iGEM 2019 — Team Aboa

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Aboa/Notebook

Aboa Navbar

![](https://static.igem.org/mediawiki/2019/a/a2/T--Aboa--Aboa_banner.png)

[![logo](https://static.igem.org/mediawiki/2019/3/32/T--Aboa--LogoVaaka.png)](https://2019.igem.org/Team%3AAboa)

* [Home](https://2019.igem.org/Team%3AAboa)
* Team
  + [Team Members](https://2019.igem.org/Team%3AAboa/Team)
  + [Collaborations](https://2019.igem.org/Team%3AAboa/Collaborations)
* Project
  + [Description](https://2019.igem.org/Team%3AAboa/Description)
  + [Design](https://2019.igem.org/Team%3AAboa/Design)
  + [Experiments](https://2019.igem.org/Team%3AAboa/Experiments)
  + [Notebook](https://2019.igem.org/Team%3AAboa/Notebook)
  + [Contribution](https://2019.igem.org/Team%3AAboa/Contribution)
  + [Results](https://2019.igem.org/Team%3AAboa/Results)
  + [Demonstrate](https://2019.igem.org/Team%3AAboa/Demonstrate)
  + [Improve](https://2019.igem.org/Team%3AAboa/Improve)
  + [Attributions](https://2019.igem.org/Team%3AAboa/Attributions)
* [Parts](https://2019.igem.org/Team%3AAboa/Parts)
* [Safety](https://2019.igem.org/Team%3AAboa/Safety)
* Human practices
  + [Human practices](https://2019.igem.org/Team%3AAboa/Human_Practices)
  + [Education & Engagement](https://2019.igem.org/Team%3AAboa/Public_Engagement)
* Awards
  + [Measurement](https://2019.igem.org/Team%3AAboa/Measurement)
* [Judging form](https://igem.org/2019_Judging_Form?team=Aboa)

# Team Aboa Notebook

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

## Table of contents

* [Work with Cell lines](#Work_with_cell_lines)
* [Producing Digoxigenin Fabs](#Producing_digox_fab)
* [Producing Dengue-NS1 Fabs](#Producing_dengue_fab)
* [Troubleshooting](#Troubleshooting)
* [Immunoassays](#Assays)

## Work with Cell lines

[#### Week 1](#collapse1)

**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.

![](https://static.igem.org/mediawiki/2019/7/78/T--ABOA--April_29th_plate.png)

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

![](https://static.igem.org/mediawiki/2019/f/f8/T--ABOA--April_29th_plate_2.png)

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.

[#### Week 2](#collapse2)

**### 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.

[#### Week 3](#collapse3)

**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.

[#### Week 4](#collapse4)

**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.

[#### Week 5](#collapse5)

**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:

![](https://static.igem.org/mediawiki/2019/b/b5/T--ABOA--gel_run_May_28th.png)

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,](https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/DNA%20clean-up/UM_PCRcleanup_Gelex_NSGelPCR.pdf)  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:

![](https://static.igem.org/mediawiki/2019/9/98/T--ABOA--gel_run_May_29th.png)

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](https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/DNA%20clean-up/UM_PCRcleanup_Gelex_NSGelPCR.pdf)
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](https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NSEP.pdf) . 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.

[#### Week 6](#collapse6)

**June 4th**

Present: Janniina, Pyry

The plasmid pLK04RCHTag was extracted using  [NucleoSpin Plasmid easyPure kit.](https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NSEP.pdf)  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.](https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NSEP.pdf)  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.

![](https://static.igem.org/mediawiki/2019/4/40/T--ABOA--Fanny_notes_img4.jpeg)

[#### Week 7](#collapse7)

**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.

![](https://static.igem.org/mediawiki/2019/a/af/T--ABOA--Gel_run_June_13th.png)

Fragments marked with red boxes were cut from gel and purified with
 [PCR clean-up Gel extraction kit,](https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/DNA%20clean-up/UM_PCRcleanup_Gelex_NSGelPCR.pdf)  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](https://www.thermofisher.com/order/catalog/product/EL0014#/EL0014)  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.](https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NSEP.pdf)  The measured DNA concentration was 271.9 ng/µL.

