Team:Kyoto/Notebook

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  • Kyoto FlorigenNote.png
  • Kyoto SecretionNote.png

Contents

Caution

Causion.gif
We devided our team into 2 groups in order to achieve Flower Fairy E.coli, Florigen and Secretion group. Florigen aims to confirm expression of florigen in E.coli, activation of R9 peptide, and function of FT made by E.coli. Secretion group aims to make a secretion system without cell death and confirm function of tatABCD and the other proteins.


Kyoto FlorigenNotebook.png

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

IMG 2177.jpg

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

IMG 2178.jpg

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

IMG 2179.jpg
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)


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

IMG 2180.jpg
2X Quick TagVF2VRMiliQTotal
25112350

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

FT(TOPO) PCR(re)

IMG 2181.jpg
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)

IMG 2182.jpg

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

IMG 2183.jpg
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

IMG 2184.jpg
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

IMG 2185.jpg
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

IMG 2186.jpg
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

IMG 2187.jpg
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

IMG 2188.jpg










Restriction enzyme processingby Takeuchi

IMG 2189.jpg
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

IMG 2190.jpg
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)

IMG 2191.jpg
BuferdNTPsMgSO4primer(-RBS)fwdprimer(-RBS)revtemplateKOD plusMilliQTotal
55311113350
94℃98℃65℃68℃cycles
2min15sec30sec30sec25

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

IMG 2192.jpg
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


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.

IMG 2193.jpg

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



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)

IMG 2194.jpg

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


IMG 2195.jpg










gelB

IMG 2196.jpg

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

IMG 2197.jpg

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

IMG 2194.jpg

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

IMG 2198.jpg

Electrophoresis(re)

IMG 2199.jpg


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

IMG 2200.jpg












September 22

mRNA extraction
FT and R9
Arabidopsis thaliana's leaves : 200mg

R9FTtotal
1.575μL33.425μL35μL
R9GFP
1.57533.42535

1. Strip cuticles on palafilm by the head of a pencil.
2. Soak plant cells in R9 & FT or R9 & GFP for 2 hours(19:45~).
3. Add PBS (21:45~)

Microscope
R9+GFP+Plant Cell ver.3
R9+

R9GFPPBStotal
0.92.2516.8520μL

R9-

R9GFPPBStotal
02.2517.6520

same as 9/20, only not on parafilm but on microscope slide from the start.

September 23

Data0923.jpg

RNA Extraction
FT- (GFP+) FT+

ISOGENchloroformelutiontotal
120011212

Reverce Transcription

gDNA wipeout bufferRNAtotal
167

at 42 degree, for 2min.

Reverce TranscriptaseRT bufferprimer mixTemplatetotal
0.520.5710

at 42 degree, for 30min.
at 95 degree, for 3min.

RT-PCR

BufferdNTPsMgSO4primerTemplateKOD PlusMilliQtotal
5521.51134.5

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

1.NC TUBULIN
2. FUL
3. SEP3
4. AP1
5.FT TUBULIN
6. FUL
7. SEP3
8. AP1

September 24

ScreeningPCR & Electrophoresis

ElectrophoresisFFE0924.jpg

Lane
1. 100bp ladder
2. T7-His:FT ① 9.2ng/μL
3. T7-His:FT ② 11.7ng/μL
4. T7-His:FT ③ 23.6ng/μL
5. T7-His:FT ④ 21.6ng/μL
6. T7-His:FT ⑤ 30.6ng/μL
7. T7-His:FT ⑥ 51.5ng/μL
8. T7-His:FT ⑦ 47.1ng/μL
9. T7-His:FT(A4) 20.0ng/μL
10. T7-His:FT(B1) 18.8ng/μL
11. final2(by_Secretion_group)
12. blank

FT Introduce

FTR9PBStotal
502.8811.1264
GFPR9PBStotal
7.22.8853.0264

We took 1 leaf out of 6 individuals of Arabidopsis thaliana that grow three weeks one by one
Leaves 30mg
1.cut off leaves
2.Inject the juice(by terumo-syringe)(Center tip、tuberculin 1nl)
3.store 20 min, then add PBS 600μL

September 25

RNA extraction
FT- (GFP+) FT+
elution 6µL

Reverce Transcription

gDNA wipeout bufferRNAtotal
167

at 42 degree, for 2min.

Reverce TranscriptaseRT bufferprimer mixTemplatetotal
0.520.5710

at 42 degree, for 30min.
at 95 degree, for 3min.

RT-PCR

BufferdNTPsMgSO4primerTemplateKOD PlusMilliQtotal
5521.51134.5

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

1.TUBULIN NC
2.TUBULIN FT
3.FUL NC
4.FUL FT
5.SEP3 NC
6.SEP3 FT
7.AP1 NC
8.AP1 FT

RT-PCR5.png

October 12

Transformation

BL21(DE3)T7-His-FTT7-His-GFP-DTtotal
1μL10μL 11μL
1μL 10μL11μL

October 16

Liquid culture
T7-His-FT 3mL

Transformation (retry)

BL21(DE3)T7-His-GFP-DTtotal
1μL10μL11μL

October 17

Moved 1mL of Liquid culture of T7-His-FT in 100mL of SOC medium -> 2 samples
Added IPTG 100μL to the medium(final conc. is 1mM) when value of OD600 is 0.649
Incubated at 20℃ for 4 hours.
Collect bacterial cells and freeze-preserved.

October 20

Liquid culture
2mL (T7-His-GFP)

October 21

Collect bacterial cells same way described above(value of OD600 is 0.8, at 37℃, for 4 hour)

Purification of GFP

Lysis bufferlysozymeTriton X-100Ni-NTA resinElution buffer
5mL0.1mL0.3mL1mL2.5mL

fraction1:31.5µM->After consentrating 47.4µM
fraction2:15.8µM->After consentrating 33.4µM
fraction3:20.1µM
(Extinction coefficient=21,890)
RNA Extraction
Added 3μL of IPTG to 3mL of E.coli and then incubated 2 hour.

ISOGENchloroformisopropanolElution buffer
1mL0.2mL0.5mL11μL

deluted to 250ng/μL

Reverce Transcription

RNA(250ng/μL)gDNA wipe out bufferdH2Ototal
0.5μL1μL5.5μL7μL
TemplateRT bufferPrimer mixRTtotal
7μL2μL0.5μL0.5μL10μL

RT-PCR Template is 20 times deluted cDNA

bufferdNTPsMgSO4primerprimerTemplateKOD-PlusMilliQtotal
5521.5(His-FT f)1.5(FT sequence r)113350
5521.5(GAPDH) 1134.550

94℃ 2min
94℃ 15sec
55℃ 30sec
68℃ 30sec
for 30 cycles

lane_1 : GAPDH
lane_2 : FT
lane_3 : 100bp ladder

October 22

Restriction

T7-His-FT(A4, 20ng/μL)T7-His-FT(B2, 73ng/μL)bufferHEcoR1Pst1MilliQtotal
10μL 2μL0.5μL0.5μL7μL20μL
5μL2μL0.5μL0.5μL12μL20μL

at 37℃ for 1 hour

lane1 : T7-His-FT(B2)
lane2 : T7-His-FT(B2, EcoR1/Pst1)
lane3 : T7-His-FT(A4)
lane4 : T7-His-FT(A4, EcoR1/Pst1)

Transformation

T7-His-FT(B2)BL21(DE3)total
11011

October 23

RNA Extraction from plant cell

ISOGENchloroformisopropanolHigh salt solutionElution buffer
1mL0.25mL0.25mL0.25mL11μL

notes: After the homogenization with ISOGEN, sample 1 was incubated at 50℃ for 10min.
Sample1. 855.8ng/µL
Sample2. 812.4ng/µLL

October 24

Collect bacterial cells(T7-6His:FT) same way described above(value of OD600 is 0.722, at 37℃, for 4 hour)
2mL of culture was used for RNA extraction.
Purification of FT

Lysis bufferlysozymeTriton X-100Ni-NTA resinElution buffer
5mL0.15mL0.3mL0.5mL1.2mL

fraction1:65.1µM
fraction2:42.1µM
fraction3:10.9µM
fraction4:6.8µM
(Ectinction coefficient=21,430)

RNA extraction

ISOGENchloroformisopropanolElution buffer
1mL0.2mL0.5mL11μL

diluted to 250ng/μL

Reverce Transcription

RNA(250ng/μL)gDNA wipe out bufferdH2Ototal
0.5μL1μL5.5μL7μL
TemplateRT bufferPrimer mixRTtotal
7μL2μL0.5μL0.5μL10μL

RT-PCR Template is 20 times deluted cDNA

bufferdNTPsMgSO4primerprimerTemplateKOD-PlusMilliQtotal
5521.5(His-FT f)1.5(FT sequence r)113350
5521.5(GAPDH) 1134.550

94℃ 2min
94℃ 15sec
55℃ 30sec
68℃ 30sec
for 30 cycles

lane_1 : 100bp ladder
lane_2 : GAPDH
lane_3 : FT

Assay of FT activation

protein-R9 solution mixFTGFPR9PBS
sample1 (FT-) - 200µL 0.11mg 100µL
sample2 (FT+) 230µL - 0.21mg 70µL

100mg of Arabidopsis thaliana leaves were harvested for each samples and injected the solution mix.
incubation at room temperature for 13h in dark

October 25

sample1
sample2
ISOGENchloroformisopropanolHigh salt solutionElution buffer
1mL0.25mL0.25mL0.25mL11μL

Sample1. 682.7ng/µL
Sample2. 859.3ng/µLL

diluted to 250ng/μL

Reverce Transcription

RNA(250ng/μL)gDNA wipe out bufferdH2Ototal
1μL2μL11μL14μL
TemplateRT bufferPrimer mixRTtotal
14μL4μL1μL1μL20μL

RT-PCR Template is 20 times diluted cDNA

bufferdNTPsMgSO4primerTemplateKOD-PlusMilliQtotal
5521.51134.550

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

Kyoto SecretionNotebook.png

February 7

Preculture

We started preculture at 12:10.

February 8

Liquid culture

We start culturing with 300mL of LB medium.

timeOD600
12:00start
14:100.019
14:450.154
15:050.267
15:210.64

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, LacP colony transformed on February 8
colony of competent cell made on February 8
B0040 1.4k pSB1A2 B0034 1.2M pSB1A2(from iGEM parts plate)

Making Competent cells

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

timeOD600
11:45start
13:300.048
14:300.168
15:030.256
15:200.405
15:350.459
at last0.576

February 11

Checking Transformation efficiency

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

February 13

Transformation

Const promoter J23110, J23109, J23100

DNACompetent cellTotal
1μL2021

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

DNAconcentration[μg/μL]
LacP339.6
LacP440.8
LacP528.9
RBS128.2
RBS257.4
RBS313.2
DT369.7
DT464.4
DT561.5
GFP164.0
GFP250.5
GFP366.0

Restrictive Digestion

Const promoter J23100

DNASpe1Pst1Buffer2BSAMilliQTotal
200.50.530.55.530

at 37℃ for overnight

February 15

Making gel

1% Agarose gel

AgaroseTAE
1.6g160mL

Electrophoresis

No.1

Gel No.1

Restriction productloading dye
5μL1

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





No.2

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 enzymeBufferBSAMilliQTotal
100.530.51630

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

February 16

Electrophoresis

Electrophoresis021601.JPG

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 Plus2mM dNTPs25mM MgSO410μM primer-f10μM primer-r158ng/μL Genomic DNAKOD plusMilliQTotal
5531.51.5113250

Electrophoresis

Electrophoresis021602.JPG

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)

DNAcompetent cell
1μL10μL

February 17

PCR

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

Genomic PCR

BufferdNTPsMgSO4primer-fprimer-rgenomic DNAKOD plusMilliQTotal
2.52.550.750.750.50.51650

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

Electrophoresis

Electrophoresis021701.JPG

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





Restrictive Digestion

J23100Spe1Pst1Buffer2BSAMilliQTotal
100.50.530.515.530

Genomic PCR

BufferdNTPsMgSO4primer-fprimer-rgenomic DNAKOD plusMilliQTotal
TMAO reductase2.52.530.750.750.50.515.525
TatABCD2.52.520.750.750.50.515.525
TatABCD2.52.520.750.7550.510.525

Electrophoresis

Electrophoresis021702.JPG

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



Checking of restriction enzyme

DTenzymeBufferBSAMilliQTotal
20.530.52430

at 37℃ for overnight
We checked EcoR1 and Xba1.

