Team:Kyoto/Secretion/Notebook

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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 at feb.8
competent cell onfeb.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
Cpnpetent 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 feb.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

Electrophoresis021501.JPG
Restriction productloading dye
5μL1

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

Electrophoresis021502.JPG

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, Pst2, and Pst3 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
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
pSBIAT3-1701.661.83
pSBIAT3-21001.521.54

diluted to 25 times

Liquid culture
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(2/19)

number of colony  0 177 590
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
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

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
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
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
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
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 (27)

Restriction

pSB3C5-5EcoR1Pst1BufferHBSAMilliQtotal
200.50.530.55.530

at 37℃, for 2 hour

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

Restriction
GFP1,2,3

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
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
(28)

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 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

①    1kb 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

Transformation

  • lacP+torA+GFP+DT
  • BBa_K11704

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)
Restriction
J23113(218ng/μL)Spe1Pst1BufferHBSAMilliQtotal
51130.519.530