP3 µl
2	INPNC-6His-scFv 300 µl＋PBS3 µl
3	INPNC-6His-ctl300 µl＋VLP3 µl
4	BclA-6His-scFv 300 µl＋VLP3 µl
5	BclA-6His-scFv 300 µl＋PBS3 µl
6	BclA-6His-ctl 300 µl＋VLP3 µl
7	VLP0.5 µl
8	VLP0.2 µl
9

Transfer ;60 min 100 mA(CC)
Blocking ;1h
1’antibody ;anti-NoV pAb(mouse)
1:1000 with blocking buffer O/N
2’antibody ;anti-mouse HRP　
1:2500 with blocking buffer 1 h
Detection time　80 sec
(ECL Prime)

9/20

Transformation

Uchino

Sample Name	Sample Volume (µl)	Competent Cells/(µl)	Medium
BclA-clos glycerol stock	-	DH5α /20	LB(CP)
INP-clos glycerol stock	-	DH5α /20	LB(CP)

9/21

PCR (Steps)

Uchino

Steps	Temparature	Time	Cycle
Predenature	95℃	5min	1
Denature	98℃	20sec	30
Annealing	65℃	15sec	30
Extension	72℃	30sec	30
Final Elongation	72℃	3min	1
Final Holding	8℃	-	-

9/21

PCR (Target)

Uchino

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	Buffer/(µl)	MilliQ/(µl)	Any Other/(µl)	Total/(µl)
2/8 miniprep INP-His9 /0.2	VF /0.75	m.scFv Rv /0.75	-	10.8	KAPA HiFi Ready Mix /12.5	25
2/8 miniprep INP-His9 /0.2	m.scFv Fw /0.75	VR /0.75	-	10.8	KAPA HiFi Ready Mix /12.5	25

VF 10µM VR 10µM

9/21

Gel Extraction

Uchino

Lane	Restriction Enzyme Digestion Product	Volume/(µl)
1	1kb ladder	5
2	100bp ladder	5
3	9/21 PCR INP-His9-1	25
4	9/21 PCR INP-His9-2	25

9/21

Liquid Culture

Ohara, Sassa

Name	medium
9/19 Liquid culture BAN-scFv 3C5[2]	LB(CP)
9/19 Liquid culture BAN-CTL	LB(CP)
9/19 Liquid culture INP-scFv 3C5	LB(CP)
9/19 Liquid culture INP-CTL	LB(CP)
9/15 BAN- scFv 1C3	LB(CP)
9/15 BAN-CBDcex	LB(CP)

9/21

Western Blotting

Ohara, Sassa

lane	sample
Marker	M.Marker
1	INPNC-6His-scFv 200 µl＋VLP2 µl
2	INPNC-6His-scFv 200 µl＋PBS2µl(NC)
3	INPNC-6His-ctl200 µl＋VLP2 µl(NC)
4	BclA-6His-scFv200 ul(Vector pSB3C5)＋VLP2 ul
5	BclA-6His-scFv 200 µl＋VLP2 µl (NC)
6	BclA-6His-scFv(Vector pSB1C3)＋VLP2 ul
7	BclA-6His-CBDcex 200 ul ＋ VLP2 ul (NC)
8	PBS200 ul ＋VLP2 ul (NC)
9	VLP1 ul (PC)

Transfer ;60 min 100 mA(CC)
Blocking ;1h
1’antibody ;anti-NoV pAb(mouse)
1:1000 with blocking buffer O/N
2’antibody ;anti-mouse HRP　
1:2500 with blocking buffer 1 h
Detection time　60 sec
(ECL Prime)

9/21

Gel Extraction(Measurement)

Uchino

Sample/(µl)	Concentration(µg/µl)	260/280	260/230
9/21 PCR INP-His9-1	19.3	1.63	0.54
9/21 PCR INP-His9-2	48.6	1.82	0.24

9/21

PCR (Steps)

Uchino

Steps	Temparature	Time	Cycle
Predenature	95℃	5min	1
Denature	98℃	20sec	30
Annealing	65℃	15sec	30
Extension	72℃	30sec	30
Final Elongation	72℃	3min	1
Final Holding	8℃	-

9/21

PCR (Target)

Uchino

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	Buffer/(µl)	MilliQ/(µl)	Any Other/(µl)	Total/(µl)
9/21 Gel Extract INP-His9-1 /1µl+9/21 Gel Extract INP-His9-2 /0.5µl	VF /0.75	VR /0.75	-	9.5	KAPA HiFi /12.5	25

9/22

growth carve

Sassa

sample	0h	0.5h	1h	1.5h	2h	3h	4h
BAM scFv　[1]	-0.048	-0.047	-0.048	1.245	-0.317	-0.308	-0.149
BAM scFv　[2]	-0.036	1.512	-0.830	-0.309	-0.314	-0.127
INP scFv　[1]	-0.047	-0.050	-0.644	1.045	-0.307	-0.153	-0.142
INP scFv　[2]	-0.038	-0.o25	-0.186	-0.280	0.000	-0.289
BAN CBDcex　[1]	-0.045	-0.048	-0.044	1.089	-0.322	-0.284	-0.296
BAN CBDcex　[2]	-0.034	1.204	-1.057	-0.311	0.268	-0.285
BAN CBDclos　[1]	0.035	-0.037	-0.035	1.261	-0.323	-0.160	-0.287
BAN CBDclos　[2]	-0.018	1.306	-0.050	-0.282	-0.128	-0.293
INP CBDcex　[1]	-0.020	-0.027	-0.016	1.245	-0.295	-0.263	-0.288
INP CBDcex　[2]	-0.027	1.608	0.016	-0.287	-0.224	-0.294
INP CBDclos　[1]	-0.034	-0.034	-0.032	1.026	-0.318	-0.235	-0.270
INP CBDclos　[2]	-0.026	1.415	0.012	-0.297	-0.047	-0.272
BAN CTL 　[1]	-0.039	-0.039	-0.036	1.274	-0.314	-0.230	-0.271
BAN CTL 　[2]	-0.056	1.530	-0.060	-0.284	-0.084	-3.11
INP CTL　[1]	-0.042	-0.043	-0.042	1.134	-0.235	-0.181	-0.296
INP CTL　[2]	-0.042	-1.488	-0.030	-0.288	-0.109	-0.295
E1010　[1]	-0.041	-0.040	-0.036	1.171	-0.322	-0.252
E1010　[2]	-0.030	1.563	-0.061	-0.267	0.286	-0.267
blank [1]/[2]				/-0.307	-0.306/-0.305	-0.307/-0.271	0.306/

9/22

Liquid Culture

Sassa, Miyazaki

Name	medium
9/21 liquid culture BAN-CBDcex	LB(AMP)
9/15 liquid culture INP-ctl	LB(AMP)
9/15 liquid culture INP-CBDcex	LB(AMP)
9/15 liquid culture INP-CBDclos	LB(AMP)
9/15 liquid culture BAN-scFv 3C5	LB(AMP)
9/15 liquid culture INP-scFv 3C5	LB(AMP)
9/19 liquid culture BAN-CBDclos	LB(AMP)
9/15 liquid culture BAN-ctl	LB(AMP)
9/16 transformation J23105-RFP	LB(AMP)

9/23

SEM

Miyazaki

number	sample(µl)	VLP(µl)	PBS	centrifugal force
1	INP scFv /25	2.5	0	×3000g
2	INP CTL /25	2.5	0	×3000g
3	BclA scFv /25	2.5	0	×3000g
4	BclA CTL /25	2.5	0	×3000g
5	BclA scFv /25	0	2.5	×3000g
6	INP scFv /25	2.5	0	×200g
7	INP CTL /25	2.5	0	×200g
8	BclA scFv /25	2.5	0	×200g
9	BclA CTL /25	2.5	0	×200g
10	BclA scFv /25	0	2.5	×200g
11	0	2.5	0	×200g

9/23

RNA extraction

Isoda

number	sample name
1	BclA-CBD cex
2	BclA-CBDclos
3	INP-CBDcex
4	INP-CBDclos
5	BclA-scFv
6	INP-scFv

