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Team Aboa Notebook

On this page, our workflow is described in a more detailed manner

Work with Cell lines

April 29th

Present: Fanni, Pyry

To get started with our iGEM project, the ordered cell lines described below, were cultured. E. coli cells containing pEVOL-pAzF (Chloramphenicol selection) and cell line C321.ΔA.exp (Zeocin selection) were plated to LA-plates containing 0.5 % Glucose, and 35 µg/mL chloramphenicol or 50 µg/mL zeocin. Controls with tetracycline, ampicillin and a plate without antibiotics were also prepared for the detection of contamination. Plates were incubated overnight at 30 °C. In addition, extra zeocin and chloramphenicol plates were made for later use.

Figure 1. C321.ΔA.exp cells growing on LA plate containing zeosin for selection.

Figure 2. pEVOL-pAzF cells growing on LA plate containing chloramphenicol for selection.

As predicted, there were no colonies in tetracycline plates after overnight culturing. Plates were stored in our laboratory’s fridge.

Testing the amber suppression of E.coli 321A and pEVOL- pAzF with BBa_K567011 (GFP-tag-RFP)

May 9th

Present: Pyry, Suski, Jutta, Robin, Janniina, Riku

Following LA-plates were made:
MM1:
LA-plates containing: chloramphenicol, 1 mM pAzF, 0.2 % arabinose, 0.5 mM IPTG
LA-plates containing: tetracycline, 1 mM pAzF 0.2 % arabinose, 0.5 mM IPTG
LA-plates containing: zeocin, 1 mM pAzF 0.2 % arabinose, 0.5 mM IPTG
LA-plates containing: chloramphenicol, 1 mM pAzF, 0.2 % arabinose
LA-plates containing: chloramphenicol, 1 mM pAzF, 0.5 mM IPTG

MM2:
LA-plates containing: chloramphenicol, 0.2 % arabinose, 0.5 mM IPTG
LA-plates containing: tetracycline, 0.2 % arabinose, 0.5 mM IPTG
LA-plates containing: zeocin, 0.2 % arabinose, 0.5 mM IPTG

25 µL cells and 1 µL BBa_K567011 (GFP-tag-RFP) was electroporated in 1 mm electroporation cuvette with 1.8 kV voltage in BioRad Gene Pulser Xcell. After electroporation cells were put into 5 mL SOC medium in 37 °C for 40 min.
After incubation cells were plated as follows: 50 µL SOC, 50 µL 1/10 and 50 µL 1/100 dilutions were plated as follows:

Antibiotic Chloramphenicol plate Chloramphenicol Chloramphenicol Zeocin for 321.A, Tetracycline for XL-1
Plasmid BBa (GFP-RFP) pAzF-tRNA plasmid Both None (Control)
321.A strain Red/Green < 1 None Red/Green =1 None
XL1 Blue strain Green None Green None

XL1-Blue 321.deltaA.exp
Cmp, IPTG Cmp, IPTG
Cmp, Ara Cmp, Ara

Plates were left in 37 °C o/n

May 10th

No fluorescence in any of the plates.

Preparing 321.A + pAzF for glycerol preps and doing glycerol preps

May 16th

Present: Janniina, Oskari, Fanny, Riku

A colony from a 321.A + pAzF plate was replated onto an MM2C plate and incubated o/n at 37 °C.

May 17th

A single colony was picked and used to inoculate 4 mL LB medium containing 33 µg/mL chloramphenicol o/n at 30 °C and 150 rpm.

May 18th

20 % glycerol preps were made by adding 500 µL of growth to 167 µL of an 80 % glycerol stock. The glycerol preps were stored at -80 °C.

May 24th

Present: Janniina

5 mL of LB containing 5 µL of 25 mg/mL chloramphenicol was inoculated with a colony picker from a plate containing 321.A (BBa_K567018) and put in Infors Multitron (200 rpm, 26 °C) at 10:10 O'clock.
Raised the temperature from 26 °C to 37 °C at 13 O'clock.
Replated 3 colonies in MM2 Chloramphenicol plates at 14:49 O'clock

May 25th

Present: Riku

Glyserol preps 321.A (BBa_K567018)
Four glycerol preps were made from yesterdays cultures. 500 µL LB of E.coli 321.A with BBa_K567018 plasmid culture was mixed with 170 µL 80 % glyserol. Preps were put in -70 °C freezer.

May 27th

Present: Janniina, Riku, Pyry

Our 321.A and XL-1 Blue E.coli strains do not express T7 polymerase so they were unable to express the original BBa_K567018 BioBrick.

Digestion of BBa_K567018 and pUC19-plasmid with EcoRI
Digestion was made as follows:

BBa_K567018 (µL) pUC19 (µL)
H2O 12 16
10x buffer 2 2
DNA 5 1
EcoRI (fermentas, expired 8/2019) 1 1
V. Tot. 20 20

incubation at 37 °C started: 15:40, stopped: 16:09
Samples stored in 4 °C.

May 28th

Present: Janniina, Riku

Yesterday’s EcoRI restrictions were mixed with 0.5 µL MidoriGreen Direct (Nippon Genetics) and 1 µL Midori Green Direct was added in 5 µL 1kb DNA Ladder (nippon Genetics).
The ladder, samples and the sample from entry "Making of pLK04R-Histag" is loaded into the 1 % gel as follows:

1 kb ladder pLK04R (SfiI + HindIII) pLK04R (SfiI + HindIII) pUC19 (EcoRI) pSB1C3(BBa_K567018) (EcoRI)

Gel run: 1h with 70 V
Results:

The faint longer bands are probably undigested plasmids in lanes 2, 3 and 5. pLK04 is cut from around 4 kb from the lanes 2 and 3. The linearized pUC19 (lane 4, marked with red) is cut from the gel around 3 kb. The GFP- TAG-RFP fragment (lane 5, marked with red) is cut from the gel close to 2 kb even though it is supposed to be 1569 bp long and should be closer to 1.5 kb strands.
The DNA was purified with Nucleospin Gel and PCR Clean- up kit, according to the manufacturer's protocol.
Purified DNA concentrations were measured with ThermoScientific NanoDrop ND-1000
Results:
pUC19: 1.2 ng/µL
GFP-tag-RFP: 14.8 ng/µL
pLK04R: 2.7 ng/µL

May 29th

Present: Riku, Janniina, Pyry

Due to DNA purification low yields digestion was done again similarly as yesterday.
Digestion incubation at 37 °C started at 10:09. Stopped at 10:41 and put on ice to wait. The reaction tubes were put in 65 °C at 11:28 to inactivate the EcoRI enzyme and taken from inactivation at 11:50.
After digestion each reaction mix was mixed with 0.5 µL Midori Green direct and 1 µL Midori green Direct was added to 5 µL 1kb DNA ladder. Samples were loaded in 1% agarose gel as follows:

5 µL 1 kb ladder + 1 µL midori direct 20 µL pUC19 (EcoRI) + 0.5 µL Midori direct 20 µL BBa_K567018 (EcoRI) + 0.5 µL midori direct 20 µL pLK04 (SfiI + HindIII)+ 0.5 µL midori direct

Starting gel electrophoresis at 12:00 using 70 V.
After gel run, gel was removed from the tray and imaged under blue light with Xiaomi Redmi Note 5 Pro:

pUC19 backbone (red box on the left) and BBa_K567018 (red box on the right) were cut from the gel and purified with PCR clean-up Gel extraction kit with 2 15 µL eluation in different tubes.

1st eluation 2nd eluation
pLK04R Sfil+Hindll 7.5 ng/µL 7.7 ng/µL
pUC19 EcoRI 5.4 ng/µL 2.7 ng/µL
GFP-tag-RFP (BBa_K567018) 5.0 ng/µL 3.4 ng/µL

GFP-tag-RFP-gene was ligated on pUC19 -backbone in a following ligation reaction:

milli-Q 5.8
pUC19 4
GFP-tag-RFP 8
T4 ligase buffer 2
T4 Ligase 0.2
Total volume (µL) 20

Enzyme and buffer was made by ThermoFischer.
Ligation started 15:40. Ended: 15:50.
1 µL ligation mix was mixed with 35 µL electrocompetent E.coli 321.A cells. Electroporation was done with 1mm electroporation cuvette using BioRad Gene Pulsen Xcell electroporation device with 1.8 kV voltage.
After electroporation cells were put into 5 mL prewarmed (37 °C) LB-medium and put into 37 °C, 250 rpm shaking for 40 minutes. Then cells were plated on a LA+chloramphenicol plate and the plate was put to 37 °C o/n.

Multiplying pUC19 and pLK04RCH-MLCR plasmids

Electrocompetent 321.A cells were electroporated with pUC19 or pLK04R plasmid. The cell mixture was transferred to LB medium and incubated on Multritron shaker at 37 °C, 300 rpm for 45 minutes.

May 30th

Present: Janniina

Lot’s of colonies in o/n plate, but no fluorescence. This might be due to wrong gene. In the agarose gel, the gene was too big for what it should have been.

Multiplying pUC19 and pLK04RCH-MLCR plasmids

One colony from each plate was used to inoculate 5 mL of LB with 100 µg/mL ampicillin and incubated on Multitron shaker at 37 °C, 300 rpm o/n.

May 31st

Multiplying pUC19 and pLK04RCH-MLCR plasmids

Present: Janniina, Riku, Fanni, Pyry

The pLK04RCH+MLCR and pUC19 plasmids were purified from the cells using NucleoSpin Plasmid easyPure kit (Macherey-Nagel) according to the manufacturer’s instructions . DNA concentrations were measured with Nanodrop.

DNA concentrations:
pUC19: 380.5 ng/µL
pLK04 original: 115.7 ng/µL

Glycerol preps were also made from both pLK04RCH+MLCR and pUC19 cultures. 167 µL of 80 % glycerol stock was added to 500 µL of cell suspension. The glycerol preps were stored at -80 °C.

June 4th

Present: Janniina, Pyry

The plasmid pLK04RCHTag was extracted using NucleoSpin Plasmid easyPure kit. The DNA-concentration measured with Nanodrop was 280.7 ng/µL.

321.A electrocompetent cells were electroporated with pLK04RCHTag(MLCR). 1 µL of DNA (stock concentration 104 ng/µL) was added to 30 µL of cell suspension and the mixture was electroporated. As a negative control we added MQ H2O instead of DNA to the cell mixture. The cell mixture was transferred to LB medium and incubated at 37 °C at 300 rpm for 1 hour. The cell mixture was then plated onto selective plates and incubated o/n at 37 °C.

June 5th

Present: Pyry

5 colonies were inoculated in LB medium and incubated o/n at 37 °C at 300 rpm.

June 6th

Present: Janniina, Riku, Pyry, Linnea

The shaker had stopped during the night so only 3 culture tubes had visible growth in them. Plasmids were extracted from the overnight cultures with visible growth using NucleoSpin Plasmid easyPure kit. The DNA concentration was measured with Nanodrop.

DNA concentration of the extracted pLK04RCHTag-MLCR:
pLK04RCHTag-MLCR 1: 103.9 ng/µL
pLK04RCHTag-MLCR 2: 98.4 ng/µL
pLK04RCHTag-MLCR 3: 88 ng/µL

We digested the pLK04RCHTag-MLCR plasmid with SfiI restriction enzyme:

SfiI digestion mixture:

 

µL

MQ

12

Purified DNA

5

G Buffer

2

SfiI

1

Total volume

20

The reaction mixture was incubated at 50 °C for 15 minutes. The samples were run on an agarose gel at 70 V for 1 hour.

June 11th

Present: Riku, Fanni, Pyry

We got a new pUC19 plasmid because we were unsure what was in our old tube. We transformed the vector into XL-1electrocompetent cells, incubated on a shaker at 300 rpm, 37 °C for 1 hour and then plated the cell mixture onto selective plates and incubated o/n at 37 °C.

June 12th

A single colony was picked and inoculated with 3 mL of LB with ampicillin and incubated o/n at 37 °C, 300 rpm.

June 13th

Present: Janniina

A modified GFP-tag-RFP was ordered from IDT. Original part was BBa_K567018, but we ordered it without the T7 promoter.

BamHI and AvaI digestion of pUC19 and the ordered GFP-tag-RFP:

pUC19 and GFP-tag-RFP were digested with ThermoFischer BamHI and AvaI digestion ezymes. Reaction mixtures were as follows:

pUC19 GFP-TAG-RFP
MQ 15 7
DNA 2 10
10x Tango buffer 2 2
BamHI 0.5 0.5
AvaI 0.5 0.5
V. tot. (µL) 20 20

The reactions are put on 37 °C at 11:18 and stopped at 12:16.

0.5 µL Midori Green Direct was put on digestion reaction mixes and 1 µL Midori Green Direct was added to 5 µL 1kb DNA ladder. Samples were loaded on 1 % agarose gel. as follows:

5 µL 1 kb ladder + 1 µL midori direct 20 µL pUC19 (BamHI % AvaI) + 0.5 µL Midori direct 20 µL GFP-TAG-RFP (BamHI % AvaI) + 0.5 µL Midori direct

Gel run:
Voltage:100 V
Start at 12:27
Stop at 13:15.

Fragments marked with red boxes were cut from gel and purified with PCR clean-up Gel extraction kit, according to manufacturer's guide.
Concentrations were measured with Thermo Fischer NanoDrop ND-1000:

ng/µL
pUC19 9.1
GFP-TAG-RFP 2.6

Ligation of pUC19 backbone and GFP-tag-RFP insert

Purified fragments were ligated with ThermoFischer T4 ligase in following ligation reaction:

µL
MQ 1.8
pUC19 3
GFP-TAG-RFP 13
10x ligase buffer 2
Ligase 0.2
V. tot. 20

The ligation reaction was incubated for 10 min.

