Contents 1 Secretion 2 Florigen 2.1 August 2 2.2 August 13 2.3 August 14 2.4 August 15 2.5 August 16 2.6 August 17 2.7 August 20 2.8 August 21 2.9 August 22 2.10 August 23 2.11 August 24 2.12 August 27 2.13 August 28 2.14 August 29 3 Golden Gate Assembly 3.1 August 10

Secretion

Florigen

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°C 2min, (98°C 10sec, 68°C 4min)x4cycles, 4°C Hold

Dpn1 Digestion

PCR product	Dpn1
50	2

37°C,1h incubate

Self-ligation

product	MilliQ	Ligase	T4 Kinase	Total
2	7	5	1	15

16°C, 1h incubate

Transformation

competent cell	DNA
20	2

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

August 13

Liquid culture of FT 37°C, overnight

August 14

Miniprep of FT : by Sato, Takeuchi
The concentrarion 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 of 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 (FT) 4ml by Nobeyama

August 28

Mutaion 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 HighllT4 kinaselltotal
2µL	7µL	5µL	1µL	15µL

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

Liquid culture(I746902) 3mL

Golden Gate Assembly

August 10

Golden Gate Assembly method This method helps us to constract some genes quickly and we can design the order of constractions. We are trying to construct a gene cycle composed of 6 genes( pSB1A3, lacI, GFP, RFP, GFP, DT). After this experiment is confirmed to work, we will so some experiment to check how the numbers of promorters influences the strength of transcription.