9/23

growth carve

sample	0h	0.5h	1h	1.5h	2h	3h	4h	5h	6h	7h	8h	9h	10h
9/19 Liquid culture BAN scFv[1]	0.003	0.001	0.000	0.008	0.015	0.009	0.013	0.016	0.015	0.022	0.062	0.020	0.027
9/19 Liquid culture BAN scFv[2]	0.013	0.012	0.000	0.000	0.000	0.000	0.001	0.011	0.011	0.015	0.022	0.027	0.057
9/19 Liquid culture INP scFv[1]	0.012	0.000	0.000	0.011	0.006	0.006	0.007	0.008	0.013	0.025	0.036	0.098	0.076
9/19 Liquid culture INP scFv[2]	0.007	0.005	0.000	0.002	0.003	0.002	0.012	0.012	0.019	0.029	0.034	0.047	0.072
9/19 Liquid culture BAN cex[1]	0.014	0.005	0.010	0.008	0.011	0.009	0.014	0.014	0.016	0.029	0.038	0.043	0.083
9/19 Liquid culture BAN cex[2]	0.011	0.011	0.010	0.008	0.004	0.004	0.020	0.021	0.036	0.047	0.065	0.087	0.109
9/19 Liquid culture BAN clos[1]	0.000	0.000	0.000	0.002	0.009	0.003	0.009	0.014	0.019	0.014	0.016	0.024	0.046
9/19 Liquid culture BAN clos[2]	0.005	0.007	0.000	0.000	0.000	0.003	0.012	0.022	0.020	0.035	0.055	0.086	0.117
9/19 Liquid culture INP cex[1]	0.187	0.000	0.001	0.007	0.011	0.006	0.013	0.016	0.020	0.023	0.037	0.048	0.068
9/19 Liquid culture INP cex[2]	0.002	0.009	0.000	0.001	0.001	0.002	0.015	0.017	0.023	0.036	0.053	0.080	0.111
9/19 Liquid culture INP clos[1]	0.006	0.000	0.000	0.023	0.011	0.003	0.011	0.013	0.016	0.022	0.033	0.058	0.068
9/19 Liquid culture INP clos[2]	0.029	0.028	0.000	0.009	0.000	0.016	0.001	0.007	0.008	0.014	0.020	0.029	0.074
9/19 Liquid culture BAN CTL[1]	0.004	0.000	0.000	0.000	0.002	0.000	0.002	0.012	0.015	0.012	0.027	0.055	0.081
9/19 Liquid culture BAN CTL[2]	0.002	0.002	0.001	0.000	0.001	0.005	0.017	0.024	0.031	0.042	0.059	0.073	0.113
9/19 Liquid culture INP CTL[1]	0.011	0.000	0.032	0.013	0.017	0.010	0.015	0.033	0.031	0.035	0.037	0.019	0.026
9/19 Liquid culture INP CTL[2]	0.013	0.020	0.000	0.001	0.000	0.003	0.009	0.016	0.021	0.026	0.044	0.059	0.136
9/19 Liquid culture E1010[1]	0.015	0.016	0.014	0.011	0.016	0.018	0.032	0.066	0.108	0.173	0.258	0.361	0.393
9/19 Liquid culture E1010[2]	0.064	0.010	0.007	0.011	0.012	0.019	0.029	0.041	0.061	0.097	0.150	0.180

9/23

Electrophoresis

Uchino

Lane	sample/(µl)	Length(bp)
1	1kb ladder/5
2	9/21 OEPCR/25

9/23

OEPCR

Uchino

sample1 /µl	sample2 /µl	primer Fw /µl	primer Rv /µl	milliQ /µl	KAPA HiFi /µl	total
9/21 Gel Extract1 /1	9/21 Gel Extract2 /0.5	VF/ 0.75	VR /0.75	9.5	12.5	25

9/23

Electrophoresis

Uchino

Lane	sample/(µl)	Length(bp)
1	1kb ladder/3
2	9/23 OEPCR1 /25
3	9/23 OEPCR2 /25

9/23

PCR (Target)

Yamamoto

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	Buffer/(µl)	MilliQ/(µl)	Total/(µl)
2/8 miniprep INP-His-scFv9 /0.4	m.scFv Fw/1.25	VR/1.25	KAPA2GFAST/12.5	9.6	25
2/8 miniprep INP-His-scFv9 /0.4	m.scFv Fw/1.25	VR/1.25	KAPA2GFAST/12.5	9.6	25
2/8 miniprep INP-His-scFv9 /0.4	VF/1.25	m.scFv Rv/1.25	KAPA2GFAST/12.5	9.6	25
2/8 miniprep INP-His-scFv9 /0.4	VF/1.25	m.scFv Rv/1.25	KAPA2GFAST/12.5	9.6	25
9/23 Gel Extraction INP-scFv m.PCR/1.5	VF/1.25	VR/1.25	KAPA2GFAST/12.5	8.5	25

9/26

Electrophoresis

Uchino

Lane	sample/(µl)	Length(bp)
1	1kb ladder/4
2	9/23PCR1 /25
3	9/23PCR2 /25
4	9/23PCR3 /25
5	9/23PCR4 /25
6	9/23PCR5 /25

9/26

Gel Extraction

Uchino

Lane	Restriction Enzyme Digestion Product	Volume/(µl)
9/26 electrophoresis lane 4,5		0.357g
9/26 electrophoresis lane 2,3		0.354g

9/26

PCR (Target)

Uchino

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	Buffer/(µl)	MilliQ/(µl)	Any Other/(µl)	Total/(µl)
9/26 Gel Extract lane4,5 /2	VF/0.75	VR/0.75	2×KAPA HiFi Hotstart ReadyMix/12.5	7		25
9/26 Gel Extract lane2,3 /2	VF/0.75	VR/0.75	2×KAPA HiFi Hotstart ReadyMix/12.5	7		25

9/26

Liquid Culture

Ohara

Name	medium
INP-scFv/30ml	LB
INP-CTL/30ml	LB
BAN-scFv/30ml	LB
Ban CTL/30ml	LB

9/26

Transformation

Yamada

Sample Name	Competent Cells/(µl)	Medium
Bcla-cex 8/5 miniprep	BL21	LB(CP)
Bcla-CTL 8/4 miniprep	BL21	LB(CP)
Bcla-cex 8/5 miniprep	BL21	LB(CP)
Bcla-CTL 8/4 miniprep	BL21	LB(CP)

9/26

RNA extraction

Isoda

number	sample	conc.µg/µl	260/280	260/230
4	BclAcex	74.73	1.94	1.61
2	INPcex	83	1.97	1.38
5	INPclos	120.21	1.99	1.94
3	INPclos	58.23	1.98	1.84
6	BclAscFV	74.76	1.96	1.84
7	INPscFv	80.6	1.97	1.97

9/27

Liquid Culture

Yamada

Name	medium
BclA-cex /250ml	LB(CP)
BclA-CTL /250ml	LB(CP)

9/27

growth carve

Sassa, Takimoto

sample	0h	0.5h	1h	1.5h	2h	3h	4h	5h	6h	7h	8h
9/21Liquid culture BAN scFv	0.113	0.170	0.266	0.408	0.599	0.893	0.164(1.64)	0.169(1.69)	0.232(2.32)	0.234(2.34)	0.221(2.21)
9/22Liquid culture BAN cex	0.109	0.147	0.219	0.374	0.515	0.873	0.146(1.46)	0.160(1.60)	0.176(1.76)	0.231(2.31)	0.260(2.60)
9/19Liquid culture BAN clos	0.097	0.109	0.184	0.338	0.486	0.744	0.130(1.30)	0.173(1.73)	0.199(1.99)	0.203(2.03)	0.274(2.74)
9/21Liquid culture BAN CTL	0.106	0.170	0.252	0.304	0.553	0.797	0.177(1.77)	0.155(1.55)	0.216(2.16)	0.236(2.36)	0.257(2.57)
9/21Liquid culture INP scFv	0.088	0.083	0.101	0.122	0.299	0.409	0.073(0.73)	0.121(1.21)	0.140(1.40)	0.143(1.43)	0.195(1.95)
9/15Liquid culture INP cex	0.102	0.116	0.145	0.207	0.320	0.626	0.170(1.70)	0.143(1.43)	0.176(1.76)	0.188(1.88)	0.238(2.38)
9/22Liquid culture INP clos	---	0.133	0.209	0.373	0.572	0.930	0.156(1.56)	0.201(2.01)	0.246(2.46)	0.237(2.37)	0.293(2.93)
9/21Liquid culture INP CTL	0.112	0.155	0.237	0.409	0.582	0.955	0.153(1.53)	0.209(2.09)	0.268(2.68)	0.288(2.88)	0.274(2.74)
9/21Liquid culture E1010	0.073	0.091	0.149	0.265	0.396	0.706	0.157(1.57)	0.182(1.82)	0.222(2.22)	0.222(2.22)

