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

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)

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

=>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

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

Electrophoresis

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

September 19

Transformation(re) by NAKAGAWA,TAKEUCHI
competent cell:20µL
DNA(No RBS FT):1µL
plate  :CP+

Transformation(re;re) by NAKAGAWA,TAKEUCHI
competent cell:10µL
DNA(No RBS FT):1µL
LB  :100µL
plate  :CP+

Adjusted GM Agar Medium making by TAKEUCHI

Ingredient of MS medium(SIGMA M5519): 0.88g
MES  : 0.1g
ion exchanged water  : 200mL
NaOH  : 26µL(adjust to pH5.6)
Agar medium  : 1.6g

Autoclave 120℃

Colony PCR(re)

gelA
Lane1: 100bp Ladder
Lane2: No RBS FT colony number 9
Lane3: No RBS FT colony number 10
Lane4: No RBS FT colony number 11
Lane5: No RBS FT colony number 12
Lane6: No RBS FT colony number 13
Lane7: empty
Lane8: empty

gelB
Lane1: 100bp Ladder
Lane2: No RBS FT colony number 14
Lane3: No RBS FT colony number 15
Lane4: No RBS FT colony number 16
Lane5: No RBS FT colony number 17
Lane6: No RBS FT colony number 18
Lane7: No RBS FT colony number 19
Lane8: empty

gelC
Lane1: 100bp Ladder
Lane2: No RBS FT colony number 20
Lane3: No RBS FT colony number 21
Lane4: No RBS FT colony number 22
Lane5: No RBS FT colony number 23
Lane6: No RBS FT colony number 24
Lane7: empty
Lane8: empty

September 20

Liquid culture by NAKAGAWA
No RBS FT x8 (by using September 18)
Plate: CP+

Colony PCR
No RBS FT by using September19

Lane1 : 100bp ladder
Lane2 : colony number 1
Lane3 : colony number 2
Lane4 : colony number 3
Lane5 : colony number 4
Lane6 : colony number 5
Lane7 : colony number 6
Lane8 : colony number 7
Lane9 : colony number 8
Lane10: empty
Lane11: empty
Lane12: 100bp ladder

Liquid culture
A-11, A-12

Miniprep No RBS FT(by using September20) 1.-10.4µg/mL
2.-4.7µg/mL
3.-3.6µg/mL
4.-7.6µg/mL
5.-4.5µg/mL
6.-8.4µg/mL
7. 1.8µg/mL(average)
8. 18.1µg/mL
Restriction enzyme processing

DNA(No RBS FT)x2	E.coli	BufferH	MilliQ	total
30µL	1µL	4µL	5µL	40µL

in 37℃,2hours

Electrophoresis
DNA(No RBS FT) sample7,8: 10µL
Loading Dye  : 2µL

Lane1: 1kb ladder
Lane2: empty
Lane3: No RBS FT(E) sample7
Lane4: empty
Lane5: No RBS FT(E) sample8
Lane6: empty

Electrophoresis(re)

RNA Extraction
5 leaves: 100mg
ISOGEN  : 1mL
Elution : 20µL

cDNA Synthesis(1/4)

gDNA wipeout buffer	template RNA	H2O	Total
1µL	0.5µL	5.5µL	7µL

at 42℃, 2min

Reverse Transcriptase	RT buffer	primer mix	template	Total
0.5µL	2µL	0.5µL	7µL	10µL

at 42℃, 2min and 95℃,3min

RT PCR

buffer	dNTPs	MgSO4	primer f	primer r	Template	KODplus	MilliQ	Total
5	5	2	1.5	1.5	1	1	34.5	50

94℃	94℃	54℃	68℃	cycles
2min	15sec	30sec	10sec	30

1.TUBULIN
2.FUL
3.SEP3
4.AP1

February 7

Preculture
We started preculture at 12:10.

February 8

Culture
We start culturing with 300[mL] of LB medium.

time	OD600
12:00	start
14:10	0.019
14:45	0.154
15:05	0.267
15:21	0.64

Making Competent Cell
We made competent cells.

Transformation
pGEM_TAP
Lacp (BBa_R0011)
DT (BBa_B0015)

Making Culture Medium Plates
We made 200mL of ampicillin culture, kanamycin culture, and chloramphenicol culture.

Transformation
GFP(BBa_E0040) in pSB1A2
DT(BBa_B0015) in pSB1AK3
ara(BBa_I0500) in pSB2K3
Lacp(BBa_R0011) in pSB1A2

February 9

transformation
BBa-E0040(GFP)(Mr.Fujita)

Liquid culture
DT.leap colony transformed on February 8
colony of competent cell made on February 8

February 10

Miniprep
DT2 43.9μg/ml(1.34 260/230 1.74 260/280)
lacp1 17.1μg/ml(1.54 260/280 0.83 260/230)
lacp2 18.0μg/ml(1.58 260/280 0.87 260/230)

transformation
B0040 1.4k PsB1A2 B0034 1.2M pSB1A2(from iGEM parts plate)

Competent cell
We did preculture for overnight. We put 1.5mL of preculture on 150mL of LB culture.

time	OD600
11:45	start
13:30	0.048
14:30	0.168
15:03	0.256
15:20	0.405
15:35	0.459
at last	0.576

February 11

Checking Transformation efficiency
Conpetent cell's transformation efficiency is 1.3x10^4colonys/μg

February 13

Transformation
Const promoter J23110,J23109,J23100

DNA	Competent cell	total
1μL	20	21

No colony was there on February 14

Liquid culture
lacP,DT,RBS(BBa_B0034),GFP
start at 20:00
in Plus grow with Ampicilin 3mL

February 14

Miniprep
concentration[μg/μL]

lacP3	39.6
lacP4	40.8
lacP5	28.9
RBS1	28.2
RBS2	57.4
RBS3	13.2
DT3	69.7
DT4	64.4
DT5	61.5
GFP1	64.0
GFP2	50.5
GFP3	66.0

Restriction
Const promoterJ23100

DNA	Spe1	Pst1	Buffer2	BSA	MilliQ	total
20	0.5	0.5	3	0.5	5.5	30

at 37℃ for overnight

February 15

Making gel
1% Agarose gel

Agarose	TAE
1.6g	160mL

Electrophoresis

Gel No.1

Restriction product	loading dye
5μL	1

The marker was 1kb ladder
It seemed that this restriction product was not cut.

Gel No.2
Lane1 : 1kb ladder
Lane2 : J23100 2μL + 6*Loading dye 1μL
Lane3 : J23100(Spe1,Pst1) 5μL
Lane4 : J23100(Spe1,Pst1) 2μL

• There were bands on lane_2 and we cannot identify these bands because the sample of lane_2 was not cut with any restriction enzyme.
  
• There must have been bands at 2100bp and 883bp on lane_3 and lane_4.
  
  

Testing whether restriction enzyme were deactivated or not

DNA(DT)	restriction enzyme	Buffer	BSA	MilliQ	total
10	0.5	3	0.5	16	30

at 37℃ for Oveernight
Restriction enzyme means Spe1(1,2) Pst1(1,2,3) in this time.

February 16

Electrophoresis

1. 1kb ladder
2. DT2
3. DT3
4. DT2 (Spe1-1)
5. DT2 (Spe1-2)
6. DT2 (Pst1-1)
7. DT2 (Pst1-2)
8. DT2 (Pst1-3)
9. DT3 (Pst1-4)
10. 1kb ladder

Pst1-1, Pst1-2, and Pst1-3 did not cut DNAs. They seemed to be deactivated.

Genomic PCR

10*Buffer for KOD Plus	2mM dNTPs	25mM MgSO4	10μM primer-f	10μM primer-r	158ng/μL Genomic DNA	KOD plus	MilliQ	total
5	5	3	1.5	1.5	1	1	32	50

Electrophoresis

1. 1kb ladder
2. tatABCD (2.5kb)
3. TAMO reductase (2.7kb)
4. Negative control
We got bands of tatABCD but there were nonspecific amplification products.
We failed amplification of TAMO reductase.







Transformation
Constitutive promoter (BBa_J23107 , BBA_J23117)
High copy plasmid (pSB1AT3)

DNA	competent cell
1μL	10μL

February 17

PCR
We did PCR to amplify products of PCR that we had done yesterday but we could not amplify tatABCD.

Genomic PCR

Buffer	dNTPs	MgSO4	primer-f	primer-r	genomic DNA	KOD plus	MilliQ	total
2.5	2.5	5	0.75	0.75	0.5	0.5	16	50

Predenature 94℃, 2min
Denature 98℃, 10sec
Annealing 57℃, 30sec
Extension 68℃, 2.5min
(30cycles)

Electrophoresis

1. 1kb ladder
2. TAMO reductase
3. Negative control

Restriction

J23100	Spe1	Pst1	Buffer2	BSA	MilliQ	total
10	0.5	0.5	3	0.5	15.5	30

Genomic PCR

	Buffer	dNTPs	MgSO4	primer-f	primer-r	genomic DNA	KOD plus	MilliQ	total
TMAO reductase	2.5	2.5	3	0.75	0.75	0.5	0.5	15.5	25
tatABCD	2.5	2.5	2	0.75	0.75	0.5	0.5	15.5	25
tatABCD	2.5	2.5	2	0.75	0.75	5	0.5	10.5	25

Electrophoresis

1. 1kb ladder
2.3. TAMO reductase
4. tatABCD
5. tatABCD(10 times amount of genome)

Checking of restriction enzyme

DT	enzyme	Buffer	BSA	MilliQ	total
2	0.5	3	0.5	24	30

at 37℃ for overnight
We checked EcoR1 and Xba1.

February 18

Miniprep

	μg/mL	260/280	230/260
JS3117-1	135	1.5	2.06
JS3117-2	75	1.6	1.63
JS3109-1	115	1.5	1.88
JS3109-2	75	1.65	1.71
pSB1AT3-1	70	1.66	1.83
pSB1AT3-2	100	1.52	1.54

diluted to 25 times

Competent cell
We put 3mL of preculture product on yesterday onto 300mL of LB medium

time	OD600
10:30	start
12:10	0.118
13:00	0.270
13:30	0.502

Transformation

pSB1AT3-2	competent cell	MilliQ	total
0	20	10	30
2	20	8	30
10	20	0	30

• Results(on Feb. 19)
  

pSB1AT3-2	number of colony
0	0
2	177
10	590

Transformation efficiency 7.4x10^4 colonys/μg

February 20

Restriction
sample 1

DT plasmid	EcoR1	Xba1	Buffer	BSA	MilliQ	total
7.5	0.5	0.5	3	0.5	18	30

sample2

GFP plasmid	EcoR1	Spe1	Buffer	BSA	MilliQ	total
10	0.5	0.5	3	0.5	15.5	30

Electrophoresis

• sample1
  

a. sample1 2μL + MilliQ 3μL + 6×Loading Dye 1μL
b. sample1 5μL + 6×Loading Dye 1μL
lane_1 1kb ladder
lane_2 a
lane_3 b
lane_4 a
lane_5 b
lane_6 a
lane_7 b
lane_8 1kb ladder

• sample2
  

c. sample2 2μL + MilliQ 3μL + 6×Loading Dye 1μL
d. sample2 5μL + 6×Loading Dye 1μL
lane_1 1kb ladder
lane_2 c
lane_3 d
lane_4 1kb ladder

PCR

	Buffer	dNTPs	MgSO4	primer-f	primer-r	genomic DNA	KOD plus	MilliQ	total
tatABCD1	2.5	2.5	1.5	0.75	0.75	0.5	0.5	16	25
tatABCD2	2.5	2.5	1.5	0.75	0.75	0.5	0.5	16	25
TMAO reductase1	2.5	2.5	2	0.75	0.75	0.5	0.5	15.5	25
TMAO reductase2	2.5	2.5	2	0.75	0.75	5	0.5	10.5	25

Predenature 94℃ 2min
Denature 98℃ 10sec
Annealing 59℃ 30sec
Extension 68℃ 2.5min
→30cycles

	Buffer	dNTPs	MgSO4	primer-f	primer-r	PCR products	genomic DNA	KOD plus	MilliQ	total
tatABCD1	2.5	2.5	1.5	0.75	0.75	0	0.5	0.5	16	25
tatABCD2	2.5	2.5	1.5	0.75	0.75	1	0	0.5	15.5	25
TMAO reductase1	2.5	2.5	2	0.75	0.75	0	0	0.5	16	25
TMAO reductase2	2.5	2.5	2	0.75	0.75	0	0	0.5	16	25

Predenature 94℃ 2min
Denature 98℃ 10sec
Annealing 59℃ 30sec
Extension 68℃ 2.5min
→35cycles