February 18

Miniprep

μg/mL260/280230/260
JS3117-11351.52.06
JS3117-2751.61.63
JS3109-11151.51.88
JS3109-2751.651.71
pSB1AT3-1701.661.83
pSB1AT3-21001.521.54

diluted to 25 times

Making Competent cells

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

timeOD600
10:30start
12:100.118
13:000.270
13:300.502

Transformation

pSB1AT3-2competent cellMilliQTotal
0201030
220830
1020030
  • Results(on Feb. 19)
pSB1AT3-2number of colony
00
2177
10590

Transformation efficiency 7.4x10^4 colonys/μg

February 20

Restrictive Digestion

sample 1

DT plasmidEcoR1Xba1BufferBSAMilliQTotal
7.50.50.530.51830

sample2

GFP plasmidEcoR1Spe1BufferBSAMilliQTotal
100.50.530.515.530

Electrophoresis

  • sample1
Electrophoresis022001 .JPG

a. sample1 2μL + MilliQ 3μL + 6×Loading Dye 1μL
b. sample1 5μL + 6×Loading Dye 1μL
lane1. 1kb ladder
lane2. a
lane3. b
lane4. a
lane5. b
lane6. a
lane7. b
lane8. 1kb ladder

  • sample2
Electrophoresis022302.JPG

c. sample2 2μL + MilliQ 3μL + 6×Loading Dye 1μL
d. sample2 5μL + 6×Loading Dye 1μL
lane1. 1kb ladder
lane2. c
lane3. d
lane4. 1kb ladder

PCR

BufferdNTPsMgSO4primer-fprimer-rgenomic DNAKOD plusMilliQTotal
TatABCD12.52.51.50.750.750.50.51625
TatABCD22.52.51.50.750.750.50.51625
TMAO reductase12.52.520.750.750.50.515.525
TMAO reductase22.52.520.750.7550.510.525

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

BufferdNTPsMgSO4primer-fprimer-rPCR productsgenomic DNAKOD plusMilliQTotal
TatABCD12.52.51.50.750.7500.50.51625
TatABCD22.52.51.50.750.75100.515.525
TMAO reductase12.52.520.750.75000.51625
TMAO reductase22.52.520.750.75000.51625

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

Electrophoresis

Electrophoresis022003.JPG
Electrophoresis022004.JPG

lane1.1kb ladder
lane2.TatABCD
lane3.TatABCD
lane4.TAMO1
lane5.TAMO2
lane6.1kb ladder




lane1.1kb ladder
lane2.TatABCD1
lane3.TatABCD2
lane4.TAMO
lane5.TAMO
lane6.1kb ladder

February 21

PCR (Advantage HF protocol)

bufferdNTPsprimer-fprimer-rgDNAPCR productsDWpolymeraseTotal
TatABCD2.52.50.750.7510170.525
TMAO2.52.50.750.7501170.525

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

Electrophoresis

Electrophoresis0221.JPG

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

BufferdNTPsMgSO4prefix primer-fsuffix primer-rproduct of gel extractproduct of PCR purification(1ng/μL)KOD plusMilliQTotal
15541.51.50.50132.550
25541.51.510132.550
35541.51.500.5132.550
45541.51.501132.550

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

Electrophoresis

Electrophoresis022201.JPG

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

BufferdNTPsMgSO4prefix primer-fsuffix primer-rproduct of PCR purification(1ng/μL)KOD plusMilliQTotal
15531.51.50.5132.550
25531.51.5113250
35531.51.5213150
45531.51.5313050
55531.51.51012950
65531.51.5013350

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

Electrophoresis

Electrophoresis022202.JPG








Checking Dpn1

BufferLacP(28.7ng/μL)Dpn1MilliQTotal
2100.57.520
2100517

February 23

Colony PCR

TatABCD(2 samples)

BufferdNTPsMgSO4primer-fprimer-rKOD plusMilliQTotal
5531.51.513350

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

Electrophoresis

Electrophoresis022301.JPG

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

Electrophoresis022302.JPG

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

Buffer2GFP2BSAMilliQDpn1
330.3231

Colony PCR

bufferdNTPsMgSO4Primer-fPrimer-rMilliQKOD PlusTotal
Colony PCR(2 samples)5531.51.533150
Negative control5531.51.534050

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

Electrophoresis

Electrophoresis0227.JPG
laneDNAsampleLoading DyeMilliQ
11kb ladder200
2product of PCR1510
3product of PCR2510
4product of PCR(Negative control)510
5product of PCR(2/23)510
6GFP2(DPN1)1020
7GFP2327
81kb ladder200

Results of liquid culture

We measure this after dilute it to 10 times.

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

PCR

bufferdNTPsMgSO4Primer-f(prefix)Primer-r(suffix)PCR purification product(1ng/µL)MilliQKOD PlusTotal
15531.51.50.232.8150
25531.51.50.532.5150
  • 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

Electrophoresis022801.JPG

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

Restrictive Digestion

Buffer2plasmid(?)enzymeMilliQTotal
220.215.820

incubate 1 hour at 37℃

Electrophoresis

Electrophoresis022802.JPG
Electrophoresis022803.JPG

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 Mixprimer-fprimer-rtemplateMilliQTotal
251.01.00.522.550

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

Restrictive Digestion

BufferHTatABCDEcoR1Spe1MilliQTotal
250.20.212.620

PCR purification

We eluted the product for 30µL MilliQ

Ligation

Insert(TatABCD)Vector(pSB1C3)Ligation HighTotal
101516

4℃, overnight

February 29

Transformation

TatABCDcompetent cellTotal
11011

Checking Restriction enzyme

plasmid seems to be 1-18C promoterEnzymeBufferMilliQTotal
20.2215.820

Checking TatABCD

TatABCDHind3BufferMilliQTotal
50.2212.820

PCR

bufferdNTPsMgSO4Primer-fPrimer-rColE1(6.5ng/µL) / TMAOMilliQKOD Plus NeoTotal
Kil2.52.51.50.750.750.5160.525
TMAO2.52.51.50.750.750.5160.525

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

Electrophoresis

38f7cf5b-20b9-42d0-a497-1885f1f92bbe.jpg

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

BuffergNTPsMgSO4Primer-fPrimer-rKOD Plus NeoTemplate gDNAproduct of gel extractionDWTotal
12.52.51.50.750.750.50.501625
22.52.51.50.750.750.50214.525

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

  • Kil
BufferdNTPsMgSO4primer-fprimer-rcolE1(6.5ng/µL)KOD Plus NeoMilliQTotal
2.52.51.50.750.750.5160.525

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

Electrophoresis

Electrophoresis030101.JPG

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




PCR

  • kil
BufferdNTPsMgSO4primer-fprimer-rProduct of PurificationKOD Plus NeoMilliQTotal
5531.51.5132150
Electrophoresis030102.JPG

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


Restrictive Digestion

KilEcoR1Spe1BufferHMilliQTotal
50.20.2212.620

incubate at 37℃, for 1.5 hours

PCR Purification

Ligation

KilpSB1C3Ligation HighTotal
5139

at 4℃, for overnight

March 2

Ligation

KilpSB1C3Ligation High Ver.2Total
5139
TatABCDpSB1C3LigationTotal
101516

at 16℃ for 1 hour

Transformation

KilKil(3/1,Ligation)TatABCDcompetet cellTotal
1001011
0101011
0011011

PCR

Quick Taqprimer-rprimer-ftemplateMilliQTotal
25110.522.550

Electrophoresis

Electrophoresis030227.JPG





Restrictive Digestion

pSB3C5-5EcoR1Pst1BufferHBSAMilliQTotal
200.50.530.55.530

at 37℃, for 2 hour

Electrophoresis

Electrophoresis030225.JPG

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

Restrictive Digestion

GFP1 ,2 ,3

GFPEcoR1Pst1BufferBSAMilliQTotal
100.50.530.515.530

at 37℃, for 2.5 hours

Restrictive Digestion

DTEcoR1Xba1BufferMBSAMilliQTotal
100.20.230.316.330
Constitutive PromoterSpe1Pst1BufferMBSAMilliQTotal
150.20.230.311.330

at 37℃, 2 hours

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

Checking TMAO

something seems to be TMAOBuffer2EcoR1MilliQTotal
1020.57.520

at 37℃, for 1 hour

Electrophoresis

Electrophoresis030226.JPG

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 Taqprimer-fprimer-rMilliQTotal
25112350

Electrophoresis

Electrophoresis030228.JPG









March 3

PCR

templatebufferdNTPsMgSO4VFVRKOD plusMilliQTotal
115531.51.513250
225531.51.513150

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

Miniprep

14.2µg/mL, 15.0µg/mL

March 4

Sequence of TatABCD

Quick Taqprimer-fpromer-r sequencetemplateMilliQTotal
251112350
Quick Taqprimer-f sequenceprimer-rtemplateMilliQTotal
251112350

Colony PCR of TMAO

bufferdNTPsNgSO4primer-fprimerr-rKOD plusMilliQTotal
5541.51.513250

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

Electrophoresis

34 percent 20~2.JPG

1. 1kb ladder
2. TatABCD1
3. TatABCD2
4. TMAO
5. 1kb ladder

Restrictive Digestion

TMAOEcoR1BufferHBSAMilliQTotal
100.230.316.530
TMAOXba1Pst1BudderMBSAMilliQTotal
100.20.230.316.330

at 37℃ for 1 hour

Electrophoresis

34 percent 20~1.JPG








Transformation

pSB1C3competent cell(made at 2/8)Total
5100105

March 5

Restrictive Digestion

pSB1C3(Xba1, Spe1)Pst1BufferHBSAMilliQTotal
100.220.27.620
pSB1C3(Xba1, Spe1)EcoR1BufferHBSAMilliQTotal
100.220.27.620

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

Ligation

Kil(EcoR1, Spe1)pSB1C3(EcoR1)Ligation HighTotal
5139

at 16℃ for 1 hour

Transformation

Kilcompetent cellTotal
11011

We used commercially available competent cells in this time.

PCR

TMAO

bufferdNTPsMgSO4Primer-fPrimer-rTemplateKOD plusMilliQTotal
5531.51.5113250

Electrophoresis

35percent20~2.JPG






Restrictive Digestion

LacPpSB3C5EcoR1Pst1BufferHBSAMilliQTotal
12000.50.530.55.530
20200.50.530.55.530

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

Ligation

LacPpSB3C5Ligation HighTotal
102618

at 4℃ for overnight

Transformation

LacP+pSB3C5competent cellTotal
11011

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

Restrictive Digestion

GFP PlasmidEcoR1Spe1Buffer2BSAMilliQTotal
100.50.530.515.530

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
We did Colony PCR to pspA

BufferdNTPsMgSO4Primer-fPrimer-rtemplate(TMAO)KOD plusMilliQTotal
2.52.51.50.750.750.50.51625

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

electrophoresis

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

March 6

Restrictive Digestion

GFPEcoR1Spe1BufferMBSAMilliQTotal
120.50.530.513.530

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

PCR

bufferdNTPsMgSO4Primer-fPrimer-rtemplateKOD plus neoMilliQTotal
5531.51.50.5132.550

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.

