August 2

Mutation of FT

by Sato
FT gene has two BioBrick restriction enzyme sites, EcoR1 and Pst1 which is next to each other.
So we tried to delete both at once by using two primers with mutation.

Inverse PCR

10xBufer	2mM dNTPs	primer fwd	primer rev	template	polymerase	MilliQ	Total
5	5	1.5	1.5	0.5	1	35.5	50

94℃ 2min, (98℃ 10sec, 68℃ 4min)x4cycles, 4℃ Hold

Dpn1 Digestion

PCR product	Dpn1
50	2

37℃,1h incubate

Self-ligation

product	MilliQ	Ligase	T4 Kinase	Total
2	7	5	1	15

16℃, 1h incubate

Transformation

competent cell	DNA
20	2

Cells were stored on ice for 30min.
After 42℃ 60sec heat shock, cells were stored on ice for 2min.
Then cells were precultureed at 37℃ for 1hr, plated to Kanamycin plate.

August 13

Liquid culture

FT at 37°C, for overnight.

August 14

Miniprep of FT

by Sato, Takeuchi
The concentration was 81.5ng/uL

Restriction digestion and Electrophoresis

by Sato
To check wheter mutation was succeed, we did restriction enzyme digestion.

DNA(FT,80ng/uL)	10xBuferH	EcoR1	Pst1	MilliQ	Total
5	2	1	-	12	20
5	2	-	1	12	20

37°C 2h incubate
We did electrophoresis of three FT plasmids, non-cutted, EcoR1 cutted, Pst1 cutted.
However, we couldn't get any bands (data not shown.)

Liquid culture

FT (4mL)

August 15

Miniprep of FT

by Sato, Takeuchi, Hyungcheol
We couldn't get enough concentration of plasmids.

Electrophoresis

by Sato
We retried electrophoresis of three samples same as yesterday.
However, we couldn't get any bands as well.

August 16

Transformation

by Takeuchi, Ota

Name	Well	Sample	Competent Cells	Total	Plate	Colony
FT	-	1 µL	10	11	LB (Kan+)	×
pSB1C3	1-3-A	1	10	11	LB (CP+)	○
I719005	1-15-N	1	10	11	LB (Amp+)	○

August 17

Transformation

by Takeuchi

Name	Well	Sample	Competent Cells	MilliQ	Total	Plate	Colony
FT	-	2	2	18	22	LB (Kan+)	×

We found that mutation of FT was not successful.

August 20

We decided to do PCR using FT specific primers before mutation.

PCR of FT

by Sato

10xBufer	dNTPs	MgSO4	primer fwd	primer rev	template	polymerase	MilliQ	Total
5	5	3	1	1	1(130ng/µL)	1(KOD plus neo)	33	50

94°C 2min, (98°C 10sec, 68°C 15sec)x30cycles, 4°C Hold
After the ethanol precipitation, we diluted in 30µL of MilliQ.
The concentration was 203ng/µL.

====Electrophoresis====

Lane	Name	length(bp)
1	1kb ladder	-
2	FT	600

August 21

Restriction digestion

by Sato

DNA(FT,203ng/µL)	10xBuferM	Xba11	Pst1	MilliQ	Total
10	4	1	1	24	40

37°C, 5h incubate
After the ethanol precipitation, we diluted in 30µL of MilliQ.
The concentration was 54,9ng/µL.

Ligation

by Sato

Vector		Insert		Ligation High Ver.2
pSB1C3	1	FT	10	5.5

Liquid culture

T7 promoter, pSB1C3 (4mL)

August 22

Miniprep

by Sato

T7 promoter	pSB1C3
85.3ng/µL	82.93ng/µL

August 23

Ethanol Precipitation

diluted in 20µL 79.3ng/µL

mada dekite nai

August 24

Restriction enzyme processing

T7 promoter(85.3ng/µL)	Spel	Pstl	buffer M	MiliQ	Total
10	1	1	2	6	20

->purifying column 33.4ng/µL(dissolution 40µL)

pSB1C3(82.9ng/µL)	Xbal	Spel	buffer M	MiliQ	Total
20	1	1	4	14	40

->gene clean2 39.9ng/µL(dissolution 40µL)