[#### Week 8](#collapse8)

**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](https://www.thermofisher.com/order/catalog/product/EL0014#/EL0014)  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,](https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NSEP.pdf)  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,](https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NSEP.pdf)  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 |

[#### Week 9](#collapse9)

**June 24th**

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

Digestion reactions as follows ( [ThermoFisher](https://www.thermofisher.com/order/catalog/product/BY5#/BY5) ): 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,](https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/DNA%20clean-up/UM_PCRcleanup_Gelex_NSGelPCR.pdf)  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:](https://www.thermofisher.com/order/catalog/product/EL0014#/EL0014)

|  |  |  |
| --- | --- | --- |
| 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:

![](https://static.igem.org/mediawiki/2019/7/78/T--ABOA--June_26th_plate.png)

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.](https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NSEP.pdf)  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

[#### Week 10](#collapse10)

**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:

![](https://static.igem.org/mediawiki/2019/d/d5/T--ABOA--July_6th_plate.png)

**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.

[#### Week 11](#collapse11)

**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

[#### Cloning Digoxigenin Fabs into pLK04RCHTag](#collapse100)

**### 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**

![](https://static.igem.org/mediawiki/2019/3/33/T--ABOA--Ville_notes_img10.png)

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

![](https://static.igem.org/mediawiki/2019/6/69/T--ABOA--Ville_notes_img11.png)

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](https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/DNA%20clean-up/UM_PCRcleanup_Gelex_NSGelPCR.pdf)  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

![](https://static.igem.org/mediawiki/2019/b/b9/T--ABOA--Ville_notes_img12.png)

Imaging with invitrogen Safe Imager(tm)

Purifying with  [Macherey-Nagel NucleoSpin Gel and PCR Clean-up -kit,](https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/DNA%20clean-up/UM_PCRcleanup_Gelex_NSGelPCR.pdf)
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](https://nebiocalculator.neb.com/#!/ligation was used for calculations)  and

 [Thermo T4 DNA ligase protocol](https://www.thermofisher.com/order/catalog/product/EL0014)  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.](https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/DNA%20clean-up/UM_PCRcleanup_Gelex_NSGelPCR.pdf)

**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.](https://www.thermofisher.com/order/catalog/product/EL0014#/EL0014)

**### 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,](https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/DNA%20clean-up/UM_PCRcleanup_Gelex_NSGelPCR.pdf)  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](https://www.thermofisher.com/order/catalog/product/EL0014#/EL0014 )  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](https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NSEP.pdf%20)  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](https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NSEP.pdf)  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.](https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0012585_FastDigest_SfiI_UG.pdf)
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.](https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0012585_FastDigest_SfiI_UG.pdf)  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:

![](https://static.igem.org/mediawiki/2019/4/48/T--ABOA--Oskari_notes_img1.png)

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.](https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/DNA%20clean-up/UM_PCRcleanup_Gelex_NSGelPCR.pdf)  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.](https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NSEP.pdf)
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:

![](https://static.igem.org/mediawiki/2019/2/23/T--ABOA--Oskari_notes_img2.png)

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.](https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NSEP.pdf)

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.](https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NSEP.pdf)
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.](https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/DNA%20clean-up/UM_PCRcleanup_Gelex_NSGelPCR.pdf)  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](https://www.thermofisher.com/order/catalog/product/EL0014#/EL0014)  according to manufacturer’s protocol. Ligated fragments were transformed into XL-1 Blue (chemical competent) cells according to  [the protocol from Addgene.org](https://www.addgene.org/mol-bio-reference/) . 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.](https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NSEP.pdf)  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](https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NSEP.pdf)  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.](https://nebiocalculator.neb.com/#!/ligation)

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.](https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NSEP.pdf)  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](https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NSEP.pdf)
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](https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NSEP.pdf)  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.