Electrophoresis

lane_1.1kb ladder
lane_2.tatABCD
lane_3.tatABCD
lane_4.TAMO1
lane_5.TAMO2
lane_6.1kb ladder

lane_1.1kb ladder
lane_2.tatABCD1
lane_3.tatABCD2
lane_4.TAMO
lane_5.TAMO
lane_6.1kb ladder

February 21

PCR (Advantage HF protocol)

	buffer	dNTPs	primer-f	primer-r	gDNA	PCR products	DW	polymerase	total
tatABCD	2.5	2.5	0.75	0.75	1	0	17	0.5	25
TMAO	2.5	2.5	0.75	0.75	0	1	17	0.5	25

Predenature 94℃ 1min
Denature 94℃ 30sec
Annealing 58℃ 30sec
Extension 68℃ 3min
→25cycles

Electrophoresis

1. 1kb ladder 2μL
2. tatABCD 5μL + 6×Loading Buffer 1μL
3. TAMO 5μL + 6×Loading Buffer 1μL
4. 1kb ladder 2μL

Liquid culture
pSB3C5-1,2
pSB4K5-1,2

February 22

Gel extraction
lane 1 of the gel 45.0μg/mL

PCR purification
product 38.2μg/mL

PCR
TMAO reductase

	Buffer	dNTPs	MgSO4	prefix primer-f	suffix primer-r	product of gel extract	product of PCR purification(1ng/μL)	KOD plus	MilliQ	total
1	5	5	4	1.5	1.5	0.5	0	1	32.5	50
2	5	5	4	1.5	1.5	1	0	1	32.5	50
3	5	5	4	1.5	1.5	0	0.5	1	32.5	50
4	5	5	4	1.5	1.5	0	1	1	32.5	50

94℃, 2min
98℃, 10sec
59℃, 30sec
68℃, 3min
→25cycles

Electrophoresis

1. 1kb ladder
2. TAMO1
3. TAMO2
4. TAMO3
5. TAMO4
6. constructive promoter 1-18C
7. constructive promoter Spe1
8. constructive promoter Pst1
9. 1kb ladder
PCR

	Buffer	dNTPs	MgSO4	prefix primer-f	suffix primer-r	product of PCR purification(1ng/μL)	KOD plus	MilliQ	total
1	5	5	3	1.5	1.5	0.5	1	32.5	50
2	5	5	3	1.5	1.5	1	1	32	50
3	5	5	3	1.5	1.5	2	1	31	50
4	5	5	3	1.5	1.5	3	1	30	50
5	5	5	3	1.5	1.5	10	1	29	50
6	5	5	3	1.5	1.5	0	1	33	50

94℃, 2min
98℃, 10sec
59℃, 30sec
68℃, 3min
→25cycles

Electrophoresis

Checking Dpn1

Buffer	lacp(28.7ng/μL)	Dpn1	MilliQ	total
2	10	0.5	7.5	20
2	10	0	5	17

February 23

Colony PCR
tatABCD(2 samples)

Buffer	dNTPs	MgSO4	primer-f	primer-r	KOD plus	MilliQ	total
5	5	3	1.5	1.5	1	33	50

Predenature 94℃ 1min
Denature 98℃ 10sec
Annealing 59℃ 30sec
Extension 68℃ 3min
→25cycles

Electrophoresis

1. 1kb ladder 2μL
2. tatABCD 1 5μL + 6×Loading Buffer 1μL
3. tatABCD 2 5μL + 6×Loading Buffer 1μL
4. 1kb ladder 2μL

Miniprep
pSB4K5 and pSB3C5
deluted it to 25 times and then measured it
pSB4K5 1 : 60.0 μg/ml 1.67(260/280) 1.98(260/230)
pSB4K5 2 : 55.0 μg/ml 1.49(260/280) 1.62(260/230)
pSB3C5-3 : 3.6 μg/ml 1.57(260/280) 3.00(260/230)
pSB3C5-4 : 1.3 μg/ml 1.44(260/280) 1.04(260/230)

Liquid culture
pSB3C5-3,4

Electrophoresis

1. 1kb ladder 2μL
2. pSB3C5-3 5μL, 6×loading dye 1μL
3. pSB3C5-4 5μL, 6×loading dye 1μL
4. 1kb ladder 2μL

February 27

Test of Dpn1

Buffer2	GFP2	BSA	MilliQ	Dpn1
3	3	0.3	23	1

Colony PCR

	buffer	dNTPs	MgSO4	Primer-f	Primer-r	MilliQ	KOD Plus	total
Colony PCR(2 samples)	5	5	3	1.5	1.5	33	1	50
Negative control	5	5	3	1.5	1.5	34	0	50

Predenature 94℃,2min
Denature 98℃,10sec
Annealing 59℃,30sec
Extension 68℃,3min
→25cycles

Electrophoresis

	sample	Loading Dye	MilliQ
1.1kb ladder	2	0	0
2.product of PCR1	5	1	0
3.product of PCR2	5	1	0
4.product of PCR(Negative control)	5	1	0
5.product of PCR(2/23)	5	1	0
6.GFP2(DPN1)	10	2	0
7.GFP2	3	2	7
8.1kb ladder	2	0	0

Results of liquid culture
We measure this after dilute it to 10 times.

pSB3C5-5	pSB3C5-6	pSB3C5-5(1% glucose)	pSB3C5-6(1% glucose)
8.5[µg/ml]	-1.8	-17.9	-18.2

PCR

	buffer	dNTPs	MgSO4	Primer-f(prefix)	Primer-r(suffix)	PCR purification product(1ng/µL)	MilliQ	KOD Plus	total
1	5	5	3	1.5	1.5	0.2	32.8	1	50
2	5	5	3	1.5	1.5	0.5	32.5	1	50

• PCR purification product was that purification product(75ng/µL) of electrophoresis-3 deluted to 1ng/µL
  

Predenature 94℃,2min
Denature 98℃,10sec
Annealing 59℃,30sec
Extension 68℃,3min
→25cycles

February 28

Electrophoresis

1. 1kb ladder
2. PCR1 →Product of gel extraction : tatABCD with prefix and suffix 105[ng/µL]
3. PCR2

Restriction

Buffer2	plasmid(?)	enzyme	MilliQ	total
2	2	0.2	15.8	20

incubate 1 hour at 37℃

Electrophoresis

1. 1kb ladder
2. Control (without enzymes)
3. EcoR1
4. Xba1 (crystallized)
5. Xba1 (with seal)
6. Spe1
7. Pst1
8. 1kb ladder

PCR and Electrophoresis

Quick Taq Dye Mix	primer-f	primer-r	template	MilliQ	total
25	1.0	1.0	0.5	22.5	50

Predenature 94℃,2min
Denature 94℃,30sec
Annealing 59℃,30sec
Extension 68℃,3min
→25cycles

Restriction

BufferH	tatABCD	EcoR1	Spe1	MilliQ	total
2	5	0.2	0.2	12.6	20

PCR purification
We eluted the product for 30µL MilliQ

Ligation

Insert(tatABCD)	Vector(pSB1C3)	Ligation High	total
10	1	5	16

4℃, overnight

February 29

Transformation

tatABCD	competent cell	total
1	10	11

Checking Restriction enzyme

plasmid seems to be 1-18C promoter	Enzyme	Buffer	MilliQ	total
2	0.2	2	15.8	20

Checking tatABCD

tatABCD	Hind3	Buffer	MilliQ	total
5	0.2	2	12.8	20

PCR

	buffer	dNTPs	MgSO4	Primer-f	Primer-r	ColE1(6.5ng/µL) / TMAO	MilliQ	KOD Plus Neo	total
Kil	2.5	2.5	1.5	0.75	0.75	0.5	16	0.5	25
TMAO	2.5	2.5	1.5	0.75	0.75	0.5	16	0.5	25

Predenature 94℃,2min
Denature 98℃,10sec
Annealing 60℃,30sec
Extension 68℃,3min
→30cycles

Electrophoresis

• evernoteに写真なし！至急求む

1. 1kb ladder
2. Kil (649bp)
3. TMAO (2720bp)
4. TMAO (Quick Taq)
5. tatABCD (Quick Taq)
6. tatABCD (Hind3)
7. 1kb ladder
+

March 1

PCR

• TMAO
  

Template is gDNA and product of colony PCR gel extraction

	Buffer	gNTPs	MgSO4	Primer-f	Primer-r	KOD Plus Neo	Template gDNA	product of gel extraction	DW	total
1	2.5	2.5	1.5	0.75	0.75	0.5	0.5	0	16	25
2	2.5	2.5	1.5	0.75	0.75	0.5	0	2	14.5	25

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 1.5min
→25cycles

• Kil

Buffer	dNTPs	MgSO4	primer-f	primer-r	colE1(6.5ng/µL)	KOD Plus Neo	MilliQ	total
2.5	2.5	1.5	0.75	0.75	0.5	16	0.5	25

94℃, 2min
98℃, 10sec
61℃, 30sec
68℃, 1min
→20cycles

Electrophoresis

1. 1kb ladder
2. TMAO1 (gDNA)
3. TMAO2 (product of gel extraction)
4. Kil

PCR

Buffer	dNTPs	MgSO4	primer-f	primer-r	Product of Purification	KOD Plus Neo	MilliQ	total
5	5	3	1.5	1.5	1	32	1	50

94℃, 2min
98℃, 10sec
61℃, 30sec
68℃, 30sec
20cycles
→Purification 230ng/µL

Restriction

Kil	EcoR1	Spe1	BufferH	MilliQ	total
5	0.2	0.2	2	12.6	20

incubate at 37℃, for 1.5 hours

PCR Purification

Ligation

Kil	pSB1C3	Ligation High	total
5	1	3	9

at 4℃, for overnight

March 2

Ligation

Kil	pSB1C3	Ligation High Ver.2	total
5	1	3	9

tatABCD	pSB1C3	Ligation	total
10	1	5	16

at 16℃ for 1 hour

Transformation

Kil	Kil(3/1,Ligation)	tatABCD	competet cell	total
1	0	0	10	11
0	1	0	10	11
0	0	1	10	11

PCR

Quick Taq	primer-r	primer-f	template	MilliQ	total
25	1	1	0.5	22.5	50

Electrophoresis

Restriction

pSB3C5-5	EcoR1	Pst1	BufferH	BSA	MilliQ	total
20	0.5	0.5	3	0.5	5.5	30

at 37℃, for 2 hour

Electrophoresis

1. 1kb ladder 2µL
2. pSB3C5 5µL + 6×Loading Buffer 1µL
・product of gel extraction(about 2700bp)
－30.9µg/mL

Restriction
GFP1,2,3

GFP	EcoR1	Pst1	Buffer	BSA	MilliQ	total
10	0.5	0.5	3	0.5	15.5	30

at 37℃, for 2.5 hours

Restriction

DT	EcoR1	Xba1	BufferM	BSA	MilliQ	total
10	0.2	0.2	3	0.3	16.3	30

Constitutive Promoter	Spe1	Pst1	BufferM	BSA	MilliQ	total
15	0.2	0.2	3	0.3	11.3	30

at 37℃, 2 hours

• J23117-1:135ng/µL, J23107-1:115ng/µL
  
• DT3→PCR Purification
  
• Promoter→Gel Extraction
  
  

Checking TMAO

something seems to be TMAO	Buffer2	EcoR1	MilliQ	total	10	2	0.5	7.5	20

at 37℃, for 1 hour

Electrophoresis

1. 1kb ladder
2. GFP1 that had been cut by restriction enzyme
3. GFP2 that had been cut by restriction enzyme
4. GFP3 that had been cut by restriction enzyme
5. GFP1
6. GFP2
7. GFP3
8. TMAO (control)
9. TMAO (EcoR1)
10. DT (control)
11. DT (EcoR1, Xba1)
12. 1kb ladder

Checking tatABCD

Quick Taq	primer-f	primer-r	MilliQ	total
25	1	1	23	50

Electrophoresis

March 3

PCR

	template	buffer	dNTPs	MgSO4	VF	VR	KOD plus	MilliQ	total
1	1	5	5	3	1.5	1.5	1	32	50
2	2	5	5	3	1.5	1.5	1	31	50

94℃, 2min
98℃, 10sec
50℃, 30sec
68℃, 1min
→30cycles

Miniprep

March 4

Sequence of tatABCD

Quick Taq	primer-f	promer-r sequence	template	MilliQ	total
25	1	1	1	23	50

Quick Taq	primer-f sequence	primer-r	template	MilliQ	total
25	1	1	1	23	50

Colony PCR of TMAO

buffer	dNTPs	NgSO4	primer-f	primerr-r	KOD plus	MilliQ	total
5	5	4	1.5	1.5	1	32	50

→ethanol precipitation
94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 2.5min
→25cycles

Electrophoresis

1. 1kb ladder
2. tatABCD1
3. tatABCD2
4. TMAO
5. 1kb ladder

Restriction

TMAO	EcoR1	BufferH	BSA	MilliQ	total
10	0.2	3	0.3	16.5	30

TMAO	Xba1	Pst1	BudderM	BSA	MilliQ	total
10	0.2	0.2	3	0.3	16.3	30

at 37℃ for 1 hour

Electrophoresis

Transformation

pSB1C3	competent cell(made at 2/8)	total
5	100	105

March 5

Restriction

pSB1C3(Xba1, Spe1)	Pst1	BufferH	BSA	MilliQ	total
10	0.2	2	0.2	7.6	20
pSB1C3(Xba1, Spe1)	EcoR1	BufferH	BSA	MilliQ	total
10	0.2	2	0.2	7.6	20

at 37℃ for 1 hour
→Then we did ethanol precipitation

Ligation

Kil(EcoR1, Spe1)	pSB1C3(EcoR1)	Ligation High	total
5	1	3	9

at 16℃ for 1 hour

Transformation

Kil	competent cell	total
1	10	11

We used commercially available competent cells in this time.