rpm 160
total10mL
CP 2mL/1L
measured 10 fold dilution after 4h

9/27

Western Blotting

Ohara

lane	sample
Marker	M.Marker(Wako)
1	BclA6His-scFv 300 µl＋supernatant of NoVLP 3 µl (washed by PBS)
2	BclA6His ctl 300 µl＋supernatant of NoVLP 3 µl (NC) (washed by PBS)
3	INPNC6His-scFv 300 µl＋supernatant of NoVLP 3 µl (washed by PBS)
4	INPNC6His ctl 300 µl＋supernatant of NoVLP 3 µl (NC) (washed by PBS)
5	BclA6His-scFv 300 µl＋supernatant of NoVLP 3µl (washed by PBST (0.1% tween20))
6	BclA6His ctl 300 µl＋supernatant of NoVLP 3µl (NC) (washed by PBST (0.1% tween20))
7	INPNC6His-scFv 300 µl＋supernatant of NoVLP 3µl (washed by PBST (0.1% tween20))
8	INPNC6His ctl 300 µl＋supernatant of NoVLP 3µl (NC) (washed by PBST (0.1% tween20))
9	PBS 300µl＋supernatant of NoVLP 3 µl (NC) (washed by PBS)

Transfer ;60 min 100 mA(CC)
Blocking ;1h
1’antibody ;anti-NoV pAb(mouse)
1:1000 with blocking buffer O/N
2’antibody ;anti-mouse HRP　
1:2500 with blocking buffer 1 h
Detection time　60 sec
(ECL Prime)

9/27

Western Blotting

Ohara, Sassa

lane	sample
Marker	M.Marker(Wako) 5µl
1	BBa_E1010(NC)
2	BclA-6His-CBDclos (17 kDa)
3	INPNC-6His-CBDclos(47 kDa)
4	BclA-6His-CBDcex (17 kDa)
5	INPNC-6His-CBDcex (47 kDa)
6	BclA-6His-scFv (33 kDa)
7	INPNC-6His-scFv (63 kDa)
8	BBa_K875004 (PC) Lpp-OmpA-scFv-6His (43kDa)
Marker	M.Marker(Wako) 3µl
9	BBa_K875004 (PC) Lpp-OmpA-scFv-6His(43 kDa)

Used membrane fraction from 100ml culture at logarithmic growth phase.

Transfer ;60min 100mA(CC)
Blocking ;1h
1’antibody ;anti6His(mouse)
1:1000 with blocking buffer O/N
2’antibody ;anti-mouse HRP　
1:2500 with TBST 1h
Detection time　380sec
(ECL Prime)

9/27

Reverse Transcription

Yamada

number	Template/µl	gDNA wipeout buffer/µl	RNAase free water/µl	total/µl
A1	BclAcex/12	2	0	14
A2	BclAclos/12	2	0	14
A3	INPcex/8.32	2	3.68	14
A4	INPclos/12	2	0	14
A5	BclAscFv/12	2	0	14
A6	INPscFv/12	2	0	14
B1	BclAcex/12	2	0	14
B2	BclAclos/12	2	0	14
B3	INPcex/8.32	2	3.68	14
B4	INPclos/12	2	0	14
B5	BclAscFv/12	2	0	14
B6	INPscFv/12	2	0	14

9/27

Reverse Transcription

Yamada

number	template/µl	RTase/µl	RNAase free water/µl	RT buffer/µl	Primer Mix/µl	Total
A1	BclAcex/14	1	0	4	1	20
A2	BclAclos/14	1	0	4	1	20
A3	INPcex/14	1	0	4	1	20
A4	INPclos/14	1	0	4	1	20
A5	BclAscFv/14	1	0	4	1	20
A6	INPscFv/12	1	0	4	1	20
B1	BclAcex/14	0	1	4	1	20
B2	BclAclos/14	0	1	4	1	20
B3	INPcex/14	0	1	4	1	20
B4	INPclos/14	0	1	4	1	20
B5	BclAscFv/14	0	1	4	1	20
B6	INPscFv/12	0	1	4	1	20

9/27

Electrophoresis

Uchino

Lane	sample/(µl)	Length(bp)
1	1kb ladder/5
2	9/26 PCR INP- His scFv/25
3	9/26 PCR INP- His scFv/25

9/27

Gel Extraction

Uchino

Lane	Restriction Enzyme Digestion Product	Volume/(µl)
9/27 electrophoresis lane2,3		0.7660g

nanodrop
17.8ng/nl
260/280 1.79
260/230 0.31

9/27

CBD

Yamada

15th	null	null	null
sample	before	after	wash
BclAcex+ (CB buffer)	0.181/0.212/0.204	0.254/0.255/0.254	0/0/0
BclAcex- (CB buffer)	0.208/0.275/0.228	0.160/0.171/0.162
BclActl+ (CB buffer)	0.178/0.176/0.171	0.082/0.085/0.082	0.022/0.02/0.02
BclActl- (CB buffer)	0.218/0.214/0.216	0.161/0.160/0.159
BclAcex+ (PBS buffer)	0.212/0.211/0.210	0.333/0.334/0.333	0.022/0.022/0.021
BclAcex- (PBS buffer)	0.203/0.202/0.201	0.178/0.178/0.180
BclActl+ (PBS buffer)	0.182/0.181/0.180	0.253/0.246/0.246	0.021/0.021/0.021
BclActl- (PBS buffer)	0.208/0.205/0.208	0.192/0.194/0.194

9/29

PCR (Target)

Uchino

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	Buffer/(µl)	MilliQ/(µl)	Any Other/(µl)	Total/(µl)
9/27 Gel Extract/2	VF/0.75	VR/0.75	2×KAPA HiFi Hotstart ReadyMix/12.5	9		25
	VF/0.75	VR/0.75	2×KAPA HiFi Hotstart ReadyMix/12.5	9		25

9/29

Liquid Culture

Ohara

Name	medium
BAN scFv	LB
INP clos	LB
BAN clos	LB
INP cex	LB
BAN cex	LB
K875004	LB

9/29

Electrophoresis

Uchino

Lane	sample/(µl)	Length(bp)
1	1kb ladder/2
2	9/29 PCR INP-His-scFv/2
3

9/29

PCR (Purification)

Uchino

Sample Name/(µl)	Concentration(ng/µl)	260/280	260/230
9/29 PCR INP-His-scFv	31.9	2.02	1.38

9/30

Electrophoresis

Uchino, Miyazaki

Lane	sample/(µl)	Length(bp)
1	1kb ladder
2	BclA clos/4.0
3	INP clos/5.5
4	BclA cex/4.5
5	INP cex/2.5
6	BclA scFv/4.5
7	INP scFv/4.0

10/1

RNA purification

Ohara

BclAcex	0.504
BclAscFv	0.788
INPscFv	0.837
INPclos	1.564
BclAclos	0.817
INPcex	0.839

10/1

RNA extraction

Uchino

number	sample	conc(ng/µl)	260/280	260/230	diluted solution volume(200ng/µl)
1	INPclos	359.2	2.07	1.21	11.94
2	BclAcex	230.9	2.03	0.85	2.31
3	INPcex	1069.0	2.11	1.43	65.17
4	INPscFv	320.0	2.10	0.93	9
5	BclAclos	386.2	2.04	0.97	13.97
6	BclAcex	441.0	2.04	0.83	18

10/1

Reverse Transcription

Uchino

name	copy(high or low)	RT
3 nega	high copy	-
3 posi	high copy	+
4 nega	low copy	-
4 posi	low copy	+

10/1

PCR (Target)