![](https://static.igem.org/mediawiki/2019/4/4a/T--ABOA--Linnea_notes_img1.png)

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.

![](https://static.igem.org/mediawiki/2019/f/f2/T--ABOA--Linnea_notes_img2.png)

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](https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NSEP.pdf)  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.](https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0012585_FastDigest_SfiI_UG.pdf)  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](https://www.addgene.org/protocols/bacterial-transformation/)

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,](https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NSEP.pdf)  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 |

![](https://static.igem.org/mediawiki/2019/f/fe/T--ABOA--Linnea_notes_img3.png)

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.

![](https://static.igem.org/mediawiki/2019/d/da/T--ABOA--Linnea_notes_img4.png)

**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,](https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/DNA%20clean-up/UM_PCRcleanup_Gelex_NSGelPCR.pdf)  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:

|  |  |
| --- | --- |
| **Culture** | OD600 |
| Lig 1 | 3.23 || 2 | 2.90 |
| 3 | 4.15 |
| 4 | 2.87 |

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.

|  |  |
| --- | --- |
| **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 |

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

|  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 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.

|  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1st gel |1/1  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

![](https://static.igem.org/mediawiki/2019/9/9c/T--ABOA--Linnea_notes_img5.png)

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](https://nebiocalculator.neb.com/#!/ligation)  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,](https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NSEP.pdf)  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](https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NSEP.pdf)  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 Digoxigenin Fabs](#collapse104)

### **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:

![](https://static.igem.org/mediawiki/2019/a/a4/T--ABOA--Jutta_notes_img3.png)

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:

![](https://static.igem.org/mediawiki/2019/4/48/T--ABOA--Jutta_notes_img4.png)

**### 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.](https://2019.igem.org/Team%3AAboa/Experiments)

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

[#### Cloning DengueFabs to pLK04RCH/Tag](#collapse201)

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:

![](https://static.igem.org/mediawiki/2019/0/01/T--ABOA--Fanny_notes_img2.png)

 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:

![](https://static.igem.org/mediawiki/2019/0/05/T--ABOA--Fanny_notes_img3.png)

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.

[#### Test production of dengue Fabs](#collapse202)

**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

[#### Testing all previously restricted plasmids](#collapse204)

**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.

![](https://static.igem.org/mediawiki/2019/a/af/T--ABOA--Janniina_notes_img1.png)

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.

[#### Changing signal sequence to pLK04RCHTag+Fab0 digox](#collapse205)

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

[#### Fab activity determination on Goat Anti-Human plates](#collapse1096)

**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.

[#### Coating DBCO beads with NHS-containing Fab molecules](#collapse1099)

**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](https://www.jenabioscience.com/click-chemistry/click-reagents-by-chemistry/dbco-reagents/dbco-magnetic-beads/clk-1037-dbco-magnetic-beads)

**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.

[#### Relative Fab activity determination on DBCO beads](#collapse1094)

**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.

[#### Optimization of click immunomagetic bead (CIMB) concentration in assay](#collapse1095)

**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

[#### Biotinylation of Fabs with DBCO Biotin](#collapse1097)

**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 |

[#### Determination of Streptavidin plate binding capacity with Fabs biotinylated with different biotinylation strategies](#collapse1098)

**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](https://2019.igem.org/Team%3AAboa/Experiments#collapse106) 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 % |

![](https://static.igem.org/mediawiki/2019/4/43/T--Aboa--SAcapacity.png)

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 % |


[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Aboa/Experiments

Aboa Navbar

![](https://static.igem.org/mediawiki/2019/a/a2/T--Aboa--Aboa_banner.png)

[![logo](https://static.igem.org/mediawiki/2019/3/32/T--Aboa--LogoVaaka.png)](https://2019.igem.org/Team%3AAboa)