PCR

TMAO

buffer	dNTPs	MgSO4	Primer-f	Primer-r	Template	KOD plus	MilliQ	total
5	5	3	1.5	1.5	1	1	32	50

Electrophoresis

(31)

 

Restriction

	Lacp	pSB3C5	EcoR1	Pst1	BufferH	BSA	MilliQ	total
1	20	0	0.5	0.5	3	0.5	5.5	30
2	0	20	0.5	0.5	3	0.5	5.5	30

at 37℃ for 1 hour
1→Ethanol precipitation　45.4µg/mL
2→Gel extraction　38.7µg/mL

Ligation

LacP	pSB3C5	Ligation High	total
10	2	6	18

at 4℃ for overnight

Transformation

Lacp+pSB3C5	competent cell	total
1	10	11

on ice for 30 mins.
heat shock at 42℃ for 60secs
on ice for 2 mins.
After we incubate with 200µL of SOC culture for 1 hour, we did plating on LB culture with CP

Restriction

GFP Plasmid	EcoR1	Spe1	Buffer2	BSA	MilliQ	total
10	0.5	0.5	3	0.5	15.5	30

at 37℃ for 2 hours

Electrophoresis

1. Ladder 2µL
2. GFP Plasmid (already restricted) 2µL + Loading Dye 2µL + MilliQ 8µL
3. Ladder 2µL

PCR

torA signal and pspA pspAはコロニーPCR

Buffer	dNTPs	MgSO4	Primer-f	Primer-r	template(TMAO)	KOD plus	MilliQ	total
2.5	2.5	1.5	0.75	0.75	0.5	0.5	16	25

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 30sec

electrophoresis

1. 100bp Ladder
2. torA signal (272bp)
3. pspA (969bp)
4. 100bp Ladder

March 6

Restriction

GFP	EcoR1	Spe1	BufferM	BSA	MilliQ	total
12	0.5	0.5	3	0.5	13.5	30

We did incubate at 37℃ for 1.5hours.
And we did gel extraction on 3/7 and get 40.0μg/mL GFP.

PCR

buffer	dNTPs	MgSO4	Primer-f	Primer-r	template	KOD plus neo	MilliQ	total
5	5	3	1.5	1.5	0.5	1	32.5	50

94℃　2min
98℃　10sec
60℃　30sec
68℃　(torA 10sec / pspA 30sec) 25 cycles
We used product of PCR on 3/5 of torA and pspA as template.

Restriction

Kil	EcoR1	Spe1	BufferM	BSA	MilliQ	total
10	0.3	0.3	3	0.3	16.1	30

DT	EcoR1	Xba1	Buffer2	BSA	MilliQ	total
10	0.5	0.5	3	0.5	15.5	30

at 37℃ for 2 hours
→ purification 37.7ng/μL

Ligation

Kil	pSB1C3	Ligation High Ver.2	total
4	2	3	9

• Kil : 350fmol
  
• pSB1C3 : 29fmol
  

at 16℃ for overnight

March 7

Electrophoresis

1. 1kb ladder 2µL
2. pspA (PCR product)2.5µL + Loading Dye 0.5µL
3. GFP 30µL + Loading Dye 6µL
4. 1kb ladder 2µL

• The GFP was gel extracted on 3/6 and concentrated by Vacuum in 150µg/mL
  
  

Ligation

VectorDNA	GFP	Ligation High Ver.2	total
5	15	10	30

Restriction

torA	EcoR1	Spe1	bufferM	BSA	MilliQ	total
10	0.3	0.3	3	0.3	16.1	30

pspA	EcoR1	Spe1	bufferM	BSA	MilliQ	total
5	0.3	0.3	3	0.3	21.1	30

at 37℃ for 1.5 hours

Purification

torA→31.8ng/µL
pspA→49.3ng/µL

Ligation

torA	pSB1C3	Ligation High Ver.2	total
3	3	3	9

pspA	pSB1C3	Ligation High Ver.2	total
4	2	3	9

at 4℃, for overnight

• torA→31.8ng/µL×3µL=95.4ng=0.529pmol
  
• pSB1C3→19.4ng/µL×3µL=58.2ng=0.042pmol
  
• pspA→49.3ng/µL×4µL=197.2ng=0.308pmol
  
• pSB1C3→19.4ng/µL×2µL=38.8ng=0.029pmol
  

March 8

Restriction

pSB4K5	EcoR1	Spe1	BufferM	BSA	MilliQ	total
20	0.2	0.2	3	0.2	6.4	30

at 37℃ for 1 hour.
→Purification : 36.6ng/µL

Ligation

Kil	pSB4K5	Ligation High Ver.2	total
10	1	5	16

at 4℃ for overnight

• Kil→37.7ng/µL×10µL=377ng=879fmol
  
• pSB4K5→36.6ng/µL×1µL=36.6ng=86fmol
  
  

Liquid culture

Lacp + pSB3C5 -1, 2

Transformation

torA	pspA	competent cell	total
1	0	10	11
0	1	10	11

We use commercially available competent cells in this time.

March 9

Restriction

tatABCD	Xba1	Pst1	BufferM	BSA	MilliQ	total	10	0.2	0.2	3	0.3	16.3	30

at 37℃ for 1hour
→purification : 75.0μg/mL (1.11 260/280 , 0.81 260/230)

Miniprep

lacP + pSB3C5 1 80.5μg/mL (1.78 260/280 , 2.00 260/230)
lacP + pSB3C5 2 107.2μg/mL (1.83 260/280 , 1.90 260/230)

Colony PCR

Quick Taq	VF	VR	MilliQ	total
25	1	1	23	50

94℃ 2min
94℃ 30sec
55℃ 30sec
68℃ 6sec
25cycles

Ligation

tatABCD	constP J23107	Ligation High Ver.2	total
5	1	3	9

tatABCD : 227fmol
constP J23107 : 21fmol

Transformation

	pspA	torA	Kil	competent cell
1	1	0	0	10
2	0	1	0	10
3	0	0	1	10

Miniprep

4mL of plusgrow which had been cultured for overnight.
pSB4K5 : 80.5μg/mL

March 10

Screening PCR

Quick Taq	VF	VR	MilliQ	total
25	1	1	23	50

Then we did electrophoresis to confirm.
1.1kb ladder
2.kil (649bp)
3,4,5, pspA (969bp)
6,1kb ladder

1, 100bp ladder
2,3,4, torA signal

Restriction

LacP-pSB3C5	Spe1	Pst1	BufferM	BSA	MilliQ	total
10	0.2	0.2	2	0.2	7.4	20

for 2.5 hours at 37℃
→purification 29.0ng/μL

torA	Xba1	Pst1	BufferM	BSA	MilliQ	total
10	0.2	0.2	2	0.2	7.4	20

for 2.5 hours at 37℃
→purification 91.8ng/μL

Ligation

torA	Lacp-pSB3C5	Ligation High Ver.2	total
4	2	3	9

torA : 929fmol
Lacp-pSB3C5 : 284fmol
for overnight at 4℃

March 11

Miniprep

We used 3μL of plus grow that we had cultured for overnight.
torA : 62.8ng/μL

Screening PCR

Quick Taq	VF	VR	MilliQ	total
25	1	1	23	50

Electrophoresis

1. 1kb ladder
2〜11. pspA (969bp)
12. 1kb ladder

The results were shown as photograph in the right.

It seemed that there were shorter sample than expected sample, so we did electrophoresis with pspA which was product of PCR and pspA which had already cut with EcoR1 and Spe1.

1.1kb ladder
2. pspA (PCR product)
3, pspA (Eco, Spe)
4〜6, pspA (colony PCR)
7,1kb ladder

The results were shown as photograph in the right.

March 12

Transformation

DT(1ng/μL)	DT(0.1ng/μL)	Kil	lacP-torA	MilliQ	competent cell	total
1	0	0	0	0	20	21
0	1	0	0	0	20	21
0	0	5	0	0	50	51
0	0	0	5	0	50	51
0	0	0	0	1	20	21

Restriction

pspA	EcoR1	Spe1	BufferM	BSA	MilliQ	total
10	0.5	0.5	3	0.3	15.7	30

at 37℃ for 4 hours
→ We did purification and got 48.3ng/μL pspA.

Ligation

pspA	pSB1C3	MilliQ	Ligation High Ver.2	total
4	2	0	3	9
2	2	0	2	6
0	2	2	2	6

March 13

Miniprep

pSB1C3 74.2µg/mL 1.65 (260/280) 1.31 (260/230)

March 14

Restriction

pSB1C3	EcoR1	Spe1	BufferM	BSA	MilliQ	total
20	0.2	0.2	4	0.4	15.2	40

We did gel extraction and got 47.2ng/µL pSB1C3 but we did not cut out RFP.

Ligation

torA	pSB1C3	Ligation High Ver.2	total
4	2	3	9
MilliQ	pSB1C3	Ligation High Ver.2	total
4	2	3	9

at 16℃, for 1 hour

• torA : 0.707pmol
  
• pSB1C3 : 0.068pmol
  
  

Liquid culture

We cultured Lacp-pSB3C5 1,2,3,4,5 (CP tolerance)on culture with Amp.
→Only 4 which did not be cultured succeeded.

March 15

Liquid culture

We cultured Lacp-pSB3C5 6,7,8,9,10 on culture with ampicillin.
→6,8,9,10 were succeeded.

Restriction

GFP	EcoR1	Spe1	Buffer2	BSA	MilliQ	total
10	0.5	0.5	3	0.5	15.5	30

DT	EcoR1	Xba1	Buffer2	BSA	MilliQ	total
10	0.5	0.5	3	0.5	15.5	30

for 2 hours at 37℃.

Miniprep

pspA (pSB1C3) 40.5ng/µL

Ligation

pspA	DT	Ligation High Ver.2	total
5	1.5	3	9.5

• pspA : 385fmol
  
• DT : 36fmol
  
  

Transformation

J23107-tatABCD	DT (0.1ng/µL)	DT (0.01ng/µL)	pspA-DT	competent cells on 3/15	total
2	0	0	0	20	22
0	2	0	0	20	22
0	0	2	0	20	22
0	0	0	2	20	22

Screening PCR

Kil, pspA and torA

Quick Taq	Primer-r	Primer-f	MilliQ	total	25	1	1	23	50

March 16

Miniprep

torA (pSB1C3) 68.8ng/µL
Kil (pSB4K5) 92.7ng/µL
torA was red for some reason. We do not know why.

Colony PCR

Quick Taq	Primer-r	Primer-f	MilliQ	total
25	1	1	23	50

94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 6sec
→25cycles

Electrophoresis

The results were shown as photograph in the right.

Checking Transformation Efficiency

competent cells that were made on March 15.
DNA : 0.02ng → 668 colonies Transformation Efficiency : 3.3×10^7
DNA : 0.2ng → 1739 colonies Transformation Efficiency : 8.7×10^6

Restriction

pSB1C3	EcoR1	Spe1	BSA	BufferM	BufferH	MilliQ	total
20	0.2	0.2	0.3	3	0	6.3	30
5	0.2	0	0.2	0	2	12.6	20

at 37℃, for 2 hours.
We did gel extraction for product with EcoR1, Spe1. We got 46.1ng/µL pSB1C3.