<<picture1,2>>

Ligation

FT(600bp, 79.3ng/µL)	pSB1C3(2000bp,39.9ng/µL)	Ligation High Ver.2
3µL => 597fmol	2µL => 60fmol	2.5µL

FT(600bp, 79.3ng/µL)	T7(2100bp,33.4ng/µL)	Ligation High Ver.2
2.4µL => 478fmol	2µL => 48fmol	2.2µL

=> 16℃,1hr incubate

August 27

Colony PCR

2X Quick Tag	VF2	VR	MiliQ	Total
25	1	1	23	50

Lane1: 1kb ladder
Lane2~16: FT(pSB1C3) about 800bp

FT(TOPO) PCR(re)

buffer	dNTPs	MgSO4	primer f	primer r	Template(130ng/µL)	KODplus neo	MilliQ	Total
5	5	3	1	1	1	1	33	50

94℃	98℃	68℃	cycles
2min	10sec	15sec	30

Liquid culture

by Nobeyama
FT 4ml

August 28

Mutation of FT (re)

inverse PCR

MilliQ	buffer	dNTP	primer f	primer r	FT(130ng/µL)	KODplus	Total
35.5	5	5	1.5	1.5	0.5	1	50

94℃	98℃	68℃	cycles
2min	10sec	4min	18

Lane1: 1kb ladder
Lane2: FT

====Miniprep FT(TOPO====)
158ng/µL

Tranformation

competent cell: 20
BBa.I746902  : 2
(plate 316f)
(GFP generator of pBAD/araC-mut3 GFP:6His-DT)

August 29

Mutaion of FT(re;re)

Inverse PCR

first

MilliQ	buffer	dNTP	primer f	primer r	FT(130ng/µL)	KODplus	Total
35.5	5	5	1.5	1.5	0.5	1	50

second

MilliQ	buffer	dNTP	primer f	primer r	FT(52ng/µL)	KODplus	Total
35	5	5	1.5	1.5	1	1	50

94℃	98℃	68℃	cycles
2min	10sec	4min	30

Lane1: 1kb ladder
Lane2: first Template 130ng/µL
Lane3: second Template 53ng/µL

first sample	Dpnl	total
45µL	2µL	47µL

in 37℃, 1 hour

Self-Ligation

PCR products	MilliQ	Ligation High	T4 kinase	total
2µL	7µL	5µL	1µL	15µL

in 16℃, 1.5hour incubate
Transformation
competent cell: 20
DNA  : 2

Liquid culture(I746902): 3mL

August 30

Liquid culture(FT) 4mL x2

August 31

Miniprep(FT)

(1) 64.9ng/µL
(2) 52.6ng/µL

Restriction enzyme processing (Mutation checking)

FT(52.6ng/µL)	bufferH	E.coli	Pst1	MilliQ	total
1µL	5µL	0.5µL	0.5µL	3.5µL	10µL

in 37℃,1.5hour

PCR(RBS primer)

buffer for KODplus neo	dNTPs	MgSO4	primer f	primer r	Template(52.6ng/µL)	KODplus neo	MilliQ	Total
5	5	3	1	1	1	1	33	50

94℃	98℃	68℃	cycles
2min	10sec	15sec	30

Lane1: 1kb ladder
Lane2: FT
Lane3: FT(E.coli)
Lane4: FT(Pst1)
Lane5: FT PCR

PCR(re)

buffer	dNTPs	MgSO4	primer f	primer r	Template	KOD neo	MilliQ	Total
5	5	3	1	1	1	1	33	50

94℃	98℃	68℃	cycles
2min	10sec	15sec	35

Lane1: 1kb ladder
Lane2: FT

PCR(re)

buffer	dNTPs	MgSO4	primer f	primer r	Template	KOD neo	MilliQ	Total
5	5	2	1	1	1	1	34	50