Transformation

3 µL of ligation mix were mixed with 100 µL chemically competent E.coli XL-1 Blue cells. Transformation was incubated 2 min in ice and then put to 45 seconds in 42 °C heat block. After heatshock cells were put on ice for 2 mins and then they were transferreed to 5 mL LB medium.
Cells were incubated in 37 °C 300 rpm shaker for 40 min and then transferred to two LA+ampicillin plates. 200 µL LB growth in first one and 1 mL to second one. Plates were incubated 37 °C overnight.

Multiplying pUC19 and pLK04RCH-MLCR plasmids

The pUC19 plasmid was extracted from the XL-1 electrocompetent cells using the Nucleospin plasmid Easypure kit. The measured DNA concentration was 271.9 ng/µL.

June 17th

There were colonies in LA+ampicillin plates, but no GFP fluorescence. We forgot to do ligation and transformation control for work done on Thursday 13th of June, so we had no idea why GFP-tag-RFP gene did not work.
We started pUC19 and GFP-tag-RFP ligation all over again, but this time with ligation control. Ligation was done with ThermoFischer T4 ligase enzyme as follows:

pUC19 + GFP-tag-RFP pUC19 transformation control
MQ 1.8 14.8
pUC19 3 3
GFP-TAG-RFP 13 0
10x ligase buffer 2 2
Ligase 0.2 0.2
V. tot. (µL) 20 20

Incubation started at 11:35 and ended at 11:45

Electroporation mix was done by adding 1 µL ligation mix in 35 µL E.coli XL-1 blue electrocompetent in 1 mm electroporation cuvette (BioRad). Electroporation was done with BioRad Gene Pulser Xcell with 1.8 kV voltage

Transformations were put to prewarmed (37 °C) LB medium and 300 rpm shaking in 37 °C.
Start: 12:06
End: 12:55

Both transformations were centrifuged 2000 g, 5 min. Supernatant was thrown away and cells were resuspended in 300 µL LB medium. Medium was divided both 1 mM and 0.5 mM IPTG plates (LA plates, with ampicillin) and incubated in 37 °C o/n

June 18th

Colonies from all the plates were counted, but still no fluorescence.

pUC19 + GFP-tag- RFP pUC19 control
1 mM 32 7
0.5 mM 68 6

Starting cultures for plasmid extraction

2 colonies were picked from 0.5 mM pUC19+GRP-tag-RFP plate and put in LB with ampicillin. Tubes were put in 37 °C 300 Rpm for o/n.

June 19th

Plasmids were extracted from o/n cultures with Nucleospin Plasmid Easypure kit, according to manufacturer's protocol.

Yields were measured with NanoDrop ND-1000 and are as follows:

Yield (ng/µL)
1/2 77.8
2/2 62.5

June 20th

Present: Riku

GFP-tag-RFP Transformation in E.coli 321.A with pEVOL-pAzF

Transformation was done with electroporation in electrocompetent E.coli 321.A. Each transformation mix contained either 1 or 2 plasmids as follows:

35 µL E.coli 321A cells with:

pEVOL- pAzF (µL) pUC19 - GFP-tag-RFP (µL)
0.5 0.5
0.5 0
0 0.5

Transformation was done in 1 mm electroporation cuvette (BioRad) with 1,85 kV voltage (BioRad Gene Pulser Xcell)

After electroporation cells were transferred in 5 mL LB medium and incubated in 37 °C 250 rpm shaking. Incubation started: 11:25 and ended: 12:35

Transformed cells was plated as follows:

LA + Ampicillin, Cloramphenicol, Zeocin LA + ampicillin
pEVOL, pAzF+pUC19, GFPtagRFP 200 µL and 1/100 200 µL
pEVOL, pAzF 200 µL
pUC19, GFPtagRFP 200 µL 50 µL

At this point, we noticed that the pUC19 that was given us by University of Turku was actually pUC18. That might explain quite a lot of struggles we have had with it. We acquired a new pUC19 from University of Turku.

Transforming confirmed pUC19 in E.coli XL-1 Blue strain

Before we could start work with pUC19 it had to be cloned as we got only 2 µL of commercial pUC19.

Transforming mixes as follows:

XL-1 pUC19 XL-1 mQ control
MQ 0 µL 1 µL
pUC19 (100 pg/µL) 1 µL 0 µL
Cells 35 µL 35 µL

Transformation was done in 1 mm electroporation cuvette using 1.8 kV with BioRad Gene Pulser Xcell.
XL-1 Blue pUC19: T: 4.8
XL-1 Blue mQ control T: 5.0

After electroporation, cells were transferred to 5 mL LB medium and incubation in 37 °C, 250 rpm shaking was started: 16:20 and ended: 17:10.

3 plates were plated:

1: 200 µL E.coli XL-1 Blue pUC19 transformant
2: 100 µL E.coli XL-1 Blue pUC19 transformant
3: 100 µL E.coli XL-1 Blue Control/background

Plates were put to 37 °C o/n

June 21st

Colonies from the o/n plates were counted as follows:

1: 25
2: 14
3: 0

2 colonies from plate 1 and 1 colony from plate 2 was used to start cultures in 5 mL LB medium with 100 µg/mL ampicillin.

Cultures were put in 30C 300 rpm for o/n incubation at 11:20.

June 22nd

O/n cultures were taken out at 10:30 and and glyserol preps were done as follows:

500 µL growth
167 µL 80 % glycerol

Preps were mixed and put to -70 freezer.
The rest of the cells were centrifuged in 2000g for 10 min.

Plasmids were extracted with Nucleospin EasyPure kit, according to manufacturer’s protocol. Yield was measured with Nanodrop ND-1000

Results:

Culture (Plate/colony) DNA concentration (ng/µL)
1/1 190.6
1/2 138.7
2/1 156.8

June 24th

AvaI + BamHI digestion of pUC19 and GFP-tag-RFP

Digestion reactions as follows ( ThermoFisher ): pUC19 reaction was done for 1/1, 1/2 and 2/1 pUC19 vectors:

GFP-tag-RFP (in pUC18) (µL) pUC19 (µL)
MQ 13 7
DNA 4 10
10xTango buffer 2 2
BamHI 0.5 0.5
AvaI 0.5 0.5
V. tot. 20 20

Incubation in 37 °C started: 11:31 end: 11:30.

0.5µL Midori Green Direct was added to all the digestion reactions and 1 µL Midori Green Direct was added to 1 kb DNA Ladder (Nippon Genetics).

All 7 samples were loaded on 1% agarose gel as follows:

Ladder pLk04RCH SfiI digested pUC19 1/1 BamHI + AvaI digested pUC19 2/1 BamHI + AvaI pUC19 BAmHI + AvaI GFP-TAG-RFP BamHI + AvaI pLK04 Fab0

Gel run: Voltage: 70 V. Start: 12:56. End:?.

No picture was saved from this gel run due to smartphone malfunction. Correct bands were cut from gel and DNA was purified with PCR clean-up Gel extraction kit, according to manufacturer’s protocol. All pUC19 containing gel pieces were pooled together.

DNA concentrations were measured with ThermoFischer NanoDrop ND-1000.

June 25th

Following ligation reactions were made with ThermoFischer T4 ligase:

MQ 14.3µL 17.3µL
GFP-tag-RFP insert (17.1 ng/µL) 3 0
pUC19 backbone (59.2 ng/µL) 0.5 0.5
10x T4 buffer 2 2
T4 ligase 0.2 0.2
V. tot. (µL) 20 20

Ligation started: 13:56.
Ended: 14:07.

Control ligation started: 14:22.
Ended: 14:34.

Transformation of pUC19-GFP-tag-RFP in E.coli XL-1 Blue

Chemically competent XL-1 Blue tube was put on ice to melt. When it was melted 50 µL cells were put in 2 centrifugetubes that rested on ice. 5 µL previously ligated (pUC19 + GFP-tag-RFP and pUC19 + nothing) reactions was mixed in with cells.

Reaction mixes were incubated on ice for 20-30 min.
Start: 14:45.
End: 14:10.

Cells were put on 42 °C heatblock for 45 s and then put back into ice.

After approx. 2 mins cells were put on 5 mL prewarmed to 37 °C LB medium and put into shaking for 250 rpm in 37 °C.

Incubation started: 15:15.
End: 15:45.

Cells were centrifuged with 2000 g for 7 min. Supernatant was thrown away and cells were resuspensed in residual medium.

Cells were plated on 4 different LA+ampicillin (100 µg/mL) dishes as follows:

Amount (µL) Ligation transform.
200 pUC19 + GFP-tag-RFP
100 (1/20) pUC19 + GFP-tag-RFP
100 (1/400) pUC19 + GFP-tag-RFP
100 pUC19 + no insert

Plates were left o/n in 37 °C

June 26th

Colonies in o/n plates were counted. Results as follows:

Amount plated (µL) Plate Colonies
200 pUC19 + GFP-tag-RFP 121
100 (1/20) pUC19 + GFP-tag-RFP 2
100 (1/400) pUC19 + GFP-tag-RFP 0
100 pUC19 + no insert 6

No fluorescence was observed. This might be due to XL-1 Blue poor expression combined with pUC19 being poor expression vector.

Incubation in 37 °C overnight.

June 27th

The 1/20 plate has 2 clearly GFP expressing colonies:

In plate 2 there are 2 colonies with fluorescence. 1 is picked in 5 mL liquid culture (LB + ampicillin 100µg/ml) for o/n incubation in 37C 300 rpm.

The reason why only the small plate had GFP expression is due to larger plate containing also glucose, which inhibits the lacZ operator.

June 28th

o/n culture was centrifuged in 2000 g for 10 min and supernatant was thrown away. New liquid culture for glycerol stock was started by inoculating from pellet into 5 mL LB+ampicillin.

Rest of the pellet was used to extract plasmid with NucleoSpin Plasmid -kit. Elution was done 2x with 25 µL Elution buffer and 5 min 70 °C incubation.

Concentration was measured with ND-1000:
pUC19 + GFP-TAG-RFP: 579,5 ng/µL

July 2nd

Present: Janniina, Ossi

Transformation of E.coli 321.A with pUC19+GFP-tag-RFP and pEVOl+pAzF

321.A was transformed with 0.5 µL of pEVOL-pAzF and pUC19+GFP-tag-RFP plasmids.

Actual transformation Control 1 Control 2
321.A cells 35 35 35
pAzF-pEVOL 0.5 0.5 0
pUC19-GFP-tag-RFP 0.5 0 0.5
MQ 0 0.5 0.5

Electroporation was done using 1 mm electroporation cuvette (BioRad) in BioRad Gene Pulser Xcell.

After electroporation cells were put into LB medium and shaking 300 rm in 37 °C for 40 min.

The transformations were plated on LA plates containing zeocin, ampicillin and chloramphenicol.

July 3rd

No green, red nor yellow fluorescent proteins detected on the plates.

2 growths from o/n LA plates were each used to inoculate 4 mL LB with ampicillin, chloramphenicol, zeocin and glucose. They were then put to grow o/n in 37°C 300rpm.

July 4th

No growth observed.

321.A with pEVOL-pAzF and pUC19(GFP-TAG-RFP) was re-plated in order to get separate colonies. Plates with 1/100 (large plate) and 1/1000 (small plate) dilutions were incubated o/n at 37 °C.

July 5th

Present: Ossi, Riku

From 321.A with pEVOL-pAzF and pUC19(GFP-TAG-RFP) LA plate, 3 colonies were picked to inoculate 4 mL LB with ampicillin, chloramphenicol, zeocin and 0.5 % glucose to grow o/n in 37 °C and 300 rpm.

XL-1 Blue was transformed with pEVOL-pAzF and pUC19-GFP-tag-RFP as follows:

E.coli XL-1 Blue cells (µL) pEVOL-pAzF (µL) pUC19-GFP-tag-RFP (µL)
Actual 35 0.5 0.5
Control 1 35 0.5
Control 2 35 0.5

Transformation was done via electroporation in 1 mm electroporation cuvette (BioRad) by 1.8 kV with BioRad Gene Pulser Xcell.
All transformation T's were 5.0.
After transformation cells were put into 5 mL LB in 37 °C, 300 rpm shaking for 1 hour.

After incubation cells were plated in LA+cloramphenicol+ampicillin+ Zeocin dishes as follows:

Transformation No dilution (µL) 1/100 (µL)
pUC19 + pEVOL 200 100
pEVOL 100
pUC19 100

100 µL XL-1 blue + pUC19-GFP-tag-RFP transformation was put into LA+ampicillin+tetracyclin+IPTG dish.

All dished were put into 37 °C o/n.

July 6th

No growth was observed in A+cloramphenicol+ampicillin+ Zeocin dishes, due to zeocin that killed XL-1.

Nice green glow was observed in XL-1 Blue + pUC19-GFP-tag-RFP plate:

July 7th

2 colonies 321.A pEVOL+pAzF - pUC19+GFP-tag-RFP and 2 colonies XL-1 GFP-tag-RFP was inoculated into LB culture with ampicillin, chloramphenicol and zeocin (321.A strain) or tetracycline (XL-1 Blue strain) in 30 °C 300 rpm for o/n.