Kil(pSB4K5)	EcoR1	Pst1	BSA	BufferH	MilliQ	total
5	0.2	0.2	0.2	2	12.4	20
5	0.2	0	0.2	2	12.6	20

for overnight at 37℃.
We did this to confirm.

pspA (pSB1C3)	EcoR1	Pst1	BSA	BufferH	MilliQ	total
5	0.2	0.2	0.2	2	12.4	20
5	0.2	0	0.2	2	12.6	20

for overnight at 37℃.
We did this to confirm.

Ligation

torA	pSB1C3	Ligation High Ver.2	total
4	2	3	9

MilliQ	pSB1C3	Ligation High Ver.2	total
4	2	3	9

at 16℃, for overnight

• torA→767fmol
  
• pSB1C3→68fmol
  

March 17

Miniprep

J23107-tatABCD 72.7ng/µL
pspA-DT 50.5ng/µL

Checking the Insert

J21037-tatABCD	EcoR1	Pst1	BSA	BufferH	MilliQ	total
5	0.2	0.2	0.2	2	12.4	20
5	0.2	0	0.2	2	12.6	20

Success.

pspA-DT	EcoR1	Pst1	BSA	BufferH	MilliQ	total
5	0.2	0.2	0.2	2	12.4	20
5	0.2	0	0.2	2	12.6	20

Failed.

March 19

Restriction

DT	EcoR1	Xba1	BSA	BufferM	MilliQ	total
10	0.2	0.2	0.3	3	16.3	30

We did Gel extraction and got 17.2ng/µL of DT.

Ligation

Kil	DT	Ligation High Ver.2	total
10	2	6	18

We did this for an hour at 16℃.

Restriction

GFP	EcoR1	Spe1	BSA	BufferM	MilliQ	total
10	0.5	0.5	0.5	3	15.5	30

We did this for 4 hours at 37℃

Ligation

pspA	DT	Ligation High Ver.2	total
5	5	5	15

pspA	pSB1C3	Ligation High Ver.2	total
4	1	3	8

We did these for an hour at 16℃.

• pspA (5µL)→377fmol
  
• DT→39fmol
  
• pspA (4µL)→339fmol
  
• pSB1C3→34fmol
  
  

Transformation

pspA-DT, pspA (pSB1C3), torA (pSB1C3) and GFP-DT

March 20

Screaning PCR

Quick Taq	Primer-R	Primer-F	MilliQ	total
25	1	1	23	50

• pspA→○
  
• pspA-DT→○
  
• GFP-DT→○
  
• torA→×
  
• Kil-DT 6 of 8 sumples→○
  

Quick Taq	VR	VF	MilliQ	total
25	1	1	23	50

• pspA→○
  
• pspA-DT→×
  
  

Restriction

torA	EcoR1	Spe1	BSA	BufferM	MilliQ	total
10	0.3	0.3	0.3	3	16.1	30

DT	EcoR1	Xba1	BSA	BufferM	MilliQ	total
10	0.2	0.2	0.3	3	16.3	30

We did this for overnight at 37℃. And we did purification.
torA 34.2ng/µL
DT 28.0ng/µL

March 21

Miniprep

GFP-DT-1 : 77.7ng/µL
GFP-DT-2 : 67.6ng/µL

Restriction

pSB1C3	EcoR1	Spe1	BSA	BufferM	MilliQ	total
10	0.2	0.2	0.3	3	16.3	30
5	0.2	0	0.2	2	12.6	20

We did this for 3 hours at 37℃, and then we did gel extraction. We got 25.1ng/µL pSB1C3

GFP-DT	EcoR1	Pst1	Xba1	BSA	BufferM	MilliQ	total
5	0.2	0.2	0	0.2	2	12.4	20
5	0.2	0	0	0.2	2	12.6	20
10	0.2	0	0.2	0.3	3	16.3	30

We did this for 3 hours at 37℃, and then we did Purification. We got 30.7ng/µL GFP-DT.

Ligation

	torA	pSB1C3	pspA	DT	GFP-DT	Ligation High Ver.2	total
1	4	1	0	0	0	3	8
2	0	1	7	0	0	4	12
3	0	0	5	3	0	4	12
4	3	0	0	0	5	4	12

We did this for an hour at16℃.

March 22

PCR

We did PCR to amplify torA_signal that was product of PCR at March 5 with redesigned primers.

Buffer	dNTPs	MgSO4	Primer-F	Primer-R	Template	MilliQ	KOD plus neo	total
5	5	3	1.5	1.5	0.5	32.5	1	50

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 10sec
→30cycles
→Purification 110.7ng/µL

Restriction

torA	EcoR1	Spe1	BSA	BufferM	MilliQ	total
10	0.2	0.2	0.3	3	16.3	30

Ligation

	torA	pSSB1C3	pspA	DT	GFP-DT	Ligation high	total
1	4	3	0	0	0	4	11
2	3	0	0	0	3	3	9
3	0	0	5	5	0	5	15

• torA (4µL)→512fmol
  
• pSB1C3→54fmol
  
• torA (3µL)→384fmol
  
• GFP-DT→36fmol
  
• pspA→377fmol
  
• DT→65fmol
  
  

March 23

Screening PCR

torA (pSB1C3), torA-GFP-DT and pspA-DT

Quick Taq	VF	VR	MilliQ	total
25	1	1	23	50

E.coli in liquid culture that had pspA(pSB1C3) also expressed RFP.

Restriction

pspA	EcoR1	Spe1	BufferM	BSA	MilliQ	total
5	0.3	0.3	3	0.3	21.1	30

And then we did ethanol precipitation

Ethanol precipitation

pspA 11.5ng/µL.

Miniprep

Lacp+pSB3C5-8 77.9µg/mL
Lacp+pSB3C5-10 69.0µg/mL
Kil+DT-4 58.0µg/mL
Kil+DT-7 15.2µg/mL

Restriction

Lacp+pSB3C5-8	Spe1	Pst1	Buffer2	BSA	MilliQ	total
20	0.5	0.5	3	0.5	5.5	30

Kil+DT-4	Xba1	Pst1	Buffer2	BSA	MilliQ	total
20	0.5	0.5	3	0.5	5.5	30

at 37℃ for 1.5 hours
And then we did Gel extraction.

Gel Extraction

Lacp+pSB3C5-8 40.4µg/mL
Kil+DT-4 26.8µg/mL

March 26

Miniprep

torA(pSB1C3) 18.5[ng/µL]
torA-GFP-DT 20.0[ng/µL]

Restriction

torA(pSB1C3)	EcoR1	Pst1	BufferH	BSA	MilliQ	total
5	0.2	0.2	2	0.2	12.4	20
5	0.2	0	2	0.2	12.6	20

torA-GFP-DT	EcoR1	Xba1	Pst1	BufferH	BufferM	BSA	MilliQ	total
5	0.2	0	0.2	2	0	0.2	12.4	20
5	0.2	0	0	2	0	0.2	12.6	20
20	0	0.2	0.2	0	3	0.3	6.3	30

We did Gel extraction and then got ??? 28.7[ng/µL]

Ligation

Lacp (pSB3C5)	torA-GFP-DT	Ligation High Ver.2	total
1	5	3	9

• Lacp : 22fmol
  
• torA-GFP-DT : 197fmol
  

pspA	pSB1C3	Ligation High Ver.2	total
10	1	5	16

pspA	DT	Ligation High Ver.2	total
10	1	5	16

• pspA : 180fmol
  
• pSB1C3 : 18fmol
  
• DT : 16fmol
  

LacP(pSB3C5)	Kil-DT	Ligation High Ver.2	total
1	5	3	9

for 2 hours at 16℃

Transformation

Lacp-Kil-DT	competent cell	total
1	10	11

March 27

Miniprep

We retryed miniprep of torA(pSB1C3).
We got torA and its concentration was 39.3[ng/µL].

Transformation

Name	Well	Sample	Competent Cells	Total	Plate	Colony
BBa_K117004	14J(2011 plate2)	5	20	?	?	?

We added 100[µL] of culture medium before we started culturing the E.coli.

Screening PCR

Quick Taq	VF2	VR	MilliQ	Total
25	1	1	23	50

Lacp-torA-GFP-DT 1, 3, 5, 6, 8, 9 was successful.
pspA-DT was failed.
E.coli that had pspA(pSB1C3) did not make any colony.
Lacp-Kil-DT 1,2,3,4,5,6,7,8 was failed.

Liquid culture

Lacp-torA-GFP-DT

July 23

Transformation

DT(64.4ng/μl)	Competent cell	Plating in SOC medium	Total
1	20	100	121

July 24

Plating in SOC medium

July 25

Transformation

• BBa_J23113 from iGEM Kit
• LacP from iGEM Kit

July 25

July 26

July 27

Restriction Enzyme Processing

Kil +DT (44.0 ng/μl)	10xBuffer2	BSA	Xbal	MilliQ	Total
13.6	3.0	0.5	0.5	11.9	30.0

LacP(28.7ng/μl)	10xBuffer2	BSA	Spe1	Pst1	Total
20.9	3.0	0.5	0.5	4.6	30.0

July 30

Ligation

kil+DT(Xba1,Pst1)	LacP(Spe1,Pst1)	Ligation High	Total
30	6	36	72

July 31

Restriction Enzyme Processing

torAGFP+DT	10×M Buffer	BSA	MilliQ	Xba1	Pst1	Total
2.5	3.0	0.5	23.4	0.3	0.3	30.0

Liquid Culture

J23113(backbone J61002)

August 1

Restriction Enzyme Processing

lacP+kil+DT	BSA	10×H Buffer	EcoR1	Pst1	MilliQ	Total
5.0	0.5	3.0	0.5	0.5	20.5	30.0

37℃ 2h

lacP middle copy	BSA	10×H Buffer	EcoR1	Pst1	MilliQ	Total
10.0	0.5	3.0	0.5	0.5	15.5	30.0

37℃ 2h

MIniprep

J23113(backbone J61002)

Liquid culture

J23113(backboneJ61002) Three test tubes of 4 mL LB medium with ampicillin 37℃ overnight

August 2

August 3

Restriction Enzyme processing

pSB4K5	BSA	10×H Buffer	EcoR1	Pst1	MilliQ	Total
8.0	0.5	3.0	0.5	0.5	17.5	30.0

B0034	BSA	10×H Buffer	Spe1	Pst1	MilliQ	Total
12.0	0.5	3.0	0.5	0.5	13.5	30.0

37℃ 2h

DNA purification

Ligation

lacP+kil+DT	pSB4K5	ligation high	Total
15	15	15	45

16℃ 1h

Miniprep

• J23113(backbone J61002) 218.0μg/mL
• J23113(backbone J61002) 252.5μg/mL
• J23113(backbone J61002) 201.0μg/mL
• pSB3C5 45.0μg/ml 1.60 260/280 1.80 260/230
• pSB4C5 213.0μg/mL 1.77 260/280 4.40 260/230

August 4

August 5

August 6

August 7

August 8

August 9

August 10

Ligation

B0034 Spe1 Pst1	GFP+DT Xba1 Pst1	ligation high	Total
2	3	4	9

16℃ 1h

Transformation

• RBS+GFP+DT
• lacP+kil+DT
• RBS（for control）

To put 2ng DNA in 20μL competent cell and leave it on ice for 30min

Heat shock for 60s at 42℃

To leave on ice for 2min

To spread on ampicillin LB plate

August 11

August 12

August 13

Liquid culture

B0034 3mL ×3 　　　

37℃　overnight

August 14

Miniprep

B0034 0μg/mL

B0034 235.5μg/mL

B0034 76.5μg/mL

Colony PCR

lacP+kil+DT ×5

2×Quick Taq	VF	VF	MilliQ	Total
25	1	1	23	50

25cycles
pspA

10×Buffer for KOD-Plus-Ver2	2mM dNTPs	25mM MgSO4	Primer PsPA f-p	Primer PsPA r-s	KOD-Plus-	MilliQ	Total
5	5	3	1.5	1.5	1	33	50

Electrophoresis

positive control(GFP+DT)

negative control(MilliQ)

① ～⑥ colony PCR lacP+kil+DT

次の写真

① 　　　1kb ladder

② 　　　Positive control (GFP+DT)

③ 　　　Negative control (MilliQ)