Uchino

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	Buffer/(µl)	MilliQ/(µl)	Total/(µl)
10/1 RT×20 3RT- /1.0µl	rRNA16s forward primer1 /0.4µl	rRNA16s reverse primer1 /0.4µl	2×KAPA HiFi /5µl	3.2µl	10µl
10/1 RT×20 3RT+ /1.0µl	rRNA16s forward primer1 /0.4µl	rRNA16s reverse primer1 /0.4µl	2×KAPA HiFi /5µl	3.2µl	10µl
10/1 RT×20 4RT- /1.0µl	rRNA16s forward primer1 /0.4µl	rRNA16s reverse primer1 /0.4µl	2×KAPA HiFi /5µl	3.2µl	10µl
10/1 RT×20 4RT+ /1.0µl	rRNA16s forward primer1 /0.4µl	rRNA16s reverse primer1 /0.4µl	2×KAPA HiFi /5µl	3.2µl	10µl
9/27 RT×1 A-6 /1.0µl	rRNA16s forward primer1 /0.4µl	rRNA16s reverse primer1 /0.4µl	2×KAPA HiFi /5µl	3.2µl	10µl
9/27 RT×1 A-5 /1.0µl	rRNA16s forward primer1 /0.4µl	rRNA16s reverse primer1 /0.4µl	2×KAPA HiFi /5µl	3.2µl	10µl
9/27 RT×1 B-6 /1.0µl	rRNA16s forward primer1 /0.4µl	rRNA16s reverse primer1 /0.4µl	2×KAPA HiFi /5µl	3.2µl	10µl
9/27 RT×1 B-5 /1.0µl	rRNA16s forward primer1 /0.4µl	rRNA16s reverse primer1 /0.4µl	2×KAPA HiFi /5µl	3.2µl	10µl

10/1

PCR (Target)

Uchino

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	Buffer/(µl)	MilliQ/(µl)	Total/(µl)
10/1 RT×20 3RT- /1.0	INP primer for CBD /0.4	CBD cex primer Rv /0.4	2×KAPA HiFi /5	3.2	10
10/1 RT×20 3RT+ /1.0	INP primer for CBD /0.4	CBD cex primer Rv /0.4	2×KAPA HiFi /5	3.2	10
10/1 RT×20 4RT- /1.0	scFv primer1 /0.4	scFv primer2 /0.4	2×KAPA HiFi /5	3.2	10
10/1 RT×20 4RT+ /1.0	scFv primer1 /0.4	scFv primer2 /0.4	2×KAPA HiFi /5	3.2	10
9/27 RT×1 A-6 /1.0	INP primer for CBD /0.4	CBD cex primer Rv /0.4	2×KAPA HiFi /5	3.2	10
9/27 RT×1 A-5 /1.0	INP primer for CBD /0.4	CBD cex primer Rv /0.4	2×KAPA HiFi /5	3.2	10
9/27 RT×1 B-6 /1.0	scFv primer1 /0.4	scFv primer2 /0.4	2×KAPA HiFi /5	3.2	10
9/27 RT×1 B-5 /1.0	scFv primer1 /0.4	scFv primer2 /0.4	2×KAPA HiFi /5	3.2	10

10/1

Electrophoresis

Uchino

Lane	sample/(µl)	Length(bp)
1	3RT- target /10
2	3RT+ target /10
3	3RT- 16s rRNA /10
4	3RT+ 16s rRNA /10
5	4RT- target /10
6	4RT+ target /10
7	4RT- 16s rRNA /10
8	4RT+ 16s rRNA /10
9	100bp ladder /5
10	A-6 target /10
11	A-5 target /10
12	A-6 16s rRNA /10
13	A-5 16s rRNA /10
14	B-6 target /10
15	B-5 target /10
16	B-6 16s rRNA /10
17	B-5 16s rRNA /10

10/1

Transformation

Ohara

Sample Name	Sample Volume (µl)	Competent Cells/(µl)	Medium
K165002		DH5α
BclA scFv		DH5α
INP scFv		DH5α
BclA cex		DH5α
INP cex		DH5α
BclA clos		DH5α
INP clos		DH5α
K165002		BL21
BclA scFv		BL21
INP scFv		BL21
BclA cex		BL21
INP cex		BL21
BclA clos		BL21
INP clos		BL21

10/4

Transformation

Yamada

Sample Name	Sample Volume (µl)	Competent Cells/(µl)	Medium
INP CTL 8/4 miniprep		DH5α/20	LB(CP)

10/5

Liquid Culture

Yamada

Name	medium
INP CTL 10/4 transformation

10/5

Western Blotting

Ohara

lane	sample
Marker	M.Marker(Wako) 5µl
1	BBa_E165002(NC)
2	BclA-6His-scFv (33 kDa)
3	INPNC-6His-scFv (63 kDa)
4	BclA-6His-CBDcex (17 kDa)
5	INPNC-6His-CBDcex(47kDa)
6	BclA-6His-CBDclos (17 kDa)
7	INPNC-6His-CBDclos (47 kDa)
8	BBa_K875004(PC) Lpp-OmpA-scFv-6His (43 kDa)

Used pelet from 400 µl overnight culture.

Transfar ;80 min 200 mA(CC)
Blocking ;O/N
1’antibody ;anti-6His(mouse)
1:1000 with blocking buffer 1h
2’antibody ;anti-mouse HRP　1:2500 with TBST 1h
Detection time　240 sec
(ECL Prime)

10/6

Liquid Culture

Uchino

Name	medium
10/5 liquid culture INP CTL /2.5ml	LB(CP) /47.5ml
10/5 liquid culture INP CTL /2.5ml	LB(CP) /47.5ml
10/5 liquid culture INP CBD cex(5) /2.5ml	LB(CP) /47.5ml
10/5 liquid culture INP CBD cex(5) /2.5ml	LB(CP) /47.5ml

10/6

Liquid Culture

Yamada

Name	medium
10/5 liquid culture INP CTL /100ml	LB(CP) /100ml
10/3 liquid culture INP CBD cex /100ml	LB(CP) /200ml

10/7

CBD 16th

Yamada

sample	before	after	wash
INPcex+	0.208/0.208/0.211	0.224/0.221/0.226	0.013/0.012/0.010
INPcex-	0.210/0.216/0.204	0.232/0.231/0.232
INPctl+	0.196/0.196/0.191	0.220/0.217/0.212	0.023/0.024/0.025
INPctl-	0.194/0.195/0.196	0.207/0.199/0.208
cellulose only	0.015/0.013/0.015	0.031/0.031/0.032	0.018/0.017/0.019

10/11

Transformation

Yamada

Sample Name	Sample Volume (µl)	Competent Cells/(µl)	Medium
2/25 miniprep INPcex	3	DH5α /20	LB(CP)×3
2/8 miniprep INP His 12 (INPscFv)	3	DH5α /20	LB(CP)×3
2/25 miniprep INPcex	3	BL21 /20	LB(CP)×3
2/8 miniprep INP His 12 (INPscFv)	3	BL21 /20	LB(CP)×3

10/11

SEM(CBD)

Miyazaki

sample/ml	before	after	wash1	wash2
INP CBDcex 10/1 liquid culture /90	0.550	0.392	0.074/0.084	-0.004/-0.018
INP CBDcex 10/1 liquid culture /45	0.550	0.432	0.002/0.000	-0.001/0.000

10/12

Colony PCR (typeA)

Yamada

Templates/(µl)	Primers/(µl)	MilliQ/(µl)	PreMix/(µl)	Total/(µl)
INP-His-scFv-INP-His-CBDcex DH5α colony1	VF/0.5 VR/0.5	11.5	Quick Taq/12.5	25
INP-His-scFv-INP-His-CBDcex DH5α colony2	VF/0.5 VR/0.5	11.5	Quick Taq/12.5	25
INP-His-scFv-INP-His-CBDcex DH5α colony3	VF/0.5 VR/0.5	11.5	Quick Taq/12.5	25
INP-His-scFv-INP-His-CBDcex DH5α colony4	VF/0.5 VR/0.5	11.5	Quick Taq/12.5	25
INP-His-scFv-INP-His-CBDcex DH5α colony5	VF/0.5 VR/0.5	11.5	Quick Taq/12.5	25
INP-His-scFv-INP-His-CBDcex DH5α colony6	VF/0.5 VR/0.5	11.5	Quick Taq/12.5	25
INP-His-scFv-INP-His-CBDcex DH5α colony7	VF/0.5 VR/0.5	11.5	Quick Taq/12.5	25
INP-His-scFv-INP-His-CBDcex DH5α colony8	VF/0.5 VR/0.5	11.5	Quick Taq/12.5	25
INP-His-scFv-INP-His-CBDcex DH5α colony9	VF/0.5 VR/0.5	11.5	Quick Taq/12.5	25
INP-His-scFv-INP-His-CBDcex DH5α colony10	VF/0.5 VR/0.5	11.5	Quick Taq/12.5	25
INP-His-scFv-INP-His-CBDcex DH5α colony11	VF/0.5 VR/0.5	11.5	Quick Taq/12.5	25
INP-His-scFv-INP-His-CBDcex DH5α colony12	VF/0.5 VR/0.5	11.5	Quick Taq/12.5	25
INP-His-scFv-INP-His-CBDcex DH5α colony13	VF/0.5 VR/0.5	11.5	Quick Taq/12.5	25
INP-His-scFv-INP-His-CBDcex DH5α colony14	VF/0.5 VR/0.5	11.5	Quick Taq/12.5	25
INP-His-scFv-INP-His-CBDcex DH5α colony15	VF/0.5 VR/0.5	11.5	Quick Taq/12.5	25
INP-His-scFv-INP-His-CBDcex DH5α colony16	VF/0.5 VR/0.5	11.5	Quick Taq/12.5	25
INP-His-scFv-INP-His-CBDcex DH5α colony17	VF/0.5 VR/0.5	11.5	Quick Taq/12.5	25
INP-His-scFv-INP-His-CBDcex DH5α colony18	VF/0.5 VR/0.5	11.5	Quick Taq/12.5	25
INP-His-scFv-INP-His-CBDcex DH5α colony19	VF/0.5 VR/0.5	11.5	Quick Taq/12.5	25
INP-His-scFv-INP-His-CBDcex DH5α colony20	VF/0.5 VR/0.5	11.5	Quick Taq/12.5	25