* [Home](https://2019.igem.org/Team%3AAboa)
* Team
  + [Team Members](https://2019.igem.org/Team%3AAboa/Team)
  + [Collaborations](https://2019.igem.org/Team%3AAboa/Collaborations)
* Project
  + [Description](https://2019.igem.org/Team%3AAboa/Description)
  + [Design](https://2019.igem.org/Team%3AAboa/Design)
  + [Experiments](https://2019.igem.org/Team%3AAboa/Experiments)
  + [Notebook](https://2019.igem.org/Team%3AAboa/Notebook)
  + [Contribution](https://2019.igem.org/Team%3AAboa/Contribution)
  + [Results](https://2019.igem.org/Team%3AAboa/Results)
  + [Demonstrate](https://2019.igem.org/Team%3AAboa/Demonstrate)
  + [Improve](https://2019.igem.org/Team%3AAboa/Improve)
  + [Attributions](https://2019.igem.org/Team%3AAboa/Attributions)
* [Parts](https://2019.igem.org/Team%3AAboa/Parts)
* [Safety](https://2019.igem.org/Team%3AAboa/Safety)
* Human practices
  + [Human practices](https://2019.igem.org/Team%3AAboa/Human_Practices)
  + [Education & Engagement](https://2019.igem.org/Team%3AAboa/Public_Engagement)
* Awards
  + [Measurement](https://2019.igem.org/Team%3AAboa/Measurement)
* [Judging form](https://igem.org/2019_Judging_Form?team=Aboa)

# Experiments

This page describes what we did in our research project and all the protocols we
used. If you want to read more about our project, please read our project
[description](https://2019.igem.org/Team%3AAboa/Description)
.

This page contains only the succesful part of the project. If you want to know exactly
what we did, please read our
[notebook](https://2019.igem.org/Team%3AAboa/Notebook)
.

## Contents

* [Building plasmids](#DNA)
* [Cell culturing & Protein production](#Cell_culturing)
* [Protein purification and analysis](#Protein)
* [Testing Site-specific immobilization](#Testing)

## Building plasmids

When we recieved our ordered DNA from the iGEM sponsors, we digested them and the plasmids
we recieved from University of Turku with SfiI and HindIII restriction enzymes (ThermoFisher).
Digestion was done according to the manufacturer's protocol. After digestion, DNA
was purified with agarose gel electrophoresis. We used the Macherey-Nagel Gel and PCR
cleanup-kit to extract the right DNA bands from the agarose gel.

The next step was to ligate the purified genes and plasmid backbones together. We used T4
ligase (ThermoFisher), and followed the manufacturer’s protocol. Following ligation, we used
electroporation
to transform the plasmid to E.coli XL-1 Blue -strain, which we used for cloning our plasmids.

Several colonies of transformed cells were picked from plates and grown
overnight for plasmid extraction. Plasmids were extracted with the Macherey-Nagel NucleoSpin
Plasmid EasyPure -kit and sent to Eurofins Genomics for sequencing.

When the sequencing results came, we picked the verified plasmids and continued our work with them.

Protocols used are described below.

[#### Agarose gel](#collapse1)

Here is a protocol for 60 mL agarose gel. The protocol is fully scalable for larger
gels.

1. 0.6 g Agarose or 1.2 g for a 2% gel
2. Add TBE-buffer to a final volume of 60 ml
3. Heat the mix until the agarose fully dissolves in the TBE buffer. DO NOT LET IT
   BOIL OVER!
4. Optional step: Wait until the solution has cooled down to 50 C and add the DNA
   stain
   to the solution.- Cast the solution into a casting tray and put the well comb in its place.
   - Wait until the solution solidifies. (It takes about 15 min in +4 C and 30 min
     in RT

[#### Agarose gel electrophoresis](#collapse2)

1. Place the gel into the gel box.
2. Cover the gel with TBE-buffer.
3. Add loading buffer to your samples. (We used Nippon Genetics Midori Green Direct
   stain + loading buffer)
4. Place your sample carefully in the gel well. Do not let air bubles enter the well.
5. Connect the gel box to a voltage source.
6. Set the voltage to 70 V - 100 V and run the gel until the loading buffer has
   reached the bottom
   fourth of your gel.
7. Turn the power off and disconnect the voltage source from the gel box.
8. Remove the gel from the gel box and visualize the DNA bands according to the
   manufacturer’s instructions for your particular DNA
   stain.