④ ～⑨　lacP+kil+DT　①～⑥

⑩～⑫　 pspA 10μL ,6×buffer 2μL

⑬　　　　1kb ladder

August 15

Colony PCR

pspA

2×Quick Taq	pspA r-s	pspA f-p	MilliQ	Total
25	1	1	23	50

94℃, 2min
94℃, 30sec
56℃, 30sec
68℃, 1min
→35cycles

August 16

August 17

Colony PCR

pspA

10×Buffer for KOD-Plus-Ver2	2mM dNTPs	25mM MgSO4	Primer PsPA f	Primer PsPA r	KOD-Plus-	MilliQ	Total
5	3	3	1.5	1.5	1	35	50
5	3	5	1.5	1.5	1	33	50

negative control(MilliQ 50μL) 94℃, 2min
94℃, 15sec
55℃, 30sec
68℃, 1min
→35cycles

Electrophoresis

①1kb ladder

②, ③　　MgSO4 3μL, pspA

④, ⑤　　MgSO4 5μL, pspA

⑥ negative control(MilliQ)

⑧ 1kb ladder

August 18

August 19

August 20

Restriction

J23113(201.0ng/μL)	Xba1	Pst1	BufferM	BSA	MilliQ	total
10	1	1	3	0.5	14.5	30

August 21

Colony PCR

pspA

10×Buffer	2mM dNTPs	25mM MgSO4	Primer PsPA f	Primer PsPA r	KOD Plus Neo	MilliQ	Total
5	3	5	1.5	1.5	1	33	50
5	3	7	1.5	1.5	1	31	50
5	3	10	1.5	1.5	1	28	50

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 30sec
→25cycles

Restriction

J23113(218ng/μL)	Spe1	Pst1	BufferH	BSA	MilliQ	total
10	1	1	3	0.5	14.5	30

torA-GFP-DT(20ng/μL)	Xba1	Pst1	BufferM	BSA	MilliQ	total
5	0.6	0.6	3	0.5	20.3	30

Electrophoresis

① 　　　1kb ladder

②, ③　　torA-GFP-DT(Xba1,Pst1)

④, ⑤　　J23113(Spe1,Pst1)

Transformation

• lacP-torA-GFP-DT(Backbone pSB3C5)
• BBa_K11704(Backbone pSB1A2)

Colony PCR

• pspA

10×Buffer	2mM dNTPs	25mM MgSO4	Primer PsPA f	Primer PsPA r	KOD Plus Neo	MilliQ	Total
5	5	3	1.5	1.5	1	33	50

10×Buffer	2mM dNTPs	25mM MgSO4	Primer PsPA f-p	Primer PsPA r-s	KOD Plus Neo	MilliQ	Total
5	5	3	1.5	1.5	1	33	50

• negative control for pspA

We did PCR without a template.

• GFP-DT

10×Buffer	2mM dNTPs	25mM MgSO4	VF	VR	KOD Plus Neo	MilliQ	Total
5	5	3	1.5	1.5	1	33	50

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 30sec

Transformation

• Lacp-torA-GFP-DT(Backbone pSB3C5)
• J23107-tatABCD

PCR

kil, torA-GFP

10×Buffer	2mM dNTPs	25mM MgSO4	VF	VR	KOD Plus Neo	MilliQ	DNA	Total
5	5	3	1.5	1.5	1	32	1	50

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 30sec
→25cycles

August 22

Restriction

pSB3C5	EcoR1	Pst1	BufferH	BSA	MilliQ	total
13.3	0.5	0.5	3	0.5	12.2	30

Gel extraction

pSB3C5(EcoR1, Pst1)
27.2μg/mL

Transformation(the second time)

• Lacp-torA-GFP-DT(Backbone pSB3C5)
• J23107-tatABCD

Electrophoresis

写真お願いします。
See "August 21 -Colony PCR-"
1. 1kb ladder
2. pspA(Primer pspA f&r)
3. negative control for 2
4. GFP-DT
5. pspA(Primer pspA f-p&r-s)
6. pspA(Primer pspA f-p&r-s)
7. negative control for 5&6

PCR

Lacp-GFP-DT(a kit of biological parts)

10×Buffer	2mM dNTPs	25mM MgSO4	VF	VR	KOD Plus Neo	MilliQ	DNA	Total
5	5	3	1.5	1.5	1	32	1	50

Colony PCR

pspA

10×Buffer	2mM dNTPs	25mM MgSO4	Primer pspA f	Primer pspA r	KOD Plus Neo	MilliQ	Total
5	5	3	1.5	1.5	1	33	50

10×Buffer	2mM dNTPs	25mM MgSO4	Primer pspA f-p	Primer pspA r-s	KOD Plus Neo	MilliQ	Total
5	5	3	1.5	1.5	1	33	50

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 30sec
→30cycles

Electrophoresis

写真お願いします。
1. 1kb ladder
2. pspA(Primer pspA f&r)
3. pspA(Primer pspA f-p&r-s)
4. negative control for pspA(Primer pspA f&r)
5. negative control for pspA(Primer pspA f-p&r-s)

Transformation

Kil

August 23

Colony PCR

J23107-tatABCD
VF and VR were used for amplifying approximately 2800bp

10×Buffer	2mM dNTPs	25mM MgSO4	VF	VR	KOD Plus Neo	MilliQ	DNA	Total
5	5	3	1.5	1.5	1	32.5	0.5	50

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 90sec
→25cycles

August 24

Purification of PCR products

pspA(Primer pspA f-p&r-s)
84.1μg/mL
→See "August 22 -Colony PCR-"

Restriction

pspA	EcoR1	Pst1	BufferH	BSA	MilliQ	total
20	0.5	0.5	3	0.5	5.5	30

pspA	Xba1	Pst1	BufferM	BSA	MilliQ	total
20	0.5	0.5	3	0.5	5.5	30

at 37℃ for 1h

Gel extraction

• pspA(EcoR1, Pst1) 30.7μg/mL
• pspA(Xba1, Pst1) 44.0μg/mL

Restriction

pSB1C3(82.9μg/mL)	EcoR1	Pst1	BufferH	BSA	MilliQ	total
20	0.5	0.5	3	0.5	5.5	30

→Gel extraction

Electrophoresis

1. 1kb ladder
2. J23107-tatABCD →See "August 23 -PCR-"
3. negative control for J23107-tatABCD
4. pSB1C3(EcoR1, Pst1)

Ligation

pSB3C5(EcoR1, Pst1)	pspA(EcoR1, Pst1)	Ligation High Ver.2	total
1	5	3	9

16℃, overnight

August 25

August 26

August 27

Ligation

pSB1C3(EcoR1, Pst1)	pspA(EcoR1, Pst1)	Ligation High Ver.2	total
1	5	3	9

16℃, overnight

Colony PCR

Kil (1-7)

10×Buffer	2mM dNTPs	25mM MgSO4	VF	VR	KOD Plus Neo	MilliQ	Total
5	5	3	1.5	1.5	1	33	50

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 30sec
→30cycles

Purification of PCR products

torA-GFP →See "August 21 -PCR-"

Restriction

Lacp(Backbone:pSB3C5)	Spe1	Pst1	BufferH	BSA	MilliQ	total
20	0.5	0.5	3	0.5	5.5	30

at 37℃ for 1h
→Gel extraction

torA-GFP	Xba1	Pst1	BufferM	BSA	MilliQ	total
20	0.5	0.5	3	0.5	5.5	30

at 37℃ for 1h
→Purification

Purification of PCR products

J23107-tatABCD
42.0μg/mL
→See "August 23 -PCR-"

Restriction

J23107-tatABCD	Spe1	EcoR1	BufferM	BSA	MilliQ	total
20	0.5	0.5	3	0.5	5.5	30

August 28

Colony PCR

Kil (1-16)

10×Buffer	2mM dNTPs	25mM MgSO4	VF	VR	KOD Plus Neo	MilliQ	Total
5	5	3	1.5	1.5	1	33	50

Transformation

• pspA-pSB1C3
• pspA-pSB3C5

Liquid culture

• Kil-3 →Miniprep 100.4μg/mL
• Kil-6
• Kil-7 →Miniprep 77.7μg/mL
• Lacp-torA-GFP-1 →Miniprep 88.3μg/mL
• Lacp-torA-GFP-2 →Miniprep 85.0μg/mL
• Lacp-torA-GFP-3 →Miniprep +++

August 29

Kil assay

evernoteに結果のグラフがあります。

Liquid culture

• pspA-pSB1C3(1-3)
• pspA-pSB3C5(1-3)

Restriction

Lacp-Kil-DT(134.7ng/μL)	EcoR1	Spe1	BufferM	MilliQ	total
10	1	1	3	15	30

37℃, overnight

Colony PCR

• Lacp-torA-GFP-DT (1-5)
• pspA-pSB1C3 (1-5)
• pspA-pSB3C5 (1-6)

10×Buffer	2mM dNTPs	25mM MgSO4	VF	VR	KOD Plus Neo	MilliQ	Total
5	5	3	1.5	1.5	1	33	50

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 40sec
→30cycles

Gel extraction

J23107-tatABCD(EcoR1, Pst1) -5.9μg/mL

August 30

Ligation

J23107-tatABCD(EcoR1, Spe1)22.2μg/mL	DT(EcoR1, Xba1)37.5μg/mL	Ligation High Ver.2	total
10	1	9	20

at 16℃ for 1h

Transformation

J23107-tatABCD-DT (Amp resistance)

Electrophoresis

1. Lacp-torA-GFP-DT-1
2. Lacp-torA-GFP-DT-2
3. Lacp-torA-GFP-DT-3
4. Lacp-torA-GFP-DT-4
5. Lacp-torA-GFP-DT-5
6. pspA-pSB1C3-1
7. pspA-pSB1C3-2
8. pspA-pSB1C3-3
9. pspA-pSB1C3-4
10.pspA-pSB1C3-5
11.pspA-pSB3C5-1
12.pspA-pSB3C5-2
13.pspA-pSB3C5-3
14.pspA-pSB3C5-4
15.pspA-pSB3C5-5
16.pspA-pSB3C5-6

Miniprep

• pspA-pSB3C5-1 176μg/mL
• pspA-pSB3C5-2 214μg/mL
• pspA-pSB3C5-3 461μg/mL
• pspA-pSB1C3-2 79μg/mL

Colony PCR

Lacp-torA-GFP-DT (6-13)

2×Quick Taq	VF	VR	MilliQ	Total
25	1	1	23	50

94℃, 2min
94℃, 30sec
56℃, 30sec
68℃, 1min
→30cycles

Restriction

pspA-pSB1C3	EcoR1	Spe1	BufferM	BSA	MilliQ	total
15	0.5	0.5	5	0.5	28.5	50

at 37℃ for 1h

Electrophoresis

1. 1kb ladder
3. pspA-pSB1C3(EcoR1, Spe1)

August 31

Gel extraction

pspA-pSB1C3(EcoR1, Spe1) 8.0μg/mL

Purification

Lacp-GFP-DT -0.7μg/mL

Ligation

pspA(EcoR1, Spe1)	DT(EcoR1, Xba1)	Ligation High Ver.2	total
25	1	13	39

at 16℃ for 1h

Transformation

pspA-DT (Amp resistance)

September 1

September 2

Colony PCR

J23107-tatABCD-DT (1-8)

2×Quick Taq	VF	VR	MilliQ	Total
25	1	1	23	50

94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 3min
→25cycles

pspA-DT (2-8)

2×Quick Taq	VF	VR	MilliQ	Total
25	1	1	23	50

Extension, 10sec
→25cycles

Restriction

LacP-torA-GFP-DT	EcoR1	Spe1	BufferH	MilliQ	total
10	1	1	2	6	20

at 4℃ for overnight

Electrophoresis

1. 1kb ladder
3. J23107-tatABCD-DT-1
4. J23107-tatABCD-DT-3
5. J23107-tatABCD-DT-4
6. J23107-tatABCD-DT-5
7. J23107-tatABCD-DT-6
8. J23107-tatABCD-DT-7
9. J23107-tatABCD-DT-8

1. 1kb ladder
3. J23107-tatABCD-DT-1
4. J23107-tatABCD-DT-3
5. J23107-tatABCD-DT-4
6. J23107-tatABCD-DT-5
7. J23107-tatABCD-DT-6
8. J23107-tatABCD-DT-7
9. J23107-tatABCD-DT-8
11. LacP-pSB3C5(Spe1, Pst1)

1. 1kb ladder
3. pspA-DT-2
4. pspA-DT-3
5. pspA-DT-4
6. pspA-DT-5
7. pspA-DT-6
8. pspA-DT-7
9. pspA-DT-8

September 3

PCR

tatABCD-DT

10×Buffer	2mM dNTPs	25mM MgSO4	VF	VR	KOD Plus Neo	MilliQ	DNA	Total
5	5	3	1.5	1.5	1	32	1	50

Gel extraction

LacP-torA-GFP-DT →See "September 5 -Gel extraction-"

Restriction

torA-GFP-DT	Xba1	Pst1	BufferM	MilliQ	total
20	2	2	4	12	40

Purification

J23107-tatABCD 29.6μg/mL 1.56(260/280) 1.60(260/230)

Electrophoresis

1. 1kb ladder
2. J23107-tatABCD

Liquid culture

• LacP-kil-DT(1)
• pspA-DT(4, 7)

PCR

J23107-tatABCD

10×Buffer	2mM dNTPs	25mM MgSO4	VF	VR	KOD Plus Neo	MilliQ	DNA	Total
5	5	3	1.5	1.5	1	29	4	50

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 10sec
→30cycles

Gel extraction

LacP-torA-GFP-DT 31μg/mL 1.28(260/280) 0.49(260/230) LacP-torA-GFP-DT 8μg/mL 1.32(260/280) 0.83(260/230)

Transformation

J23107-tatABCD (Amp resistance)
J23113-kil-DT (Amp resistance)
J23100-kil-DT (CP resistance)
J23101-kil-DT (CP resistance)
J23106-kil-DT (CP resistance)
J23115-kil-DT (CP resistance)→We didn't transform because of lack of culture medium.
J23105-kil-DT (CP resistance)→We didn't transform because of lack of culture medium.