Restrictive Digestion

KilEcoR1Spe1BufferMBSAMilliQTotal
100.30.330.316.130
DTEcoR1Xba1Buffer2BSAMilliQTotal
100.50.530.515.530

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

Ligation

KilpSB1C3Ligation High Ver.2Total
4239
  • Kil : 350fmol
  • pSB1C3 : 29fmol

at 16℃ for overnight

March 7

Electrophoresis

Electrophoresis0307.JPG

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

VectorDNAGFPLigation High Ver.2Total
5151030

Restrictive Digestion

torAEcoR1Spe1bufferMBSAMilliQTotal
100.30.330.316.130
pspAEcoR1Spe1bufferMBSAMilliQTotal
50.30.330.321.130

at 37℃ for 1.5 hours

Purification

torA→31.8ng/µL
pspA→49.3ng/µL

Ligation

torApSB1C3Ligation High Ver.2Total
3339
pspApSB1C3Ligation High Ver.2Total
4239

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

Restrictive Digestion

pSB4K5EcoR1Spe1BufferMBSAMilliQTotal
200.20.230.26.430

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

Ligation

KilpSB4K5Ligation High Ver.2Total
101516

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

torApspAcompetent cellTotal
101011
011011

We use commercially available competent cells in this time.

March 9

Restrictive Digestion

TatABCDXba1Pst1BufferMBSAMilliQTotal
100.20.230.316.330

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 TaqVFVRMilliQTotal
25112350

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

Ligation

TatABCDconstP J23107Ligation High Ver.2Total
5139

TatABCD : 227fmol
constP J23107 : 21fmol

Transformation

pspAtorAKilcompetent cell
110010
201010
300110

Miniprep

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

March 10

Screening PCR

Quick TaqVFVRMilliQTotal
25112350
Electrophoresis0310.JPG

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

Restrictive Digestion

LacP-pSB3C5Spe1Pst1BufferMBSAMilliQTotal
100.20.220.27.420

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

torAXba1Pst1BufferMBSAMilliQTotal
100.20.220.27.420

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

Ligation

torALacP-pSB3C5Ligation High Ver.2Total
4239

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 TaqVFVRMilliQTotal
25112350

Electrophoresis

Electrophoresis031101.JPG
Electrophoresis031102.JPG

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)KilLacP-torAMilliQcompetent cellTotal
100002021
010002021
005005051
000505051
000012021

Restrictive Digestion

pspAEcoR1Spe1BufferMBSAMilliQTotal
100.50.530.315.730

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

Ligation

pspApSB1C3MilliQLigation High Ver.2Total
42039
22026
02226

March 13

Miniprep

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

March 14

Restrictive Digestion

pSB1C3EcoR1Spe1BufferMBSAMilliQTotal
200.20.240.415.240

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

Ligation

torApSB1C3Ligation High Ver.2Total
4239
MilliQpSB1C3Ligation High Ver.2Total
4239

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.

Restrictive Digestion

GFPEcoR1Spe1Buffer2BSAMilliQTotal
100.50.530.515.530
DTEcoR1Xba1Buffer2BSAMilliQTotal
100.50.530.515.530

for 2 hours at 37℃.

Miniprep

pspA (pSB1C3) 40.5ng/µL

Ligation

pspADTLigation High Ver.2Total
51.539.5
  • pspA : 385fmol
  • DT : 36fmol

Transformation

J23107-TatABCDDT (0.1ng/µL)DT (0.01ng/µL)pspA-DTcompetent cells on 3/15Total
20002022
02002022
00202022
00022022

Screening PCR

Kil, pspA and torA

Quick TaqPrimer-rPrimer-fMilliQTotal
25112350

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 TaqPrimer-rPrimer-fMilliQTotal
25112350

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

Electrophoresis

Electrophoresis0316.JPG

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

Restrictive Digestion

pSB1C3EcoR1Spe1BSABufferMBufferHMilliQTotal
200.20.20.3306.330
50.200.20212.620

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

Kil(pSB4K5)EcoR1Pst1BSABufferHMilliQTotal
50.20.20.2212.420
50.200.2212.620

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

pspA (pSB1C3)EcoR1Pst1BSABufferHMilliQTotal
50.20.20.2212.420
50.200.2212.620

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

Ligation

torApSB1C3Ligation High Ver.2Total
4239
MilliQpSB1C3Ligation High Ver.2Total
4239

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-TatABCDEcoR1Pst1BSABufferHMilliQTotal
50.20.20.2212.420
50.200.2212.620

Success.

pspA-DTEcoR1Pst1BSABufferHMilliQTotal
50.20.20.2212.420
50.200.2212.620

Failed.

March 19

Restrictive Digestion

DTEcoR1Xba1BSABufferMMilliQTotal
100.20.20.3316.330

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

Ligation

KilDTLigation High Ver.2Total
102618

We did this for an hour at 16℃.

Restrictive Digestion

GFPEcoR1Spe1BSABufferMMilliQTotal
100.50.50.5315.530

We did this for 4 hours at 37℃

Ligation

pspADTLigation High Ver.2Total
55515
pspApSB1C3Ligation High Ver.2Total
4138

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 TaqPrimer-RPrimer-FMilliQTotal
25112350
  • pspA→○
  • pspA-DT→○
  • GFP-DT→○
  • torA→×
  • Kil-DT 6 of 8 sumples→○
Quick TaqVRVFMilliQTotal
25112350
  • pspA→○
  • pspA-DT→×

Restrictive Digestion

torAEcoR1Spe1BSABufferMMilliQTotal
100.30.30.3316.130
DTEcoR1Xba1BSABufferMMilliQTotal
100.20.20.3316.330

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

Restrictive Digestion

pSB1C3EcoR1Spe1BSABufferMMilliQTotal
100.20.20.3316.330
50.200.2212.620

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

GFP-DTEcoR1Pst1Xba1BSABufferMMilliQTotal
50.20.200.2212.420
50.2000.2212.620
100.200.20.3316.330

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

Ligation

torApSB1C3pspADTGFP-DTLigation High Ver.2Total
14100038
201700412
300530412
430005412

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.

BufferdNTPsMgSO4Primer-FPrimer-RTemplateMilliQKOD plus neoTotal
5531.51.50.532.5150

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

Restrictive Digestion

torAEcoR1Spe1BSABufferMMilliQTotal
100.20.20.3316.330

Ligation

torApSSB1C3pspADTGFP-DTLigation highTotal
143000411
23000339
300550515
  • 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 TaqVFVRMilliQTotal
25112350

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

Restrictive Digestion

pspAEcoR1Spe1BufferMBSAMilliQTotal
50.30.330.321.130

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

Restrictive Digestion

LacP+pSB3C5-8Spe1Pst1Buffer2BSAMilliQTotal
200.50.530.55.530
Kil+DT-4Xba1Pst1Buffer2BSAMilliQTotal
200.50.530.55.530

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]

Restrictive Digestion

torA(pSB1C3)EcoR1Pst1BufferHBSAMilliQTotal
50.20.220.212.420
50.2020.212.620
torA-GFP-DTEcoR1Xba1Pst1BufferHBufferMBSAMilliQTotal
50.200.2200.212.420
50.200200.212.620
2000.20.2030.36.330

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

Ligation

LacP (pSB3C5)torA-GFP-DTLigation High Ver.2Total
1539
  • LacP : 22fmol
  • torA-GFP-DT : 197fmol
pspApSB1C3Ligation High Ver.2Total
101516
pspADTLigation High Ver.2Total
101516
  • pspA : 180fmol
  • pSB1C3 : 18fmol
  • DT : 16fmol
LacP(pSB3C5)Kil-DTLigation High Ver.2Total
1539

for 2 hours at 16℃

Transformation

LacP-Kil-DTcompetent cellTotal
11011

March 27

Miniprep

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

Transformation

NameWellSampleCompetent CellsTotalPlateColony
BBa_K117004 14J(2011 plate2)520???

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

Screening PCR

Quick TaqVF2VRMilliQTotal
25112350

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 cellPlating in SOC mediumTotal
120100121

July 24

Plating in SOC medium

July 25

Transformation

  • BBa_J23113 from iGEM Kit
  • LacP from iGEM Kit

July 27

Restrictive Digestion

Kil +DT (44.0 ng/μl)10xBuffer2BSAXbalMilliQTotal
13.63.00.50.511.930.0
LacP(28.7ng/μl)10xBuffer2 BSASpe1Pst1Total
20.93.00.50.54.630.0

July 30

Ligation

kil+DT(Xba1,Pst1)LacP(Spe1,Pst1)Ligation High Total
3063672

July 31

Restriction Enzyme Processing

torA-GFP+DT10×M BufferBSAMilliQXba1Pst1Total
2.53.00.523.40.30.330.0

Liquid Culture

J23113(backbone J61002)

August 1

Restrictive Digestion

LacP+kil+DT BSA10×H BufferEcoR1Pst1MilliQTotal
5.00.53.00.50.520.530.0
37℃ 2h
LacP middle copy BSA10×H BufferEcoR1Pst1MilliQTotal
10.00.53.00.50.515.530.0
37℃ 2h

MIniprep

J23113(backbone J61002)

Liquid culture

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

August 3

Restrictive Digestion

pSB4K5BSA 10×H BufferEcoR1Pst1MilliQTotal
8.00.53.00.50.517.530.0
B0034BSA10×H BufferSpe1Pst1MilliQTotal
12.00.53.00.50.513.530.0

37℃ 2h

DNA purification

Ligation

LacP+kil+DTpSB4K5ligation highTotal
15151545
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 10

Ligation

B0034 Spe1 Pst1GFP+DT Xba1 Pst1ligation highTotal
2349
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 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 TaqVFVFMilliQTotal
25112350

25cycles
pspA

10×Buffer for KOD-Plus-Ver22mM dNTPs25mM MgSO4Primer PsPA f-pPrimer PsPA r-sKOD-Plus-MilliQTotal
5531.51.513350

August 15

Colony PCR

pspA

2×Quick TaqpspA r-spspA f-pMilliQTotal
25112350

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

August 16

Electrophoresis

Electrophoresis081601.jpg
0814.jpg

positive control(GFP+DT)
negative control(MilliQ)
① ~⑥ colony PCR LacP+kil+DT





①1kb ladder
②Positive control (GFP+DT)
③Negative control (MilliQ)
④~⑨LacP+kil+DT ①~⑥
⑩~⑫pspA 10μL ,6×buffer 2μL
⑬1kb ladder

August 17

Colony PCR

pspA

10×Buffer for KOD-Plus-Ver22mM dNTPs25mM MgSO4Primer PsPA fPrimer PsPA rKOD-Plus-MilliQTotal
5331.51.513550
5351.51.513350

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

Electrophoresis

0820.JPG

① 1kb ladder
②, ③ MgSO4 3μL, pspA
④, ⑤ MgSO4 5μL, pspA
⑥ negative control(MilliQ)
⑧ 1kb ladder



August 20

Restrictive Digestion

J23113(201.0ng/μL)Xba1Pst1BufferMBSAMilliQTotal
101130.514.530

August 21

Colony PCR

pspA

10×Buffer2mM dNTPs25mM MgSO4Primer PsPA fPrimer PsPA rKOD Plus NeoMilliQTotal
5351.51.513350
5371.51.513150
53101.51.512850