[#### Making of electrocompetent cells](#collapse3)

This guide is for 2 x 250 mL cultures

1. Inoculate 1 colony from an o/n culture of the strain to be made into
   electrocompetent cells
   into 10 mL of SB in a 125 mL flask. Incubate for 16-18 hours at 37 oC and 250
   rpm.
2. Have ready two 1 L flasks containing 250 mL each of SB medium pre-warmed to 37°C.
   Add
   two drops of the o/n culture to each of the flasks.
3. Shake at 37°C and 250 rpm until the cultures reach an OD600 of 0.5-0.7. Place 1
   L of 10% glycerol on ice.
4. Place the cultures on ice for 15 minutes. From this point on, the cultures must be
   kept
   ice cold. Pour each of the 250 mL culture into chilled 500 mL (or 1000 mL)
   centrifuge
   bottles.
5. Centrifuge at 5000 rpm for 10 minutes. Discard the supernatant and aspirate any
   residual broth.
6. Add 250 mL of glycerol to each of the centrifuge bottles and completely resuspend
   the cells by pipetting up and down.
7. Centrifuge at 5000 rpm for 10 minutes. Discard the supernatant (it is not
   necessary
   to aspirate). Completely resuspend the cells in 250 mL glycerol and centrifuge
   again
   for 10 minutes at 5000 rpm.
8. Discard the supernatant and resuspend the cells in the residual glycerol by
   pipetting
   up and down.
9. At this point, you can electroporate or freeze the cells. To freeze them, add 100
   µl of the culture to a microcentrifuge tube on ice. Once you have used all of the
   culture, transfer the tubes to dry ice or liquid nitrogen for 10 minutes. Once the
   cultures are frozen, transfer them to a -80°C freezer. The cultures should be good
   for >6 months.

[#### Liquid LB medium (400 mL)](#collapse4)

1. 4 g NaCl
2. 4 g Tryptone
3. 2 g Yeast extract
4. Add dH2O to a final volume of 400 mL. Autoclave to sterilize.

[#### Liquid SB medium, 1 L](#collapse5)

SB medium can be used as an alternative to LB medium.

1. 30 g Tryptone
2. 20 g Yeast extract
3. 10 g MOPS (3-[N-morpholino]-propanesulfonic acid)
4. Add 1 L dH2O. Autoclave to sterilize.

[#### Electroporation - transformation of cells](#collapse6)

1. Prepare 14 mL round-bottom culture tubes at RT. Place LB medium in a 37°C water
   bath. Pre-warm selective plates at 37°C for 1 hour.
2. Place the electroporation cuvettes (1 mm) and the microcentrifuge tubes on ice.
3. Thaw the electrocompetent cells on ice for 10 - 20 minutes and mix the cells by
   flicking
   the tube gently. Transfer 25 μl of the cell suspension (or the amount specified
   for
   the cuvettes) to a chilled microcentrifuge tube. Add 1 μl of the DNA solution.
4. Carefully transfer the cell/DNA mixture into a chilled cuvette, without
   introducing
   bubbles, and make sure that the cells deposit across the empty crevasse of the
   cuvette.
   Electroporate using the following conditions for
    [Bio-Rad GenePulser electroporators:](http://www.bio-rad.com/en-fi/product/gene-pulser-xcell-electroporation-systems?ID=b1a35eb3-d55c-47b3-aaf3-95e4d1d85848#fragment-doc)
   1,8 kV.
5. Record time constants.
6. Immediately add 1 ml of 37°C LB to the cuvette, gently mix up and down twice, then
   transfer to the round-bottom culture tube.
7. Shake at 250 rpm at 37°C for 45 min.
8. Dilute the cells as appropriate (1/10, 1/100) then spread 100-200 μl onto pre-
   warmed
   selective plates.
9. Incubate the plates for 8 hours to overnight at 37°C.