Electrophoresis

1. 1kb ladder
2-7. LacP-torA-GFP-DT

Colony PCR

LacP-torA-GFP-DT (14-21) J23113-kil-DT (Amp resistance, 1-4) J23113-kil-DT (CP resistance, 1-4)

2×Quick Taq	primer-f	primer-r	MilliQ	Total
25	1	1	23	50

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

September 4

Liquid culture

• LacP-kil-DT(4, 5)(Amp resistance)
• pspA-DT(3, 6, 7)(Amp resistance)

PCR

We did PCR 10 more cycles to amplify products of PCR that we had done yesterday because we could not amplify J23107-tatABCD on September 3.
94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 1.5min
→30cycles

Electrophoresis

1-8. Lacp-torA-GFP-DT

1-4. J23113-kil-DT (Amp resistance)
5-8. J23113-kil-DT (CP resistance)

Liquid culture

• LacP-torA-GFP-DT
• J23113-kil-DT
• LacP-kil-DT(with IPTG)
• LacP-kil-DT(without IPTG)

After 20 hour, we quantified OD600.

plasmid	OD600
LacP-kil-DT(with IPTG)	2.116
LacP-kil-DT(without IPTG)	2.320

We cultured CP tolerance plasmid and give an antibiotic(Amp or Kan or none) after 2 hours. We quantify OD600.

time	1	2	3
2hours	0.131	0.100	0.606
add an antibiotic	Amp	Kan	none
7hours	0.491	0.988	2.634
19hours	2.253	0.815	2.742

September 5

Colony PCR

J23107-tatABCD (1-7)

buffer	2mM dNTPs	25mM MgSO4	primer-f	primer-r	KOD Plus Neo	DW	Total
5	5	3	1.5	1.5	1	33	50

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 1.25min
→30cycles

Gel extraction

by Kato
See "September 3 -Gel extraction-" LacP-torA-GFP-DT 15.9μg/mL 1.29(260/280) 0.38(260/230) LacP-torA-GFP-DT 17.2μg/mL 1.23(260/280) 0.48(260/230)

Miniprep

• J23113-kil-DT 85.7μg/mL 1.76(260/280) 1.97(260/230)
• LacP-torA-GFP-DT 126.6μg/mL 1.81(260/280) 2.09(260/230)
• LacP-kil-DT-4 62.5μg/mL 1.79(260/280) 1.99(260/230)
• LacP-kil-DT-5 56.0μg/mL 1.79(260/280) 2.01(260/230)
• pspA-DT-3 322μg/mL 1.45(260/280) 1.42(260/230)
• pspA-DT-6 147.6μg/mL 1.25(260/280) 1.37(260/230)
• pspA-DT-7 145.5μg/mL 1.24(260/280) 1.27(260/230)

Restriction

pspA-DT-6	Xba1	Pst1	BufferM	BSA	MilliQ	total
20	0.5	0.5	3	0.5	5.5	30

at 37℃ for 1h

Electrophoresis

J23107-tatABCD (1-7)

Colony PCR

J23107-tatABCD (8-23)

2×Quick Taq	VF2	VR	MilliQ	Total
25	1	1	23	50

94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 1.5min
→30cycles

Gel extraction

pspA-DT 10.2μg/mL 1.42(260/280) 0.76(260/230)

Ligation

pspA-DT(Xba1, Pst1)	LacP(pSB3C5)(Spe1, Pst1)	Ligation High Ver.2	total
5	1	3	9

at 16℃

Electrophoresis

Lacp-torA-GFP-DT(8-15)

Lacp-torA-GFP-DT(9-23)

Ligation

Lacp-torA-GFP-DT(EcoR1, Spe1)	DT 17.2μg/mL(Xba1, Pst1)	Ligation High Ver.2	total
15	25	20	60

Liquid culture

• J23107-tatABCD-8
• J23107-tatABCD-20
• Lacp-torA-GFP-DT-21

September 6

Miniprep

• J23107-tatABCD-8 82.4μg/mL 2.06(260/280) 2.95(260/230)
• J23107-tatABCD-20 102.9μg/mL 2.04(260/280) 2.94(260/230)

Restriction

J23107-tatABCD 102.9μg/mL	Spe1	Pst1	BufferH	BSA	MilliQ	total
10	0.5	0.5	3	0.5	15.5	30

at 37℃ for 1h

Transformation

LacP-pspA-DT(pSB3C5)

DNA	Competent cell	total
2	20	22

Gel extraction

J23107-tatABCD(Spe1, Pst1) →We failed gel extraction because we mistook steps of experiment.

Electrophoresis

LacP-torA-GFP-DT-pspA-DT

Restriction

by Terasaka

J23107-tatABCD	Spe1	Pst1	BufferH	BSA	MilliQ	total
10	0.5	0.5	3	0.5	15.5	30

Gel extraction

J23107-tatABCD(Spe1, Pst1) →See "September 7 -Gel extraction-"

September 7

Liquid culture

• Lacp-torA-GFP-DT-1 (CP resistance)
• Lacp-torA-GFP-DT-21 (CP resistance)

preculture for 1 hour

Colony PCR

Lacp-torA-GFP-DT (1-8) Lacp-pspA-DT (1-8)

2×Quick Taq	VF2	VR	MilliQ	Total
25	1	1	23	50

94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 1.5min
→30cycles

Restriction

by Terasaka

J23107-tatABCD	EcoR1	Spe1	BufferM	BSA	MilliQ	total
10	0.5	0.5	3	0.5	15.5	30

GFP-DT	Xba1	Pst1	BufferM	BSA	MilliQ	total
15	0.5	0.5	3	0.5	10.5	30

Electrophoresis

① -⑧. LacP-pspA-DT
1-8. LacP-torA-GFP-DT

Gel extraction

• J23107-tatABCD(EcoR1, Spe1)
• J23107-tatABCD(Pst1, Spe1) ①57.6μg/mL ②23.4μg/mL
• GFP-DT(Xba1, Pst1)

Restriction

pspA-DT(145.5μg/mL)	Xba1	Pst1	BufferM	BSA	MilliQ	total
20	0.5	0.5	3	0.5	5.5	30

37℃, overnight

Liquid culture

• Lacp-torA-GFP-DT-7 (LB 2mL)
  

+IPTG 0mM, 0.05mM, 0.1mM, 0.15mM, 0.2mM, 0.3mM

• Lacp-torA-GFP-DT-7 (LB 5mL)

September 8

Check the effect of Amp

We used 5mL of the liquid culture medium with Lacp-torA-GFP-DT-7.
→See "September 7 -Liquid culture-"
We diluted it with LB until OD600=0.444, and dispensed it.
The dispense volume was 1.3mL.
We added 0/13/130μL of Amp(50mg/mL) to it.

5.5h after incubating at 37℃

Amp	0μL	13μL	130μL
OD600	2.457	2.696	0.310

Observation through a confocal microscope

We used the liquid culture media with Lacp-torA-GFP-DT + 13μL of Amp(50mg/mL) + IPTG 0/0.1/0.15mM.
Their OD600 was 0.47.
3h after incubating at 37℃, we eliminated each supernatant using a centrifuge, and diluted them with LB to which Amp was added.
2.5h after incubating at 37℃, we observed them through a confocal microscope.
We failed to observe it.

Gel extraction

pspA-DT -5.5μg/mL

Restriction

pspA-DT-3(322μg/mL)	Xba1	Pst1	BufferM	BSA	MilliQ	total
10	1	1	3	3	12	30

at 37℃ for 1h

September 9

Restriction

J23107-tatABCD(102.9μg/mL)	EcoR1	Spe1	BufferM	MilliQ	total
10	1	1	3	15	30

Electrophoresis

1. J23107-tatABCD(EcoR1, Spe1)
2. J23107-tatABCD(before restriction)
3. pspA-DT(Xba1, Pst1)
4. pspA-DT(before restriction)

Gel extraction

• J23107-tatABCD(EcoR1, Spe1) 26μg/mL
• pspA-DT(Xba1, Pst1) 199μg/mL

Liquid culture

• J23100/J23101/J23106-1
• pspA-DT-5 ×2
• Lacp-torA-GFP-DT-21
• GFP generator-2
• Lacp-pspA-DT-6

September 10

Miniprep

• J23100-1 105.5μg/mL
• J23101-1 76.0μg/mL
• J23106-1 90.9μg/mL
• pspA-DT-5 ①104.1μg/mL ②80.0μg/mL
• Lacp-torA-GFP-DT-21 79.6μg/mL
• GFP generator-2 84.1μg/mL
• Lacp-pspA-DT-6 99.8μg/mL

Ligation

• J23107-tatABCD-DT

J23107-tatABCD(EcoR1, Spe1)	DT(EcoR1, Xba1)	Ligation High Ver.2	total
10	5	7.5	22.5

• Negative control for J23107-tatABCD-DT

DT(EcoR1, Xba1)	Ligation High Ver.2	total
5	5	10

• J23107-tatABCD-pspA-DT

J23107-tatABCD(Spe1, Pst1)	pspA-DT(Xba1, Pst1)	Ligation High Ver.2	total
3	6	9	18

• Negative control for J23107-tatABCD-pspA-DT

J23107-tatABCD(Spe1, Pst1)	Ligation High Ver.2	total
3	3	6

Restriction

DNA	Spe1	Pst1	BufferH	MilliQ	total
15	1	1	3	10	30

DNA=J23100(105.5μg/mL), J23101(76.0μg/mL), J23106(90.9μg/mL)
at 37℃

Gel extraction

J23100 27.1μg/mL
J23101 50.2μg/mL
J23106 16.1μg/mL

Ligation

• J23100/J23101/J23106-kil-DT

J23100/J23101/J23106(Spe1, Pst1)	kil-DT(Xba1, Pst1)	Ligation High Ver.2	total
1	5	3	9

• Negative control for J23100/J23101/J23106-kil-DT

J23100/J23101/J23106(Spe1, Pst1)	MilliQ	Ligation High Ver.2	total
1	5	3	9

Transformation

J23100/J23101/J23106-kil-DT

September 11

Colony PCR

• J23107-tatABCD-DT (1-8)
• J23107-tatABCD-pspA-DT (1-8)

2×Quick Taq	VF2	VR	MilliQ	Total
25	1	1	23	50

94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 3.5min
→30cycles

Electrophoresis

• J23107-tatABCD-DT (1-8)
• J23107-tatABCD-pspA-DT (1-8)

Restriction

GFP generator(84.1μg/mL)	Xba1	Pst1	BufferM	BSA	MilliQ	total
15	1	1	3	3	7	30

Lacp-torA-GFP-DT(85.0μg/mL)	Spe1	Pst1	BufferH	MilliQ	total
15	1	1	3	10	30

Lacp-pspA-DT(99.8μg/mL)	Xba1	Pst1	BufferM	BSA	MilliQ	total
15	1	1	3	3	7	30

Liquid culture

pSB1C3(self-ligation) ×5 in LB 3mLCP 3μL
When OD600=approximately 0.6, we added 0/30/60/150/300μL of Amp(50mg/mL) to each liquid culture medium.
1h and 5h after incubating at 37℃, we measured OD600.