July 8th

Present: Riku, Janniina

OD600 was measured from 1/10 dilutions of o/n cultures and then multiplying measured OD600 by 10. Results were:

Strain and plasmids OD600
XL-1 + pUC19-GFP-tag-RFP 2.5
321.A + pUC19-GFP-tag-RFP + pEVOL-pAzF 2.21

To make 0.1 OD600 cultures from o/n cultures, 160 µL of 321.A culture and 180 µL of XL-1 Blue culture were transferred to 4 mL LB containing ampicillin, 0.5 % glucose and tetracycline (for XL-1 Blue) or chloramphenicol (for 321.A). Glucose was added to inhibit Lac-Promoter.

The growths were put in 37 °C 300 rpom at 10:25.

Cultures OD600 was measured at 14:30 o’clock like previously and results were:

Strain and plasmids OD600
XL-1 + pUC19-GFP-tag-RFP 2
321.A + pUC19-GFP-tag-RFP + pEVOL-pAzF 1.3

1.9 mL of XL-1 culture and 1.5 mL of 321.A culture was put in 2 separate tubes to start new test and control cultures so that in total there were 4 tubes. Cultures were centrifuged 2000g for 15 min and pellets were resuspended into 4 mL of growth medium that contained: LB, ampicillin and tetracycline (for XL-1 Blue) or chloramphenicol (for 321.A). In addition to antibiotics on tube containing XL-1 Blue and One tube containing 321.A had 0.1 mM pAzF and 1 % arabinose in medium. After about 10 min of 26 °C, 300 rpm incubation 0.1 M IPTG was added to test cultures to total IPTG concentration of 0.1 mM.

July 9th

The OD was measured from the o/n growths by making 1/10 dilutions and measuring dilutions OD600 and multiplying results by 10.

OD in 1:1
321.A test 3.24
321.A control 3.64
XL-1 test 4.06
XL-1 control 4.03

The cells were centrifuged (16100 g, 2 min) and supernatant was transferred to other microcentrifuge tube. Pellets and supernatants were stored at -20 °C.

Producing Digoxigenin Fabs

Digoxigenin-Fab-TAG cloning of ordered variants

May 20th

Present: Janniina, Riku, Ville, Linnea, Pyry and Oskari

Gel electrophoresis

We made 500 mL of 1 x TBS (Medicago AB, Uppsala) and 1 L of 1 x TBE (Medicago).

We made 1 % and 2 % agarose gels into 60 mL of 1 x TBE. 3,6 µL of Midori Green Advance DNA Stain was added into each 60 mL of agarose gel.

Restrictions and ligations

The DNA was centrifuged to get the pellet into the bottom. 20 µL of MQ was added into each tube containing the ordered DNA.

Ordered DNA:

1.45 kb fragments:
SfiI restriction (1 % agarose gel)

Digox CL C-terminal TAG
Digox VL Lys 108->TAG
Digox Fab 0
Digox CL Lys 191->TAG

0.1 kb fragments:
SfiI and HindIII restriction (2 % agarose gel)

pEB04 Histag TAG2
pEB04 Histag Cys-TAG
pEB04 Histag TAG

 

SfiI (µL)

SfiI + HindIII (µL)

 

Water

17

15

 

10 x FastDigest Buffer

3

3

 

DNA

10

10

 

FastDigest Enzyme (SfiI)

1

1

 

FastDigest Enzyme (HindIII)

0

2

 

Total volume

31

31

 

Done according the instructions of manufacturer:
Single digestion for 1.45kb fragments:
15 min at 50 °C for SfiI
Stored at RT until gel electrophoresis

Double digestion for 0.1kb fragments:
15 min at 50 °C for SfiI
1h at 37 °C for HindIII
20 min at 80 °C to inactivate HindIII

Thermo Scientific FastDigest SfiI #FD1824
Thermo Scientific HindIII #ER0505

Electrophoresis:
1.45 kb fragments with 5 µL Loading dye (Thermo Fisher)
Ladder: FastGene 50bp DNA Ladder (NIPPON Genetics Europe GmbH, lot:10 1028 2807 01)
70 V
1x TBE buffer

1 % gel

1.45kb

DNA Ladder

Digox CL Lys 191->TAG (gel sample run order number 1)

Digox VL Lys 108->TAG (2)

Digox Fab 0 (3)

Digox CL C-terminal TAG (4)

2 % gel

0.1kb

DNA Ladder

pEB04 Histag TAG2

pEB04 Histag Cys-TAG

pEB04 Histag TAG

 

Results

Agarose gel 1 %. Midori green stain. Samples: Ladder, 1, 2, 3, 4

Agarose gel 2 %. Sample order (50 bp ladder, 1, 2, 3)

May 21st

Present: Janniina, Riku, Ville, Linnea

Insert purification from gel
DNA bands from gel 1 at 1.5kb and 2 at 0.1kb were purified with Macherey-Nagel NucleoSpin Gel and PCR Clean-up -kit with manufacturer’s guide. Elution was done twice for samples 1-4 from 1% agarose gel, first time using 30 µL and second time using 15 µL elution buffer. For samples from 2 % agarose gel elution was done only once with 30 µL elution buffer.

Preparing 1% agarose gel for plasmid
0.6 g agarose
0.6 mL TBE
6 µL MidoriGreen

Plasmid restriction

Restriction reactions as follows:

 

Sfil

Sfil + HindIII

Water

22,5 µL

19.5 µL

10 x Tango buffer

3 µL

3 µL

Plasmid DNA (350 ng/µL)

3 µL

3 µL

Sfil

1.5 µL

1.5 µL

Hindll

0 µL

3 µL

Total volume

30 µL

30 µL

Sfil and Sfil + Hindlll incubated at 50 °C, for 15 min (start: 09:49, end: 10:04) Sfil+Hindll incubated 1h 10 min (start: 10:04, end: 11:14)

Plasmid purification
Agarose gel sample preparation:
5 µL of loading dye was added to restriction reaction mixes

Agarose gel was loaded as follows:

5 µL 1 kb Ladder (nippon genetics)

20 µL Sfil pEBO4CH (1.)

15 µL Sfil pEBO4CH (2.)

20 µL Sfil + HindIII pEBO4CH (3.)

15 µL Sfil + HindIII pEBO4CH (4.)

Electrophoresis:
Voltage: 70 V
Start: 11:24
Stop: 12:42

Imaging with invitrogen Safe Imager(tm)

Purifying with Macherey-Nagel NucleoSpin Gel and PCR Clean-up -kit, with manufacturer’s guide with 2 eluation steps using 30 µL elution buffer both times.

Tubes 1 and 2 were pooled as well as tubes 3 and 4

May 22nd

Riku, Janniina, Ville, Linnea, Pyry

DNA sample concentration measurement ThermoFischer NanoDrop 1000

Sample

ng/µL

pEB04 Sfil

8.0

pEB04 Sfil + Hindlll

8.4

Digox 191TAG

9.9

Digox Lys108 -> TAG

6.9

Digox Fab0

7.3

Digox C-term TAG

7.0

pEB04 Histag TAG2

7.3

pEB04 Histag TAG

9.5

Ligation

 

1.5 kb insert

0.1 kb insert (µL)

Vector DNA

2.5 µL

2.5 µL

Insert DNA

3 µL

1 µL

10 x ligase buffer

2 µL

2 µL

ligase

0.5 µL

0.5 µL

water

12 µL

14 µL

total

20 µL

20 µL

nebiocalculator and
Thermo T4 DNA ligase protocol are used as ligation protocols

Name

Insert

pEB04CH (SfiI)

pEB04CH (SfiI + HindIII)

reaction mix

Lig1.23.05.-19/ABOA

Digox 191 TAG

x

 

using 1.5 kb column in previous table

Lig2.23.5.-19/ABOA

Digox Lys 108

x

 

using 1.5 kb column in previous table

Lig3.23.5.-19/ABOA

Digox Fab 0

x

 

using 1.5 kb column in previous table

Lig4.23.5.-19/ABOA

Digox C-term TAG

x

 

using 1.5 kb column in previous table

Lig5.23.5.-19/ABOA

pEB04 Histag TAG2

 

x

using 0.1 kb column in previous table

Lig6.23.5.-19/ABOA

pEB04 Histag TAG

 

x

using 0.1 kb column in previous table

Lig7.23.5.-19/ABOA

hrFab SfiI DIGD12 harm

x

 

using 1.5 kb column in previous table

Ligation reaction mixtures are left to incubate for approximately an hour. We got a plasmid pLK04R which has AmpR gene in it. Our plan is to use that for production of our Fab fragments:
We will make following constructs for inserts pLK04RCH, a plasmid with a histidine tag; pLK04RCH-TAG, a plasmid with a histidine tag and a TAG codon for insertion of ncAA's and pLK04RCH-TAG2 with a Histidine tag and a TAG in a slightly different position.

Restriction of pLK04R
Reaction mix as follows:

 

Sfil + Hindlll

Water

12.5 µL

10x Tango Buffer

3 µL

Plasmid DNA (104 ng/µL)

10 µL

Sfil

1.5 µL

Hindll

3 µL

Total volume

30 µL

Restriction incubation:
50 °C: started 11:50 ended 12:50
37 °C: started 12:54 ended

Gel purification according to Macherey-Nagel gel purification kit protocol.

Ligation

We will insert ~100 bp ordered "pEB04-Histag" fragments (SfiI/HindIII restriction) to the pLK04CH backbone. At the same ligation reaction we can add SfiI restricted Fab gene into the correct backbones to get Histidine tagged TAG-Fabs and Histidine-TAg tagged unmodified Fabs.

Ligation according to Thermo T4 DNA ligase protocol.

One-Pot Triple fragment cloning of pEB04CH SfiI/SfiI and SfiI/HindIII fragments to replace existing pLK04 Histag

May 25th

Present: Ville, Linnea, Pyry

Introduction

The idea is to clone our Fab fragments into Amp (ampicillin) resistant pLK04 vector. However, the pLK04 vector has a histidine tail that contains a double stop codon of TAG-TAA, so we need to replace this Histidine tail from a pEB04CH where the Histidine tail ends only on one TAA stop. We can do this cloning in a single reaction without gel purifying our insert because the acceptor backbone has an Amp resistance and the donor backbone Cmp (chloramphenicol). The resulting construct can only be assembled correctly in one way to the pLK04 vector. All transformed pEB04 constructs will die in Amp.

Procedure

12,5 µL

MQ

2 µL

Buffer Tango

3 µL

DNA (pLK04)

2.5 µL

Enzyme (1 µL SfiI + 1.5 HindIII)

20 µL

Total vol

0 µL

MQ

6 µL

Buffer Tango

30 µL

DNA (pEB04) (8ng/µL)

3 µL

Enzyme (SfiI)

39 µL

Total vol

Gel Run at 100V 50mA:
Sample order:
1kb ladder (Nippon); pEB04CH HindIII SfiI; pLk04 HindIII SfiI; pEB04CH Hind SfiIII; pLk04 HindIII SfiI

Purifying with NucleoSpin Gel and PCR Clean-up -kit, according to the manufacturer’s guide with 1 eluation step using 30 µL elution buffer.

Concentration of the samples were:
pEB04CH HindIII SfiI (done on saturday) 0.5 ng/µL
pLk04 HindIII SfiI (done on saturday) 0.8 ng/µL
pEB04CH HindIII SfiI (done on friday) 0.9 ng/µL
pLk04 HindIII SfiI (done on friday) 1.6 ng/µL

Ligation

Digestion mix was added 4.5 µL to 5 µL of vector. Control without digestion mix also done. Thermo T4 DNA ligase instructions were followed.

Afterwards, the ligation reaction was electroporated.

Ligation reactions (1-4 and Bg) that were left over were stored to freezer after incubation for 1h

May 27th

Present: Pyry

Plates 1 and 3 has significantly more growth than 2, 4 or bg.

5 mL LB culturing (LB medium with 100 µg/ml ampicillin) were started from growth plates. 3 colonies were picked from plate 3 and 1 colony from plate 1. Cultures were incubated o/n 37 °C 200 rpm incubator.

May 28th

Present: Janniina, Riku

o/n cultures were fuged in 3000 rpm for 5 minutes. Supernatant was tossed away.

New 5 mL cultures were started by inoculating pellet into a new 5 mL LB+ampicillin (100 µg/ml) medium. Cultures were set to incubate in 37 °C 200 rpm shaking.

Rest of the pellet was used to extract plasmid from the cells with NucleoSpin Plasmid EasyPure -kit with manufacturer’s instruction.

DNA concentrations are measured with Nanodrop ND-1000 (elution buffer as blank) after the purification.

 

c (µg/mL)

1/1

98.5

3/1

172.9

3/2

74

3/3

202.1

May 29th

Present: Pyry

20% Glycerol preps were made from o/n cultures.
500 µL culture medium
80% 170 µL Glycerol
Tubes were vortexed gently and put to -70 °C freezer

Cloning Digoxigenin fabs into pLK04RCHTag

June 3rd

Digestion and purification of plasmid backbone of pLK04RCHTag+MCLR

Present: Janniina, Linnea

Extraction of the plasmid backbone

pLK04RCHTag plasmid containing gene for microcystin LR (MCLR) (BTEK/Tuomas) was purified from C321.ΔA.exp-cells with NucleoSpin Plasmid EasyPure kit and its DNA concentration was measured with nanodrop.

 

ng/µL

1st tube

61.7

2nd tube

183.7

Purified plasmids were digested with SfiI according to manufacturer's protocol. Digested plasmid backbones were separated from MCLR-genes on 1 % agarose gel using Mupid-One 70 V.

The desired fragment (pLK04RCHTag) should be 3753 bp long, but in the 2nd tube there was a contamination of chromosomal DNA and anyway so little DNA that no clear bands were seen, only a greenish line.