10/12

Colony PCR (typeA)

Yamada

Templates/(µl)	Primers/(µl)	MilliQ/(µl)	PreMix/(µl)	Total/(µl)
INP-His-scFv-INP-His-CBDcex DH5α colony21	VF/0.5 VR/0.5	11.5	Quick Taq/12.5	25
INP-His-scFv-INP-His-CBDcex DH5α colony21	VF/0.5 VR/0.5	11.5	Quick Taq/12.5	25
INP-His-scFv-INP-His-CBDcex DH5α colony21	VF/0.5 VR/0.5	11.5	Quick Taq/12.5	25
INP-His-scFv-INP-His-CBDcex DH5α colony21	VF/0.5 VR/0.5	11.5	Quick Taq/12.5	25
2/8 miniprep INP-His-scFv /0.5	VF/0.5 VR/0.5	11.5	Quick Taq/12.5	25
2/25 miniprep INP-His-CBDcex /0.5	VF/0.5 VR/0.5	11.5	Quick Taq/12.5	25

10/12

Electrophoresis

Yamada

Lane	sample/(µl)	Length(bp)
1	INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony1 /5
2	INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony2 /5
3	INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony3 /5
4	INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony4 /5
5	INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony5 /5
6	INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony6 /5
7	INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony7 /5
8	INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony8 /5
9	INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony17 /5
10	INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony18 /5
11	INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony19 /5
12	INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony20 /5
13	INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony21 /5
14	INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony22 /5
15	INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony23 /5
16	INPNC-His-CBDcex 10/12 colony PCR /5
17	1kbp ladder /10

10/12

PCR (Target)

Uchino

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	Buffer/(µl)	MilliQ/(µl)	Any Other/(µl)	Total/(µl)
2/8 INP-His 12 /0.1	INP His coding Fw /0.75	INP His coding Rv /0.75	2×KAPA /12.5	10.9	0	25

10/13

PCR (Target)

Uchino

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	Buffer/(µl)	MilliQ/(µl)	Any Other/(µl)	Total/(µl)
2/8 INP-His 12 /0.1	VF /0.75	VR /0.75	2×KAPA /12.5	10.9	0	25

10/13

Colony PCR

Yamada

sample /µl	primer /µl	KAPA Taq Extra Hotstart /µl	DMSO /µl	MilliQ /µl	total /µl
INP-cex + INP-scFv colony2	VF /1.25 VR /1.25	12.5	0.75	9.25	25
INP-cex + INP-scFv colony7	VF /1.25 VR /1.25	12.5	0.75	9.25	25
INP-cex + INP-scFv colony17	VF /1.25 VR /1.25	12.5	0.75	9.25	25
INP-cex + INP-scFv colony19	VF /1.25 VR /1.25	12.5	0.75	9.25	25
INP-cex + INP-scFv colony9	VF /1.25 VR /1.25	12.5	0.75	9.25	25
INP-cex + INP-scFv colony10	VF /1.25 VR /1.25	12.5	0.75	9.25	25
INP-cex + INP-scFv colony11	VF /1.25 VR /1.25	12.5	0.75	9.25	25
INP-cex + INP-scFv colony12	VF /1.25 VR /1.25	12.5	0.75	9.25	25
INP-cex + INP-scFv colony13	VF /1.25 VR /1.25	12.5	0.75	9.25	25
INP-cex + INP-scFv colony16	VF /1.25 VR /1.25	12.5	0.75	9.25	25
INP-cex + INP-scFv colony23	VF /1.25 VR /1.25	12.5	0.75	9.25	25
INP-cex + INP-scFv colony24	VF /1.25 VR /1.25	12.5	0.75	9.25	25
INP-cex + INP-scFv colony25	VF /1.25 VR /1.25	12.5	0.75	9.25	25
INP-cex + INP-scFv colony26	VF /1.25 VR /1.25	12.5	0.75	9.25	25
INP-cex + INP-scFv colony27	VF /1.25 VR /1.25	12.5	0.75	9.25	25
INP-cex + INP-scFv colony28	VF /1.25 VR /1.25	12.5	0.75	9.25	25
INP-cex + INP-scFv colony29	VF /1.25 VR /1.25	12.5	0.75	9.25	25
INP-cex + INP-scFv colony30	VF /1.25 VR /1.25	12.5	0.75	9.25	25
INP-cex + INP-scFv colony31	VF /1.25 VR /1.25	12.5	0.75	9.25	25

10/13

Colony PCR

Yamada

sample /µl	primer /µl	KAPA Taq Extra Hotstart /µl	DMSO /µl	MilliQ /µl	total /µl
INP-cex + INP-scFv colony31	VF /1.25 VR /1.25	12.5	0.75	9.25	25
INP-cex + INP-scFv colony32	VF /1.25 VR /1.25	12.5	0.75	9.25	25
INP-cex + INP-scFv colony33	VF /1.25 VR /1.25	12.5	0.75	9.25	25
INP-cex + INP-scFv colony34	VF /1.25 VR /1.25	12.5	0.75	9.25	25
INP-scFv /0.25	VF /1.25 VR /1.25	12.5	0.75	9.25	25
INP-cex /0.25	VF /1.25 VR /1.25	12.5	0.75	9.25	25

10/13

Miniprep

Uchino

DNA	Concentration/(µg/ml)	260/280	260/230
10/6 liquid culture INP-His-CBDcex	27.8	1.96	1.86

10/13

PCR (Purification)

Uchino

Sample Name/(µl)	Concentration(ng/µl)	260/280	260/230
10/12 PCR INP-His 12 (INP-His coding)	727.3	1.88	1.86
10/13 PCR INP-His 12	1132.1	1.89	2.04

10/13

Restriction Enzyme Digestion

Uchino

Sample	DNA/(µl)	EcoR1/(µl)	Xba1/(µl)	Spe1/(µl)	Pst1/(µl)	Buffer/(µl)	MilliQ/(µl)	Total/(µl)
10/13 PCR purification INP-His coding	7	2	0	0	2	10×H /6	44	60
10/13 PCR purification INP-His 12	6	0	2	0	2	10×M /6	44	60
10/13 midiprep INP-His-CBDcex	50	2	0	0	2	10×H /6	0	60
10/13 midiprep INP-His-CBDcex	50	0	0	2	2	10×H /6	0	60

10/13

Electrophoresis

Yamada

Lane	sample/(µl)	Length(bp)
1	INPcex+INPscFv colony2
2	INPcex+INPscFv colony7
3	INPcex+INPscFv colony19
4	INPcex+INPscFv colony9
5	INPcex+INPscFv colony17
6	INPcex+INPscFv colony10
7	INPcex+INPscFv colony11
8	INPcex+INPscFv colony12
9	INPcex+INPscFv colony15
10	INPcex+INPscFv colony16
11	INPcex+INPscFv colony23
12	INPcex+INPscFv colony24
13	INPcex+INPscFv colony25
14	INPcex+INPscFv colony26
15	INPscFv /5
16	INPcex /5
17	1kbp ladder

