Team:Kyoto/Florigen/Notebook

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Contents

Florigen Notebook

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
10xBufer2mM dNTPsprimer fwdprimer revtemplatepolymeraseMilliQTotal
551.51.50.5135.550

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

Dpn1 Digestion
PCR productDpn1
502
37℃,1h incubate
Self-ligation
productMilliQLigaseT4 KinaseTotal
275115

16℃, 1h incubate

Transformation
competent cellDNA
202

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)10xBuferHEcoR1Pst1MilliQTotal
521-1220
52-11220

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

NameWellSampleCompetent CellsTotalPlateColony
FT-1 µL1011LB (Kan+)×
pSB1C31-3-A11011LB (CP+)
I7190051-15-N11011LB (Amp+)

August 17

Transformation

by Takeuchi

NameWellSampleCompetent CellsMilliQTotalPlateColony
FT-221822LB (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

10xBuferdNTPsMgSO4primer fwdprimer revtemplatepolymeraseMilliQTotal
553111(130ng/µL)1(KOD plus neo)3350

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

Electrophoresis0821.png
LaneNamelength(bp)
11kb ladder-
2FT600


August 21

Restriction digestion

by Sato

DNA(FT,203ng/µL)10xBuferMXba11Pst1MilliQTotal
104112440

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

Ligation

by Sato

VectorInsertLigation High Ver.2
pSB1C31FT105.5

Liquid culture

T7 promoter, pSB1C3 (4mL)

August 22

Miniprep

by Sato

T7 promoterpSB1C3
85.3ng/µL82.93ng/µL


August 23

Ethanol Precipitation

diluted in 20µL 79.3ng/µL

August 24

Restriction enzyme processing

T7 promoter(85.3ng/µL)SpelPstlbuffer MMiliQTotal
10112620

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

pSB1C3(82.9ng/µL)XbalSpelbuffer MMiliQTotal
201141440

->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 => 597fmol2µL => 60fmol2.5µL
FT(600bp, 79.3ng/µL)T7(2100bp,33.4ng/µL)Ligation High Ver.2
2.4µL => 478fmol2µL => 48fmol2.2µL

=> 16℃,1hr incubate

August 27

Colony PCR

2X Quick TagVF2VRMiliQTotal
25112350

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

FT(TOPO) PCR(re)

bufferdNTPsMgSO4primer fprimer rTemplate(130ng/µL)KODplus neoMilliQTotal
55311113350
94℃98℃68℃cycles
2min10sec15sec30


Liquid culture

by Nobeyama
FT 4ml

August 28

Mutation of FT (re)

inverse PCR

MilliQbufferdNTPprimer fprimer rFT(130ng/µL)KODplusTotal
35.5551.51.50.5150
94℃98℃68℃cycles
2min10sec4min18

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

MilliQbufferdNTPprimer fprimer rFT(130ng/µL)KODplusTotal
35.5551.51.50.5150

second

MilliQbufferdNTPprimer fprimer rFT(52ng/µL)KODplusTotal
35551.51.51150
94℃98℃68℃cycles
2min10sec4min30

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

first sampleDpnltotal
45µL2µL47µL

in 37℃, 1 hour

Self-Ligation

PCR productsMilliQLigation HighT4 kinasetotal
2µL7µL5µL1µL15µL

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)bufferHE.coliPst1MilliQtotal
1µL5µL0.5µL0.5µL3.5µL10µL

in 37℃,1.5hour

PCR(RBS primer)

buffer for KODplus neodNTPsMgSO4primer fprimer rTemplate(52.6ng/µL)KODplus neoMilliQTotal
55311113350

94℃98℃68℃cycles
2min10sec15sec30

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

PCR(re)

bufferdNTPsMgSO4primer fprimer rTemplateKOD neoMilliQTotal
55311113350

94℃98℃68℃cycles
2min10sec15sec35

Lane1: 1kb ladder
Lane2: FT

PCR(re)

bufferdNTPsMgSO4primer fprimer rTemplateKOD neoMilliQTotal
55211113450

94℃98℃68℃cycles
2min10sec10sec30

September 2

PCR(re;re)

first

bufferdNTPsMgSO4primer fprimer rTemplate(1ng/µL)KODplus neoMilliQTotal
55311113350

second

bufferdNTPsMgSO4primer fprimer rTemplate(10ng/µL)KODplus neoMilliQTotal
55311113350

94℃98℃68℃cycles
2min10sec10sec25

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

refine first Template => 132ng/µL

September 3

Restriction enzyme processing

FT(132ng/µL)bufferMEcoRISpeIMilliQTotal
10211620

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 cellDNAplatecolony
20µLFT-DT 2µLAmpo
20µLpT7-6His:R9 2µLAmpo
10µLGFP generator 1µLAmpo
(BBa,I746915,pT7-6His:GFP)