Designing ligation reactions

Digoxigenin fabs were purified and digested with SfiI elsewhere, with following concentrations:

Sample

ng/µL

DigoxFab191TAG

9,9

DigoxFab108 -> TAG

6,9

DigoxFab0

7.3

Digox C-term TAG

7.0

June 4th

Present: Janniina, Riku, Fanni, Pyry

The pLK04RCHTag plasmid was digested with SfiI in order to ligate the digoxigenin fabs into it. The DNA concentration measured to be 66.19 ng/µL (but 280 ng/µL in reality due to an error) and SfiI digestion was done according to manufacturer's protocol. Since the digestion was made with smaller concentrations than in reality, it resulted in some extra amounts of DNA.

Digested plasmids were separated on a 1 % agarose gel:

Results were not that good, lowest band (4 kb) was over twice the size it should have been (1.7 kb, MCLR Fab).

Gel run was repeated but with success this time.

June 6th

Present: Janniina, Linnea, Oskari, Pyry, Riku

pLK04RCHTag band from the successful gel run was purified with the Gel and PCR cleanup-kit. The extracted fragment had a concentration of 4.1 ng/µL. Purified pLK04RCHTag and DigoxFab0 were ligated together using T4 ligase, with MQ water and normal Digoxigenin as controls. Most of the purified vector was used for the DigoxFab0 sample so that only 0.5 µL was left for MQ and normal Digoxigenin controls.

Ligated plasmids were transformed in to 30 µL CaCl competent XL-1 Blue cells by incubating 20 min on ice following with 45 s at 42 °C and a further 2 min on ice. 1 mL of LB was added to each mixture and the mixtures were left to grown at 37 °C, 300 rpm Multitron. After 1 h of incubation cells were plated and left to grow o/n at 37 °C.

June 7th

Present: Oskari, Pyry

Colonies from yesterday’s plates were counted:
Background (MQ): 21 colonies
pLK04RCHTag + Digox: 70 colonies
pLK04RCHTag + DigoxFab0: 30 colonies

Five colonies from DigoxFAb0 were picked giving a 15 % chance of picking a background colony.
For normal Digox the chance was 0.02 %.

June 10th

Present: Janniina, Jutta, Linnea

10 colonies from the plate containing XL-1 with pLK04RCHTag+DigoxFab0 were used to inoculate 5 mL of LB with ampicillin. Same was done to 3 colonies from the plate containing 100 µL of XL-1 with pLK04RCHTag+normal Digoxigenin and 2 colonies from the plate containing 900 µL of the same transformant. All of the inoculations were left to grow at 37 °C, 300 rpm Multitron. Mistakenly chloramphenicol was added instead of ampicillin resulting in no growth.

New try with 10 colonies from the plate containing XL-1 with pLK04RCDTag+Digox Fab0, 5 colonies from the plate containing 100 µL of XL-1 with pLK04RCHTag+harmonized digox Fab and 1 colony from the plate containing 900 µL of the same transformant. All of the inoculations were left to grow at 37 °C, 300 rpm Multitron.

June 11th

Present: Janniina, Pyry

The plasmids were extracted from the cells using the EasyPure kit. The DNA-concentrations were measured with nanodrop.

sample of DigoxFab0

ng/µL

1/10

26.3

2/10

20.6

3/10

19.7

4/10

12.9

5/10

12.9

6/10

9.7

7/10

29.2

8/10

15.6

9/10

12.5

10/10

29.2

sample of normal Digox

ng/µL

1/5

19.1

2/5

16

3/5

10.5

4/5

57.6

5/5

78.2

To pretest the presence of a 1.5 kb fragment before sequencing, a SfiI digestion and gel run (1 % agarose, 135 V) were made to separate the DNA fragments:

Since only the fifth of the pLK04RCHTag+harm Digox Fab had a band at 1.5 kb, it was saved and others discarded. 5 colonies from the plate containing
pLK04RCHTag+DigoxFab0 were used to inoculate 3.5 mL of LB with ampicillin and left to grow at 37 °C. The plate was also left in 37 °C for further growth.

June 12th

Present: Janniina, Pyry, Riku

The OD600 was measured from the most opaque growth (1/4) and it was about 4 (1:10 dilution). 3/4 was the clearest, when visually observed. After taking glycerol preps (20 %), the plasmids were extracted using the EasyPure kit and the concentration was measured with Nanodrop.

sample

ng/µL

pLK04RCHTag+Fab0 Digox 1/4

13.3

pLK04RCHTag+Fab0 Digox 2/4

17.0

pLK04RCHTag+Fab0 Digox 3/4

217.5

pLK04RCHTag+Fab0 Digox 4/4

49.4

As it is seen from the table above, the 3rd colony yielded the highest amount of plasmid, which may be due to the smaller amount of cells.

The presence of a 1.5 kb was tested with SfiI digestion and gel electrophoresis. The gel showed that sample 3/4 yielded some plasmid, but the insert was longer than DigoxFab0 would be, so it was unsure if it really was DigoxFab0. It could also have been due to 135 V. The 3/4 colony was taken to be sequenced.

June 17th

Present: Janniina, Jutta, Pyry

The sequencing results came and since the reverse primer was actually forward, we only checked the forward sequence. The normal Digoxigenin had some mutations in the recognition site, so the transformant was unusable. We thought have 20 glycerol preps.

All 10 glycerol preps made on 11th June were used to inoculate 3 mL of LB with ampicillin, 1 % glucose and left to grow at 37 °C and 300 rpm.

June 18th

Present: Pyry, Riku

Plasmid extraction was done for 2 mL o/n cultures with the NucleoSpin Plasmid -kit.
Yields were as follows (measured with ND-1000 with elution buffer as blank):

Sample

ng/µL

1

8.7

2

10.1

3

8.8

4

9.1

5

5.6

6

8.4

7

8.3

8

8.6

9

7.7

10

10.6

Yields showed no DNA from extractions. 3 mL of LB and 3 µL of ampicillin were added to every culture and growth was continued for a further 3 h. Plasmids were extracted as before and concentrations measured with nanodrop.

Sample nr.

ng/µL

1

6.2

5

3.1

Again no DNA in samples.

New cultures (3 mL of LB) were started from glycerol preps.

June 19th

Present: Fanny, Janniina, Linnea, Oskari, Pyry, Riku, Susanna

2 mL of each culture was used to extract plasmid from cells. Extraction was done with NucleoSpin Plasmid Kit. Plasmid concentrations were measured with Nanodrop.

Sample

ng/µL

1

7.5

2

8.7

3

7.0

4

7.0

5

8.6

6

8.8

7

9.6

8

8.4

9

8.4

10

9.6

Again no good yield in any of the growths. This might be due to DigoxFabs being toxic to cells.

June 25th

Present: Linnea, Pyry, Riku

pLK04RCHTag was digested with SfiI to allow for ligation with Digoxigenin fabs and the digested fragments were separated with agarose gel electrophoresis (1 % agarose, 70 V). Separated fragments were purified with NucleoSpin Gel and PCR Clean-up kit. Concentration of purified pLK04RCHTag fragments was measured to be 18.2 ng/µL (NanoDrop ND-1000).

June 26th

Present: Janniina, Pyry

Purified DigoxFab0 was ligated to the purified pLK04RCHTag. Ligation was done with T4 ligase according to manufacturer’s protocol. Ligated fragments were transformed into XL-1 Blue (chemical competent) cells according to the protocol from Addgene.org . Transformed cells were plated and grown o/n at 37 °C, 300 rpm.

June 28th

Present: Riku

Colonies from plates were counted but the control plate had more colonies so transformation was done again as before. New plates were left to grow o/n at 37 °C, 300 rpm.

June 29th

Present: Jutta, Fanni

A colony from yesterday’s plates was used to inoculate 4 mL of LB with ampicillin to grow o/n at 37 °C, 300 rpm.

June 30th

Present: Janniina, Susanna

pLK04RCHTag was extracted from the cells using NucleoSpin Plasmid kit. Concentration of purified DNA was 343.8 ng/µL. Extracted plasmids were digested with SfiI and separated on a 1 % agarose gel (70 V) together with pEB04RCH ligations.

July 4th

Present: Jutta, Linnea, Oskari, Pyry

Re-digested the vector out of pLK04RCHTag+DigoxFab0 (30.6.) with SfiI and separated it on a gel together with SfiI digested pEB04RCH+DigoxFab0. SfiI digested DigoxFab0 from pEB04RCH and SfiI digested pLK04RCHTag were cut from the gel and stored in -20 °C.

June 5th

Present: Oskari, Ossi

DigoxFab0 and pLK04RCHTag were extracted with NucleoSpin Plasmid kit and measured with Nanodrop resulting in 3.5 ng/µL and 37.7 ng/µL respectively.

Extracted DigoxFab0 was ligated to pLK04RCH and pLK04RCHTag with T4 ligase.

XL-1 Blue cells (35 µL) were electroporated with 1 µL pLK04RCHTag+DigoxFab0. Transformed cells were incubated in 5 mL of LB for 1 h at 37 °C, 300 rpm shaking. After incubation cells were plated onto LA plates with ampicillin and stored o/n at 37 °C.

July 6th

Present: Riku

The transformed cells had grown nicely overnight. 3 colonies from an undiluted plate and 1 colony form 1/100 diluted plate were incubated o/n in LB+ampicillin in 37 °C, 300 rpm.

July 7th

Present: Riku

Glycerol preps were made from 500 µL of o/n cultures and the rest of the medium was centrifuged (2000 g, 10 min) to collect plasmids.

Centrifuged supernatant was discarded and the plasmids were extracted with NucleoSpin EasyPure-kit. Elution was made twice with 25 µL of 70 °C prewarmed Elution buffer.

Concentrations were measured with NanoDrop ND-1000:

Sample

ng/µL

1/1

89.4

1/2

75.3

1/3

80.6

100/1

59.1

Cloning Synthesised Digox Fabs into pLK04RCH

June 21st

Present: Janniina, Linnea

A colony was picked from glycerol prep (321.a with pLK04RCH*) and inoculated to 4 mL of LB with 100 µg/mL ampicillin. It was then put to Multitron overnight in 37°C, 300 rpm. This vector has been checked by sequencing, it’s from colony 1/1.

June 22nd

Present: Riku, Linnea

We noticed that yesterday's growth had been in RT over night.

pLK04RCH was purified from the growth with NucleoSpin Plasmid EasyPure -kit according to manufacturer's instructions.

Measured concentration: 16.2 ng/µL

Due to low yield, a new growth was started in 5 mL LB with 100 µg/ml ampicillin.

June 23rd

Present: Riku, Linnea

Growth was taken out and fuged at 2000 g for 10 minutes to pellet cells. Plasmids were extracted with NucleoSpin Plasmid EasyPure -kit according to manufacturer’s instructions. Yield was measured with Nanodrop ND-1000.

Results: 75.9 ng/µL
260/280: 1.99
260/230: 1.33

June 24th

Present: Janniina, Riku

Digestion of PLK04RCH was made with ThermoFisher FastDigest SfiI according to manufacturers instructions and run on gel.

Since there is no DNA visible in the second lane a, new SfiI digestion was made according to the following table.

µL
MQ 7
FastDigest Buffer 2
DNA 10
SfiI FastDigest 1
Total volume 20

June 25th

Present: Riku, Linnea

5 µL 1 kb ladder pLK04RCH + Sfi1
1 µL Midori Direct 0.5 µL Midori Direct

Samples A and B were loaded to 1 % agarose gel and run with 70 V.

pLK04RCH doesn't show up on the gel because the endonucleases in 321.a have disintegrated it completely. Conclusion: we need to produce the vector in XL-1 instead of 321.a.

35 µL of electrocompetent XL-1 cells and 1 µL of pLK04RCH (from 22.6.) were used for transformation. Transformants were put in LB-medium. Incubation was started in 37 °C, 300 rpm at 13:05 and stopped at 13:50.

20 and 200 µL of growth were spread on LA plates with ampicillin and incubated in 37 °C overnight.

New 5 mL culture of 321.A with pLK04RCH was started in 37 °C 250 rpm.

June 26th

Present: Janniina, Riku, Linnea

Cells in previous days culture was centrifuged in 2000 g for 10 min. Supernatant was thrown away and plasmids were extracted with NucleoSpin Plasmid -kit according to manufacturers instructions with additional AW-buffer wash. Concentration was measured with ND-1000.

Result: 13.4 ng/µL

Digestion was made with ThermoFisher FastDigest SfiI according to manufacturer’s instructions. Digested plasmid was run on gel.

Results: Vector has dimerized and cannot be used.

pLK04RCH from tubes 3/2 and 3/1 from 28.5.-19 were transformed into XL-1 according to this protocol from Addgene

After transformation, cells were put into 5 mL LB for 45 min 37 °C 250 rpm shaking. After shaking cells were put into LA + ampicillin plate.

A colony from previous days transformation plate (20 µL) was used to inoculate 4 mL SB medium containing ampicillin and put on 37 °C 300 rpm overnight at 13:40.

June 27th

Present: Janniina, Riku

There was a good growth in all transformation plates except mQ-control. 2 colonies were picked from each transformation (3/1: 1/100 dilution & 3/2: no dilution) plate. and put into 5 mL LB 100µg/ml ampicillin. Cultures were put into 37 °C 250 rpm shaking overnight.

Since the 1/1 was forgotten, a new overnight growth was made with 1 colony into 4 mL of LB with ampicillin. Put in 37 °C at 17.

June 28th

Present: Janniina, Riku, Ossi

Overnight cultures (1/1, 3/1.1, 3/1.2, 3/2.1 & 3/2.2) were centrifuged in 2000 g for 10 min and supernatant was thrown away. New liquid cultures were started by inoculating from pellet.