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

Restrictive Digestion

J23113(218ng/μL)Spe1Pst1BufferHBSAMilliQTotal
101130.514.530
torA-GFP-DT(20ng/μL)Xba1Pst1BufferMBSAMilliQTotal
50.60.630.520.330

Electrophoresis

0821e.jpg

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)

Electrophoresis

0821r.jpg

laneA-C. pspA
laneD. pspA(without primer)



Colony PCR

  • pspA
10×Buffer2mM dNTPs25mM MgSO4Primer PsPA fPrimer PsPA rKOD Plus NeoMilliQTotal
5531.51.513350
  • negative control for pspA
10×Buffer2mM dNTPs25mM MgSO4Primer PsPA f-pPrimer PsPA r-sKOD Plus NeoMilliQTotal
5531.51.513350
  • GFP-DT(positive control)
10×Buffer2mM dNTPs25mM MgSO4VFVRKOD Plus NeoMilliQTotal
5531.51.513350

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

Transformation

  • LacP-torA-GFP-DT(Backbone pSB3C5)
  • J23107-TatABCD

PCR

kil, torA-GFP

10×Buffer2mM dNTPs25mM MgSO4VFVRKOD Plus NeoMilliQDNATotal
5531.51.5132150

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

August 22

Restrictive Digestion

pSB3C5EcoR1Pst1BufferHBSAMilliQTotal
13.30.50.530.512.230

Gel extraction

pSB3C5(EcoR1, Pst1)
27.2μg/mL

Transformation(the second time)

  • LacP-torA-GFP-DT(Backbone pSB3C5)
  • J23107-TatABCD

Electrophoresis

Electrophoresis082202.JPG

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×Buffer2mM dNTPs25mM MgSO4VFVRKOD Plus NeoMilliQDNATotal
5531.51.5132150

Colony PCR

pspA

10×Buffer2mM dNTPs25mM MgSO4Primer pspA fPrimer pspA rKOD Plus NeoMilliQTotal
5531.51.513350
10×Buffer2mM dNTPs25mM MgSO4Primer pspA f-pPrimer pspA r-sKOD Plus NeoMilliQTotal
5531.51.513350

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

Electrophoresis

Electrophoresis082201.jpg

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×Buffer2mM dNTPs25mM MgSO4VFVRKOD Plus NeoMilliQDNATotal
5531.51.5132.50.550

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-"

Restrictive Digestion

pspAEcoR1Pst1BufferHBSAMilliQTotal
200.50.530.55.530
pspAXba1Pst1BufferMBSAMilliQTotal
200.50.530.55.530

at 37℃ for 1h

Gel extraction

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

Restrictive Digestion

pSB1C3(82.9μg/mL)EcoR1Pst1BufferHBSAMilliQTotal
200.50.530.55.530

→Gel extraction

Electrophoresis

Electrophoresis082401.jpg

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.2Total
1539

16℃, overnight

August 25

August 26

August 27

Ligation

pSB1C3(EcoR1, Pst1)pspA(EcoR1, Pst1)Ligation High Ver.2Total
1539

16℃, overnight

Colony PCR

Kil (1-7)

10×Buffer2mM dNTPs25mM MgSO4VFVRKOD Plus NeoMilliQTotal
5531.51.513350

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

0827c.jpg

Purification of PCR products

torA-GFP →See "August 21 -PCR-"

Restrictive Digestion

LacP(Backbone:pSB3C5)Spe1Pst1BufferHBSAMilliQTotal
200.50.530.55.530

at 37℃ for 1h
→Gel extraction

torA-GFPXba1Pst1BufferMBSAMilliQTotal
200.50.530.55.530

at 37℃ for 1h
→Purification

Purification of PCR products

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

Restrictive Digestion

J23107-TatABCDSpe1EcoR1BufferMBSAMilliQTotal
200.50.530.55.530

August 28

Colony PCR

Kil (1-16)

10×Buffer2mM dNTPs25mM MgSO4VFVRKOD Plus NeoMilliQTotal
5531.51.513350

IMG 2207.jpg

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

After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600.

IPTG 0 0.001mM 0.01mM 0.1mM 1mM
0.5h 0.202 0.207 0.201 0.207 0.200
1h 0.406 0.428 0.424 0.402 0.421
1.5h 0.796 0.813 0.779 0.751 0.802
2h 1.107 1.129 1.141 1.092 1.124
2.5h 1.546 1.565 1.541 1.532 1.578
3h 1.912 1.933 1.890 1.883 1.940
3.5h 2.300 2.295 2.238 2.247 2.259
4h 2.546 2.545 2.528 2.490 2.520
4.5h 2.688 2.663 2.603 2.633 2.666
5h 2.699 2.826 2.787 2.593 2.673
5.5h 2.863 2.742 2.754 2.741 2.756
6h 2.831 2.876 2.758 2.759 2.728
20h 2.671 2.706 2.680 2.606 2.619

Liquid culture

  • pspA-pSB1C3(1-3)
  • pspA-pSB3C5(1-3)

Restrictive Digestion

LacP-Kil-DT(134.7ng/μL)EcoR1Spe1BufferMMilliQTotal
101131530

37℃, overnight

Colony PCR

  • LacP-torA-GFP-DT (1-5)
  • pspA-pSB1C3 (1-5)
  • pspA-pSB3C5 (1-6)
10×Buffer2mM dNTPs25mM MgSO4VFVRKOD Plus NeoMilliQTotal
5531.51.513350

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

Gel extraction

J23107-TatABCD(EcoR1, Pst1) -5.9μg/mL

0829.jpg

August 30

Ligation

J23107-TatABCD(EcoR1, Spe1)22.2μg/mLDT(EcoR1, Xba1)37.5μg/mLLigation High Ver.2Total
101920

at 16℃ for 1h

Transformation

J23107-TatABCD-DT (Amp resistance)

Electrophoresis

0830e.jpg

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 TaqVFVRMilliQTotal
25112350

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

Restrictive Digestion

pspA-pSB1C3EcoR1Spe1BufferMBSAMilliQTotal
150.50.550.528.550

at 37℃ for 1h

Electrophoresis

0830r.jpg

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

August 31

Gel extraction

0831e.jpg

pspA-pSB1C3(EcoR1, Spe1) 8.0μg/mL

Purification

LacP-GFP-DT -0.7μg/mL

Ligation

pspA(EcoR1, Spe1)DT(EcoR1, Xba1)Ligation High Ver.2Total
2511339

at 16℃ for 1h

Transformation

pspA-DT (Amp resistance)

September 2

Colony PCR

J23107-TatABCD-DT (1-8)

2×Quick TaqVFVRMilliQTotal
25112350

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

pspA-DT (2-8)

2×Quick TaqVFVRMilliQTotal
25112350

Extension, 10sec
→25cycles

Restrictive Digestion

LacP-torA-GFP-DTEcoR1Spe1BufferHMilliQTotal
10112620

at 4℃ for overnight

Electrophoresis

0902.jpg

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

0902-2.jpg

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)

0902-3.jpg

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×Buffer2mM dNTPs25mM MgSO4VFVRKOD Plus NeoMilliQDNATotal
5531.51.5132150

Gel extraction

0903.jpg

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




Restrictive Digestion

torA-GFP-DTXba1Pst1BufferMMilliQTotal
202241240

Purification

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

Electrophoresis

0903-2.jpg

1. 1kb ladder
2. J23107-TatABCD




Liquid culture

  • LacP-kil-DT(1)
  • pspA-DT(4, 7)

PCR

J23107-TatABCD

10×Buffer2mM dNTPs25mM MgSO4VFVRKOD Plus NeoMilliQDNATotal
5531.51.5129450

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

Gel extraction

IMG 2135.jpg

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

IMG 2136.jpg

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 Taqprimer-fprimer-rMilliQTotal
25112350

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

IMG 2137.jpg
1-8. LacP-torA-GFP-DT

IMG 2138.jpg
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.

plasmidOD600
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.

time123
2hours0.1310.1000.606
add an antibioticAmpKannone
7hours0.4910.9882.634
19hours2.2530.8152.742

September 5

Colony PCR

J23107-TatABCD (1-7)

buffer2mM dNTPs25mM MgSO4primer-fprimer-rKOD Plus NeoDWTotal
5531.51.513350

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)

Restrictive Digestion

pspA-DT-6Xba1Pst1BufferMBSAMilliQTotal
200.50.530.55.530

at 37℃ for 1h

Electrophoresis

IMG 2139.jpg

J23107-TatABCD (1-7)







Colony PCR

J23107-TatABCD (8-23)

2×Quick TaqVF2VRMilliQTotal
25112350

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.2Total
5139

at 16℃

Electrophoresis

IMG 2140.jpg
LacP-torA-GFP-DT(8-15)

IMG 2141.jpg
LacP-torA-GFP-DT(9-23)

Ligation

LacP-torA-GFP-DT(EcoR1, Spe1)DT 17.2μg/mL(Xba1, Pst1)Ligation High Ver.2Total
15252060

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)

Restrictive Digestion

J23107-TatABCD 102.9μg/mLSpe1Pst1BufferHBSAMilliQTotal
100.50.530.515.530

at 37℃ for 1h

Transformation

LacP-pspA-DT(pSB3C5)

DNACompetent cellTotal
22022

Gel extraction

IMG 2142.jpg

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





Electrophoresis

IMG 2143.jpg


LacP-torA-GFP-DT-pspA-DT




Restrictive Digestion

by Terasaka

J23107-TatABCDSpe1Pst1BufferHBSAMilliQTotal
100.50.530.515.530

Gel extraction

IMG 2144.jpg

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 TaqVF2VRMilliQTotal
25112350

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

Restrictive Digestion

by Terasaka

J23107-TatABCDEcoR1Spe1BufferMBSAMilliQTotal
100.50.530.515.530
GFP-DTXba1Pst1BufferMBSAMilliQTotal
150.50.530.510.530

Electrophoresis

IMG 2145.jpg

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




Gel extraction

IMG 2146.jpg

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



Restrictive Digestion

pspA-DT(145.5μg/mL)Xba1Pst1BufferMBSAMilliQTotal
200.50.530.55.530

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℃
Amp0μL13μL130μL
OD6002.4572.6960.310

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

IMG 2147.jpg

pspA-DT -5.5μg/mL


Restrictive Digestion

pspA-DT-3(322μg/mL)Xba1Pst1BufferMBSAMilliQTotal
1011331230

at 37℃ for 1h

September 9

Restrictive Digestion

J23107-TatABCD(102.9μg/mL)EcoR1Spe1BufferMMilliQTotal
101131530

Electrophoresis

IMG 2148.jpg

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


Gel extraction

IMG 2149.jpg

  • 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.2Total
1057.522.5
  • Negative control for J23107-TatABCD-DT
DT(EcoR1, Xba1)Ligation High Ver.2Total
5510
  • J23107-TatABCD-pspA-DT
J23107-TatABCD(Spe1, Pst1)pspA-DT(Xba1, Pst1)Ligation High Ver.2Total
36918
  • Negative control for J23107-TatABCD-pspA-DT
J23107-TatABCD(Spe1, Pst1)Ligation High Ver.2Total
336

Restrictive Digestion

IMG 2151.jpg
DNASpe1Pst1BufferHMilliQTotal
151131030

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

Gel extraction

IMG 2152.jpg IMG 2153.jpg
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.2Total
1539
  • Negative control for J23100/J23101/J23106-kil-DT
J23100/J23101/J23106(Spe1, Pst1)MilliQLigation High Ver.2Total
1539

Transformation

J23100/J23101/J23106-kil-DT

September 11

Colony PCR

  • J23107-TatABCD-DT (1-8)
  • J23107-TatABCD-pspA-DT (1-8)
2×Quick TaqVF2VRMilliQTotal
25112350

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

Electrophoresis

  • J23107-TatABCD-DT (1-8)

IMG 2156.jpg

  • J23107-TatABCD-pspA-DT (1-8)

IMG 2155.jpg

Restrictive Digestion

IMG 2154.jpg
GFP generator(84.1μg/mL)Xba1Pst1BufferMBSAMilliQTotal
151133730
LacP-torA-GFP-DT(85.0μg/mL)Spe1Pst1BufferHMilliQTotal
151131030
LacP-pspA-DT(99.8μg/mL)Xba1Pst1BufferMBSAMilliQTotal
151133730

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.