10/13

Electrophoresis

Yamada

Lane	sample/(µl)	Length(bp)
A1	INPcex+INPscFv colony2
A2	INPcex+INPscFv colony7
A3	INPcex+INPscFv colony19
A4	INPcex+INPscFv colony9
A5	INPcex+INPscFv colony17
A6	INPscFv /5
A7	INPcex /5
A8	1kbp ladder /5
B1	INPcex+INPscFv colony10
B2	INPcex+INPscFv colony11
B3	INPcex+INPscFv colony12
B4	INPcex+INPscFv colony13
B5	INPcex+INPscFv colony14
B6	1kbp ladder /5

10/13

Electrophoresis

Uchino

Lane	sample/(µl)	Length(bp)
1	1kbp ladder
2	10/13 1A
3	10/13 2A
4	10/13 3A
5	10/13 4A
6	10/13 5A
7	10/13 1B
8	10/13 2B
9	10/13 3B
10	10/13 4B
11	10/15 5B

10/13

Gel Extraction

Uchino

Lane	Restriction Enzyme Digestion Product	Volume/(µl)
1A+2A	10/13 PCR purification INP His coding
3A~5A	10/13 midiprep INP His CBDcex
1B+2B	10/13 PCR purification INP His12
3B~5B	10/13 midiprep INP His CBDcex

10/13

Fluorescence Microscopy

Uchino

sample	number	magnification
INPclos	13318	×200
INPclos	13319~13328	×600
INP CTL	13329	×200
INP CTL	13330~13338	×600
cellulose only	13339	×200
cellulose only	13340,13343~13351	×600
cellulose only	13341,13342	×1000

without filter

10/13

Fluorescence Microscopy

Uchino

E.coli	OD600	cellulose	weight	filter
-	-	powder	10	non-filtered
INPNC-His-ctl	0.2	powder	10	non-filtered
INPNC-His-CBDcex	0.2	powder	10	non-filtered
-	-	powder	10	filtered
INPNC-His-ctl	0.2	powder	10	filtered
INPNC-His-CBDcex	0.2	powder	10	filtered

10/13

Fluorescence Microscopy

Uchino

sample	number	magnification
INPcex	13353	×200
INPcex	13354~13356,13364,13366	×600
INPcex	13357,13365	×1000
INP CTL	13367	×200
INP CTL	13368~13378	×600
INP CTL	13379	×1000
cellulose only	13380,13381	×200
cellulose only	13382~13391	×600
cellulose only

with filter

10/13

Fluorescence Microscopy

Uchino

sample	number	magnification
INPcex	13392~98	×200
INPcex	13399~13408	×600
INP CTL	13309~13419	×200
bemliese	13420~13430	×200

with bemliese

10/14

Electrophoresis

Yamamoto

Lane	sample/(µl)	Length(bp)
1	1kbp ladder /3
2	10/13 1A /25
4	10/13 2A /25
5	10/13 3A /25
6	10/13 4A /25
7	10/13 5A /25
8	10/13 1B /25
9	10/13 2B /25
10	10/13 3B /25
11	10/13 4B /25
12	10/13 5B /25

with bemliese

10/14

SEM(CBD)

Miyazaki

sample	membrane shape
INPcex + cellulose	square
INP CTL + cellulose	triangle
cellulose only	pentagon

10/14

Electrophoresis

Uchino

Lane	sample/(µl)	Length(bp)
1	10/14 gel extract e2 /5

10/14

Gel Extraction

Uchino

Lane	Restriction Enzyme Digestion Product	Volume/(µl)
10/14 electrophoresis 1B longer + 2B longer e1	10/13 restriction enzyme design INP His 12
10/14 electrophoresis 1B longer + 2B longer e2	10/13 restriction enzyme design INP His12

10/14

Electrophoresis

Uchino

Lane	sample/(µl)	Length(bp)
1	1kbp ladder /3
2	1A + 2A /3
3	3A + 4A + 5A /3

10/14

Gel Extraction

Uchino

Lane	Restriction Enzyme Digestion Product	Volume/(µl)
10/14 electrophoresis 1B + 2B	10/13 restriction enzyme design INP His12
10/14 electrophoresis 1A + 2A	10/13 restriction enzyme design INP His coding
10/14 electrophoresis 3A + 4A + 5A	INP His CBDcex

10/14

Ligation

Uchino

sample1 1A + 2A /µl	sample2 3A~5A /µl	Ligation high ver.2	total
5	2.5	7.5	15
3.75	3.75	7.5	15

10/14

Transformation

Uchino

Sample Name	Sample Volume (µl)	Competent Cells/(µl)	Medium
INP His coding ligationA	2	DH5α /20	LB(CP)
INP His coding ligationA	4	DH5α /20	LB(CP)
INP His coding ligationB	2	DH5α /20	LB(CP)
INP His coding ligationB	4	DH5α /20	LB(CP)

10/15

PCR (Purification)

Sample Name/(µl)	Concentration(ng/µl)	260/280	260/230
10/15 INP-His coding i	207.4	1.87	2.11
10/15 INP-His coding G	174.7	1.88	1.09
10/15 INP-His coding E	175.5	1.08	2.10
10/15 INP-His coding M	212.9	1.88

10/15

CBD　Binding Assay

Ohara

sample	Buffer	OD
INP cex	CBC buffer	1.002
simultaneous expression	CBC buffer	0.994
INP CTL	CBC buffer	1.004
INP CTL	PBS	0.964
simultaneous expression	PBS

10/15

Restriction Enzyme Digestion

Sample	DNA/(µl)	EcoR1/(µl)	Pst1/(µl)	Buffer/(µl)	MilliQ/(µl)	Total/(µl)
10/15 PCR purification INP His coding i	29.0	2	2	6	21	60
10/15 PCR purification INP His coding G	34.3	2	2	6	15.7	60
10/15 PCR purification INP His coding E	34.2	2	2	6	15.8	60
10/15 PCR purification INP His coding M	28.2(6000ng)	2	2	6	21.8	60
10/13 midiprep INP-His-CBDcex	50	2	2	6	0	60
10/13 midiprep INP-His-CBDcex	50	2	2	6	0	60
10/13 midiprep INP-His-CBDcex	50	2	2	6	0	60
10/13 midiprep INP-His-CBDcex	50	2	2		0	60

1.5　Dr.Kitabatake
2.6 Agarose S ＋　Promega Gel Extraction
3.7 Agarose S ＋　Dr.Nasuda
4.8　stock

10/15

Gel strecking

Yamada

sample	null
INPNc-His-scFv	(CP)(LB)plate×3
INP-His-CBDcex

10/15

PCR (Target)

Uchino

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	Buffer/(µl)	MilliQ/(µl)	Total/(µl)
2/22 INP-His scFv colony3 /0.1	INP His coding Fw /0.75	INP His coding Rv /0.75	2×KAPA /12.5	10.9	25
2/22 INP-His scFv colony3 /0.1	INP His coding Fw /0.75	INP His coding Rv /0.75	2×KAPA /12.5	10.9	25
2/22 INP-His scFv colony3 /0.1	INP His coding Fw /0.75	INP His coding Rv /0.75	2×KAPA /12.5	10.9	25
2/22 INP-His scFv colony3 /0.1	INP His coding Fw /0.75	INP His coding Rv /0.75	2×KAPA /12.5	10.9	25
2/22 INP-His scFv colony3 /0.1	INP His coding Fw /0.75	INP His coding Rv /0.75	2×KAPA /12.5	10.9	25
2/22 INP-His scFv colony3 /0.1	INP His coding Fw /0.75	INP His coding Rv /0.75	2×KAPA /12.5	10.9	25
2/22 INP-His scFv colony3 /0.1	INP His coding Fw /0.75	INP His coding Rv /0.75	2×KAPA /12.5	10.9	25
2/22 INP-His scFv colony3 /0.1	INP His coding Fw /0.75	INP His coding Rv /0.75	2×KAPA /12.5	10.9	25
2/22 INP-His scFv colony3 /0.1	INP His coding Fw /0.75	INP His coding Rv /0.75	2×KAPA /12.5	10.9	25
2/22 INP-His scFv colony3 /0.1	INP His coding Fw /0.75	INP His coding Rv /0.75	2×KAPA /12.5	10.9	25
2/22 INP-His scFv colony3 /0.1	INP His coding Fw /0.75	INP His coding Rv /0.75	2×KAPA /12.5	10.9	25
2/22 INP-His scFv colony3 /0.1	INP His coding Fw /0.75	INP His coding Rv /0.75	2×KAPA /12.5	10.9	25
2/22 INP-His scFv colony3 /0.1	INP His coding Fw /0.75	INP His coding Rv /0.75	2×KAPA /12.5	10.9	25
2/22 INP-His scFv colony3 /0.1	INP His coding Fw /0.75	INP His coding Rv /0.75	2×KAPA /12.5	10.9	25
2/22 INP-His scFv colony3 /0.1	INP His coding Fw /0.75	INP His coding Rv /0.75	2×KAPA /12.5	10.9	25
2/22 INP-His scFv colony3 /0.1	INP His coding Fw /0.75	INP His coding Rv /0.75	2×KAPA /12.5	10.9	25