## Cell culturing & Protein production

Once we had confirmed that our plasmids contained the right genes, we continued to produce
antibodies. We transformed the E.coli 321.deltaA -strain (C321.ΔA; a gift from George
Church (Addgene plasmid # 48998)), which we used as a production strain, with plasmids
containing the antibody gene and a plasmid containing a gene for p-azidophenylalanine tRNA
synthetase (pEVOL-pAzF (Addgene plasmid # 31186 ; http://n2t.net/addgene:31186 ;
RRID:Addgene\_31186)).

First, we made a small scale production, just to confirm that our plasmids and E.coli
strain work as they're supposed to. Then, we moved on to large scale productions.

Protocols used are below.

[#### Antibody production](#collapse101)

**Materials**

- E. coli XL1 Blue cells containing the appropriate vector (e.g. pEB06H-scFv or pLK06H-scFv)
- LA plate (Tryptone 10 g/l, Yeast extract 5 g/l, NaCl 5 g/l, Agar 15 g/l) with with
  0.5% glucose and appropriate antibiotics
- SB medium
- D-Glucose, stock solution 20% (w/v) in H2O, autoclaved
- Appropriate antibiotic
- IPTG: isopropyl-b-D-thio-galactoside, stock solution 100 mM in H2O, sterilized by
  filtering Ø0.22 µm
- Benzonase
- Lysotsyme

**Preculture**

- Inoculate cells from a plate or glycerol prep to 4 ml (test production) or 20 ml (big production) SB medium with
  0.5% glucose and appropriate antibiotics.
- Incubate at 37°C o/n.

**Main culture**

- In the morning dilute the preculture into OD600 of 0.1 in SB with 0.05 % glucose and appropriate antibiotics, i.e. 2 ml of o/n culture in
  200 ml of medium in a 1L Erlenmeyer flask.
- Incubate at 37°C, 300 rpm, until OD600 is about 0.8 (0.6–1.0).

**Protein production**

- Induce tRNA production by addition of 0.5 % arabinose and 1 mM pAzF.
- After 10 min induce Fab production by addition of 500 µM IPTG.
- Culture at 26°C, 250 rpm, o/n.

[#### Antibiotics](#collapse102)

The antibiotics we used in our work were zeocin, tetracycline, ampicillin and chloramphenicol.
Their working concentrations in growth mediums and plates are listed below:

|  |  |
| --- | --- |
|  | working concentration (µg/ml) |
| Zeocin | 50 |
| Tetracycline | 15 |
| Ampicillin | 100 |
| Chloramphenicol | 25 |

[#### Making 50 mM pAzF](#collapse103)

MW of pAzF is 206,1 g/mol

50 ml of 100 mM stock, where 100 mmol/l is 0,1 mol/l:

the amount of pAzF is 0,1 mol/l x 0,05 l = 0,005 mol.

0,005 mol is 206,1 g/mol x 0,005 mol= 1,0305 g of pAzF.

Made 50mM pAzF stock solution, 2 g weighed.

100 ml of 0,1 M NaOH

100 ml of NaOH

## Protein purification and analysis

After profuction, antibodies need to be purified and it needs to be verified that p-azidophenylalanine
has been incorporated into the antibody. Purifying was done with Ni-NTA. Fab0 (antibody
with no p-azidophenylalanine) was also purified with Size Exclusion Chromatography.

Purified antibody yield was measured with an immunoassay. We used Goat Anti-Human IgG
coated plates (University of Turku) as a capture surface and Europium-labeled 2A11-IgG
(HyTest) as a label.

