Team:Kyoto/Secretion/Notebook

From 2012.igem.org

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Revision as of 01:02, 25 September 2012

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Contents

Secretion Notebook

February 7

Preculture
We started preculture at 12:10.

February 8

Culture
We start culturing with 300[mL] 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.leap colony transformed on February 8
colony of competent cell made on February 8

February 10

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

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

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

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
concentration[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

Restriction
Const promoterJ23100

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

Restriction

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

Competent cell
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

Restriction
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
lane_1 1kb ladder
lane_2 a
lane_3 b
lane_4 a
lane_5 b
lane_6 a
lane_7 b
lane_8 1kb ladder

  • sample2
Electrophoresis022302.JPG

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

PCR

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

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




Electrophoresis022004.JPG

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





February 21

PCR (Advantage HF protocol)

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
sampleLoading DyeMilliQ
1.1kb ladder200
2.product of PCR1510
3.product of PCR2510
4.product of PCR(Negative control)510
5.product of PCR(2/23)510
6.GFP2(DPN1)1020
7.GFP2327
8.1kb 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

Restriction

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

Restriction

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

  • evernoteに写真なし!至急求む

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

March 1

PCR

  • TMAO

Template is gDNA and product of colony PCR gel extraction

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
Electrophoresis030102.JPG

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

PCR

BufferdNTPsMgSO4primer-fprimer-rProduct of PurificationKOD Plus NeoMilliQtotal
5531.51.5132150

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

Restriction

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



Restriction

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

Restriction
GFP1,2,3

GFPEcoR1Pst1BufferBSAMilliQtotal
100.50.530.515.530

at 37℃, for 2.5 hours

Restriction

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

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

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

Restriction

TMAOEcoR1BufferHBSAMilliQtotal
100.230.316.530
TMAOXba1Pst1BudderMBSAMilliQtotal
100.20.230.316.330

at 37℃ for 1 hour

Electrophoresis


Transformation

pSB1C3competent cell(made at 2/8)total
5100105

March 5

Restriction

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

(31)

Restriction

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

Restriction

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 pspAはコロニーPCR

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

Restriction

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.

Restriction

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

Restriction

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

Restriction

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

Restriction

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

Restriction

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

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.





Electrophoresis031102.JPG

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

Restriction

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

Restriction

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.

Restriction

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

Restriction

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

Restriction

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℃.

Restriction

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→×

Restriction

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

Restriction

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

Restriction

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.

Restriction

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

Restriction

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]

Restriction

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 25

July 26

July 27

Restriction Enzyme Processing

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(XbaI,PstI)LacP(SpeI,PstI)Ligation High Total





3063672











July 31

Restriction Enzyme Processing

torAGFP+DT 10×M BufferBSA MilliQXbaIPstITotal






2.53.00.523.40.30.330.0











Liquid Culture

J23113(backbone J61002)






August 1

Restriction Enzyme Processing

lacP+kil+DT BSA10×H BufferEcoRIPstIMilliQTotal






5.00.53.00.50.520.530.0






37℃ 2h






lacP middle copy BSA10×H BufferEcoRIPstIMilliQTotal






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 2

August 3

Restriction Enzyme processing

pSB4K5BSA 10×H BufferEcoRIPstIMilliQTotal






8.00.53.00.50.517.530.0











B0034BSA10×H BufferSpeIPstIMilliQTotal






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 4

August 5

August 6

August 7

August 8

August 9

August 10

Ligation

B0034 SpeI PstIGFP+DT XbaI PstIligation 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 11

August 12

August 13

Liquid culture

B0034 3mL ×3    






37℃ overnight






August 14

Miniprep

B0034 0μg/mL






B0034 235.5μg/mL






B0034 76.5μg/mL






Colony PCR

lacP+kil+DT ×5





2×Quick TaqVFVFMilliQTotal






25112350






25cycles






pspA






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






5531.51.513350
















Electrophoresis

写真貼ってください






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

次の写真






①    1kb ladder
②    Positive control (GFP+DT)
③    Negative control (MilliQ)
④ ~⑨ lacP+kil+DT ①~⑥
⑩~⑫  pspA 10μL ,6×buffer 2μL
⑬    1kb ladder

August 15

Colony PCR

pspA






2×Quick TaqpspA r-spspA f-pMilliQTotal






25112350











94℃, 2min






94℃, 30sec






56℃, 30sec






68℃, 1min






→35cycles






August 16

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

写真貼ってください






①1kb ladder
②, ③  MgSO4 3μL, pspA
④, ⑤  MgSO4 5μL, pspA
⑥ negative control(MilliQ)
⑧ 1kb ladder