10/15

Colony PCR (typeA)

Yamamoto

Templates/(µl)	Primers/(µl)	MilliQ/(µl)	PreMix/(µl)	Total/(µl)
A1 10/14 INPcex + INPscFv colony1	VF/1.25 VR/1.25	8.75	KAPA2G /12.5 DMSO/1.25	25
A2 10/14 INPcex + INPscFv colony2	VF/1.25 VR/1.25	8.75	KAPA2G /12.5 DMSO/1.25	25
A3 10/14 INPcex + INPscFv colony3	VF/1.25 VR/1.25	8.75	KAPA2G /12.5 DMSO/1.25	25
A4 10/14 INPcex + INPscFv colony4	VF/1.25 VR/1.25	8.75	KAPA2G /12.5 DMSO/1.25	25

10/15

Western Blotting

Ohara

lane	sample
Marker	M.Marker
1	BBa_K875004(PC) Lpp-OmpA-scFv-6His 43kDa
2	INPNC-6His-CBDcex 47kDa
3	INPNC-6His-scFv 63kDa
4	INPNC-6His-CBDcex/INPNC-6His-scFv
5	BBa_K165002(NC)

Used pelet from 500 µl overnight culture, adjested OD600 to 3.1 .

Transfar ;80 min 200 mA(CC)
Blocking ;O/N
1’antibody ;anti-6His(mouse)
1:1000 with blocking buffer 1h
2’antibody ;anti-mouse HRP　1:2500 with TBST 1h
Detection time　900 sec
(ECL Prime)

10/16

Electrophoresis

Yamamoto

Lane	sample/(µl)	Length(bp)
1kb ladder
10/15 colony PCR colony 1	3
10/15 colony PCR colony 2	25
10/15 colony PCR colony 3	25
10/15 colony PCR colony 4

10/16

Cellulose binding Assay

Uchino

Cellulose/(mg)	E.coli/(mg)	buffer
1	cex/1	carbonate-bicarbonate buffer
1	ctl/1	carbonate-bicarbonate buffer
0.5	cex/1	carbonate-bicarbonate buffer
0.5	ctl/1	carbonate-bicarbonate buffer
0.5	CBC/1	carbonate-bicarbonate buffer
0.1	cex/1	carbonate-bicarbonate buffer
0.1	ctl/1

RT,-3h 500rpm 15:45~21:15 binding
fix with 4×1/100 PFA 10min→centrifuge→PBS→centrifuge
→PBS→centrifuge→PBS→centrifuge→DAP1→centrifuge
→PBS→centrifuge→PBS→through filter→PBS into filter
→centrifuge→PBSinto filter→centrifuge
→suspend with PBS→centrifuge
→suspend with?discoloration inhibitor

10/16

Dual Display Assay for Electron Microscope

E.coli	Cellulose	VLP	buffer
A.Dual 1mL	1mg	10µL	PBS
B.Dual 1mL	1mg		PBS
C.Dual 1mL		10µL	PBS
D.Dual 1mL			PBS
E.INPctl	1mg	10µL	PBS
F.INPctl	1mg		PBS
G.INPctl		10µL	PBS
H.INPctl			PBS
I	1mg		PBS
J		10µL

RT,ih incubate,850rpm
Dul=1.020
ctl=1.056

10/16

Electrophoresis

Lane	sample/(µl)	Length(bp)
1	1 kb ladder	5
2	10/15 Digestion INP His Coding 2	30 /0.94 上0.04下0.10
3	10/15 Digestion INP His Coding 2	30 /0.94 上0.04下0.19
4	10/13 miniprep INP-His-CBDcex 6	30 /0.94 0.11
5	10/13 miniprep INP-His-CBDcex 6	30 /0.94

Uchino 16:08~
ABEF→centrifuge→resupend with PBS →centrifuge→through filter→resupend with PBS→through filter→centrifuge→respend with PBS→centrifuge→resupend with PBS→centrifuge→resupend with PBS
IJ　Michimori

10/16

Electrophoresis

Uchino

Lane	sample/(µl)	Length(bp)
1	1kb ladder
2	10/15 colony PCR colony1
3	10/15 colony PCR colony2
4	10/15 colony PCR colony3
5	10/15 colony PCR colony

10/16

PCR (Target)

Sassa

Templates/(µl)	Primer1/(µl)	Primer2/(µl)	Buffer/(µl)	MilliQ/(µl)	Any Other/(µl)	Total/(µl)
2/22 scFv col.3 0.1	VF 0.75	VR 0.75	KAPA HoFi 12.5	10.9		25
2/22 scFv col.3 0.1	VF 0.75	VR 0.75	KAPA HoFi 12.5	10.9

95℃ 5min
98℃ 20s×35
55℃ 15s ×35
72℃ 30s×35
4℃ infinity

10/16

Miniprep

Uchino, Yamamoto

DNA	Concentration/(µg/ml)	260/280	260/230
10/15 Gel Extraction INP His coding 長	9.6	1.88	0.27
10/15 Gel Extraction INP His coding 短	74.4	1.87	0.84
10/15 Gel Extraction INP His CBDcex	22.0	1.97	0.33
Insert MK	64.5	1.92	1.17
Vector MK	56.6	2.37

10/16

Ligation

Yamamoto

Insert	Vector	MilliQ	Ligation High
10/15 Insert MK/2µL	10/15 Gel Extract INP His CBDcex 5.5µl	0	7.5
10/15 Insert MK/1µL	10/15 Gel Extract INP His CBDcex 6µL	0	7.5
10/15 Insert MK/1µL	10/15 Gel Extract INP His CBDcex 0.5µL	6	7.5
10/15 Digestion INP His coding/5µL	10/15 Digestion INP His CBDcex 2.5µL	0	7.5
10/15 Digestion INP His coding/6µL	10/15 Digestion INP His CBDcex 1.5µL	0	7.5
10/15 Digestion INP His coding/4.5µL	10/15 Digestion INP His CBDcex 3µL	0	7.5
10/14Gel Extract INP His coding/5.5	10/15 Digestion INP His CBDcex 2µL	0	7.5
10/14Gel Extract INP His coding/6	10/15 Digestion INP His CBDcex 1.5µL	0	7.5
10/15 Digestion INP His coding 3.5µL	10/15 Digestion INP His CBDcex 4µL	0	7.5
10/15 Digestion INP His coding 2.5µL	10/15 Digestion INP His CBDcex 5µL	0	7.5
10/15 Digestion INP His coding 3.75	10/15 Digestion INP His CBDcex 3.75	0

10/16

Transformation

Yamamoto

Sample Name	Sample Volume (µl)	Competent Cells/(µl)
10/15Ligation Insert MK＋Gel Extract1 2µL	2	DH5α/2
10/15Ligation Insert MK＋Gel Extract2 2µL	2	DH5α/2
10/15Ligation Digestion＋Digestion3 2µL	2	DH5α/2
10/15Ligation Digestion＋Digestion4 2µL	2	DH5α/2
10/15Ligation Digestion＋Digestion5 2µL	2	DH5α/2
10/15Ligation Digestion＋Digestion6 2µL	2	DH5α/2
10/15Ligation Gel Extract＋Digestion7 2µL	2	DH5α/2
10/15Ligation Gel Extract＋Digestion8 2µL	2	DH5α/2
10/15Ligation Digestion＋Digestion9 2µL	2	DH5α/2
10/15Ligation Digestion＋Digestion10 2µL	2	DH5α/2
10/15Ligation Digestion＋Digestion11 2µL	2	DH5α/2
10/14 Ligation PuroductA 2µL	2	DH5α/2
10/14 Ligation PuroductB 2µL	2

10/16

PCR (Purification)

Yamamoto

Sample Name/(µl)	Concentration(ng/µl)	260/280	260/230
10/15 PCR scFv INP His codingFw＋INP His RV	284.4	1.88