	1h	5h
0	-0.020	0.403
30	-0.046	0.544
60	0.263	0.007
150	0.061	0.839
300	0.093	1.441

Ligation

pSB3C5(EcoR1, Pst1)	pspA-DT(Xba1, Pst1)	J23107-tatABCD(EcoR1, Spe1)	Ligation High Ver.2	total
1	3	5	4.5	13.5

4℃, overnight

Colony PCR

• J23100-kil-DT (1-6)
• J23101-kil-DT (1-7)
• J23106-kil-DT (1-2)

2×Quick Taq	VF	VR	MilliQ	Total
23	1	1	25	50

Gel extraction

• GFP generator (Xba1, Pst1) 14.1μg/mL
• lacp-torA-GFP-DT (Spe1, Pst1) 42.9μg/mL 1.13(260/280) 1.19(260/230)
• lacp-pspA-DT (Xba1, Pst1) 20.0μg/mL 1.01(260/280) 1.30(260/230)

Electrophoresis

• J23107-tatABCD-DT (1-8)
• J23106-kil-DT (1, 2)
• J23100-kil-DT (1-4)

Liquid culture

• Lacp-torA-GFP-DT
• pSB1C3

Colony PCR

We did PCR 10 more cycle to amplify products of PCR on 9/11 of J23107-tatABCD-pspA-DT and J23107-tatABCD-DT.

September 12

Colony PCR

J23107-tatABCD-pspA-DT (9-15) 94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 4min
→26cycles

Check the effect of Amp

We cultured pSB1C3 for overnight.
→See "September 7 -Liquid culture-"
We diluted it with LB, and dispensed it.
We added Amp(50mg/mL) to it until density of Amp was 1/10, 1/30, 1/50 .

Amp	start	5 hours
1/10	0.557	0.077
1/30	0.628	0.166
1/50	0.472	0.055
0	0.594	2.585

Electrophoresis

• J23107-tatABCD-pspA-DT

• J23101-kil-DT (1-7)

Restriction

pspA(3C5) (106μg/mL)	EcoR1	Pst1	BufferH	MilliQ	total
15	1	1	3	10	30

pspA-DT-3	Xba1	Pst1	BufferM	BSA	MilliQ	total
15	1	1	3	3	7	30

J23107-tatABCD (102.4μg/mL)	EcoR1	Spe1	BufferM	MilliQ	total
15	1	1	3	10	30

Electrophoresis

• pspA(3C5)(EcoR1, Pst1)
• pspA-DT(Xba1, Pst1)
• J23107-tatABCD(EcoR1, Spe1)
• pSB1C3(EcoR1, Spe1)

Transformation

J23107-tatABCD-pspA-DT(pSB3C5)

DNA	Competent cell	total
2	20	22

on ice for 30 minites heatshock at 42℃ for 1 hour on ice 2 minites then add SOC medium 200μl preculture at 37℃ for 1 hour incubate CP culture plate

Gel extraction

• pspA(3C5)(EcoR1, Pst1) 26.8μg/mL 1.27(260/280) 0.32(260/230)
• pspA-DT(Xba1, Pst1) 26.1μg/mL 1.13(260/280) 0.55(260/230)
• J23107-tatABCD(EcoR1, Spe1) 1.5μg/mL 1.99(260/280) 0.06(260/230)

Ligation

pSB3C5(EcoR1, Pst1) 26.8μg/mL	pspA-DT(Xba1, Pst1) 26.1μg/mL	J23107-tatABCD(EcoR1, Spe1) 1.5μg/mL	Ligation High Ver.2	total
1	3	6	5	15

pSB3C5(EcoR1, Pst1) 26.8μg/mL	MilliQ	Ligation High Ver.2	total
1	9	5	15

LacP(pSB3C5)(Spe1, Pst1) 10μg/mL	GFP-DT(Xba1, Pst1) 7.0μg/mL	Ligation High Ver.2	total
1	5	3	9

LacP(pSB3C5)(Spe1, Pst1) 10μg/mL	MilliQ	Ligation High Ver.2	total
1	5	3	9

4℃, overnight

Observation through a confocal microscope

Evernoteに上がっている写真をお願いします。

September 13

Miniprep

• J23107-tatABCD-pspA-DT(pSB3C5)-1 130μg/mL 1.37(260/280) 0.79(260/230)

Restriction

J23107-tatABCD-pspA-DT(pSB3C5) (130μg/mL)	Spe1	Pst1	BufferH	MilliQ	total
10	1	1	3	15	30

Transformation

• LacP-GFP generater

• LacP
• J23107-tatABCD-pspA-DT(pSB3C5)
• J23107-tatABCD

Colony PCR

J23107-tatABCD-pspA-DT

→See "September 14 -Colony PCR-"

September 14

Colony PCR

See "September 13 -Colony PCR-"
J23107-tatABCD-pspA-DT

Colony PCR

• J23107-tatABCD-pspA-DT (1-6)

2×Quick Taq	VF	VR	MilliQ	Total
23	1	1	25	50

94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 4min
→30cycles

September 15

Liquid culture

• J23107-tatABCD-pspA-DT-1
• J23107-tatABCD-pspA-DT-2
• J23107-tatABCD-pspA-DT-3

Miniprep

• J23107-tatABCD-pspA-DT-1 22.9μg/mL
• J23107-tatABCD-pspA-DT-2 51.8μg/mL 1.57(260/280) 1.01(260/230)
• J23107-tatABCD-pspA-DT-3 28.5μg/mL 1.57(260/280) 0.94(260/230)

Restriction

J23107-tatABCD-pspA-DT (51.8μg/mL)	EcoR1	Spe1	BufferM	MilliQ	total
15	0.5	0.5	3	11	30

LacP-torA-GFP-DT (79.6μg/mL)	EcoR1	Xba1	BufferM	BSA	MilliQ	total
10	0.5	0.5	3	0.5	15.5	30

LacP-kil-DT (56.0μg/mL)	EcoR1	Xba1	BufferM	BSA	MilliQ	total
15	0.5	0.5	3	0.5	10.5	30

J23107-tatABCD-pspA-DT (51.8μg/mL)	EcoR1	Pst1	BufferM	MilliQ	total
15	0.5	0.5	3	11	30

37℃, 2 hours

Electrophoresis

①. 1kb ladder
②. J23107-tatABCD-pspA-DT
③. J23107-tatABCD-pspA-DT(EcoR1, Spe1)
④. J23107-tatABCD-pspA-DT(EcoR1, Pst1)
⑤. LacP-kil-DT
⑥. LacP-kil-DT(EcoR1, Xba1)
⑦. LacP-torA-GFP-DT
⑧. LacP-torA-GFP-DT(EcoR1, Xba1)

Gel extraction

①. 1kb ladder
②. J23107-tatABCD-pspA-DT(EcoR1, Spe1)
③. J23107-tatABCD-pspA-DT(EcoR1, Spe1)
④. J23107-tatABCD-pspA-DT(EcoR1, Pst1)
⑤. J23107-tatABCD-pspA-DT(EcoR1, Pst1)
⑥. 1kb ladder

• J23107-tatABCD-pspA-DT(EcoR1, Spe1) 14.2μg/mL 1.28(260/280) 0.54(260/230)
• J23107-tatABCD-pspA-DT(EcoR1, Pst1) 7.3μg/mL 1.55(260/280) 0.38(260/230)

Purification

• LacP-torA-GFP-DT(EcoR1, Xba1) 7.7μg/mL 1.41(260/280) 0.99(260/230)
• LacP-kil-DT(EcoR1, Xba1) →failed

Ligation

LacP-torA-GFP-DT(EcoR1, Xba1)	J23107-tatABCD-pspA-DT(EcoR1, Spe1)	Ligation High Ver.2	total
4	10	7	21

pSB1C3(EcoR1, Pst1)	J23107-tatABCD-pspA-DT(EcoR1, Pst1)	Ligation High Ver.2	total
4	10	7	21

at 16℃ for overnight

September 16

Transformation

LacP-torA-GFP-DT-J23107-tatABCD-pspA-DT
J23107-tatABCD-pspA-DT(pSB1C3)

September 17

Colony PCR

J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT (1-6)
J23107-tatABCD-pspA-DT(pSB1C3) (1-6)
J23107-kil-DT (6-10)

Restriction

LacP-torA-GFP-DT (126μg/mL)	EcoR1	Xba1	BufferM	BSA	MilliQ	total
10	1	1	3	3	12	30

LacP-torA-GFP-DT (126μg/mL)	Xba1	BufferM	BSA	MilliQ	total
3	1	3	3	20	30

Electrophoresis

A1-A6. J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT (1-6)
B1-B6. J23107-tatABCD-pspA-DT(pSB1C3) (1-6)

Liquid culture

• J23107-tatABCD-pspA-DT(pSB1C3)
• J23107-tatABCD-pspA-DT(pSB3C5)
• LacP-torA-GFP-DT(pSB3C5)

Electrophoresis

lane1. 1kb ladder
lane2. J23101-kil-DT-6
lane3. J23101-kil-DT-7
lane4. J23101-kil-DT-8
lane5. J23101-kil-DT-10
lane6. LacP-torA-GFP-DT
lane7. LacP-torA-GFP-DT(EcoR1, Xba1)
lane8. LacP-torA-GFP-DT(Xba1)

Miniprep

• J23106-kil-DT 11.7μg/mL 1.93(260/280) 1.46(260/230)

September 18

Gel extraction

• LacP-torA-GFP-DT(EcoR1, Xba1) 36.9μg/mL 1.37(260/280) 0.82(260/230)

Ligation

J23107-tatABCD-pspA-DT(EcoR1, Spe1) 14.2μg/mL	LacP-torA-GFP-DT(EcoR1, Xba1) 36.9μg/mL	Ligation High Ver.2	total
8	1.5	9.5	19

LacP-torA-GFP-DT(EcoR1, Xba1) 36.9μg/mL	Ligation High Ver.2	total
1.5	1.5	3

at 4℃ for 1 hour

Transformation

• J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT
• LacP-torA-GFP-DT(EcoR1, Xba1)

Observation through a confocal microscope

We made sample for observation through a confocal microscope We incubated sample with 0mM/0.5mM IPTG for 1.5 hours. Then, we incubated onto medium without IPTG for 2.5 hours.

IPTG(mM)	medium
0	LB
0.1	LB
0.1	LB with 0.1mM IPTG
0	LB with 2 percent glucose
0.1	LB with 2 percent glucose
0	LB with 5 percent glucose
0.1	LB with 5 percent glucose
0	LB with 2 percent glucose
0	LB ここ途中
0	LB
0	LB

Restriction enzyme processing

J23101-Kil-DT	BufferM	Xba1	Pst	BSA	MilliQ	total
15	3	1	1	3	7	30

J23101-Kil-DT	BufferM	Xba1	BSA	MilliQ	total
3	1	0.5	1	4.5	10

J23101-Kil-DT	BufferH	Pst	MilliQ	total
3	1	0.5	5.5	10

37℃

September 19

GeneClean II

LacP-torA-GFP-DT
4k5

Ligation

LacP-torA-GFP-DT(X,P) 9.7μg/mL	pSB4K5(E,P) 9.6μg/mL	J23107-tatABCD-PsPA-DT(E,S) 14.2μg/mL	Ligation High Ver.2	total
7	2	7	8	24μL

pSB4K5(E,P) 9.6μg/mL	Ligation High Ver.2	MilliQ	total
2	1	21	24μL

incubate at 37℃ for 2h

Electrophoresis

J23101-kil-DT(X,P) (X) (P)
Lane1: 1kb ladder
Lane2: empty
Lane3: plasmid
Lane4: X
Lane5: P
Lane6: X,P

Liquid Culture

We did liquid culture J23106-kil-DT(9/10, Amp)'s 6,7 at LB mediumAmp.