September 4

Colony PCR

Quick TagVF2VRMilliQTotal
25112350
94℃98℃55℃68℃cycles
2min30sec30sec1min25

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)XbaIPstIbufferMMiliiQtotal
201141440

at 37℃, 2 hours

Electrophoresis by Takeuchi

Restriction enzyme processingby Takeuchi

FT-DT(88.8ng/µL)bufferMXbaI/SpeMiliiQtotal
410.54.510
FT-DTbufferHPst/EcoMiliiQtotal
410.54.510

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

MilliQbufferdNTPprimer fprimer rFT(52ng/µL)KODplusTotal
35551.51.51150
94℃98℃68℃cycles
2min10sec4min20

→ GeneClean II 32.6ng/µL

Dpn1 processing

TA bufferDNADpn1Total
3.331236.3

Ligation

DNAMiliiQLigation high Ver.2T4 kinaseTotal
275115

Transformation competent cell: 10
DNA  : 1

September 10

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

FT①/②bufferHlEcoRIPstIMiliQTotal
410.50410
4100.5410

PCR

BuferdNTPsMgSO4primer(+/-RBS)fwdprimer(+/-RBS)revtemplate①/②KOD plus neoMilliQTotal
55311113350
94℃98℃68℃cycles
2min10sec10sec25

->purifying column 17.4ng/µL

Restriction enzyme processing

FT(RBS+)32.6ng/µLXbalPstlbuffer MBSATotal
30114440
T7-His:R9(62.6ng/µL)SpeIPstlMilliQbuffer MTotal
150.50.52220

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 cellDNA
20µLT7-FT 2µL

PCR(FT without RBS retry)

BuferdNTPsMgSO4primer(-RBS)fwdprimer(-RBS)revtemplateKOD plusMilliQTotal
55311113350
94℃98℃65℃68℃cycles
2min15sec30sec30sec25

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-)XbaIPstIbufferMBSAtotal
30114440

at 37℃, 2 hours
=> 6.2ng/μL

GFP-DTXbaIPstIbufferMBSAMilliQtotal
151133730

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 cellDNAplate
20µLT7-R9-GFP-DT 2µLAmp
20µLT7-R9-FT 2µLAmp
BL21CDE3 10µLT7-FT 1µLAmp


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)EcoRIPstIbuffer HMilliQtotal
20113530

at 37℃, 2 hours
=> 20.7ng/μL

T7-R9-FT (160ng/µL)EcoRISpeIbuffer MMilliQtotal
20113530

at 37℃, 2 hours
=> 20.1ng/μL

Transformation

competent cell BL21(DE3)DNAplate
10µLT7-R9-GFP-DT 1µLAmp
10µLT7-R9-FT 1µLAmp


Restriction enzyme processing

Buffer HBSAEcoRIPstIDpnⅠMilliQtotal
550.50.50.513.525

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

2× Master MixpSB1C3(Linerarized Plasmid Backbone)
44

at 37℃, 30 minutes
80℃, 30 minutes

PCR(Insert His-tag)

MilliQBuffer for iPCRdNTPsprimer fwdprimer revtemplate(T7-FT 37.1ng/μL)KOD plusTotal
35551.51.51150
94℃94℃56℃68℃cycles
2min15sec30sec2.5min15

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)Dpn1Total
45247

=>37℃, 1 hour

Self Ligation

PCR productsLigation HighT4 KinaseMilliQTotal
251715

=>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)EcoRIPstI10× buffer HMilliQtotal
100.50.52720



Colony PCR

Quick TagVFVRMilliQTotal
25112350
94℃94℃55℃68℃cycles
2min30sec30sec1min25


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 cellDNAplate
20µLPSB1C3 FT without RBS 1µLCP+

Verification of R9 function by TAKEUCHI

R9(20µg/µL)0.9µL
GFP(1.2mg/mL)2.23µL
RBS16.85µL
total20µL

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.

123456
R9oooxoo
cuticleooooxx
soak in GFP5min15min30min5min5min30min

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
gelA
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)x2E.coliBufferHMilliQtotal
30µL1µL4µL5µL40µL

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 buffertemplate RNAH2OTotal
1µL0.5µL5.5µL7µL
at 42℃, 2min
Reverse TranscriptaseRT bufferprimer mixtemplateTotal
0.5µL2µL0.5µL7µL10µL
at 42℃, 2min and 95℃,3min

RT PCR

bufferdNTPsMgSO4primer fprimer rTemplateKODplusMilliQTotal
5521.51.51134.550
94℃94℃54℃68℃cycles
2min15sec30sec10sec30

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