1h5h
0-0.0200.403
30-0.0460.544
600.2630.007
1500.0610.839
3000.0931.441

Ligation

pSB3C5(EcoR1, Pst1)pspA-DT(Xba1, Pst1)J23107-TatABCD(EcoR1, Spe1)Ligation High Ver.2Total
1354.513.5

4℃, overnight

Colony PCR

  • J23100-kil-DT (1-6)
  • J23101-kil-DT (1-7)
  • J23106-kil-DT (1-2)
2×Quick TaqVFVRMilliQTotal
23112550

Gel extraction

IMG 2159.jpg

  • 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

IMG 2160.jpg

  • 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 .

Ampstart5 hours
1/100.5570.077
1/300.6280.166
1/500.4720.055
00.5942.585

Electrophoresis

IMG 2162.jpg

  • J23107-TatABCD-pspA-DT

IMG 2161.jpg

  • J23101-kil-DT (1-7)

Restrictive Digestion

pspA(pSB3C5) (106μg/mL)EcoR1Pst1BufferHMilliQTotal
151131030
pspA-DT-3Xba1Pst1BufferMBSAMilliQTotal
151133730
J23107-TatABCD (102.4μg/mL)EcoR1Spe1BufferMMilliQTotal
151131030

Electrophoresis

IMG 2165.jpg
  • pspA(pSB3C5)(EcoR1, Pst1)
  • pspA-DT(Xba1, Pst1)
  • J23107-TatABCD(EcoR1, Spe1)
  • pSB1C3(EcoR1, Spe1)




Transformation

J23107-TatABCD-pspA-DT(pSB3C5)

DNACompetent cellTotal
22022

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

IMG 2166.jpg
  • pspA(pSB3C5)(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/mLpspA-DT(Xba1, Pst1) 26.1μg/mLJ23107-TatABCD(EcoR1, Spe1) 1.5μg/mLLigation High Ver.2Total
136515
pSB3C5(EcoR1, Pst1) 26.8μg/mLMilliQLigation High Ver.2Total
19515
LacP(pSB3C5)(Spe1, Pst1) 10μg/mLGFP-DT(Xba1, Pst1) 7.0μg/mLLigation High Ver.2Total
1539
LacP(pSB3C5)(Spe1, Pst1) 10μg/mLMilliQLigation High Ver.2Total
1539

4℃, overnight

Observation through a confocal microscope

left:E.coli sample stained with Hoechstunder and observed with 352nm wavelength
right:E.coli having Lacp-torA-GFP-DT and observed with 489nm wavelength

September 13

Miniprep

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


Restrictive Digestion

J23107-TatABCD-pspA-DT(pSB3C5) (130μg/mL)Spe1Pst1BufferHMilliQTotal


101131530

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

IMG 2169.jpg

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






Colony PCR

  • J23107-TatABCD-pspA-DT (1-6)
2×Quick TaqVFVRMilliQTotal
23112550

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)

Restrictive Digestion

J23107-TatABCD-pspA-DT (51.8μg/mL)EcoR1Spe1BufferMMilliQTotal
150.50.531130
LacP-torA-GFP-DT (79.6μg/mL)EcoR1Xba1BufferMBSAMilliQTotal
100.50.530.515.530
LacP-kil-DT (56.0μg/mL)EcoR1Xba1BufferMBSAMilliQTotal
150.50.530.510.530
J23107-TatABCD-pspA-DT (51.8μg/mL)EcoR1Pst1BufferMMilliQTotal
150.50.531130

37℃, 2 hours

Electrophoresis

IMG 2170.jpg

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

IMG 2171.jpg IMG 2172.jpg
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.2Total
410721
pSB1C3(EcoR1, Pst1)J23107-TatABCD-pspA-DT(EcoR1, Pst1)Ligation High Ver.2Total
410721

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)

Restrictive Digestion

LacP-torA-GFP-DT (126μg/mL)EcoR1Xba1BufferMBSAMilliQTotal
1011331230
LacP-torA-GFP-DT (126μg/mL)Xba1BufferMBSAMilliQTotal
31332030

Electrophoresis

IMG 2173.jpg

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

IMG 2174.jpg

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/mLLacP-torA-GFP-DT(EcoR1, Xba1) 36.9μg/mLLigation High Ver.2Total
81.59.519
LacP-torA-GFP-DT(EcoR1, Xba1) 36.9μg/mLLigation High Ver.2Total
1.51.53

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
0LB
0.1LB
0.1LB with 0.1mM IPTG (positive control)
0LB with 2 percent glucose
0.1LB with 2 percent glucose
0LB with 5 percent glucose
0.1LB with 5 percent glucose
0LB with 2 percent glucose (make medium new after incubate for 1.5 hours)
0LB with 2 percent glucose (make medium new after incubate for 1.5 hours)

Restrictive Digestion

pSB4K5EcoR1Pst1BufferHMilliQTotal
101131530
pSB4K5EcoR1BufferHMilliQTotal
30.515.530
pSB4K5Pst1BufferHMilliQTotal
30.515.510
LacP-torA-GFP-DTXba1Pst1BufferMBSAMilliQTotal
1011331230
LacP-torA-GFP-DTXba1BufferMBSAMilliQTotal
2111510
LacP-torA-GFP-DTPst1BufferHMilliQTotal
211610

37℃, 2 hours

Gel extraction

  • LacP-torA-GFP-DT(Xba1, Pst1) 9.7μg/mL 1.34(260/280) 0.62(260/230)
  • pSB4K5(EcoR1, Pst1) 9.6μg/mL 1.28(260/280) 0.32(260/230)

Miniprep

  • J23107-TatABCD-psp-DT(pSB1C3) 78μg/mL
  • J23107-TatABCD-psp-DT(pSB1C3) 48μg/mL
  • LacP-torA-GFP-DT 29μg/mL
  • J23101-kil-DT 25μg/mL

Restrictive Digestion

J23101-Kil-DT 25µg/mlBufferMXba1Pst1BSAMilliQTotal
406116660
J23101-Kil-DT 25µg/mlBufferMXba1BSAMilliQTotal
310.514.510
J23101-Kil-DT 25µg/mlBufferHPst1MilliQTotal
310.55.560

at 37℃ for 2 hours

Electrophoresis

Electrophoresis091801.jpg

①. pSB4K5
②. pSB4K5(Pst1)
③. pSB4K5(EcoR1)
④. pSB4K5(EcoR1, Pst1)
⑤. LacP-torA-GFP-DT
⑥. LacP-torA-GFP-DT(Xba1)
⑦. LacP-torA-GFP-DT(Pst1)
⑧. LacP-torA-GFP-DT(Xba1, Pst1)

Electrophoresis091802.jpg

①. 1kb ladder
②. J23101-Kil-DT(Xba1, Pst1)
③. J23101-Kil-DT(Xba1, Pst1)
④. J23101-Kil-DT(Xba1, Pst1)
⑤. J23101-Kil-DT(Xba1, Pst1)
⑥. 1kb ladder

Restrictive Digestion

J23101-Kil-DTBufferMXba1Pst1BSAMilliQTotal
153113730
J23101-Kil-DTBufferMXba1BSAMilliQTotal
310.514.510
J23101-Kil-DTBufferHPst1MilliQTotal
310.55.510

37℃

September 19

Gel extraction

lane1. 1kb ladder
lane2. pSB4K5(EcoR1, Pst1)
lane3. pSB4K5(EcoR1, Pst1)
lane4. empty
lane5. LacP-torA-GFP-DT(Xba1, Pst1)
lane6. LacP-torA-GFP-DT(Xba1, Pst1)
GelEx0919.jpg GelEx091901.jpg

Ligation

LacP-torA-GFP-DT(Xba1,Pst1) 9.7μg/mLpSB4K5(EcoR1,Pst1) 9.6μg/mLJ23107-TatABCD-PsPA-DT(EcoR1,Spe1) 14.2μg/mLLigation High Ver.2Total
727824μL
pSB4K5(EcoR1,Pst1) 9.6μg/mLLigation High Ver.2MilliQTotal
212124μL

incubate at 37℃ for 2h

Electrophoresis

CheckingElectrophoresis0919.jpg

J23101-kil-DT (Xba1,Pst1), (Xba1), (Pst1)
Lane1: 1kb ladder
Lane2: empty
Lane3: plasmid
Lane4: (Xba1)
Lane5: (Pst1)
Lane6: (Xba1, Pst1)

Liquid Culture

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

Transformation

J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT
pSB4K5 negative control

Liquid culture

J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT:1~5

Colony PCR

J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT

VF2VRQuick tagMilliQTotal
11252350

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

Restrictive Digestion

LacP-kil-DT(4) 62.5μg/mLXba1Pst110xBSAMilliQBufferMTotal
1011312330
LacP-kil-DT(4)EcoR1Pst1MilliQBufferHTotal
101115330
LacP-kil-DTXba110xBSAMilliQBufferMTotal
20.515.5110
LacP-kil-DTEcoR1MilliQBufferHTotal
20.56.5110
LacP-kil-DTPst1MilliQBufferHTotal
20.56.5110

at 37℃, 2 hours

Electrophoresis

Electrophoresis0919.jpg

LacP-kil-DT
Lane1: 1kb ladder
Lane2: empty
Lane3: plasmid
Lane4: (EcoR1)
Lane5: (Xba1)
Lane6: (Pst1)
Lane7: (EcoR1, Pst1)
Lane8: (Xba1, Pst1)


Electrophoresis091901.jpg

J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT(1-5)
Lane1: ladder
Lane2: 1
Lane3: 2
Lane4: 3
Lane5: 4
Lane6: 5

Ligation

LacP-torA-GFP-DT(Xba1,Pst1) 9.7μg/mLpSB4K5(EcoR1,Pst1) 9.6μg/mLJ23107-TatABCD-PsPA-DT(EcoR1,Spe1) 14.2μg/mLLigation High Ver.2Total
747927μL

(Negative control)

pSB4K5(EcoR1,Pst1) 9.6μg/mLLigation High Ver.2MilliQTotal
491427μL

Restrictive Digestion

torA-R9 101.8μg/mLbufferHMilliQEcoR1Pst1Total
103151130
torA-R9 101.8μg/mLbufferHMilliQEcoR1Pst1Total
315.50.5010
torA-R9 101.8μg/mLbufferHMilliQEcoR1Pst1Total
315.500.510

PCR

torA-R9MilliQ10xPCR buffer for KOD plus neo2mM dNTP325mM MgSO4M13KOD plus neoTotal
0.5345531.5150

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

Electrophoresis

CheckingElectropjoresis091901.jpg

torA-R9 plasmid, (EcoR1), (Pst1), (EcoR1,Pst1)
Lane1: 1kb ladder
Lane2: empty
Lane3: plasmid
Lane4: EcoR1
Lane5: Pst1
Lane6: EcoR1,Pst1
Lane7: empty
Lane8: 1kb ladder

Liquid culture

LacP-TatA-GFP-DT (9/4)

PCR purification

torA-R9 68.0μg/mL

September 20

Gel extraction

LacP-kil-DT (EcoR1,Pst1) (Xba1,Pst1)

Restrictive Digestion

torA-R9 68.0μg/mLbufferMSpe1MilliQTotal
2031630

at 37℃, 2h

Ligation

LacP-kil-DT(Xba1,Pst1) 12.4μg/mLpSB1C3(EcoR1,Pst1) 34.2μg/mLJ23107-TatABCD-PsPA-DT(EcoR1,Spe1) 14.2μg/mLLigation High Ver.2Total
747927μL
16℃, 1h

(Negative control)

pSB1C3(EcoR1,Pst1) Ligation High Ver.2MilliQTotal
491427μL

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

Restrictive Digestion

GFP-DT 84.1μg/mLbufferM10xBSABbaIMilliQTotal
15331830

at 37℃, 2 hours

Colony PCR

We made J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT's 1~8 samples.