94℃	98℃	68℃	cycles
2min	10sec	10sec	30

September 2

PCR(re;re)

first

buffer	dNTPs	MgSO4	primer f	primer r	Template(1ng/µL)	KODplus neo	MilliQ	Total
5	5	3	1	1	1	1	33	50

second

buffer	dNTPs	MgSO4	primer f	primer r	Template(10ng/µL)	KODplus neo	MilliQ	Total
5	5	3	1	1	1	1	33	50

94℃	98℃	68℃	cycles
2min	10sec	10sec	25

Lane1: first Template 1ng
Lane2: second Template 10ng
Lane3: 1kb ladder

refine first Template => 132ng/µL

September 3

Restriction enzyme processing

FT(132ng/µL)	bufferM	EcoRI	SpeI	MilliQ	Total
10	2	1	1	6	20

37℃,overnight => refinement 31.6ng/µL (Elution 40µL)

Ligation
Insert(FT: 31.6ng/µL, 600bp ): 2µL = 26fmol
Vector(DT: 28.0ng/µL, 3300bp): 3µL = 240fmol
Ligation High ver.2  :2.5µL
=> 16℃, 2 hours

Transformation

competent cell	DNA	plate	colony
20µL	FT-DT 2µL	Amp	o
20µL	pT7-6His:R9 2µL	Amp	o
10µL	GFP generator 1µL	Amp	o

(BBa,I746915,pT7-6His:GFP)

September 4

Colony PCR

Quick Tag	VF2	VR	MilliQ	Total
25	1	1	23	50

94℃	98℃	55℃	68℃	cycles
2min	30sec	30sec	1min	25

Lane1: 1kb ladder
Lane2: FT-DT(about 700bp)

Liquid culture(4ml) 23:30 ~
GFP generator, FT-DT

September 5

Miniprep(FT-DT) by Sato
88.8ng/µL

Restriction enzyme processing

FT-DT(88.8ng/µL)	XbaI	PstI	bufferM	MiliiQ	total
20	1	1	4	14	40

at 37℃, 2 hours

Electrophoresis by Takeuchi

Restriction enzyme processingby Takeuchi

FT-DT(88.8ng/µL)	bufferM	XbaI/Spe	MiliiQ	total
4	1	0.5	4.5	10

FT-DT	bufferH	Pst/Eco	MiliiQ	total
4	1	0.5	4.5	10

at 37℃,1hour 10min

September 6

Western blotting(BBa,I746915)

Sample making
SOC(ampt) 50ml + pre culture 1ml x2
OD600 = 0.5~0.7 incubate at 37℃ (OD 0.642)
add IPTG final concentration is 1mM (negative control)
incubate at 37℃,4 hours

SDS-PAGE
Do spin down E.coli and make suspension put E.coli into 1mL 1x sample buffer
95℃,10min
electrophoresis at 500V, 30mA, 50min
Lane1: 10µL (IPTG -)
Lane2: 10µL (IPTG -)
Lane3: 10µL (IPTG +)
Lane4: 5µL (IPTG -)
Lane5: 5µL (IPTG +)
Lane6: 2µL (IPTG -)
Lane7: 2µL (IPTG +)

Blotting at 50V,100mA,30min Put into blocking buffer and vibrating 30min
Incubate with Anti GFP(1/1000) 10mL at RT,1h
Washing with 10mL TBST (vibrating 10min x2)
Incubate with Anti-mouseAP(1/1000) 10mL at RT, 30min
Washing with 10mL TBST (vibrating 10min x3)
Put NBT,BCIP into dye buffer

September 9

Mutaion of FT

MilliQ	buffer	dNTP	primer f	primer r	FT(52ng/µL)	KODplus	Total
35	5	5	1.5	1.5	1	1	50