August 18

August 19

August 20

Restriction

J23113(201.0ng/μL)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











Restriction

J23113(218ng/μL)Spe1Pst1BufferHBSAMilliQtotal






101130.514.530











torA-GFP-DT(20ng/μL)Xba1Pst1BufferMBSAMilliQtotal






50.60.630.520.330











Electrophoresis

写真貼ってください






①    1kb ladder
②, ③  torA-GFP-DT(XbaI,PstI)
④, ⑤  J23113(SpeI,PstI)

Transformation

  • lacP-torA-GFP-DT(Backbone pSB3C5)






  • BBa_K11704(Backbone pSB1A2)






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











  • negative control for pspA






We did PCR without a template.






  • GFP-DT






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

Restriction

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

写真お願いします。





See "August 21 -Colony PCR-"





1. 1kb ladder





2. pspA(Primer pspA f&r)





3. negative control for 2





4. GFP-DT





5. pspA(Primer pspA f-p&r-s)





6. pspA(Primer pspA f-p&r-s)





7. negative control for 5&6









PCR

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





10×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

写真お願いします。





1. 1kb ladder





2. pspA(Primer pspA f&r)





3. pspA(Primer pspA f-p&r-s)





4. negative control for pspA(Primer pspA f&r)





5. negative control for pspA(Primer pspA f-p&r-s)





Transformation

Kil





August 23

Colony PCR

J23107-tatABCD





VF and VR were used for amplifying approximately 2800bp





10×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-"





Restriction

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





Restriction

pSB1C3(82.9μg/mL)EcoR1Pst1BufferHBSAMilliQtotal





200.50.530.55.530









→Gel extraction





Electrophoresis

写真お願いします。





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





写真お願いします。

Purification of PCR products

torA-GFP →See "August 21 -PCR-"






Restriction

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-"






Restriction

J23107-tatABCDSpe1EcoR1BufferMBSAMilliQtotal






200.50.530.55.530











August 28

Colony PCR

Kil (1-16)






10×Buffer2mM dNTPs25mM MgSO4VFVRKOD Plus NeoMilliQTotal






5531.51.513350











写真お願いします。






Transformation

  • pspA-pSB1C3






  • pspA-pSB3C5






Liquid culture

  • Kil-3 →Miniprep 100.4μg/mL






  • Kil-6






  • Kil-7 →Miniprep 77.7μg/mL






  • Lacp-torA-GFP-1 →Miniprep 88.3μg/mL






  • Lacp-torA-GFP-2 →Miniprep 85.0μg/mL





  • Lacp-torA-GFP-3 →Miniprep +++




August 29

Kil assay

evernoteに結果のグラフがあります。






Liquid culture

  • pspA-pSB1C3(1-3)






  • pspA-pSB3C5(1-3)






Restriction

Lacp-Kil-DT(134.7ng/μL)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






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

写真お願いします。





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





Restriction

pspA-pSB1C3EcoR1Spe1BufferMBSAMilliQtotal
150.50.550.528.550

at 37℃ for 1h

Electrophoresis

写真お願いします。
1. 1kb ladder
3. pspA-pSB1C3(EcoR1, Spe1)

August 31

Gel extraction

写真お願いします。
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 1

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






Restriction

LacP-torA-GFP-DTEcoR1Spe1BufferHMilliQtotal






10112620











at 4℃ for overnight






Electrophoresis

写真お願いします。






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











写真お願いします。






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(SpeI, PstI)





写真お願いします。






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

写真お願いします。






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






Restriction

torA-GFP-DTXbaIPstIBufferMMilliQtotal






202241240











Purification

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






Electrophoresis

写真お願いします。






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

写真お願いします。






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

写真お願いします。






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

写真お願いします。






1-8. Lacp-torA-GFP-DT











写真お願いします。






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)






Restriction

pspA-DT-6XbaIPstIBufferMBSAMilliQtotal






200.50.530.55.530











at 37℃ for 1h






Electrophoresis

写真お願いします。






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(XbaI, PstI)LacP(pSB3C5)(SpeI, PstI)Ligation High Ver.2total






5139











at 16℃






Electrophoresis

写真お願いします。






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











写真お願いします。






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






Ligation

Lacp-torA-GFP-DT(EcoRI, SpeI)DT 17.2μg/mL(XbaI, PstI)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)











Restriction

J23107-tatABCD 102.9μg/mLSpeIPstIBufferHBSAMilliQtotal





100.50.530.515.530










at 37℃ for 1h






Transformation

LacP-pspA-DT(pSB3C5)






DNACompetent celltotal






22022











Gel extraction

写真お願いします。






J23107-tatABCD(SpeI, PstI)






→We failed gel extraction because we mistook steps of experiment.