Transformation

J2-tat-psp-DT-LacP-torAGFP-DT
pSB4K5 negative control

Liquid culture

J2-tat-psp-DT-LacP-torAGFP-DT:1~5

Colony PCR

J2-tat-psp-DT-LacP-torAGFP-DT

VF2	VR	Quick tag	MilliQ	total
1	1	25	23	50

Predenature 94℃ 2min
Denature 94℃ 30sec
Annealing 55℃ 30sec
Extension 68℃ 5min
→30cycles

Restriction enzyme processing

LacP-kil-DT(4) 62.5μg/mL	Xba1	Pst1	10xBSA	MilliQ	BufferM	total
10	1	1	3	12	3	30

LacP-kil-DT(4)	EcoR1	Pst1	MilliQ	BufferH	total
10	1	1	15	3	30

LacP-kil-DT	Xba1	10xBSA	MilliQ	BufferM	total
2	0.5	1	5.5	1	10

LacP-kil-DT	EcoR1	MilliQ	BufferH	total
2	0.5	6.5	1	10

LacP-kil-DT	Pst1	MilliQ	BufferH	total
2	0.5	6.5	1	10

at 37℃, 2 hours

Electrophoresis

Lane1: 1kb ladder
Lane2: empty
Lane3: plasmid
Lane4: E
Lane5: X
Lane6: P
Lane7: E,P
Lane8: X,P

Electrophoresis

J23107-tatABCD-pspA-DT-LacP-torAGFP-DT
Lane1: ladder
Lane2: 1
Lane3: 2
Lane4: 3
Lane5: 4
Lane6: 5

Ligation

LacP-torA-GFP-DT(X,P) 9.7μg/mL	pSB4K5(E,P) 9.6μg/mL	J23107-tatABCD-PsPA-DT(E,S) 14.2μg/mL	Ligation High Ver.2	total
7	4	7	9	27μL

(Negative control)

pSB4K5(E,P) 9.6μg/mL	Ligation High Ver.2	MilliQ	total
4	9	14	27μL

Restriction enzyme processing

torA-R9 101.8μg/mL	bufferH	MilliQ	EcoR1	Pst	total
10	3	15	1	1	30

torA-R9 101.8μg/mL	bufferH	MilliQ	EcoR1	Pst	total
3	1	5.5	0.5	0	10

torA-R9 101.8μg/mL	bufferH	MilliQ	EcoR1	Pst	total
3	1	5.5	0	0.5	10

PCR

torA-R9	MilliQ	10xPCR buffer for KOD plus neo	2mM dNTP3	25mM MgSO4	M13	KOD plus neo	total
0.5	34	5	5	3	1.5	1	50

Predenature 94℃ 2min
Denature 98℃ 10sec
Annealing 60℃ 30sec
Extension 68℃ 20sec
→35cycles

Electrophoresis

torA-R9,plasmid,(E),(P),(E,P)
Lane1: 1kb ladder
Lane2: empty
Lane3: plasmid
Lane4: E
Lane5: P
Lane6: E,P
Lane7: empty
Lane8: 1kb ladder

Liquid culture

LacP-tatA9FT-DT (9/4)

PCR purification

torA-R9 68.0μg/mL

September 20

Gel extraction

LacP-kil-DT (E,P) (X,P)

Restriction enzyme processing

torA-R9 68.0μg/mL	bufferM	Spe1	MilliQ	Total
20	3	1	6	30

at 37℃, 2h

Ligation

LacP-kil-DT(X,P) 12.4μg/mL	pSB1C3(E,P) 34.2μg/mL	J23107-tatABCD-PsPA-DT(E,S) 14.2μg/mL	Ligation High Ver.2	total
7	4	7	9	27μL

16℃, 1h

(Negative control)

pSB1C3(E,P)	Ligation High Ver.2	MilliQ	total
4	9	14	27μL

Transformation

Using above Ligation and Negative control's product and torA-R9.

Miniprep

J23106-kil-DT 6; 12.0μg/mL, (260/280): 1.78, (260/230): 1.73
J23106-kil-DT 7; 13.6μg/mL, (260/280): 1.85, (260/230): 1.73

Restriction enzyme processing

GFP-DT 84.1μg/mL	bufferM	10xBSA	BbaI	MilliQ	total
15	3	3	1	8	30

at 37℃, 2 hours

Colony PCR

We made J2-tat-psp-DT-LacP-torAGFP-DT's 1~8 samples.

Quick tag	tat f primer	VR	MilliQ	total
25	1	1	23	50

Predenature 94℃ 2min
Denature 94℃ 30sec
Annealing 54℃ 30sec
Extension 68℃ 4min
→30cycles

Liquid culture

We did liquid culture J2-tat-psp-DT-LacP-torAGFP-DT's 1~4 samples.

Electrophoresis

J23701-tatABCD-pspA-DT-LacP-torA-GFP-DT Lane1: 1kb ladder
Lane2: 1
Lane3: 2
Lane4: 3
Lane5: 4
Lane6: 5
Lane7: 6
Lane8: 7
Lane9: 8
Lane10: 1kb ladder
Lane11: empty
Lane12: empty
torA-R9 (E,P): 31μg/mL, (280/280): 0.80, (280/230): 0.39
pSB1C3(E,P): 22μg/mL, (280/280):1.33, (280/230):0.6
torA-R9 (S): 56μg/mL, (280/280): 1.11, (280/230): 0.69
GFP-DT(X): 45μg/mL, (280/280): 1.15, (280/230): 0.88

Ligation

torA-R9 (E,P)	1C3(E,P)	Ligation High Ver.2	total
8	2	5	15μL

torA-R9 (S)	1C3(X)	Ligation High Ver.2	total
3	3	3	9μL

at 16℃, 1hour

Colony PCR

J23107-tatABCD-pspA-DT-lacP-torA-GFP-DT (4k5)
numbering 9~15

Transformation

J23107-tat-psp-DT-lacP-torA-GFP-DT (4k5)
J23107-tat-psp-DT-lacP-torA-kil-DT(1C3)
Negative control (1C3)
Negative control(4K5)
torA-R9(1C3)

Liquid culture

J23107-tat-pspA-DT-LacP-torA-GFP-DT: colony PCR(9~15)

2xQuick tag	VF	VR	MilliQ	total
25	1	1	23	50

Predenature 94℃ 2min
Denature 94℃ 30sec
Annealing 55℃ 30sec
Extension 68℃ 5min
→30cycles

PCR

torA-R9-GFP-DT	MilliQ	KOD plus ver.2 buffer	dNTP3	MgSO4	M13	VR	KOD plus	total
1	35	5	5	2	1.5	1.5	1	52

Predenature 94℃ 2min
Denature 94℃ 15sec
Annealing 55℃ 30sec
Extension 68℃ 5min
→30cycles

Miniprep

J2301-tat-psp-DT-LacP-torA-GFP-DT:2,3
2: 10.4μg/mL, (250/280): 1.45, (260/230): 0.95
3: 6.0μg/mL, (250/280): 1.32, (260/230): 0.79
(*)100μL

Electrophoresis

torA-R9-GFP-DT(after PCR)

September 21

Gel extraction

torA-R9-GFP-DT(after PCR): 10.3μg/mL, (260/280): 1.28, (220/230): 0.49

Electrophoresis

J23107-tat-psp-DT-LacP-torA-GFP-DT(kan): 9~15

Electrophoresis

J2-tat-psp-DT-LacP-torA-GFP-DT: 1,2
Lane1: empty
Lane2: empty
Lane3: 2
Lane4: 3
Lane5: 1kb ladder

Restriction enzyme processing

LacP-kil-DT(3)	Xba1	Pst1	10xBSA	MilliQ	BufferM	total
15	1	1	3	7	3	30

LacP-torA-6FP-DP(9/5)	Xba1	Pst1	10xBSA	MilliQ	BufferM	total
15	1	1	3	7	3	30

LacP-kil-DT	Xba1	10xBSA	MilliQ	BufferM	total
2	0.5	1	5.5	1	10

LacP-torA-6FP-DP	Xba1	10xBSA	MilliQ	BufferM	total
2	0.5	1	5.5	1	10

LacP-kil-DT	Pst1	MilliQ	BufferH	total
2	0.5	6.5	1	10

LacP-torA-6FP-DP	Xba1	Pst1	MilliQ	BufferH	total
2	0.5	1	6.5	1	10

J2-tat-psp-DT	Spe1	Pst1	BufferH	MilliQ	total
20	1	1	4	14	40

J2-tat-psp-DT	Spe1	BufferM	MilliQ	total
2	0.5	1	6.5	10

J2-tat-psp-DT	Pst1	BufferH	MilliQ	total
2	0.5	1	6.5	10

at 37℃, 2hours

September 22

Miniprep

torA-R9(Amp)1: 113.7μg/mL, (260/280): 1.96, (280/230): 2.42
torA-R9(Amp)2: 97.8μg/mL, (260/280): 1.67, (280/230): 1.72
torA-R9(Amp)3: 104.2μg/mL, (260/280): 1.93, (280/230): 2.67
LacP-torA-GFP-DT 6: 37.4μg/mL, (260/280): 2.24, (280/230): 3.27

Electrophoresis

LaneA: J2-tat (9/6)
LaneB: J2-tat-psp-DT 3C5 (9/13)
LaneC: J2-tat-psp-DT 1C5 (9/15)
LaneD: J2-tat-psp-DT (9/15)
LaneE: J2-tat-psp-DT 1C3 (9/18)
LaneF: J2-tat-psp-DT 3C5 (9/18)
LaneG: J2-tat-psp-DT-LacP-torA-GFP-DT:2 (9/20)
LaneH: J2-tat-psp-DT-LacP-torA-GFP-DT:3 (9/20)
LaneA~H are (E,P)

Restriction enzyme processing

J2-tat-psp-DT-LacPtorA-GFP-DT:2	BufferH	Pst	MilliQ	total
15	3	1	12	30

J2-tat-psp-DT-LacP-torAGFP-DT:3	BufferH	Pst	MilliQ	total
15	3	1	12	30

Restriction enzyme processing

LacP-torAGFP-DT(37.4μg/mL)	BufferH	EcoR1	Pst	total
25	3	1	1	30

Electrophoresis

1: J2-tat-psp-DT-LacP-torA-GFP-DT:2
2: LacP-torA-GFP-DT (E,P) (9/22)
3: J2-tatpsp-DT-LacP-torA-GFP-DT:3

September 23

Electrophoresis

写真お願いします。

• LacP-torA-GFP-DT(EcoR1, Pst1)

lane_1 1kb ladder lane_3 DNA lane_4 DNA lane_5 DNA

Gel extraction

写真お願いします。

• J23107-tatABCD-pspA-DT(pSB1C3)(Spe1, Pst1)
• LacP-torA-GFP-DT(Xba1, Pst1)

lane_1 1kb ladder lane_3 J23107-tatABCD-pspA-DT(pSB1C3)(Spe1, Pst1) lane_5 LacP-torA-GFP-DT(Xba1, Pst1)

Restriction

LacP-torA-GFP-DT (78.4μg/mL)	Xba1	Pst1	BufferM	BSA	MilliQ	total
15	1	1	3	3	7	30

LacP-torA-GFP-DT (78.4μg/mL)	Xba1	BufferM	BSA	MilliQ	total
3	0.5	1	1	4.5	10

J23107-tatABCD-pspA-DT(pSB1C3) (78μg/mL)	Spe1	Pst1	BufferH	MilliQ	total
9	1	1	2	7	20

Ligation

LacP-torA-GFP-DT(EcoR1, Pst1) (5.5μg/mL)	pSB4K5 (9.6μg/mL)	Ligation High Ver.2	total
8	4	6	18

at 16℃ for 1 hour

Liquid culture

• LacP-tatABCD-pspA-DT (2, 3, 5, 6)

Colony PCR

LacP-tatABCD-pspA-DT (1-6)

2×Quick Taq	VF2	VR	MilliQ	Total
25	1	1	23	50

94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 4min
→30cycles

Electrophoresis

写真お願いします。
①, LacP-torA-GFP-DT
②, LacP-torA-GFP-DT(Xba1, Pst1)
③, LacP-torA-GFP-DT(Xba1)
④, LacP-torA-GFP-DT(Pst1)
⑤, J23107-tatABCD-pspA-DT(Spe1, Pst1)

Liquid culture

• LacP-torA-GFP-DT (4, 7)
• LacP-GFP generater (1-3)
• T7-6His-GFP-DT (1, 2)

Gel extraction

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• LacP-torA-GFP-DT (Xba1, Pst1) 6.8μg/mL 1.27(260/280) 0.40(260/230)

Purification

• J23107-tatABCD-pspA-DT(Spe1, Pst1) 15.1μg/mL 1.49(260/280) 0.80(260/230)

Ligation

J23107-tatABCD-pspA-DT(Spe1, Pst1) (15.1μg/mL)	LacP-torA-GFP-DT (Xba1, Pst1) (6.8μg/mL)	Ligation High Ver.2	total
10	4	7	21

J23107-tatABCD-pspA-DT(Spe1, Pst1) (15.1μg/mL)	LacP-kil-DT (Xba1, Pst1) (12.4μg/mL)	Ligation High Ver.2	total
8	2	5	15

at 16℃ for 1 hour

Transformation

LacP-torA-GFP-DT(pSB4K5)
J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT
J23107-tatABCD-pspA-DT-LacP-kil-DT

DNA	competent cell	total
2	10	12

preculture at 37℃ for 1 hour culture at 37℃ for 16 hours

Electrophoresis

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J23107-tatABCD-pspA-DT(pSB1C3) (1-6)
lane_1 1kb ladder lane_2~7 DNA lane_8 1kb ladder

September 24