Rest of the pellet was used to extract plasmid with NucleoSpin Plasmid -kit, according to manufacturer's instructions.

Concentrations were measured with ND-1000.

Results:

1/1 333.8
3/1.1 652.5
3/1.2 538.0
3/2.1 390
3/2.2 484.5

Plasmids (1 µg) were SfiI digested for around 30 minutes at + 50°C.

1/1 3/1.1 3/1.2 3/2.1 3/2.2
Vol. (DNA) µL 3 1.53 1.86 2.56 2.06
MQ 17 18.47 18.14 17.44 17.94
FD buffer 2 2 2 2 2
FastDigest SfiI 1 1 1 1 1
Total volume 20 20 20 20 20

After digestion, reactions were purified in 1% agarose gel.

Gel loaded as follows:

1 kb Ladder (5 µL) + midori green (1 µL) pLK04RCH 1/1 (20 µL) + 0.5 µL Midori Green pLK04RCH 3/1.1 (20 µL) + 0.5 µL Midori Green pLK04RCH 3/1.2 (20 µL) + 0.5 µL Midori Green pLK04RCH 3/2.1 (20µL) + 0.5 µL Midori Green pLK04RCH 3/2.2 (20 µL) + 0.5 µL Midori Green

2nd highest band matches the size of pLK04RCH - no fab, so they were cut out from gel and put into freezer.

June 30th

Present: Janniina, Susanna

According to the sequencing results the pLK04RCH 3/1 is also good to use. Since we are not sure yet whether or not 1/1 has suffered from the transformation and growing in 321.A, we'll start using 3/1.

The vector backbone is needed for 5 different digoxigenin Fab ligations and since it will be purified from the gel, the yield will decrease. Assuming that 20 ng of backbone is needed for each reaction, at least 1000 ng has to be recovered from the gel. Let's say we need at least 10 µg of the backbone. Only 1 µg is recommended per 20 µL reaction, but we have used up to 2 µg with fine results. Only 1 well is free, so we'll SfiI digest only 2 µg of the 3/1 backbone.

MQ 14 µL
DNA 3 µL
FD buffer 2 µL
FD SfiI 1 µL
Total volume 20 µL

Incubation at 50 °C started at 11:27. Stopped at 11:56.
0.5 µL of Midori direct is added to the sample and separated on 1 % agarose gel.

1 µL Midori Direct + 5 µL 1 kb Ladder 0.5 µL Midori Direct + 20 µL pLK04RCH 3/1 0.5 µL Midori Direct + 10 µL pEB04RCH+lig 1/1 0.5 µL Midori Direct + 10 µL pEB04RCH+lig ½ 0.5 µL Midori Direct + 10 µL pEB04RCH+lig 2/1 0.5 µL Midori Direct + 10 µL pEB04RCH+lig 2/2 0.5 µL Midori Direct + 10 µL pEB04RCH+lig 3/1 0.5 µL Midori Direct + 10 µL pEB04RCH+lig 3/2 0.5 µL Midori Direct + 10 µL pEB04RCH+lig 4/1 0.5 µL Midori Direct + 10 µL pEB04RCH+lig 4/2 0.5 µL Midori Direct + 10 µL pEB04RCH+lig 7/1 0.5 µL Midori Direct + 10 µL pEB04RCH+lig 7/1 0.5 µL Midori Direct + 10 µL pLK04RCHTag+Fab0

Gel run was started using 70 V at 12:18. Stopped at 13:25.
Gel run stopped and started again 13:10.
Gel run stopped at 13:20.

Empty Eppendorf weighs 1.03 g.

2nd well cut out and put to -20 °C.

The bands didn't move accordingly to their size, apparently, which may be due to active SfiI. The 2nd more clear band was cut and stored in - 20 °C.

July 1st

Present: Linnea, Janniina, Ossi

Samples made with 3/1 and 3/2 from gel were combined and purified using NucleoSpin Gel and PCR Clean-up -kit, according to manufacturer’s instructions. Concentration measured with ND-1000 was 44,8 ng/µL.

All the Fabs were ligated into pLK04RCH (SfiI digested and purified from gel) according to the following reaction.

µL
Vector DNA 0.5
Insert DNA 3
10x ligase buffer 2
Ligase 0.2
H2O 14.3
Total volume 20

7 reactions of a master mix that does not have insert were made.

MQ 100.1
10x buffer 14
ligase 1.4
Total volume (µL) 115.5

16.5 µL of above prepared mix to all tubes.

1 µL of each ligation reaction and ligation vector control plus MQ transformation control were electroporated to 40 µL of electrocompetent XL-1 Blue cells.

1 and digd2 exploded in an arc during electroporation. All else were fine with time constants 4.8 – 5.0.

July 2nd

Present: Pyry

1 and digd2 were retried along with a new background control as previously. 1 exploded again twice.

July 3rd

Present: Linnea, Ossi

3 growths from plate containing XL-1 Digox Lys 191 TAG (number 1) were each used to inoculate 4 mL LB with ampicillin and tetracycline and put to Multitron to grow overnight in 37°C and 300 rpm.

Three separate colonies from 2 (Digox Lys108), 3 (Digox Fab0) and 4 (Digox C-term TAG) were each separately inoculated to 5 mL of LB with ampicillin and incubated overnight in Multitron at 37 °C and 300 rpm.

Digd2 (Digox harmonized) was re-plated as the growth was too dense and grown overnight at 37 °C.

July 4th

Janniina, Linnea, Riku, Ossi, Jutta

Overnight cultures OD was measured.

Most dense culture was chosen for od measurement:

Digd2 (Digox harmonized) was re-plated as the growth was again too dense. Cells were suspensed with 300µL of LB and 1/100 and 1/1000 dilutions were re-plated. Cells were grown overnight at 37°C.

Plasmids were extracted from 1-4 ligations (3 colonies each) with plasmid EasyPure kit according to manufacturer’s instructions.

The DNA concentration was measured with Nanodrop 1000.

Culture OD600
Lig 1 3.23
2 2.90
3 4.15
4 2.87

pLK04RCHTag was digested with Sfi1 according to manufacturer’s instructions and incubated for 2 hours at 50 °C.

sample name concentration
pLK04RCH +lig 1/1 340.4
pLK04RCH +lig 1/2 309.9
pLK04RCH +lig 1/3 28.2
pLK04RCH +lig 2/1 368.7
pLK04RCH +lig 2/2 57.4
pLK04RCH +lig 2/3 337.2
pLK04RCH +lig 3/1 61.8
pLK04RCH +lig 3/2 53
pLK04RCH +lig 3/3 51.6
pLK04RCH +lig 4/1 42.7
pLK04RCH +lig 4/2 58.1
pLK04RCH +lig 4/3 431.4
Sample 1/1 1/2 1/3 2/1 2/2 2/3 3/1 3/2 3/3 4/1 4/2 4/3
MQ 17 17 17 17 17 17 15 15 15 14 14 14
DNA 1 1 1 1 1 1 3 3 3 4 4 4
Buffer (FD-green) 1 1 1 1 1 1 1 1 1 1 1 1
FD-SfiI 1 1 1 1 1 1 1 1 1 1 1 1
Total volume (µL) 20 20 20 20 20 20 20 20 20 20 20 20

0.5 µL Midori Direct was added to 19 µL of samples and 5 µL of ladder.

1/1
1st gel 1/2 1/3 2/1 2/2 2/3 ladder 3/1 3/2 3/3 4/1 4/2 4/3
2nd gel ladder Fab0 pLK04RCHTag Sfi1

Gel run started at 13:56 and stopped 14:50

SfiI digested Fab0 from pEB04RCH vector (3/1 colony) and SfiI-digested pLK04RCHTag were cut from gel and stored at -20 °C.

July 5th

Present: Ossi, Oskari

pLK04RCH with Digox harmonized (digd2) was re-plated as the growth was again too dense. Cells were suspensed with 600 µL of LB and 1/10000 and 1/100000 dilutions were re-plated. Cells were grown overnight at 37 °C.

Miniprepkit extraction and Nanodrop measurements were made for Fab0 and plk04RCHTag: 3.5 and 37.7 ng/µL respectively.

Ligation:

 

ng/µL

V(20ng)

Fab0

Ligase

Buffer

MQ >20µL

pLK04RCHTag

37.7

0.5305039788

6.4142857143

0.2

2

10.8552103069

pLK04RCHTag ctrl

37.7

0.5305039788

 

0.2

2

17.2694960212

pLK04RCH

44.8

0.4464285714

6.4142857143

0.2

2

10.9392857143

pLK04RCH ctrl

44.8

0.4464285714

 

0.2

2

17.3535714286

nebiocalculator was used for calculations.

Ligation controls didn’t include the Fab0 sequence.

35 µL of XL-1 blue cells were transformed with 1 µL of pLK04RCH + Fab0 ligation reaction with electroporation.

Electroporated cells were incubated in 5 mL LB at 37°C and in 300 rpm shaking for 1 hour.

Cells were plated to LA plates with ampicillin and incubated overnight at 37 °C.

July 6th

Present: Riku

Digid2 1/100000 was washed with 500 µL LB medium and cells were put into another container. 1/100 and 1/10000 dilutions were made and 100 µL of dilutions was put into LB+ampicillin dish. Dishes were put 37 °C for overnight.

July 7th

Present: Riku

Glycerol preps were made from 500 µL overnight cultures and the rest of the medium was centrifuged in 2000g 10 minutes.

Supernatant was thrown away and plasmid was extracted with NucleoSpin EasyPure -kit, according to manufacturer’s instructions. Elution was made twice with 25 µL 70C prewarmed Elution buffer.

Concentrations were measured with NanoDrop ND-1000. Results as follows:

Sample ng/µL
1/1 86.0
1/2 74.4
1/3 65.4
100/1 86.7

3 LB + ampicillin cultures were started and put into 30 °C 300 rpm overnight.

July 8th

Present: Riku

Cells were centrifuged in 16 000 g for 3 mins and plasmids were extracted with NucleoSpin PlasmidEasyPure -kit according to manufacturer’s instructions. Buffers A1-A3 were used 1.5 x amount.

Concentrations were measured with NanoDrop ND-1000. Result as follows:

Sample ng/µL
1 15.4
2 16.4
3 0.4

Production of pLK04RCHTag-harmonized digoxigenin Fab

June 17th

Present: Janniina, Jutta, Pyry

50 µL 321.A with pEVOL-pAZF and/or pLK04RCHTag-harm were transformed. Digox Fab according to the following table + one control transformation with 1 µL of MQ.

pEVOL-pAzF pLK04RCHTag-harm. digox fab
x (0.5 µL) x (0.5 µL)
x (0.5 µL)
x (0.5 µL)

Reactions were put to 37 °C at 15:17, taken out at 16:00. The cells were centrifuged to the bottom, the supernatant was removed and the pellet was resuspended to 100 µL LB before dispensing it on the plates containing zeocin, chloramphenicol and ampicillin. The plates were put to 37 °C overnight.

June 18th

Present: Janniina

Previous day’s growths were replated, since the cells were really dense.

June 19th

Present: Janniina

50 mL of LB with 25 µL of zeocin (1 x), 75 µL ampicillin (1.5 x), 50 µL chloramphenicol (1 x) and 1 % glucose was made. A colony was taken from a plate containing 321.A with pLK04RCHTag+harm digox Fab and pEVOL-pAzF and put in 4 mL of above mentioned LB to grow overnight.

June 20th

Present: Janniina, Linnea

The OD 600 from the overnight precultured 4 mL growth was measured from 1:10 dilution yielding 0.145 which is 1.45 undiluted at 9:21. The growth was diluted into OD of 0.1 by adding 280 µL of the growth into 3,7 mL of LB with glucose, zeocin, 1,5 x ampicillin and chloramphenicol and put into 37 °C 230 rpm at 9:30.

OD was measured at 11:34, (200 µL of growth into 800 µL LB): 0.04 in dilution, 0.2 undiluted.

At 13:36 OD was 0.021 diluted and 0.21 undiluted.

June 24th

Janniina Jutta Pyry

4.5 mL of above prepared LB was inoculated from a glycerol prep made on 20th June and put in 37 °C.

June 25th

Janniina RIku Pyry

Calculated amount for 5 mL, OD 0.1:

 

A

B

C

D

1

C1

V1

C2

V2

2

2.3

0.2260869565

0.1

5.2

Culture was diluted into 0.1 OD600 by adding 225 µL into 5 mL LB medium with 1% glucose, 25 µg/mL cloramphenicol, 100 µg/mL ampicillin, 50 µg/mL zeocin.
Culture was put into 37 °C 300 rpm.

OD was measured as follows:
1/10 dilution is made with 100 µL growth and 900 µL LB medium.

Time

OD600

*calculated

09:34

~0.1

*

10:00

0.12

 

10:50

0.14

 

11:45

0.27

 

LB medium was changed to SB medium. Cells were centrifuged in bottom of Falcon tube and supernatant was thrown away. Cells were resuspensed in 5 mL SB medium (1% glucose, 25 µg/mL cloramphenicol, 100 µg/mL ampicillin, 50 µg/mL zeocin).

Time

OD600

 

13:06

0.42

 

13:20

0.51

 

13:40

0.72

 

Growth curve until induction:

After OD had reached 0.72 cells were centrifuged in 2000 g for 10 min. Supernatant was thrown away and cells were resuspensed in SB medium (25 µg/mL cloramphenicol, 100 µg/mL ampicillin, 50 µg/mL zeocin, 0.1 % arabinose).