94℃	98℃	68℃	cycles
2min	10sec	4min	20

→　GeneClean II　32.6ng/µL

Dpn1 processing

TA buffer	DNA	Dpn1	Total
3.3	31	2	36.3

Ligation

DNA	MiliiQ	Ligation high Ver.2	T4 kinase	Total
2	7	5	1	15

Transformation competent cell: 10
DNA  : 1

September 10

Miniprep of FT ①116.9ng/µL
② 34.5ng/µL
Restriction enzyme processing(Mutation checking)

FT①/②	bufferHl	EcoRI	PstI	MiliQ	Total
4	1	0.5	0	4	10
4	1	0	0.5	4	10

PCR

Bufer	dNTPs	MgSO4	primer(+/-RBS)fwd	primer(+/-RBS)rev	template①/②	KOD plus neo	MilliQ	Total
5	5	3	1	1	1	1	33	50

94℃	98℃	68℃	cycles
2min	10sec	10sec	25

->purifying column 17.4ng/µL

Restriction enzyme processing

FT(RBS+)32.6ng/µL	Xbal	Pstl	buffer M	BSA	Total
30	1	1	4	4	40

T7-His:R9(62.6ng/µL)	SpeI	Pstl	MilliQ	buffer M	Total
15	0.5	0.5	2	2	20

incubate at 37℃, 3hours
->purifying column 17.4ng/µL

September 11

Ligation
Vector(T7: 33.4ng/µL, 2100bp): 1µL = 24fmol
Insert(FT: 17.4ng/µL, 600bp ): 5µL = 217fmol
Ligation High ver.2  : 3µL
=> 16℃, 1 hours

Transformation

competent cell	DNA
20µL	T7-FT 2µL

PCR(FT without RBS retry)

Bufer	dNTPs	MgSO4	primer(-RBS)fwd	primer(-RBS)rev	template	KOD plus	MilliQ	Total
5	5	3	1	1	1	1	33	50

94℃	98℃	65℃	68℃	cycles
2min	15sec	30sec	30sec	25

Lane1:FT(RBS-) 600bp
Lane2:Ladder 100bp

えいどうしゃしん

=>purifying column 34.6ng/µL

Restriction enzyme processing

FT(RBS-)	XbaI	PstI	bufferM	BSA	total
30	1	1	4	4	40

at 37℃, 2 hours
=> 6.2ng/μL

GFP-DT	XbaI	PstI	bufferM	BSA	MilliQ	total
15	1	1	3	3	7	30

at 37℃, 2 hours
=> 7.0ng/μL

Ligation
Vector(T7-6His-R9: 11.6ng/µL, 2100bp): 1µL = 9fmol
Insert(FT: 6.2ng/µL, 600bp ): 5µL = 79fmol
Ligation High ver.2  : 3µL
=> 16℃, 1 hours

Vector(T7-6His-R9: 11.6ng/µL, 2100bp): 1µL = 9fmol
Insert(GFP-DT: 7.0ng/µL, 1000bp ): 9µL = 95fmol
Ligation High ver.2  : 4µL
=> 16℃, 1 hours

September 12

Transformation

competent cell	DNA	plate
20µL	T7-R9-GFP-DT 2µL	Amp
20µL	T7-R9-FT 2µL	Amp
BL21CDE3 10µL	T7-FT 1µL	Amp

Miniprep (T7-FT)
37.1ng/µL

September 13

Miniprep
①T7-R9-GFP-DT 90ng/µL
②T7-R9-FT 160ng/μL

Restriction enzyme processing

T7-R9-GFP-DT (90ng/µL)	EcoRI	PstI	buffer H	MilliQ	total
20	1	1	3	5	30

at 37℃, 2 hours
=> 20.7ng/μL

T7-R9-FT (160ng/µL)	EcoRI	SpeI	buffer M	MilliQ	total
20	1	1	3	5	30

at 37℃, 2 hours
=> 20.1ng/μL

Transformation

competent cell BL21(DE3)	DNA	plate
10µL	T7-R9-GFP-DT 1µL	Amp
10µL	T7-R9-FT 1µL	Amp