Quick tagTat f primerVRMilliQTotal
25112350

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

Electrophoresis091902.jpg
Electrophoresis091903.jpg

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

→Bands didn't appear so we do PCR for 10 more cycles.





Liquid culture

We did liquid culture J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT's 1~4 samples.

Gel extraction

torA-R9 (EcoR1,Pst1): 31μg/mL, (280/280): 0.80, (280/230): 0.39
pSB1C3(EcoR1,Pst1): 22μg/mL, (280/280):1.33, (280/230):0.6
torA-R9 (Spe1): 56μg/mL, (280/280): 1.11, (280/230): 0.69
GFP-DT(Xba1): 45μg/mL, (280/280): 1.15, (280/230): 0.88

Ligation

torA-R9 (EcoR1,Pst1)pSB1C3(EcoR1,Pst1)Ligation High Ver.2Total
82515μL
torA-R9 (Spe1)pSB1C3(Xba1)Ligation High Ver.2Total
3339μL

at 16℃, 1hour

Colony PCR

J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT (4k5)
numbering 9~15

Transformation

J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT (4k5)
J23107-TatABCD-pspA-DT-LacP-torA-kil-DT(pSB1C3)
Negative control (pSB1C3)
Negative control(pSB4K5)
torA-R9(pSB1C3)

Liquid culture

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

2xQuick tagVFVRMilliQTotal
25112350

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

PCR

torA-R9-GFP-DTMilliQKOD plus ver.2 bufferdNTP3MgSO4M13VRKOD plusTotal
1355521.51.5152

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

Miniprep

J23701-TatABCD-pspA-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
Electrophoresis0920.jpg

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
Electrophoresis0921.jpg Electrophoresis092101.jpg

Electrophoresis

Electrophoresis092102.jpg

J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT(kan): 9~15







Electrophoresis

Electrophoresis092103.jpg

J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT: 1,2
Lane1: empty
Lane2: empty
Lane3: 2
Lane4: 3
Lane5: 1kb ladder



Restrictive Digestion

LacP-kil-DT(3)Xba1Pst110xBSAMilliQBufferMTotal
151137330
LacP-torA-GFP-DP(9/5)Xba1Pst110xBSAMilliQBufferMTotal
151137330
LacP-kil-DTXba110xBSAMilliQBufferMTotal
20.515.5110
LacP-torA-GFP-DPXba110xBSAMilliQBufferMTotal
20.515.5110
LacP-kil-DTPst1MilliQBufferHTotal
20.56.5110
LacP-torA-GFP-DPXba1Pst1MilliQBufferHTotal
20.516.5110
J23107-TatABCD-pspA-DTSpe1Pst1BufferHMilliQTotal
201141440
J23107-TatABCD-pspA-DTSpe1BufferMMilliQTotal
20.516.510
J23107-TatABCD-pspA-DTPst1BufferHMilliQTotal
20.516.510

at 37℃, 2hours

Electrophoresis

Electrophoresis092104.jpg

lane1. 1kb ladder
lane2. empty
lane3. LacP-kil-DT plasmid
lane4. LacP-kil-DT(Xba1)
lane5. LacP-kil-DT(Pst1)
lane6. LacP-kil-DT(Xba1, Pst1)
lane7. empty
lane8. LacP-torA-GFP-DP plasmid
lane9. LacP-torA-GFP-DP(Xba1)
lane10. LacP-torA-GFP-DP(Pst1)
lane11. LacP-torA-GFP-DP(Xba1, Pst1)
lane12. 1kb ladder

Electrophoresis092105.jpg




lane1. 1kb ladder
lane2. empty
lane3. J23107-TatABCD-pspA-DT plasmid
lane4. J23107-TatABCD-pspA-DT(Spe1)
lane5. J23107-TatABCD-pspA-DT(Pst1)
lane6. J23107-TatABCD-pspA-DT(Spe1, Pst1)


Colony PCR

  • torA-R9(pSB1C3) (1-4)
10×Buffer2mM dNTPs25mM MgSO4VFVRKOD Plus NeoMilliQTotal
5531.51.513350

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

Electrophoresis

Electrophoresis092107.jpg

lane1. 1kb ladder
lane2. empty
lane3. torA-R9-1
lane4. torA-R9-2
lane5. torA-R9-3
lane6. torA-R9-4
lane7. empty
lane8. empty

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

Electrophoresis092108.jpg

LaneA: J23107-TatABCD (9/6)
LaneB: J23107-TatABCD-pspA-DT pSB3C5 (9/13)
LaneC: J23107-TatABCD-pspA-DT 1C5 (9/15)
LaneD: J23107-TatABCD-pspA-DT (9/15)
LaneE: J23107-TatABCD-pspA-DT(pSB1C3) (9/18)
LaneF: J23107-TatABCD-pspA-DT(pSB3C5) (9/18)
LaneG: J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT:2 (9/20)
LaneH: J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT:3 (9/20)
LaneA~H are (EcoR1,Pst1)

Restrictive Digestion

J23107-TatABCD-pspA-DT-LacPtorA-GFP-DT:2BufferHPst1MilliQTotal
15311230
J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT:3BufferHPst1MilliQTotal
15311230

Restrictive Digestion

LacP-torA-GFP-DT(37.4μg/mL)BufferHEcoR1Pst1Total
2531130

Electrophoresis

①: J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT:2
②: LacP-torA-GFP-DT(EcoR1,Pst1) (9/22)
③: J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT:3
Electrophoresis092202.jpg Electrophoresis092201.jpg

Gel extraction

LacP-torA-GFP-DT(EcoR1,Pst1) 5.5μg/ml 1.21(260/280) 0.37(260/230)

Purification

LacP-torA-GFP-DT 78.4μg/ml 1.62(260/280) 1.03(260/230)

Restrictive Digestion

J23107-TatABCD-pspA-DT(pSB1C3) 78μg/mlBufferHSpe1Pst1MilliQTotal
153111030
J23107-TatABCD-pspA-DT(pSB1C3) 78μg/mlBufferHSpe1MilliQTotal
310.55.510
J23107-TatABCD-pspA-DT(pSB1C3) 78μg/mlBufferHPst1MilliQTotal
310.55.510
LacP-torA-GFP-DT 78.4μg/mlBufferMBSAXba1Pst1MilliQTotal
153311730
LacP-torA-GFP-DT 78.4μg/mlBufferMBSAXba1MilliQTotal
3110.54.510
LacP-torA-GFP-DT 78.4μg/mlBufferHPst1MilliQTotal
310.55.510

at 37℃ for 2 hours

Electrophoresis

Electrophoresis092205.jpg

Lane1: 1kb ladder
Lane2: empty
Lane3: J23107-TatABCD-pspA-DT(pSB1C3)
Lane4: J23107-TatABCD-pspA-DT(Spe1, Pst1)
Lane5: J23107-TatABCD-pspA-DT(Spe1)
Lane6: J23107-TatABCD-pspA-DT(Pst1) (9/18)
Lane7: empty
Lane8: LacP-torA-GFP-DT
Lane9: LacP-torA-GFP-DT(Xba1, Pst1)
Lane10: LacP-torA-GFP-DT(Xba1)
Lane11: LacP-torA-GFP-DT(Pst1)
Lane12: empty
Lane13: 1kb ladder

September 23

Gel extraction

Electrophoresis092301.jpg Electrophoresis092302.jpg

  • LacP-torA-GFP-DT(EcoR1, Pst1)

lane1 1kb ladder
lane3 DNA
lane4 DNA
lane5 DNA
→failed

Electrophoresis092303.jpg Electrophoresis092304.jpg
lane1 1kb ladder
lane3 J23107-TatABCD-pspA-DT(pSB1C3)(Spe1, Pst1)
lane5 LacP-torA-GFP-DT(Xba1, Pst1)
→failed

Restrictive Digestion

LacP-torA-GFP-DT (78.4μg/mL)Xba1Pst1BufferMBSAMilliQTotal
151133730
LacP-torA-GFP-DT (78.4μg/mL)Xba1BufferMBSAMilliQTotal
30.5114.510
J23107-TatABCD-pspA-DT(pSB1C3) (78μg/mL)Spe1Pst1BufferHMilliQTotal
9112720

Ligation

LacP-torA-GFP-DT(EcoR1, Pst1) (5.5μg/mL)pSB4K5 (9.6μg/mL)Ligation High Ver.2Total
84618

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 TaqVF2VRMilliQTotal
25112350

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

Electrophoresis

Electrophoresis092305.jpg

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

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

Electrophoresis092306.jpg Electrophoresis092307.jpg

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.2Total
104721
J23107-TatABCD-pspA-DT(Spe1, Pst1) (15.1μg/mL)LacP-kil-DT (Xba1, Pst1) (12.4μg/mL)Ligation High Ver.2Total
82515

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

DNAcompetent cellTotal
21012

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

Electrophoresis

Electrophoresis092308.jpg

J23107-TatABCD-pspA-DT(pSB1C3) (1-6)
lane1 1kb ladder
lane2~7 DNA
lane8 1kb ladder





September 24

Miniprep

  • J23107-TatABCD-pspA-DT-2 139.4μg/ml 1.69(260/280) 1.83(260/230)
  • J23107-TatABCD-pspA-DT-3 134.7μg/ml 1.61(260/280) 1.69(260/230)
  • J23107-TatABCD-pspA-DT-5 139.3μg/ml 1.55(260/280) 1.45(260/230)
  • J23107-TatABCD-pspA-DT-6 138.5μg/ml 1.58(260/280) 1.40(260/230)
  • LacP-torA-GFP-DT-4
  • LacP-torA-GFP-DT-7
  • T7-6His-GFP-DT-1
  • T7-6His-GFP-DT-2

Colony PCR

LacP-GFP generater (1-3)

2×Quick TaqVF2VRMilliQTotal
25112350
Electrophoresis092309.jpg
94℃, 2min

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

①-③, LacP-GFP generater (1-3)




Liquid culture

  • J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT (1-4)
  • J23107-TatABCD-pspA-DT-LacP-kil-DT (1-3)

Colony PCR

J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT (1-4)
J23107-TatABCD-pspA-DT-LacP-kil-DT (1-3)

2×Quick TaqVF2VRMilliQTotal
25112350

94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 5min
→30cycles

Electrophoresis

Electrophoresis0924.jpg

lane1, 1kb ladder
lane2-5, J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (1-4)
lane6-8, J23107-TatABCD-pspA-DT-LacP-kil-DT(pSB1C3) (1-3)







September 25

Liquid culture

  • J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT(pSB4K5)-2

Miniprep

J23107-TatABCD-pspA-DT-LacP-kil-DT(pSB1C3)-3: 26.2μg/mL, (260/280): 1.50, (280/230): 0.65
J23107-TatABCD-pspA-DT-LacP-torA-GFP(pSB1C3)-3: 24.8μg/mL, (260/280): 1.56, (280/230): 0.76
LacP-GFP generater-1: 42.0μg/mL, (260/280): 1.58, (280/230): 0.94