Cells were incubated 26 ℃ 200 rpm for 10 min.
After incubation P-azidophenylalanine and IPTG was added in growth medium to final concentration 1mM
After 4h from induction, a 1 mL sample was taken and centrifuged to pellet cells.
After 4h incubation 1 mL sample was taken, centrifuged (16.1 g, 1 min) and the pellet put into freezer.

June 26th

Present: Linnea

5 µL of glycerol prep (321.A) was used to inoculate 5 mL of LB with 25 µg/mL chloramphenicol and 0.5 % glucose and put into Multitron (300 rpm, 37 °C) at 10:53.

Two XL1 colonies with the biobrick were used to inoculate 5 mL of LB with 25 µg/ml chloramphenicol and 0.5 % glucose and put into Multitron (300 rpm, 37 °C) at 12:20.

June 27th

The cell pellets were sonicated with biotechnology sonicator. Cells were resuspended into 1 mL of Assay Buffer per 1 mL of culture before sonication.

Sonication procedure:

1 minute, low setting, power level 40, cycle 0.5 seconds. Cells were held on ice while sonicating.

Assay procedure:

The cell lysate was diluted 1/10 and 1/100 and 200 µL was incubated 30 minutes at low shake on Goat Anti Human 96-well plate.

Plate was washed 4x with Wash Buffer (kaivogen).

2A11-Europium labeled anti human antibody was used to detect Fabs. Assay concentration was 0.25 ng/µL/well. Assay volume 200 µL Plate was washed 4x with WAsh Buffer (kaivogen) Delfia Enhancement Solution was used to develop signal for 10 min. Hidex multilabel reader was used for signal detection

Results:

Production of DigoxFab0, DigoxFab108 and DigoxFab191 in pLK04RCH

July 9th

Present: Linnea, Riku

To produce the modified Digox antibody fragments with TAG-codons in positions 0, 108 or 191 the corresponding genes in pLK04RCH vectors and a pEVOL-pAzF-vector were electroporated into C321.ΔA.exp-cells.

Electroporation reactions were mixed as follows:

Digox Fab

Digox Fab Plasmid (µL)

pEVOL-pAzF (µL)

MQ H2O (µL)

Electrocompetent C321.ΔA.exp cells (µL)

DigoxFab108

0.5

0

0.5

35

DigoxFab108

0.5

0.5

0

35

DigoxFab191

0.5

0

0.5

35

DigoxFab191

0.5

0.5

0

35

DigoxFab0

0.5

0

0.5

35

DigoxFab0

0.5

0.5

0

35

NoFabControl

0

0.5

0.5

35

Electroporation was done with 1.8 kV in 1 mm electroporation cuvette (Bio-Rad Gene Pulser Cuvette, 165-2089).

Time constants were 5.0 ms for all samples.

Electroporated cells were put into 5 mL of LB for 45 min in 37 °C and 250 rpm shaking.

Plating was done as follows:

Dilution

1/1 (µL)

1/100 (µL)

DigoxFab108

50

 

DigoxFab108 + pEVOL-pAzF

50

50

DigoxFab191

50

 

DigoxFab191 + pEVOL-pAzF

50

50

DigoxFab0

50

 

DigoxFab0 + pEVOL-pAzF

50

50

NoFabControl

50

 

Plate cultures were grown o/n at 37 °C.

July 10th

Present: Linnea

Two colonies from each 1/100 diluted plate (DigoxFab108 + pEVOL-pAzF, DigoxFab191 + pEVOL-pAzF, DigoxFab0 + pEVOL-pAzF) were used to inoculate 4 mL LB with ampicillin, chloramphenicol, zeocin and glucose. Tubes were grown o/n at 37 °C, 300 rpm.

July 11th

Present: Janniina, Oskari

OD600 was measured from each of the two replicate growths. Measurements were made from a 1:10 diluted sample. The OD600 was used to calculate the growth volume needed for inoculation of 4 mL of SB.

Growth

OD600 (1:10)

OD600 (1:1)

Volume need for dilution to OD600 of 0.1 in 4ml of SB (µL)

DigoxFab191 + pEVOL-pAzF

0.219

2.19

183

DigoxFab108 + pEVOL-pAzF

0.303

3.03

132

DigoxFab0 + pEVOL-pAzF)

0.229

2.29

175

Antibiotics and 0.05 % glucose were added to the growths for selection and increased growth. The cells were grown until OD600 of 0.6-0.8 at 37 °C, 300 rpm.

OD600 was measured again after 3 h.

DigoxFab191 + pEVOL-pAzF

DigoxFab108 + pEVOL-pAzF

DigoxFab0 + pEVOL-pAzF)

0.784

0.276

0.27

Arabinose (final concentration = 5 %) and pAzF (1 mM) was added to DigoxFab191 + pEVOL-pAzF test growth and after 10 min incubation in 37 °C 0.1 mM IPTG was added to induce production of DigoxFab191. Once induced the growth was left to grow at 26 °C, while a 20 % glycerol prep was made from the control growth.

The OD600 was measured again from DigoxFab108 + pEVOL-pAzF and DigoxFab0 + pEVOL-pAzF test growths from 1:10 dilutions (SB) resulting in unexpectedly low OD compared to the previous OD600 but the growths were induced anyway. Arabinose (5 %) and pAzF (1 mM) were added after 4.5 h. After 10 min incubation in RT 0.1 mM IPTG was added. After adding IPTG, growths were left to grow o/n at 26 °C, 300 rpm (multitron).

All growths were wrapped in tinfoil to prevent photolysis of pAzF and zeocin.

DigoxFab191 + pEVOL-pAzF

DigoxFab108 + pEVOL-pAzF

DigoxFab0 + pEVOL-pAzF)

Already induced

0.102

0.097

July 12th

Present: Fanny, Janniina, Linnea, Oskari, Pyry

An aliquot of 1 mL of overnight growths was harvested by centrifugation and resuspended to 1 mL of assay buffer. Rest of the growth was stored in -20 °C. The cells were sonicated with Braun Labsonic'u for a minute (duty cycle 0.5; output -053).

After lysis the cells were pelleted by centrifugation to collect and dilute supernatant to 1:10 ratio in AB. 200 µL of the dilution was added to each well (goat anti-human plate) with three parallel samples of each.

 

Wells 1-3

Wells 4-6

Wells 7-9

Wells 10-12

A

AB control

DigoxFab108 test

DigoxFab108 control

AB control

B

DigoxFab191 control

DigoxFab0 test

DigoxFab191 test

DigoxFab0 control

C

Harmonized DigoxFab

 

 

 

The 96-well plate was incubated for 1 h on a shaker. Europium labeled 2A11 antibody was diluted with AB to 0.25 ng/µL from stock (390 µg/mL). The dilution was filtered through ⌀ 0.22 µm filter. The plate was washed using Delfia platewasher (Wallac) 4 times. 200 µL of 2A11 (0.25 ng/µL) is added into each well. The dispenser dispensed some of the liquid that was supposed to be in well C1 (AB control) into D1 (DigoxFab108 test). So DigoxFab108 test should give lower results than what might be expected. The plate is incubated on a shaker for 30 min after which the plate was washed again 4 times. 200 µL of Delfia Enhancement Solution was dispensed into each well. After incubating for 10 minutes, the fluorescence was measured with Hidex Sense.

To transform the plasmids (pEVOL-pAzF and pLK04RCHTag + DigoxFab0 1/1) into cells (C321.ΔA.exp) for production; 0.5 µL of each plasmid was electroporated with 30 µL of cells. Three transformation controls were made; only pEVOL-pAzF, only pLK04RCHTag+DigoxFab0 1/1 (ligation date?) and MQ water only. After electroporation the cells were moved to a round-bottomed culture tube and grown for 1 h in 3 mL of SB at 37 °C.

Transformation cultures were diluted to 1/50 and plated with tetracycline, ampicillin and zeocine.

July 13th

Present: Pyry

Main plate had too many colonies to count with a couple of individual colonies at the edges. Decided to do dilutions of 1/100 or higher in future. No growth on any control plates.

July 14th

Present: Janniina

Two colonies from the plate were used to inoculate 4 mL of SB containing zeocin, ampicillin and chloramphenicol and grown at 37 °C, 300 rpm o/n. Since the plate was dense from colonies, a couple were used to restreak a new plate, which was grown at 37 °C o/n.

July 15th

Present: Janniina

The o/n cultures did not grow.

July 16th

Present: Jutta

Colonies from the plates were used to inoculate again 100 mL of SB containing zeocin, ampicillin and chloramphenicol and grown o/n at 37 °C, 300 rpm.

July 17th

Present: Janniina, Jutta, Linnea, Oskari, Pyry, Robin

The growths were diluted to 1:10 in 1 mL of SB for measurement of OD600.

OD600 of 1:10 dilutions

1:10

1:1

DigoxFab0

0.508

5.08

DigoxFab191

0.358

3.58

DigoxFab108

0.244

2.44

Growths were diluted to OD600 of 0.1 in 400 mL of SB and antibiotics in working concentrations. These growths were left to grown at 37 °C, 230 rpm. RPM was increased to 300 rpm after 2h. OD600 was measured again after 4h; giving 0.104 for DigoxFab0, 0.086 for DigoxFab191 and 0.088 forDigoxFab108. After 5h 0.05 % glucose was added to each culture to accelerate growth. OD600 measured again after 8h were: DigoxFab108: 1.01; DigoxFab191: 0.69; DigoxFab0: 0.65.

0.2 % arabinose was added to all growths. After 10 min of incubation in RT growths were induced with 0.4 mM IPTG and 1 mM pAzF. Growths were covered in foil and left to grown o/n at 26 °C and 300 rpm shaking.

Again no growth observed in the preculture. Transformed pEVOL-pAzF and pLK04RCHTag + DigoxFab0 1/1 into C321.ΔA.exp. After electroporation the cells were moved to a round-bottomed culture tube and grown for 1 h in 3 mL of SB at 37 °C. After incubation the cultures were diluted to 1/100 and plated with tetracycline, ampicillin and zeocine and grown o/n at 37 °C, 300 rpm.

July 21st

Present: Janniina

Two cultures of 20 mL of SB containing zeocin, ampicillin, chloraphenicol and 0.5 % glucose were made, with cells inoculated from the plate containing C321.ΔA.exp transformed with pEVOL-pAzF and pLK04RCHTag + DigoxFab0 1/1. Cultures grown o/n at 37 °C, 300 rpm.

July 22nd

Present: Janniina, Jutta

Glycerol prep was made of the cultures.

Cultures were diluted to maximum OD600 of 0.1 in 400 mL of SB (antibiotics: ZCA, 0.05 % glucose). When OD600 was around 0.7 added 1 % arabinose and 1 mM p-azido-L-phenylalanine (pAzF). Cultures were covered with aluminium foil and after 10 min 1 mM of IPTG was added to induce production of DigoxFab0. Cultures were grown o/m at 26 °C.

July 23rd

Present: Janniina, Ossi

Prepared histag column prior to purifications. A sample (500 µL) was taken from the growth. Cells were washed and lysed according to protocol. Weights of the two lysate pellets were 6,46 g and 6,28 g. Second pellet was resuspended into 15 mL NPI-lysis buffer. Another 15 mL was added since the pellet did not completely dissolve. Once resuspended the second tube was poured to the first one. Lysate (25 mL) were purified according to protocol and collected in 5 fractions of 1 mL.

ANTIBODY PRODUCTION AND PURIFICATION

Protein concentration was determined with Nanodrop 280 nm.

Fraction 1

1.26

Fraction 2

1.34

Fraction 3

1.11

Fraction 4

0.16

Fraction 5

0.06

Lysate

28.75

July 30th

Present: Janniina, Ossi, Pyry

Fractions 1-3 from earlier purification were further purified by buffer exchange with centripure P10 according to the manufacturer's protocol.

Last Production of Azido-Modified Fabs

August 22nd

Present: Janniina

Precultures for production were started by inoculating pLK04RCHTag (DigoxFab108Tag, DigoxFab191Tag and DigoxFab0HistagTag) and pEVOL-pAzF containing C321.ΔA.exp-cells from a glycerol prep into 20 mL of SB. Inoculated cells were incubated o/n at 37 °C, 250 rpm.

August 23rd

Present: Ossi, Pyry

Yesterdays growths were inoculated inoculated into the main culture (400 mL) so that the main culture’s OD600 was 0.1 with chloramphenicol, ampicillin and 0.05 % glucose. Inoculated main growth was left to grow at 37 °C, 250 rpm until OD600 was 0.6 - 0.8. Once ready, cells were induced with 1 mM pAzF, 0.5 mM IPTG and 0.5 % arabinose and incubated o/n 26 °C, 250 rpm.

August 24th

Present: Pyry

Cells were washed according to the protocol, with the modification of using ice cold PBS for washing. After washing the pellet was stored in -70 °C.

August 26th

Present: Pyry

Cells were lysed and Fabs purified according to our protocol.

NiNTA-column was equilibrated with NPI-10. Lysate was added to the column and the column was then capped. The resin lysate mixture was then on rotation for 1 h at 4 °C.

All the supernatant was combined to a 50ml bottle and the mixing with resin continued for 30 min.

The fractions were eluted in 1ml batches. 5 each. Preliminary results showed good yields of DigoxFab0Histag and control DigoxFab0 (Histag fraction 2 2.9mg/ml and Fab0 fraction 1 0.90 mg/ml). Other fractions had lower yields and were interfered by imidazole from elution buffer. Buffer was changed into PBS, except for the negative control. Added preservatives (0.05 % 2-methyl-4-isothiazolin-3-one) and stored at +4 °C.