10/16

Liquid Culture

Yamamoto

Name	medium
10/14 Tranceformation INPHis coding2 colony1	LB(CP)
10/14 Tranceformation INPHis coding2 colony2

10/16

non-woven felt

Ohara

sample	null
cex	13431×200
	32~41×600
ctl	13431×200

10/16

Liquid Culture

Yamamoto

Name	medium
10/14 Trance formation INP His coding 2 colony1	LB(CP)
10/14 Trance formation INP His coding 2 colony2

10/16

fluorescence microscope assay

Uchino

sample	number	magnification
cex bemliese	13431	×200
cex bemliese	13432~13441	×600
CTL bemliese	13442	×200
CTL bemliese	13443~13355	×600

10/16

Colony PCR

Matsumoto, Uchino

number	sample	Any others	Any others(µL)
1~5	10/15Ligation Insert MK＋Gel Extract1	2×Ready Mix	12.5
6~10	10/15Ligation Insert MK＋Gel Extract2	Fwd primer	1.25
11~15	10/15Ligation Insert MK＋Gel Extract3	Rev primer	1.25
16~20	10/15Ligation Digestion＋Digestion4	MilliQ	10
21~24	10/15Ligation Digestion＋Digestion5		52×mix
25~28	10/15Ligation Digestion＋Digestion6	2×Ready	650
29~32	10/15Ligation Gel Extract＋Digestion7	Fwd	65
33~36	10/15Ligation Gel Extract＋Digestion8	Rev	65
37~40	10/15Ligation Digestion＋Digestion9	MilliQ
41~44	10/15Ligation Digestion＋Digestion10
45~48	10/15Ligation Digestion＋Digestion11

×16
16Colony Picking　DMSO 0.75 12.75 12
Mix 12.5 212.5 200
Fwd 1.25 21.25 18.75 20
Rev 1.25 21.25 18.75 20
MilliQ 9.25 157.25 138.75 148
16sample colony1~14・・・P15 sample colony
Blue　　　　　　　15・・・2/8 INP-His9
16・・・8/1 miniprep INPcex colony

10/16

Electrophoresis

Uchino

Lane	sample/(µl)	Length(bp)
1	1kb ladder
2	1
3	2
4	3
5	4
6	5
7	6
8	7
9	8
10	9
11
12

10/16

Electrophoresis

Uchino

Lane	sample/(µl)	Length(bp)
1
2	1kb ladder
3	11
4	12
5	13
6	14
7	15
8	16
9	17
10	18
11	19
12	20

10/16

Fluorescence Microscopy

Uchino

E.coli	OD600	cellulose	size / weight	filter
-	-	bemliese	5mm×5mm	-
INPNC-His-ctl	1	bemliese	5mm×5mm	-
INPNC-His-CBDcex	1	bemliese	5mm×5mm	-
-	-	powder	1	filtered
INPNC-His-ctl	1	powder	1	filtered
INPNC-His-CBDcex	1	powder	1	filtered
-	-	powder	0.5	filtered
INPNC-His-ctl	1	powder	0.5	filtered
INPNC-His-CBDcex	1	powder	0.5	filtered
-	-	powder	0.1	filtered
INPNC-His-ctl	1	powder	0.1	filtered
INPNC-His-CBDcex	1	powder	0.1	filtered

10/16

fluorescence microscope assay

Uchino

sample	number	magnification
cex 1mg	13456	×200
cex 1mg	13457~13466	×600
cex 0.5mg	13467	×200
cex 0.5mg	13468~13378	×600
cex 0.5mg	13379	×1000
CTL 1mg	13380	×200
CTL 1mg	13381~13390	×600
CTL 0.5mg	13391	×200
CTL 0.5mg	13392~13401	×600
CTL 0.5mg	13402	×1000
cex 0.1mg	13403	×200
cex 0.1mg	13404,13407~13416	×600
cex 0.1mg	13405,13406	×1000
CTL 0.1mg	13417	×200
CTL 0.1mg	13418~13430	×600
CTL 0.1mg	13431	×200
cellulose 0.5mg	13432~13441	×600

10/17

Electrophoresis

Uchino

Lane	sample/(µl)	Length(bp)
1	21
2	22
3	1kb ladder
4	23
5	24
6	25
7	26
8	27
9	28
10	29
11	30
12

10/17

Electrophoresis

Uchino

Lane	sample/(µl)	Length(bp)
1	1kb ladder
2	31
3	32
4	33
5	34
6	35

10/17

Electrophoresis

Lane	sample/(µl)	Length(bp)
1	36
2	37
3	38
4	39
5	40
6	1kb ladder

10/17

Electrophoresis

Uchino

Lane	sample/(µl)	Length(bp)
1	1kb ladder
2	41
3	42
4	43
5	44
6	45

10/17

Electrophoresis

Uchino

Lane	sample/(µl)	Length(bp)
1
2	1kb ladder
3	45
4	46
5	47
6	48

10/17

Liquid Culture

Yamamoto

Name	medium
colony 1,2,3,4,5,11,12,13,14,15,18,19,22,24,26,30

total 16mL

10/17

Miniprep

Uchino

DNA	Concentration/(µg/ml)	260/280	260/230
1	65.8	1.91
2	63.2	1.90	1.84
3	84.6	1.87	1.33
4	100.8	1.92	1.67
5	84.6	1.89	1.59
11	89.3	1.84	1.43
12	87.3	1.92	1.88
13	88.0	1.89	1.59
14	79.8	1.91	1.72
15	83.3	1.90	1.82
18	102.5	1.88	1.50
19	114.9	1.91	1.59
22	109.9	1.78	1.14
24	80.4	1.93	2.00
26	64.6	1.93	1.84
30	88.8	1.80	1.07

total 16mL

10/17

Colony PCR (typeA)

Yamada

Templates/(µl)	Primers/(µl)	MilliQ/(µl)	PreMix/(µl)	Total/(µl)
10/15gel plated colony 1	VF1.25 VR1.25	9.25	KapaTag/12.5 DMSO 0.75	25
10/15gel plated colony	VF1.25 VR1.25	9.25	KapaTag/12.5 DMSO 0.75	25
10/15gel plated colony	VF1.25 VR1.25	9.25	KapaTag/12.5 DMSO 0.75	25
10/15gel plated colony	VF1.25 VR1.25	9.25	KapaTag/12.5 DMSO 0.75	25
10/15gel plated colony	VF1.25 VR1.25	9.25	KapaTag/12.5 DMSO 0.75	25
10/15gel plated colony	VF1.25 VR1.25	9.25	KapaTag/12.5 DMSO 0.75	25
10/15gel plated colony	VF1.25 VR1.25	9.25	KapaTag/12.5 DMSO 0.75	25
10/15gel plated colony	VF1.25 VR1.25	9.25	KapaTag/12.5 DMSO 0.75	25
10/15gel plated colony	VF1.25 VR1.25	9.25	KapaTag/12.5 DMSO 0.75	25
10/15gel plated colony	VF1.25 VR1.25	9.25	KapaTag/12.5 DMSO 0.75	25
10/15gel plated colony	VF1.25 VR1.25	9.25	KapaTag/12.5 DMSO 0.75	25
10/15gel plated colony	VF1.25 VR1.25	9.25	KapaTag/12.5 DMSO 0.75	25
10/15gel plated colony	VF1.25 VR1.25	9.25	KapaTag/12.5 DMSO 0.75	25
10/15gel plated colony	VF1.25 VR1.25	9.25	KapaTag/12.5 DMSO 0.75	25
INPNC-His-scFv	VF1.25 VR1.25	9.25	KapaTag/12.5 DMSO 0.75	25
INP-His-CBDcex	VF1.25 VR1.25	9.25	KapaTag/12.5 DMSO 0.75	25

VF 1.25×8=10µL
VR 1.25×8=10µL
Kapa 12.5×8=100µL
DM80 0.75×8=6µL
MilliQ 9.25×8=74µL
Total 200µL

10/17

CBD Binding Assay

Uchino

Dual 1mL OD=1.0	bemlise 5mm×5mm	VLP Narita104 10µL
Dual 1mL OD=1.0	bemlise 5mm×5mm
ctll 1mL OD=1.0	bemlise 5mm×5mm
ctl 1mL OD=1.0	bemlise 5mm×5mm

10/18

Fluorescence Microscopy

Uchino

E.coli	OD600	cellulose	size	filter
-	-	bemliese	5mm×5mm	-