Restrictive Digestion

J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (26.2ng/µL)EcoRlSpelbuffer MMiliQTotal
151131030
J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (26.2ng/µL)EcoRlbuffer MMiliQTotal
20.516.510
J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (26.2ng/µL)Spelbuffer MMiliQTotal
20.516.510
J23107-TatABCD-pspA-DT-LacP-kil-DT(pSB1C3) (24.8ng/µL)EcoRlPstlbuffer HMiliQTotal
151131030
J23107-TatABCD-pspA-DT-LacP-kil-DT(pSB1C3) (24.8ng/µL)Pstlbuffer HMiliQTotal
20.516.510
J23107-TatABCD-pspA-DT-LacP-kil-DT(pSB1C3) (24.8ng/µL)EcoRlbuffer HMiliQTotal
20.516.510

at 37℃ for 2 hours

Electrophoresis

Electrophoresis092501.jpg

lane1, 1kb ladder
lane2, J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3)
lane3, J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (EcoRl, Spel)
lane4, J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (EcoRl)
lane5, J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (Spel)
lane6, J23107-TatABCD-pspA-DT-LacP-kil-DT(pSB1C3)
lane7, J23107-TatABCD-pspA-DT-LacP-kil-DT(pSB1C3) (EcoRl, Pstl)
lane8, J23107-TatABCD-pspA-DT-LacP-kil-DT(pSB1C3) (EcoRl)
lane9, J23107-TatABCD-pspA-DT-LacP-kil-DT(pSB1C3) (Pstl)
lane10, LacP-torA-GFP-DT(EcoR1,Xba1)(9/15)
lane11, LacP-torA-GFP-DT(EcoR1,Xba1)(9/17)
lane12, 1kb ladder

→The bands didn't appear, so we did one more electrophoresis.

Electrophoresis092502.jpg

lane1, 1kb ladder
lane2, LacP-torA-GFP-DT(EcoR1,Xba1)(9/17)
lane3, J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3)
lane4, J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (EcoRl, Spel)
lane5, J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (EcoRl)
lane6, J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (Spel)

Liquid culture

  • LacP-torA-GFP-DT(pSB4K5) (1-4)

Restrictive Digestion

J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3)EcoRlSpelbuffer MMiliQTotal
151131030
J23107-TatABCD-pspA-DT-LacP-kil-DT(pSB1C3)EcoRlPstlbuffer HMiliQTotal
151131030

at 37℃ for 1 hour

Electrophoresis

J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (EcoRl, Spel)
J23107-TatABCD-pspA-DT-LacP-kil-DT(pSB1C3) (EcoRl, Pstl)
→The bands didn't appear.

Observation through a confocal microscope

① J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) ② J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) ③ J23107-TatABCD-pspA-DT-LacP-kil-DT(pSB1C3)

→We observed nothing.

October 12

Transformation

conpetent cell(3/15)J23107-TatABCD-PspA-DT (9/13) (pSB3C5)J23107-TatABCD-PspA-DT(9/18)(pSB3C5)LacP-torA-GFP-DT(9/10)total
20μL2μL 22μL
20μL 2μL 22μL
20μL 2μL22μL

preculture in SOC medium for 1 hour at 25 degree

Checking Colony PCR(negative control)

Electrophoresis1012.jpg
Quick TaqVFVRMilliQtotal
25μL1μL1μL23μL50μL

94℃ 2min
94℃ 30sec
55℃ 30sec
68℃ 5min
30 cycles

->Electrophoresis

October 15

Restrictive Digestion & Electrophoresis

Electrophoresis1015.jpg
pSB4C5Xba1Pst1BSABufferMMilliQtotal
10μL1μL1μL3μL3μL12μL30μL

at 37℃ for 1 hour

Transformation
retry of lab work that we did on 10/12

J23107-TatABCD-PspA-DTLacP-torA-GFP-DTcompetent celltotal
2μL 20μL22μL
2μL20μL22μL

preculture in 100μL of SOC medium

October 16

Colony PCR

  • 4 colonies of J23107-TatABCD-PspA-DT(9/18)
Quick TaqVF2VRMilliQtotal
25μL1μL1μL23μL50μL

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

  • 4 colonies of LacP-torA-GFP-DT(10/15)
Quick TaqVF2VRMilliQtotal
25μL1μL1μL23μL50μL

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

October 17

Electrophoresis

fig.1
fig.2
fig.3
fig.4

product of colony PCR on 10/16

  • fig.1

lane_1 : 1kb ladder
lane_2-5 : J23107-TatABCD-PspA-DT
lane_6-9 : LacP-torA-GFP-DT
lane_10 : 1kb ladder

we retried this because something is wrong with 1 kb ladder

  • fig.2

lane_1 : 1kb ladder
lane_2 : J23107-TatABCD-PspA-DT(lane_3)

Liquid culture
J23017-TatABCD-PspA-DT(10/15)

Colony PCR

  • 8 colonies of LacP-torA-GFP-DT(10/15)
Quick TaqVF2VRMilliQtotal
25μL1μL1μL23μL50μL

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

->Electrophoresis we named 8 colonies [5]~[12] because on 10/16, we did colony PCR using 4 colonies([1]~[4]) of J23107-TatABCD-PspA-DT.

  • fig.3

lane_1 : 1kb ladder
lane_2 : [5]
lane_3 : [6]
lane_4 : [7]
lane_5 : [8]

  • fig.4

lane_1 : 1kb ladder
lane_2 : [9]
lane_3 : [10]
lane_4 : [11]
lane_5 : [12]

Liquid culture [5] and [7]

October 18

Miniprep

geneng/μL
J23107-TatABCD-PspA-DT (10/17)134.0
LacP-torA-GFP-DT [5] (10/17)94.7

October 19

Restrictive Digestion

fig.1
fig.2
fig.3
  • sample_1
J23107-TatABCD-PspA-DTEcoR1Spe1BufferMMilliQtotal
15μL1μL1μL3μL10μL30μL
  • sample_2
LacP-torA-GFP-DTXba1Pst1BSABufferMMilliQtotal
10μL1μL1μL3μL3μL12μL30μL
  • sample_3
LacP-kil-DTXba1Pst1BSABufferMMilliQtotal
12μL1μL1μL3μL3μL10μL30μL

Electrophoresis
lane_1 : 1kb ladder
lane_2 : J23107-TatABCD-PspA-DT
lane_3 : LacP-torA-GFP-DT
lane_4 : LacP-kil-DT

Restrictive Digestion

T7Spe1Pst1BufferHMilliQtotal
10μL1μL1μL2μL6μL20μL

at 37℃ for 1 hour

Eledtrophoresis
lane_1 : 1kb ladder
lane_2 : sample 1
lane_3 : blank
lane_4 : sample 2
lane_5 : blank
lane_6 : sample 3
Though this figure did not indicate, there was a 1000 bp band that seems to be product of LacP-Kil-DT

October 22

Gel extraction

fig.1
fig.2
fig.3
fig.4
fig.5
fig.6
  • LacP-torA-GFP-DT
  • LacP-kil-DT
Geneng/µL260/280260/230
LacP-torA-GFP-DT4,42,020,22
LacP-kil-DT3,81,540,11

Restrictive Digestion

LacP-torA-GFP-DT(pSB3C5, 9/10)EcoR1Pst1Buffer MMilliQTotal
10µL1µL1µL2µL6µL20µL

At 37℃ for 1 hour

Electrophoresis

  • fig.1

Lane_1:1kb ladder
Lane_2:empty
Lane_3:T7(S,P)

  • fig.2

Lane_1:1kb ladder
Lane_2:LacP-torA-GFP-DT(E,P)

Gel Extraction

  • fig.3, 4

Lane_1:1kb ladder
Lane_2:blank
Lane_3:T7(S,P)

Geneng/µL260/280260/230
T7(S,P)5,41,820,23
  • fig.5, 6

Lane_1:1kb ladder
Lane_2: LacP-torA-GFP-DT(E,P)
Lane_3:pSB4C5(X,P)

Geneng/µL260/280260/230
LacP-torA-GFP-DT(E,P)4,91,660,28
pSB4C523,91,880,88

Ligation

LacP-torA-GFP-DT(X,P)T7(S,P)Ligation high Ver.2Total
10µL2µL5,5µL 17,5µL
LacP-torA-GFP-DT(E,P)pSB4K5(E,P)Ligation high Ver.2Total
10µL 2µL 5,5µL 17,5µL
LacP-torA-GFP-DT(X,P)pSB4C5(X,P)Ligation high Ver.2Total
10µL2µL 5,5µL 17,5µL

At 4℃ overnight

October 23

Transformation

  • T7-LacP-kil-DT
  • LacP-torA-GFP-DT(pSB4K5)
  • LacP-torA-GFP-DT(pSB4C5)

transformed competent cells were plated on two plates respectively.(plate a and plate b)

Ligation

pSB4C5(X,P)LacP-GFP-DT(X,P)Ligation high Ver.2Total
2µL10µL6µL18µL

At 4℃ overnight

October 24

Colony PCR

  • T7-LacP-kil-DT(a1~a8)
  • LacP-torA-GFP-DT(pSB4C5)(a1~a4,b1~b4)
  • LacP-torA-GFP-DT(pSB4K5)(a1~a4,b1,b2)
Quick TaqVF2VRMilliQTotal
25µL1µL1µL23µL50µL

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

Transformation

  • LacP-GFP-DT(pSB4C5)

Electrophoresis
Products of colony PCR

fig.1
  • fig.1

Lane_1 :1kb ladder
Lane_2 :empty
Lane_3 : T7-LacP-kil-DT(a1)
Lane_4 : T7-LacP-kil-DT(a2)
Lane_5 : LacP-torA-GFP-DT(pSB4C5)(a1)
Lane_6 : LacP-torA-GFP-DT(pSB4C5)(a2)
Lane_7 : LacP-torA-GFP-DT(pSB4C5)(b1)
Lane_8 : LacP-torA-GFP-DT(pSB4C5)(b2)
Lane_9 : LacP-torA-GFP-DT(pSB4K5)(a1)
Lane_10 : LacP-torA-GFP-DT(pSB4K5)(a2)
Lane_11 : LacP-torA-GFP-DT(pSB4K5)(b1)
Lane_12 : LacP-torA-GFP-DT(pSB4K5)(b2)

fig.2
  • fig.2

Lane_1 :1kb ladder
Lane_2 : T7-LacP-kil-DT(a3)
Lane_3 : T7-LacP-kil-DT(a4)
Lane_4 : T7-LacP-kil-DT(a5)
Lane_5 : T7-LacP-kil-DT(a6)
Lane_6 : T7-LacP-kil-DT(a7)
Lane_7 : T7-LacP-kil-DT(a8)
Lane_8 : LacP-torA-GFP-DT(pSB4K5)(a3)
Lane_9 : LacP-torA-GFP-DT(pSB4K5)(a4)
Lane_10 :1kb ladder

Liquid culture

  • LacP-torA-GFP-DT(pSB4C5)(a1,a2)
  • T7-LacP-kil-DT(a6)

October 25

Observation through a confocal microscope

  • LacP-torA-GFP-DT(pSB4C5)

We observed nothing.