Electrophoresis

写真お願いします。






LacP-torA-GFP-DT-pspA-DT






Restriction

by Terasaka






J23107-tatABCDSpeIPstIBufferHBSAMilliQtotal






100.50.530.515.530











Gel extraction

写真お願いします。






J23107-tatABCD(SpeI, PstI)






→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






Restriction

by Terasaka






J23107-tatABCDEcoRISpeIBufferMBSAMilliQtotal






100.50.530.515.530











GFP-DTXbaIPstIBufferMBSAMilliQtotal






150.50.530.510.530











Electrophoresis

写真お願いします。






① -⑧. LacP-pspA-DT






1-8. LacP-torA-GFP-DT






Gel extraction

写真お願いします。






  • J23107-tatABCD(EcoR1, Spe1)






  • J23107-tatABCD(Pst1, Spe1) ①57.6μg/mL ②23.4μg/mL






  • GFP-DT(Xba1, Pst1)






Restriction

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

pspA-DT -5.5μg/mL





Restriction

pspA-DT-3(322μg/mL)Xba1Pst1BufferMBSAMilliQtotal
1011331230

at 37℃ for 1h

September 9

Restriction

J23107-tatABCD(102.9μg/mL)EcoR1Spe1BufferMMilliQtotal
101131530

Electrophoresis

写真お願いします。
1. J23107-tatABCD(EcoR1, Spe1)
2. J23107-tatABCD(before restriction)






3. pspA-DT(Xba1, Pst1)






4. pspA-DT(before restriction)






Gel extraction

写真(2枚)お願いします。






  • 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



Restriction

写真お願いします。






DNASpe1Pst1BufferHMilliQtotal






151131030











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






at 37℃






Gel extraction

写真(2まい)お願いします。






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

写真お願いします。






  • J23107-tatABCD-DT (1-8)


  • J23107-tatABCD-pspA-DT (1-8)


Restriction

写真お願いします。






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

写真お願いします。






  • 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

写真お願いします。






  • 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

写真お願いします。






  • J23107-tatABCD-pspA-DT










写真お願いします。





  • J23101-kil-DT (1-7)


Restriction

pspA(3C5) (106μg/mL)EcoR1Pst1BufferHMilliQtotal


151131030


pspA-DT-3Xba1Pst1BufferMBSAMilliQtotal


151133730









J23107-tatABCD (102.4μg/mL)EcoR1Spe1BufferMMilliQtotal


151131030


Electrophoresis

写真お願いします。

  • pspA(3C5)(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

写真お願いします。





  • pspA(3C5)(EcoR1, Pst1) 26.8μg/mL 1.27(260/280) 0.32(260/230)





  • pspA-DT(Xba1, Pst1) 26.1μg/mL 1.13(260/280) 0.55(260/230)





  • J23107-tatABCD(EcoR1, Spe1) 1.5μg/mL 1.99(260/280) 0.06(260/230)





Ligation

pSB3C5(EcoR1, Pst1) 26.8μg/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

Evernoteに上がっている写真をお願いします。





September 13

Miniprep

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


Restriction

J23107-tatABCD-pspA-DT(pSB3C5) (130μg/mL)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

写真お願いします。





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)





Restriction

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

写真お願いします。





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

写真お願いします。
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)

Restriction

LacP-torA-GFP-DT (126μg/mL)EcoR1Xba1BufferMBSAMilliQtotal
1011331230
LacP-torA-GFP-DT (126μg/mL)Xba1BufferMBSAMilliQtotal
31332030









Electrophoresis

写真お願いします。





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

写真お願いします。





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

写真お願いします。

  • 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
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
0LB ここ途中
0LB
0LB


Restriction enzyme processing

J23101-Kil-DTBufferMXbaIPstBSAMilliQtotal
153113730
J23101-Kil-DTBufferMXbaIBSAMilliQtotal
310.514.510
J23101-Kil-DTBufferHPstMilliQtotal
310.55.510

37℃

September 19

GeneClean II

LacP-torA-GFP-DT





4k5





Ligation

LacP-torA-GFP-DT(X,P) 9.7μg/mLpSB4K5(E,P) 9.6μg/mLJ23107-tatABCD-PsPA-DT(E,S) 14.2μg/mLLigation High Ver.2total





727824μL













pSB4K5(E,P) 9.6μg/mLLigation High Ver.2MilliQtotal





212124μL














incubate at 37℃ for 2h









Electrophoresis

J23101-kil-DT(X,P) (X) (P)