To verify that p-azidophenylalanine has been incorporated into our antibody, we labeled antibodies
with DBCO-Cy5.5 (Jena Biosciences). As a negative contol we used the same method to label
Fab0. Labeled proteins were run with SDS-PAGE.

Protocols used are described below.

[#### NPI solutions and lysis buffer for histag purification](#collapse201)

2 L of modified NPI-10 was made by adding 20 ml of 1 M imidazole in 2 L of PBS.

1 L of modified NPI-20 was made by adding 20 ml of 1 M imidazole in 1 L of PBS.

1 L of modified NPI-20 was made by adding 250 ml of 1 M imidazole in 500 ml of 2xPBS
and 250 ml MQ.

10 ml of 1 M MgCl2 was added to 1 L of NPI-10 to make the lysis buffer.

1 L of NPI-10, NPI-20 and NPI-250 were adjusted to pH 8 with HCl.

60 µl of benzonase (stock 25 U/µl) was added.

NPI-500 was made to test if there is Fabs that are bound more strongly.

NPI-500:

10 ml 10 x PBS

50 ml 1 M imidazole -> 500 mM

40 ml MQ

pH 8

[#### Cell lysis and Histag purification](#collapse202)

**Cell collection**

- Take a 1 ml sample of cell culture (label properly: Fab, vector, T/C, culture, date).
- Centrifuge cellt 4,500–6,000 x g for 15 min at 4 °C. Remove supernatant.
- To wash the cells resuspend in NPI-10 and centrifuge again. Remove supernatant.
- Cell pellets may be stored at -20 °C or -80 °C until needed.

**Purification of scFv-ALP clones**

**Buffers**

- NPI-10/Equilibrium buffer: PBS: 50 mM NaH2PO4 sodium phosphate, 300 mM sodium chloride,
  10 mM imidazol; pH 8
- Lysis buffer: Equilibrium buffer with 10 mM MgCl2, 25 U/ml benzonase, 0.4 mg/ml
  lysozyme, pH 8
- Lysozyme from chicken egg white, L6876-10G, Sigma-Aldrich, USA
- MgCl2 1M -
- Benzonase, Merck, Germany, 250 U/µl
- NPI-20/Binding/Wash buffer - PBS with 20 mM imidazole, pH 8
- NPI-250/Elution buffer - PBS with 250 mM imidazole, pH 8
- **(Regeneration: if necessary, check Macherey-Nagel's protocol for this step)**

**Cell lysis**

- Thaw the cell pellet from an E. coli expression culture on ice (if frozen).
- Resuspend 1 g of pelleted, wet cells in 2–5 mL lysis buffer. Pipette up and down,
  or stir until complete resuspension without visible cell aggregates. Perform this
  step on ice.
- optional: Incubate at room temperature (RT) in rotation for 30 min.
- Freeze and thaw cells three times (20 min in -70 °C → 10 min +40 °C water bath).
- Centrifuge (Avanti J-26 XP Centrifuge, JS-25.50, Beckman Coulter) 13000 rpm, 45
  min, +4°C.
- Take a 50 µl sample of the supernatant and store it at -20 °C (label properly: Fab,
  vector, T/C, lysate, date). Proceed to protein purification with the rest of supernatant.