August 27th

Present: Pyry

Negative control was purified with SEC and SDS-PAGE was done to determine the purity and incorporation-rate of pAzF.

Producing Dengue Fabs

In this lab, we attempted to clone the antigen-binding fragment (Fab) from a Dengue virus antibody into our pLK04RCH and pLK04RCH-Tag vectors.

July 18th

Present: Oskari, Robin, Janniina

The Dengue-Fab sequences from Integrated DNA Technologies were suspended in 20 µL MQ-H2O to a final concentration of 50 ng/µL. We have four different Dengue-Fab fragments to work with, three of which have had an amino acid replaced with an amber stop codon (TAG):
DengueFab Ser221 > TAG (DengueFab221),
DengueFab Thr196 > TAG (DengueFab196)
and DengueFab Asp152 > TAG (DengueFab152).
The last one is an unmodified Dengue-Fab fragment (DengueFab0). The concentration of the previously SfiI-digested vector backbones were:

pLK04RCH: 44.8 ng/µL
pLK04RCH-Tag: 37.7 ng/µL

The Dengue-Fabs were digested with SfiI:

 

volume (µL)

MQ-H2O

7

10x FastDigest buffer

2

DNA

10

SfiI (FastDigest)

1

total volume

20

The reactions were incubated for 15 minutes at 50°C. SfiI was inactivated by addition of 20 mM EDTA.

We then attempted ligation of the Dengue-Fab inserts into the vector backbones. DengueFab0 was cloned into the pLK04RCH and the pLK04RCH-Tag vector. The DengueFab152, DengueFab196 and DengueFab221 were cloned into the pLK04RCH vector:

 

pLK04RCH

pLK04RCH-Tag

pLK04RCH ligation ctrl

pLK04RCH-Tag ligation ctrl

Vector

0.45

0.5

0.4

0.5

Insert

0.95

1

0

 

10x ligase buffer

2

2

2

2

ligase

0.5

0.5

0.5

0.5

MQ-H2O

16.1

16

17.1

17

total volume (µL)

20

20

20

20

The reactions were incubated for 1 hour at room temperature. Then 1 µL of the ligated product was electroporated into 40 µL of XL-1 electrocompetent cells, according to our electroporation protocol. Electroporation was followed by a 45-minute incubation at 37°C and 300rpm. 200 µL of the mixture was plated onto selective LA plates containing tetracyclin and ampicillin and incubated o/n at 37°C.

July 19th

Present: Oskari

Colonies were counted:

Vector + insert

number of colonies

pLK04RCHTag + DengueFab0

108

pLK04RCH + DengueFab0

0

pLK04RCH + DengueFab152

110

pLK04RCH + DengueFab196

24

pLK04RCH + DengueFab221

10

pLK04RCHTag – ligation ctrl

9

pLK04RCH - ligation ctrl

2

Transformation ctrl

0

July 21st

Present: Janniina, Jutta

40 mL of SB medium, containing ampicillin and 0.5 % glucose, was prepared. Two colonies were picked from the pLK04RCH-Tag+DengueFab0, pLK04RCH+DengueFab152, -196 and -221, and inoculated to 2x4 mL of SB medium. The growths were incubated o/n at 37°C.

July 22nd

Present: Jutta

Glycerol preps were made from the overnight cultures according to our protocol for making glycerol preps. Plasmids were extracted according to our miniprep protocol.

The DNA concentration was measured with Nanodrop:

 

miniprep 1

miniprep 2

pLK04RCH-DengueFab152

136.4 ng/µL

124.5 ng/µL

pLK04RCH-DengueFab221

120.7 ng/µL

130.1 ng/µL

pLK04RCH-DengueFab196

129 ng/µL

431.3 ng/µL

pLK04RCHTag-DengueFab0

448.1 ng/µL

137.9 ng/µL

To verify that the ligation worked, the vectors were re-digested with SfiI:

 

volume (µL)

MQ-H2O

7.5

DNA

1

FastDigest buffer

1

SfiI

0.5

total volume

20

The reaction was incubated for 30 minutes at 50 °C. 0.5 µL Midori Green Direct (Nippon Genetics) was added to each digestion reaction and 1 µL to 1 kB ladder.

The digested vectors and inserts were separated on a 1 % agarose gel:


From left to right: ladder, pLK04RCH-DengueFab152 (miniprep 1), pLK04RCH-DengueFab152 (miniprep 2), pLK04RCH-DengueFab196 (miniprep 1), pLK04RCH-DengueFab196 (miniprep 2), pLK04RCH-DengueFab221 (miniprep 1), pLK04RCH-DengueFab221 (miniprep2), pLK04RCHTag-DengueFab0 (miniprep 1), pLK04RCHTag-DengueFab0 (miniprep 2).

We repeat the ligation of DengueFab0 and the pLK04RCH vector (vector concentration: 18.8 ng/µL):

 

pLK04RCH

pLK04RCH ligation ctrl

vector

1.1

1.1

insert

1

0

10x ligase buffer

2

2

ligase

0.5

0.5

MQ-H2O

15.4

16.4

total volume (µL)

20

20

Reaction was incubated for 1 hour at room temperature. XL-1 electrocompetent cells were transformed with the ligation mixture according to our electroporation protocol, and then incubated for 45 minutes at 37 °C and 300 rpm, plated onto selective plates and incubated o/n at 37 °C.

July 23rd

Present: Ossi

Colonies from the plates were inoculated into 4 mL SB cultures containing ampicillin and 0.5 % glucose. The growths were incubated o/n at 37 °C and 300 rpm.

The pLK04RCH-DengueFab196, pLK04RCH-DengueFab221, pLK04RCH-DengueFab152 and pLK04RCH-Tag-DengueFab0 were sent for sequencing.

July 24th

Present: Ossi

Glycerol preps and minipreps were made from the o/n cultures, according to our protocols. The DNA concentration as measured with Nanodrop:

pLK04RCH-DengueFab0 (prep 1): 518.8 ng/µL
pLK04RCH-DengueFab0 (prep 2): 471.9 ng/µL

To verify if the cloning worked we re-digested the vectors with SfiI:

MQ-H2O

7.5

vector

1

FastDigest buffer

1

SfiI

0.5

total volume (µL)

10

The reaction was incubated for 10 minutes at 50 °C. The digestion products were separated on a 1 % agarose gel:

A sample of pLK04RCH-DengueFab0 was sent for sequencing.

According to the results from the previous sequencing, we decided to not use the pLK04RCH-DengueFab221 vector. The rest were OK.

321.A cells were transformed with the pEVOL-pAzF vector and one of the pLK04RCH-DengueFab196, -152 or pLK04RCH-Tag-DengueFab0 vectors. Transformation was done with electroporation according to our electroporation protocol. The cell mixtures were afterward plated onto selective LA plates containing chloramphenicol, zeocin, ampicillin and 0.5 % glucose.

Comment:
we attempted test production on a small scale of the DengueFab0 in the 321.A strain, but we soon realized that it has a mutation in a stop codon (TAA > TAG), leading to a nonsense peptide that is not able to bind anything.

July 30th

New 4 mL SB growths containing ampicillin and 0.5 % glucose were inoculated with pLK04RCH-Tag-DengueFab0 and incubated o/n at 37°C.

July 31st

Glycerol preps and minipreps were made from the o/n cultures according to our protocols. The DNA concentration in the minipreps:

pLK04RCH-Tag-DengueFab0 (prep 1): 27.9 ng/µL
pLK04RCH-Tag-DengueFab0 (prep 2): 7.9 ng/µL
pLK04RCH-Tag-DengueFab0 (prep 3): 40.8 ng/µL

The vector (prep 1 and 3) was re-digested with SfiI:

MQ-H2O

7.5

vector

1

FastDigest buffer

1

SfiI

0.5

total volume (µL)

10

The reaction was incubated for 10 minutes at 50°C, and the digestion fragments separated on a 1 % agarose gel.

There was a 8 kB band visible in the gel, which is too large to be the vector.

August 5th

Present: Janniina

2 new colonies from the plate containing XL-1 with pLK04RCHTag-Dengue Fab0 were picked and inoculated to 4 mL of SB with ampicillin and 0.5 % glucose and incubated o/n at 37 °C, 300 rpm.

August 6th

Present: Janniina

Glycerol preps and minipreps were made from the o/n cultures according to our protocols. The DNA concentration in the minipreps:

pLK04RCH-Tag-DengueFab0 (prep 1): 413.6 ng/µL
pLK04RCH-Tag-DengueFab0 (prep 2): 512.7 ng/µL

SfiI digestion:

MQ-H2O

7.5

vector

1

FastDigest buffer

1

SfiI

0.5

total volume (µL)

10

The reaction was incubated for 10 minutes at 50 °C, and the digestion fragments separated on a 1 % agarose gel.

Comment: We now had bands of the correct size in the gel.Therefore, pLK04RCH-Tag-DengueFab0 vector was sent for sequencing.

July 25th

E.coli 321.A was transformed with pEVOL-pAzF and pLK04RCH-AntiDengueFab plasmids according to electroporation protocol.

After transformation, cells were plated on LA+ampicillin+cloramphenicol plates. Plates were incubated o/n in 37 °C.

Friday July 26th

After o/n incubation, plates were put on +4 °C.

Sunday July 28th

4 mL precultures or test production was started. Precultures were done on each AntiDengueFab. Preculture was done in SB-medium containing 0.5 % glucose ampicillin, cloramphenicol and zeocin.

All precultures were put on 250 rpm shaking 37 °C for o/n.

July 28th

Each preculture was diluted to OD 0.1 with similar SB medium as before, so that the final volume of cultures were 4 mL. Cultures were put to grow in 37 °C 250 rpm shaking.

When OD reached 0.6-0.8, 20 µL of 20 % arabinose and 80 µL of 50 mM pAzF was added to growth medium. Cultures were protected from the light from this point on due to light sensitivity of p-Azidophenylalanine.

After 10 min of incubation 4 µL 0.1 M IPTG was added to growth medium and temperature was lowered to 26 °C and cultures were incubated o/n.

July 29th

Sequencing results came and revealed that one of colonies used to start preculture contained a mutation. As a result we abandoned all precultures.

Troubleshooting

June 4th

Riku, Linnea, Pyry, Janniina

We came to suspect, that at some point we have mixed at least the plasmids pUC19 and the original pLK04RCH we had been multiplying and purifying ourselves. Therefore, we decided to SfiI digest and separate on a 1 % agarose gel everything we thought might have become compromised in addition to the constructs we had just cloned and purified.

SfiI digestions were done using the enzyme from Thermo Fisher according to the manufacturer’s protocol. The digested constructs were:
pLK04RTagCH+harm. (5 colonies: ⅕-5/5)
pLK04RCH (1 colony from a plate (1/1) and 3 from another plate (3/1, 3/2 & 3/3)).
Also, two copies of the plasmid we had been multiplying in 321.A, that was thought to be the pLK04RCHTag from professor Huovinen was also SfiI digested. In the last well 1 µL of the vector gotten from Huovinen was loaded undigested by adding 10 µL of MQ.

The samples and 1 kb ladder (Nippon Genetics) were loaded with MidoriDirect (BioRad) according to the manufacturer (0.5 µL in a sample and 1 µL to a DNA ladder) to visualize the bands under blue light and orange plastic filter. After the gel electrophoresis we discovered, that the pLK04RTagCH+harm. ⅕ - ⅘ transformants as well as the pLK04RCH 3/3 did not show the supposed 3.7 and 1.5 kb bands (figure X). The 5/5, 1/1, 3/1-3/3 were chosen to be sequenced.


Figure 1. Gel electrophoresis of the vectors constructed and plasmids multiplied.

From the gel, it can also be seen that the original vector was possibly present as a double plasmid. More disturbingly, the gel run also revealed that the original pLK04RCHTag plasmid that we have thought we have been multiplying did not create the correct sized bands indicating that it was in fact some other similar sized plasmid we have been working with. We had been multiplying pUC19 at the same time to be able to clone the biobrick into it. We have also plated the GFP-TAG-RFP gene from the biobrick gotten from the iGEM plate that was cloned into pUC19.

Since both pUC19 and the pLK04 vectors we had, also had XbaI site, we decided to digest the suspicious plasmids with it (FastDigest XbaI, ThermoFisher) and run them on a gel. However, we later figured out, that all our strains have the native methylation enzymes, which create methylations sites blocking XbaI (see ____).

Since either the plasmid or MidoriDirect was forgotten from the digestion sample, there was nothing to see in the gel. Regardless, it was quite clear that the plasmids had been mixed up at some point, so we got rid of all the plasmids we thought were possibly mixed. Since we still needed pUC19 and pLK04RCH backbones, we transformed the original pLK04RCH plasmid achieved from Tuomas and the pUC19 (2nd try) we got from professor Käpylä.

Janniina & Pyry

We were encountering some difficulties with extracting pLK04-based vectors with our Fabs containing PelB signal sequence; the colonies with Fab0 we were able to pick from the plate and grow in liquid medium seemed to have disturbingly often a deletion in the PelB signal sequence according to our sequencing services purchased from Eurofins. However, the sequences containing amber stop codons showed at least one good sequence per cloning with XL-1 as the host. This could be due to the toxicity of Fabs to E. coli, since XL-1 is not able to translate the complete Fab sequence due to the amber stop codon and the absence of corresponding tRNA synthetase.

So we decided to use a sledgehammer to crack a nut and change the signal sequence with restriction enzymes XbaI and XhoI. To maximize the probability to succeed, we tried a pLK04 vector with harmonized Fab and a signal sequence obtained from utu (not shown) as the “donor”.