Lane1: 1kb ladder





Lane2: empty





Lane3: plasmid





Lane4: X





Lane5: P





Lane6: X,P









Liquid Culture

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









Transformation

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





pSB4K5 negative control









Liquid culture

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









Colony PCR

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





VF2VRQuick tagMilliQtotal





11252350













Predenature 94℃ 2min





Denature 94℃ 30sec





Annealing 55℃ 30sec





Extension 68℃ 5min





→30cycles









Restriction enzyme processing

LacP-kil-DT(4) 62.5μg/mLXbaIPstI10xBSAMilliQBufferMtotal


1011312330


LacP-kil-DT(4)EcoRIPstIMilliQBufferHtotal


101115330


LacP-kil-DTXbaI10xBSAMilliQBufferMtotal





20.515.5110













LacP-kil-DTEcoRIMilliQBufferHtotal





20.56.5110













LacP-kil-DTPstIMilliQBufferHtotal





20.56.5110













at 37℃, 2 hours









Electrophoresis

Lane1: 1kb ladder





Lane2: empty





Lane3: plasmid





Lane4: E





Lane5: X





Lane6: P





Lane7: E,P





Lane8: X,P









Electrophoresis

J2-tat-psp-DTLacP-torAGFP-DT


Lane1: ladder

Lane2: 1

Lane3: 2
Lane4: 3

Lane5: 4


Lane6: 5


Ligation

LacP-torA-GFP-DT(X,P) 9.7μg/mLpSB4K5(E,P) 9.6μg/mLJ23107-tatABCD-PsPA-DT(E,S) 14.2μg/mLLigation High Ver.2total


747927μL














(Negative control)





pSB4K5(E,P) 9.6μg/mLLigation High Ver.2MilliQtotal





491427μL














Restriction enzyme processing

torA-R9 101.8μg/mLbufferHMilliQEcoRIPsttotal





103151130













torA-R9 101.8μg/mLbufferHMilliQEcoRIPsttotal





315.50.5010













torA-R9 101.8μg/mLbufferHMilliQEcoRIPsttotal





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

torA-R9,plasmid,(E),(P),(E,P)

Lane1: 1kb ladder
Lane2: empty

Lane3: plasmid

Lane4: E

Lane5: P
Lane6: E,P
Lane7: empty
Lane8: 1kb ladder


Liquid culture

LacP-tatA9FT-DT (9/4)

PCR purification

torA-R9 68.0μg/mL


September 20

Gel extraction

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

Restriction enzyme processing

torA-R9 68.0μg/mLbufferMSpeIMilliQTotal
2031630


at 37℃, 2h

Ligation

LacP-kil-DT(X,P) 12.4μg/mLpSB1C3(E,P) 34.2μg/mLJ23107-tatABCD-PsPA-DT(E,S) 14.2μg/mLLigation High Ver.2total
747927μL





16℃, 1h









(Negative control)





pSB1C3(E,P) 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









Restriction enzyme processing

GFP-DT 84.1μg/mLbufferM10xBSABbaIMilliQtotal





15331830









at 37℃, 2 hours









Colony PCR

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





Quick tagtat f primerVRMilliQtotal





25112350













Predenature 94℃ 2min





Denature 94℃ 30sec





Annealing 54℃ 30sec





Extension 68℃ 4min





→30cycles














Liquid culture

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









Electrophoresis

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









Lane1: 1kb ladder





Lane2: 1





Lane3: 2





Lane4: 3





Lane5: 4





Lane6: 5





Lane7: 6





Lane8: 7





Lane9: 8





Lane10: 1kb ladder





Lane11: empty





Lane12: empty









torA-R9 (E,P): 31μg/mL, (280/280): 0.80, (280/230): 0.39





1C3(E,P): 22μg/mL, (280/280):1.33, (280/230):0.6





torA-R9 (S): 56μg/mL, (280/280): 1.11, (280/230): 0.69





GFP-DT(X): 45μg/mL, (280/280): 1.15, (280/230): 0.88



Ligation

torA-R9 (E,P)1C3(E,P)Ligation High Ver.2total
82515μL
torA-R9 (S)1C3(X)Ligation High Ver.2total
3339μL


at 16℃, 1hour

Colony PCR

J23107-tat-psp-DT-lacP-torA-GFP-DT (4k5)


numbering 9~15









Transformation

J23107-tat-psp-DT-lacP-torA-GFP-DT (4k5)





J23107-tat-psp-DT-lacP-torA-kil-DT(1C3)





Negative control (1C3)





Negative control(4K5)





torA-R9(1C3)