Kyoto GoldenGateAssemblyNotebook.png

September 6

PCR and Electrophoresis assay

Epa001.jpg

①psB1K3 ②lacI ③GFP ④GFP ⑤RFP ⑥RFP ⑦CFP ⑧DT

Quick Taqprimer FPrimer RDNA(①-⑧)milliQtotal
12.50.50.5110.525

94℃ 2min, (94℃ 30sec, 50℃ 30sec)x25cycles, 68℃ 50sec

September 7

PCR and Electrophoresis assay of psB1K3

Epa002.jpg
10x Buffer KOD plus2mM dNTPs25mM MgSO4F primerR primerDNA(psB1K3)KOD plusmilliQtotal
2.52.51.00.750.750.50.516.525

94℃ 2min, (94℃ 15sec, 58℃ 30sec)x30cycles, 68℃ 2min

September 10

PCR and Electrophoresis assay of CFP

Epa002.jpg
10x Buffer KOD plus2mM dNTPs25mM MgSO4F primerR primerDNA(CFP)KOD plusmilliQtotal
2.52.51.00.750.750.50.516.525

94℃ 2min, (94℃ 15sec, 48℃ 30sec)x30cycles, 68℃ 45sec

PCR and Electrophoresis assay of lacI

Epa003.jpg
10x Buffer KOD plus2mM dNTPs25mM MgSO4F primerR primerDNA(lacI)KOD plusmilliQtotal
2.52.51.00.750.750.50.516.525

94℃ 2min, (94℃ 15sec, 58℃ 30sec)x30cycles, 68℃ 30sec

PCR

①PCR product of psB1K3 ②psB1K3 ③psB1C3 ④psB1C3 ⑤PCR product of CFP

10x Buffer KOD plus2mM dNTPs25mM MgSO4F primerR primerDNA(①-⑤)KOD plusmilliQtotal
2.52.51.00.750.750.50.516.525

94℃ 2min, (94℃ 15sec, 58℃ 30sec)x30cycles, 68℃ 2min20sec

September 11

PCR and Electrophoresis assay

Epa004.jpg

①DT ②PCR product of DT ③PCR product of CFP ④CFP(2011 plate)

10x Buffer KOD plus2mM dNTPs25mM MgSO4F primerR primerDNA(①-④)KOD plusmilliQtotal
2.52.51.00.750.750.50.516.525

94℃ 2min, (94℃ 15sec, 48℃ 30sec)x30cycles, 68℃ 1min

PCR and Electrophoresis assay

Epa005.jpg

①DT 92.1ng/µl ②DT 28.0ng/µl ③DT

10x Buffer KOD plus2mM dNTPs25mM MgSO4F primerR primerDNA(①-③)KOD plusmilliQtotal
2.52.51.00.750.750.50.516.525

94℃ 2min, (94℃ 15sec, 50℃ 30sec)x30cycles, 68℃ 30sec

September 12

Purification of PCR product

psB1K3(9/10④) 55µg/ml lacI(9/10) 90µg/ml GFP(9/6) 42µg/ml RFP(9/6) 71µg/ml DT(9/11) 81µg/ml GFP 37µg/ml RFP 57µg/ml

Golden Gate Assembly

psB1K3lacIGFP(2'-3)RFP(3'-5)DT10x NEB T4 Buffer100x BSABsaINEB T4 ligasemilliQtotal
1.80.11.00.60.11.50.151.01.08.1515
psB1K3lacIGFP(2'-5)DT10x NEB T4 Buffer100x BSABsaINEB T4 ligasemilliQtotal
1.80.11.00.11.50.151.01.08.7515
psB1K3lacIGFP(2'-3)RFP(3'-5)DT10x NEB T4 Buffer100x BSABsaINEB T4 ligasemilliQtotal
2.00.21.01.00.21.50.151.01.06.9515
psB1K3lacIGFP(2'-3)DT10x NEB T4 Buffer100x BSABsaINEB T4 ligasemilliQtotal
2.00.21.00.21.50.151.01.07.9515

Transformation

①lacI-GFP(2'-3)-RFP(3'-5)-DT ②lacI-GFP(2'-5)-DT ③lacI-GFP(2'-3)-RFP(3'-5)-DT ④lacI-GFP(2'-5)-DT

September 13

Transformation

①lacI-GFP(2'-3)-RFP(3'-5)-DT ②lacI-GFP(2'-5)-DT ③lacI-GFP(2'-3)-RFP(3'-5)-DT ④lacI-GFP(2'-5)-DT

September 14

Liquid Culture

①lacI-GFP(2'-3)-RFP(3'-5)-DT ②lacI-GFP(2'-5)-DT ③lacI-GFP(2'-3)-RFP(3'-5)-DT ④lacI-GFP(2'-5)-DT

September 15

Miniprep

①lacI-GFP(2'-3)-RFP(3'-5)-DT ②lacI-GFP(2'-5)-DT ③lacI-GFP(2'-3)-RFP(3'-5)-DT ④lacI-GFP(2'-5)-DT

①4.0 µg/ml ②17 µg/ml ③15 µg/ml ④8.0 µg/ml

Restriction Enzyme Digestion

①lacI-GFP(2'-3)-RFP(3'-5)-DT ②lacI-GFP(2'-5)-DT ③lacI-GFP(2'-3)-RFP(3'-5)-DT ④lacI-GFP(2'-5)-DT

DNA(①-④)10x NEB Bufer3BSAEcoR1MilliQTotal
920.50.5820

September 17

Electrophoresis assay

Epa006.jpg

①lacI-GFP(2'-3)-RFP(3'-5)-DT ②lacI-GFP(2'-5)-DT ③lacI-GFP(2'-3)-RFP(3'-5)-DT ④lacI-GFP(2'-5)-DT

September 18

Restriction Enzyme Digestion

③lacI-GFP(2'-3)-RFP(3'-5)-DT ④lacI-GFP(2'-5)-DT

DNA(③,④)10x H BuferBSAEcoR1MilliQTotal
1530.50.51130

Colony PCR

Epa007.jpg

①lacI-GFP(2'-3)-RFP(3'-5)-DT ②lacI-GFP(2'-5)-DT ③lacI-GFP(2'-3)-RFP(3'-5)-DT ④lacI-GFP(2'-5)-DT

2x Quick Tag Dye MixVF primerVR primerMiliQTotal
25112350

September 19

DpnI Restriction Enzyme Digestion of psB1K3

psB1K3DpnITotal
100.210.2

37℃ 1h incubate

Ligation of psB1K3

psB1K3Ligation HighMiliQTH KinaseTotal
257715

16.0℃ 1.5h

September 20

Transformation of psB1K3

September 21

PCR of psB3C5 and Electrophoresis assay

Epa008.jpg
10x Buffer KOD plus2mM dNTPs25mM MgSO4F primerR primerDNA(psB3C5)KOD plusmilliQtotal
2.52.51.00.750.750.50.516.525

94℃ 2min, (94℃ 15sec, 58℃ 30sec)x30cycles, 68℃ 2min

PCR and Electrophoresis assay

Epa009.jpg

①psB3C5 ②psB1K3

10x Buffer KOD plus2mM dNTPs25mM MgSO4F primerR primerDNA(①,②)KOD plusmilliQtotal
5.05.02.01.51.51.01.03350

94℃ 2min, (94℃ 15sec, 58℃ 30sec, 68℃ 2min)x30cycles

PCR of DT and Electrophoresis assay

Epa011.jpg
10x Buffer KOD plus2mM dNTPs25mM MgSO4F primerR primerDNA(DT)KOD plusmilliQtotal
5.05.02.01.51.51.01.03350

94℃ 2min, (94℃ 15sec, 50℃ 30sec, 68℃ 30sec)x30cycles

September 22

Colony PCR (Colony plated in September 20)

Epa012.jpg
10x Buffer KOD plus2mM dNTPs25mM MgSO4F primerR primerDNAKOD plusmilliQtotal
5.05.02.01.51.51.01.03350

94℃ 2min, (94℃ 15sec, 58℃ 30sec, 68℃ 2min20sec)x25cycles

PCR of Primers and Electrophoresis assay

Epa013.jpg

①J23106(1'-2) ②J23106(2'-3) ③J23106(3'-4) ④J23106(4'-5) ⑤J23106(3'-5) ⑥J23106(2'-5) ⑦J23106(1'-5) ⑧araC(1'-6) ⑨araC(5'-6)

10x Buffer KOD plus2mM dNTPs25mM MgSO4F primerR primerDNAKOD plusmilliQtotal
2.52.51.00.750.750.50.516.525

94℃ 2min, (94℃ 15sec, 56℃ 30sec)x30cycles, 68℃ 20sec

①J23106(1'-2) 60µg/ml ②J23106(2'-3) 63µg/ml ③J23106(3'-4) 48µg/ml ④J23106(4'-5) 60µg/ml ⑤J23106(3'-5) 62µg/ml ⑥J23106(2'-5) 40µg/ml ⑦J23106(1'-5) 29µg/ml ⑧araC(1'-6) 60µg/ml ⑨araC(5'-6) 62µg/ml

Ligation

4 promoters

psB3C5①J23106 (1'-2)②J23106 (2'-3)③J23106 (3'-4)④J23106 (4'-5)⑨araC (5'-6)GFPDT10x NEB T4 Buffer100x BSABsaINEB T4 ligasemilliQtotal
2.02.02.02.52.02.03.03.01.50.151.01.07.8530

3 promoters

psB3C5①J23106 (1'-2)②J23106 (2'-3)⑤J23106 (3'-5)⑨araC (5'-6)GFPDT10x NEB T4 Buffer100x BSABsaINEB T4 ligasemilliQtotal
2.02.02.02.52.03.03.01.50.151.01.09.8530

2 promoters

psB3C5①J23106(1'-2)⑥J23106(2'-5)⑨araC(5'-6)GFPDT10x NEB T4 Buffer100x BSABsaINEB T4 ligasemilliQtotal
2.02.02.52.03.03.01.50.151.011.8530

1 promoter

psB3C5⑦J23106(1'-5)⑨araC(5'-6)GFPDT10x NEB T4 Buffer100x BSABsaINEB T4 ligasemilliQtotal
2.02.52.03.03.01.50.151.01.013.8530

0 promoter

psB3C5⑨araC(5'-6)GFPDT10x NEB T4 Buffer100x BSABsaINEB T4 ligasemilliQtotal
2.02.03.03.01.50.151.01.016.3530

September 23

Transformation of product of ligation(9/22)

September 24

Transformation of product of ligation(9/22)

September 25

Liquid Culture

colony1,2,3 of plate2,3

Ligation

4 promoters

psB3C5① J23106 (1'-2)② J23106 (2'-3)③ J23106 (3'-4)④ J23106 (4'-5)⑨ araC (5'-6)GFPDT10x NEB T4 Buffer100x BSABsaINEB T4 ligasemilliQtotal
2.00.20.30.20.20.21.00.21.50.151.01.07.0515

3 promoters

psB3C5①J23106 (1'-2)②J23106 (2'-3)⑤J23106 (3'-5)⑨araC (5'-6)GFPDT10x NEB T4 Buffer100x BSABsaINEB T4 ligasemilliQtotal
2.00.20.30.20.21.00.21.50.151.01.07.2515

2 promoters

psB3C5①J23106 (1'-2)⑥J23106 (2'-5)⑨araC (5'-6)GFPDT10x NEB T4 Buffer100x BSABsaINEB T4 ligasemilliQtotal
2.00.20.30.21.00.21.50.151.01.07.4515

1 promoter

psB3C5⑦J23106 (1'-5)⑨araC (5'-6)GFPDT10x NEB T4 Buffer100x BSABsaINEB T4 ligasemilliQtotal
2.00.60.21.00.21.50.151.01.07.3515

0 promoter

psB3C5⑨araC (5'-6)GFPDT10x NEB T4 Buffer100x BSABsaINEB T4 ligasemilliQtotal
2.00.21.00.21.50.151.01.07.9515

(37℃ 3min, 16℃ 4min)x25cycles,

Transformation of product of ligation(9/25)