Today, we transformed the donor from utu into XL-1 and digested the recipient vector using the enzymes from Thermo Scientific. The sizes of the bands were not checked with Benchling’s virtual digestion, but a band close to the size of the vector was cut from the 1 % agarose gel after electrophoresis. A colony from the transformation today was picked to inoculate 4 mL of SB containing ampicillin and grown over night for plasmid extraction.

The following day, the signal sequence donor plasmid was extracted and 1394 ng of it was digested with FastDigest XhoI and XbaI. While the digestion was incubating, using the virtual digestion attribute of benchling I checked the sizes of the insert and the vector to calculate the ligation reactions. Based on the virtual digestion we learned that instead of one XhoI and two XbaI sites there were actually two of both of them in the pLK04RCHTag+Fab0 with deletion making it too difficult to change the signal sequence by digesting and ligating. Also this meant that the fragment cut from the gel yesterday was missing all of the Fab sequence and little extra making our customary SfiI digestion just as useful. Therefore we did not continue to change the signal sequence, but to try to pick new colonies and to try to clone the correct Fab0 sequence from vector to the pLK04RCHTag backbone by SfiI digestion.

Immunoassays

16th of August
Niemelä P., Karinsalo J. & Leppänen O.

Goat-anti-human plates were used in determination of Fab amount in samples. Fab standard was made so that there was 10, 50, 100, 150, 300 ng (rhFab in TSA, 3,62 mg/mL) of fab in the wells.
Fabs and lysates were diluted 1/10, 1/100 and 1/1000 before adding to the wells in the volume or 200 µL. Each sample was added to the plate as triplicates. The samples were incubated for 30 minutes in shaking before washing of the unbounds.

200 µL of 250 ng/µL Eu-labeled antibody solution was put into each well and plate incubated in shaking for 30 minutes. Plate was washed and 200 µL of Delfia Enhancement Solution per well was added with delfia plate dispenser. After 10 min incubation, time-resolved fluorescence was measured with Hidex. As a result, it was noticed that there were no dengue fabs in the reaction.

28th of August
Karinsalo J. & Leppänen O.

SEC- Purified Fab fractions were diluted into 1/500, 1/5000 and 1/1000 in AB

Table 1. Following dilutions were made from each fab.

amount of standard Fab (ng)

ng/µL

µL of 2400 ng/µL stock

STOCK (mg/mL)

µL AB

V_tot (µL)

Add µL of standard Fab

µL AB

2400

12

1.32

3,62

399

400

1.3

399

120

0.6

50

2.40

950

1000

50

950

100

0.5

41.6

2.40

958

1000

42

958

70

0.35

29.2

2.40

971

1000

29

971

50

0.25

20.8

2.40

979

1000

21

979

20

0.1

8.3

2.40

992

1000

8.3

992

10

0.05

4.2

2.40

996

1000

4.2

996

 

200 µL of each fab dilution was incubated on GAH plates in triplicates for 30 minutes.

17 mL of Eu-2A11 0.25 ng/mL dilution was made from 20 µg/ml stock to Assay Buffer and the solution was filtered. Add 200 µL of the 2A11 dilution to each well, The tracer was incubated for 30 minutes. Unbounds were removed by washing the wells four times. 200 µL of Delfia Enhancement solution was added to each well and incubated 10 min in shaking. Time-resolved fluorescence was measured with Hidex.


Digoxigenin binding capacity

Fabs were incubated on plates for 30 min as previously. The wells were washed twice afterwards.

15 mL of biotinylated digoxigenin (0.1 ng/µL concentration) was made. 200 µL of the 0.1 ng/µL bio-dig dilution was added to each well and incubated for 30. 15 mL of 0.125 ng/µL dilution of Eu-streptavidin (stock concentration 0.1 mg/mL) was made by adding 18.8 µL of Eu-Streptavidin to 15 mL of Assay Buffer.

The wells were washed twice and 200 µL of the 0.125 ng/µL Eu-streptavidin dilution was added to each well. The tracer was incubated for 30 minutes. Unbounds were removed by washing the wells four times. 200 µL of Delfia Enhancement solution was added to each well and incubated 10 min in shaking. Time-resolved fluorescence was measured with Hidex.

15th of August
Niemelä P.

The produced Fabs were immobilized via azide groups onto magnetic beads (MB) with dibenzocyclo-octyne (DBCO). The DBCO-MBs were obtained from Jena Bioscience and the assumed amount of DBCO per 1 mg of beads was 40 nmol. 30 to 50 µg of DBCO-MBs were coated with two fold molar excess of digoxigenin Fabs 191, 108 and histag as well as Fab without any azide groups (Fab0) and Fabs with randomly attached azide groups (Fab NHS-AZ).

The total volume of the reactions was not adjusted to keep it as low as possible. Strain promoted azide-alkyne cycloaddition (SPAAC) was allowed to take place for an hour in RT while covered with aluminum foil to block light. After incubation, unbound Fabs were washed away with first 1000 µL and then 500 µL of PBS prior to washing the MBs twice with 250 µL of washing buffer (Kaivogen). The MBs were washed again with 1000 µL and then with 500 µL of PBS before resuspending into final concentration of 0.5 µg/µL of MB in PBS. The oated MBs were stored in +4 °C.

Materials

2nd of September
Karinsalo J., Leppänen O., Niemelä P.

Since the Fabs had started to decay, a new set of MBswas coated with digoxigenin Fabs 191, 108 and Histag as well as Fab NHS-AZ and Fab. 17.1 µg of MBs was used in each reaction, but the MBs were pipetted by accident in the protein stocks instead of the reaction tubes. After 5 minutes the mistake was noticed and the Fab stocks were pipetted into new tubes while holding the MBs with magnets. Assuming that there was 40 nmol of DBCO on 1 mg of MBs, 1.5 molar excess of digoxigenin Fabs was used to coat the 17.1 µg of MBs. PBS was added to the total volume of 250 µL, after which the SPAAC was incubated for 16 hours in +4 °C covered with aluminium foil. However the reaction with Fab 191 was forgotten in RT and disposed due to that.

3rd of September
Karinsalo J.

The MBs coated with digoxigenin Fab 108, Histag and NHS-AZ in addition to the MBs with Fab0 were washed first with 1000 µL of PBS. Since it took a long time for the MBs to aggregate in such a volume, the MBs were next washed with only 350 µL of PBS (500 µL last time). The MBs were washed twice with 250 µL of washing buffer (Kaivogen) before resuspending the coated MBs into 0.5 µg/µL of MBs in PBS with 0.05% 2-methyl-4-isothiazolin-3-one to preserve the Fabs. However, it was visually observed that the size of the MB precipitates varied between the coated MBs. The coated MBs were stored in +4 °C for later use.

21th of August
Karinsalo J. & Lindfors J.

This is continuation to “Coating DBCO beads with NHS-containing Fab molecules”.

0.5 ng/µL working solution of Eu-2A11 was made into Assay Buffer. The solution was filtered before use. 100 µL of 2A11 mAb working solution was added into wells. Therefore, there was 50 ng of Eu-2A11 mAb in each well.

10, 30, 60, and 100 ng of DBCO magnetic beads was added to each well in three replicates. In addition, non-coated DBCO bead control was used. 100 µL of Eu-2A11 mAb was added to wells and reaction was incubated for 1 hour in shaking.

The beads were washed three times and 200 µL of DELFIA Enhancement Solution. Enhancement solution was incubated for 10 minutes in shaking and time-resolved fluorescence was measured with Hidex.

21th of August
Karinsalo J. & Lindfors J.

1 µg of beads was added to each well/tube in 100 µL. 10 µg/mL stock from empty MB was made by adding 180 µL of the 100 µg/ml stock to 1620 µL of Assay Buffer.

Because there wasn’t enough of 10 µg/mL stock 15 µg/mL stock was diluted by adding 600 µL of beads to 300 µL of Assay Buffer. 0.01, 0.1, 1, 10, 50 ng of Eu-2A11 was added to each well in the volume of 100 µL in Assay Buffer. The Eu-226A2 solution was filtered before use to reduce %CV. The tracer was incubated at shaking for 1h.

The wells were washed three times with Kaivogen wash buffer on a magnetic plate holder. (Invitrogen). 200 µL of Delfia Enhancement solution was added to wells and incubated for 10 minutes in shaking. Time-resolved fluorescence was measured with Hidex

Table 1. Results

8th of August
Niemelä P.

1 mg of DBCO-PEG4-Biotin was dissolved to 1000 µL of DMSO

1000 µL of azido derivatized Dengue Fab0 (stock concentration 0.09 mg/mL) and Digox fab 0 (0.58 mg/mL) were biotinylated. Additionally, 500 µL of HistagTAG Digox Fab (stock concentration 0.80 mg/mL) was biotinylated with three-fold molar excess of DBCO-PEG4-Biotin.

Slide-A-Lyser 10 kDa MWCO (Thermo Scientific) Dialysis Cassette was used according to instructions to remove unreacted labels. First, biotinylated Fabs were dialysed for 2 hours and Dialyse buffer was changed to fresh. This was done twice and samples were retrieved afterwards.

2nd of September
Karinsalo K., Leppänen O.

250 µL of Fab0 was incubated with NHS-PEG4-azide (stock concentration 0.23 mg/mL).

Table 1. Contents of biotinylation reactions.

 

protein (mg/mL)

Protein (mL)

PBS (mL)

DBCO-PEG4-biotin (µL)

Fab0

0.81

0.035

0

12.4

191

0.49

0.1173

0.1326

0.26

108

0.29

0.1982

0.0517

0.26

TAG

2.47

0.0232

0.2267

0.26

 

Buffer exchange was done with Nanosep 10k Omega to remove excess DBCO label: The column was washed four times with 500 µL of PBS buffer. Fabs were eluted with two times 20 µL of PBS buffer. The total volume was around 70 µL since excess liquid was still present in the column. Concentration of the biotinylated fabs was measured with Nanobrop and 0.05% of preservative was added for storage at + 4°C protected from light.

Table 2. Protein concentrations after reaction.

 

mg/mL

Bio-Fab0

0.33

Bio-108

0.36

Bio-191

0.41

Bio-TAG

0.65

 

12th of August
Niemelä P.

This is a continuation from work "Biotinylation with DBCO-Biotin"

Biotinylated Fab was inserted into wells with increasing amounts to determine SA binding capacity. The tested Fabs were Fab0 (without any pAzF) and Fab0 Histag-TAG (where pAzF is located after 6x His tail).

Capacity determination of streptavidin plates with Fabs biotinylated with different strategies protocol was used in this immunoassay with Fab amounts of 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 30 ng per well.

Table 1. Analysis of signals.

 

Fab0-NHS-Azide-DBCO-Biotin

ng/well

CPS

average

std. Dev

CV%

0

282

331

293

302

20.99206

7 %

2

8490

8228

9278

8665.333

446.2296

5 %

4

22290

22513

27353

24052

2335.934

10 %

6

43705

43632

47703

45013.33

1902.115

4 %

8

99658

97701

110325

102561.3

5547.573

5 %

10

124563

133717

136137

131472.3

4984.527

4 %

12

179571

207073

176129

187591

13847.34

7 %

14

209872

240720

235876

228822.7

13545.28

6 %

16

278056

274925

287992

280324.3

5570.495

2 %

18

315992

287959

321476

308475.7

14679.21

5 %

20

355686

342806

347386

348626

5330.841

2 %

30

489319

534859

509428

511202

18633.9

4 %

 

 

FAb0-Histag-TAG-DBCO-Biotin

ng/well

CPS

average

std. Dev

CV%

0

460

459

404

441

26.16614

6 %

2

3655

3331

3377

3454.333

143.1301

4 %

4

6865

7504

7579

7316

320.3717

4 %

6

11920

11807

11048

11591.67

387.1884

3 %

8

16215

17734

18364

17437.67

902.0016

5 %

10

20272

21460

21218

20983.33

512.5995

2 %

12

28444

33457

29546

30482.33

2150.982

7 %

14

41178

42606

33716'

41892

714

2 %

16

55522

47123

55440

52695

3940.141

7 %

18

63181

72166

69026

68124.33

3723.109

5 %

20

71606

79041

72873

74506.67

3247.713

4 %

30

121981

132264

141396

131880.3

7930.782

6 %

 

Figure 1. Signal-to-background plotted standard curves for Fab0-NHS-DBCO and Fab0-Histag-TAG.

Table 2. The assay was repeated with broader range of Fab per well. A couple of measurement points were removed from plotted image because at 45 ng/well only protein stock was used instead of diluting Fabs first to 1000 ng/mL of AB. This caused bias in measurement points when sub-micromolar volumes of stock were used. Removed points are in bold.

ng/well

CPS

Average

stdev

CV%

1

472

547

509

509.3333

30.61953

6 %

2

1126

1178

1287

1197

67.08701

6 %

5

3912

4077

3867

3952

90.27735

2 %

10

17437

12638

13673

14582.67

2062.073

14 %

20

45686

48325

44913

46308

1460.73

3 %

25

70606

90006

75487

78699.67

8239.374

10 %

30

108469

99911

88514

98964.67

8174.031

8 %

35

154547

138559

133625

142243.7

8929.916

6 %

40

186262

213886

195294

198480.7

11500.36

6 %

45

200182

182486

195448

192705.3

7480.141

4 %

50

241520

224263

239563

235115.3

7715.237

3 %

60

310825

300665

318143

309877.7

7166.738

2 %

70

418202

449384

449434

439006.7

14711.14

3 %

100

649618

652603

624289

642170

12702.37

2 %

150

807853

820781

852370

827001.3

18698.67

2 %