Restriction enzyme processing

Buffer H	BSA	EcoRI	PstI	DpnⅠ	MilliQ	total
5	5	0.5	0.5	0.5	13.5	25

=>We define this solution "2× Master Mix"

2× Master Mix	pSB1C3(Linerarized Plasmid Backbone)
4	4

at 37℃, 30 minutes
80℃, 30 minutes

PCR(Insert His-tag)

MilliQ	Buffer for iPCR	dNTPs	primer fwd	primer rev	template(T7-FT 37.1ng/μL)	KOD plus	Total
35	5	5	1.5	1.5	1	1	50

94℃	94℃	56℃	68℃	cycles
2min	15sec	30sec	2.5min	15

Lane1:Ladder 1kb
Lane2:T7-6His:FT about 2700bp

えいどうしゃしん

September 14

Ligation
Vector(pSB1C3: 12.5/µL, 2000bp)  : 2µL = 19fmol
Insert(T7-R9-GFP-DT: 20.7ng/µL, 700bp ): 4µL = 180fmol
Ligation High ver.2  : 3µL
=> 16℃, 1 hours

Dpn1 processing

PCR products(9/13)	Dpn1	Total
45	2	47

=>37℃, 1 hour

Self Ligation

PCR products	Ligation High	T4 Kinase	MilliQ	Total
2	5	1	7	15

=>16℃, 1 hour

Ligation
Vector(DT: 17.1/µL, 2100bp)  : 2µL = 12fmol
Insert(T7-R9-FT: 20.1ng/µL, 650bp ): 5µL = 496fmol
Ligation High ver.2  : 3.5µL
=> 16℃, 1 hours

September 15

Restriction enzyme processing

FT without RBS (34.6ng/µL)	EcoRI	PstI	10× buffer H	MilliQ	total
10	0.5	0.5	2	7	20

Colony PCR

Quick Tag	VF	VR	MilliQ	Total
25	1	1	23	50

94℃	94℃	55℃	68℃	cycles
2min	30sec	30sec	1min	25

Electrophoresis
Lane1: ladder
Lane2: T7-R9-GFP-DT
Lane3: T7-R9-GFP-DT

Lane4: T7-R9-FT-DT
Lane5: T7-R9-FT-DT
Lane6: T7-R9-FT-DT
Lane7: T7-R9-FT-DT
Lane8: T7-R9-FT-DT
A member of secretion group electrophoresed DNA from Lane9 to Lane12.

Lane13: T7-His-FT
Lane14: T7-His-FT
Lane15: T7-His-FT
Lane16: T7-His-FT
Lane17: ladder

えいどうしゃしん

Liquid culture(3ml) 3:30~
T7-R9-FT-DT×2, T7-His-FT×2, T7-His-FT

September 16

Miniprep
①T7-R9-FT-DT 150ng/µL
②T7-R9-FT-DT 139ng/μL
③T7-R9-GFP-DT 67ng/µL
④T7-His-FT 153ng/μL
⑤T7-His-FT 73ng/μL

September 17

Purifying column
=>FT without RBS :36.4ng/µL

Ligation
Vector(PSB1C3: 12.5/µL, 2000bp)  : 3µL = 9fmol
Insert(FT without RBS: 36.4ng/µL, 600bp ): 3µL = 92fmol
Ligation High ver.2  : 3µL
=> 16℃, 1 hours

September 18

Transformation by NAKAGAWA

competent cell	DNA	plate
20µL	PSB1C3 FT without RBS 1µL	CP+

Verification of R9 function by TAKEUCHI

R9(20µg/µL)	0.9µL
GFP(1.2mg/mL)	2.23µL
RBS	16.85µL
total	20µL

X5

Method:
1. Peel cuticles on parafilm by using the head of pencil.(Menasha,wI,54952)
2. Put plant cells into GFP&R9 or GFP for 5~30min.
3. Put plant cells into PBS.
4. Hoechst dyeing.

	1	2	3	4	5	6
R9	o	o	o	x	o	o
cuticle	o	o	o	o	x	x
soak in GFP	5min	15min	30min	5min	5min	30min