Liquid culture

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

2xQuick 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

J2-tat-psp-DT-LacP-torA-GFP-DT:2,3





2: 10.4μg/mL, (250/280): 1.45, (260/230): 0.95





3: 6.0μg/mL, (250/280): 1.32, (260/230): 0.79





(*)100μL









Electrophoresis

torA-R9-GFP-DT(after PCR)









September 21

Gel extraction

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









Electrophoresis

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









Electrophoresis

J2-tat-psp-DT-LacP-torA-GFP-DT: 1,2









Lane1: empty





Lane2: empty





Lane3: 2





Lane4: 3





Lane5: 1kb ladder









Restriction enzyme processing

LacP-kil-DT(3)XbaIPstI10xBSAMilliQBufferMtotal





151137330









LacP-torA-6FP-DP(9/5)XbaIPstI10xBSAMilliQBufferMtotal





151137330









LacP-kil-DTXbaI10xBSAMilliQBufferMtotal





20.515.5110









LacP-torA-6FP-DPXbaI10xBSAMilliQBufferMtotal





20.515.5110









LacP-kil-DTPstIMilliQBufferHtotal





20.56.5110









LacP-torA-6FP-DPXbaIPstIMilliQBufferHtotal
20.516.5110
J2-tat-psp-DTSpeIPstIBufferHMilliQtotal
201141440
J2-tat-psp-DTSpeIBufferMMilliQtotal
20.516.510






J2-tat-psp-DTPstIBufferHMilliQtotal





20.516.510










at 37℃, 2hours









September 22

Miniprep

torA-R9(Amp)1: 113.7μg/mL, (260/280): 1.96, (280/230): 2.42





torA-R9(Amp)2: 97.8μg/mL, (260/280): 1.67, (280/230): 1.72





torA-R9(Amp)3: 104.2μg/mL, (260/280): 1.93, (280/230): 2.67





LacP-torAGFP-DT 6: 37.4μg/mL, (260/280): 2.24, (280/230): 3.27









Electrophoresis

LaneA: J2-tat (9/6)





LaneB: J2-tat-psp-DT 3C5 (9/13)


LaneC: J2-tat-psp-DT 1C5 (9/15)


LaneD: J2-tat-psp-DT (9/15)
LaneE: J2-tat-psp-DT 1C3 (9/18)
LaneF: J2-tat-psp-DT 3C5 (9/18)


LaneG: J2-tat-psp-DT-LacP-torA-GFP-DT:2 (9/20)
LaneH: J2-tat-psp-DT-LacP-torA-GFP-DT:3 (9/20)

LaneA~H are (E,P)

Restriction enzyme processing

J2-tat-psp-DT-LacPtorA-GFP-DT:2BufferHPstMilliQtotal
15311230


J2-tat-psp-DT-LacP-torAGFP-DT:3BufferHPstMilliQtotal
15311230

Restriction enzyme processing

LacP-torAGFP-DT(37.4μg/mL)BufferHEcoRIPsttotal
2531130

Electrophoresis

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

3: J2-tatpsp-DT-LacP-torA-GFP-DT:3


September 23

Electrophoresis

写真お願いします。





  • LacP-torA-GFP-DT(EcoR1, Pst1)





lane_1 1kb ladder





lane_3 DNA





lane_4 DNA





lane_5 DNA





Gel extraction

写真お願いします。





  • J23107-tatABCD-pspA-DT(pSB1C3)(Spe1, Pst1)





  • LacP-torA-GFP-DT(Xba1, Pst1)





lane_1 1kb ladder





lane_3 J23107-tatABCD-pspA-DT(pSB1C3)(Spe1, Pst1)





lane_5 LacP-torA-GFP-DT(Xba1, Pst1)





Restriction

LacP-torA-GFP-DT (78.4μg/mL)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

写真お願いします。





①, LacP-torA-GFP-DT





②, LacP-torA-GFP-DT(Xba1, Pst1)





③, LacP-torA-GFP-DT(Xba1)





④, LacP-torA-GFP-DT(Pst1)





⑤, J23107-tatABCD-pspA-DT(Spe1, Pst1)





Liquid culture

  • LacP-torA-GFP-DT (4, 7)





  • LacP-GFP generater (1-3)





  • T7-6His-GFP-DT (1, 2)





Gel extraction

写真お願いします。





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





Purification

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





Ligation

J23107-tatABCD-pspA-DT(Spe1, Pst1) (15.1μg/mL)LacP-torA-GFP-DT (Xba1, Pst1) (6.8μg/mL)Ligation High Ver.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

写真お願いします。





J23107-tatABCD-pspA-DT(pSB1C3) (1-6)





lane_1 1kb ladder





lane_2~7 DNA





lane_8 1kb ladder









September 24