**Protein purification, Ni-NTA and buffer exchange**

- [more info here](https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Protino/UM_ProtinoNi-NTA.pdf)
- **EQUILIBRIUM**
- Ni-NTA, x mL resin bed. Do this in a dark room!
**Do not expose to light or it is ruined for ever and ever and ever! Samples lost.**- Equilibrate column(s) to working temperature. Perform purifications at room temperature
  or covered with ice.
- Resuspend Protino® Ni-NTA Agarose by mixing thoroughly to achieve a homogeneous
  suspension.
- Immediately transfer an appropriate amount of suspension to an appropriate chromatography
  column, which allows slow flow rates of 0.5–1 mL/min. Pipette 2 mL of the original
  50 % suspension per 1 mL of bed volume required.
- Allow the column to drain by gravity.
- Add 10 bed volumes of NPI-10 to equilibrate the gel.
- Allow the column to drain by gravity.
- **BINDING**
- Add the clarified E.coli lysate or protein extract to the equilibrated gel.
- Allow the column to drain by gravity using a flow rate of 0.5–1 mL/min.
- **Note: If the flow rate is too high polyhistidine-tagged proteins may not bind to
  the column efficiently. Reduce the flow rate or re-apply the flow-through to improve
  binding.**
- **WASH**
- Wash the gel by adding 10 bed volumes of NPI-20.
- Allow the column to drain by gravity.
- Repeat the washing step (total wash 2 x 10 bed volumes of NPI-20).
- Take a 50 µł sample from the flowthrough (label properly: Fab, vector, T/C, wash,
  date).
- **ELUTION**
- Add 5–10 bed volumes of NPI-250 to the gel.
- Allow the column to drain by gravity and collect the eluate in fractions (manual).
- Store eluted protein on ice.
- Analyze fractions for the presence of the target protein. To determine the protein
  concentration use a Bradford protein assay (quick and easy) or measure the absorbance
  at 280 nm. Note that imidazole will also absorb at 280 nm.
- Pool fractions containing the majority of the eluted polyhistidine-tagged protein
  and remove an aliquot for SDS-PAGE analysis.
- Store protein at -70 °C or -20 °C. Note that many proteins irreversibly precipitate
  out of solution in the presence of imidazole. In this case remove imidazole prior
  to freezing. For proper storage it is recommended to remove the imidazole by ultrafiltration
  or dialysis.
- Remove imidazole by changing the buffer with a buffer exchange column into PBS.
  Store at -20 °C.
- **Note: the proteins won't last long in PBS.**
- **CLEANING THE Ni-NTA RESIN**
- Wash Protino® Ni-NTA Agarose with 15 CV of 0.5 M NaOH for 30 min to solubilize and
  desorb contaminants. When using columns adjust the flow rate accordingly. For example,
  wash a Protino® Ni-NTA Columns 1 mL by using a flow rate of 0.5 mL/min for 30 min,
  corresponding to a total volume of 15 mL.
- Remove the NaOH solution by washing with 10 bed volumes of de-ionized water.
- If you are reusing the resin directly, wash with 10 bed volumes of NPI-10/equilibrium
  buffer to equilibrate the resin.
- For storage wash with 2 CV of 30 % ethanol. Resuspend the resin in 30 % ethanol
  and store at 2–8 °C.

[#### Size Exclusion Chromatography - SEC](#collapse203)

Column used: Superdex 200 Increase 10/300 GL

1. Equilibriate the column with PBS, pH 7.4 according to manufacturer’s guide.
2. Concentrate the sample with 10 000 MWCO ultrafiltration spin column, 4000g, 3 min, +4
   C.
3. Inject samples into the column
4. Use a flow rate of 0,2 ml/min, fraction size 0,5 ml.
5. Use 280 nm light absorbance for detecting proteins.
6. Measure Fab concentration with nanodrop and pool contiguous fractions. Use a Fab activity
   assay to determine the correct fractions.

[#### Labeling antibodies with DBCO-Cy5.5](#collapse204)

1. Add 13 pmol DBCO-Cy5.5 to every µg of protein.
2. Incubate in shaker. Reaction should happen in 15 min, but we incubated o/n at +4 C

Yep, that's how easy it really is!

[#### SDS-PAGE -gel & buffer](#collapse205)

###### Separation gel

1. up to 5 ml ddH2O
2. x ml 30% acrylamide/Bis (calculate amount needed for protein size)
3. 1.25 mL 1.5 M Tris (pH 8.8)
4. 25 μl 20% SDS
5. 25 μl 10% ammonium persulfate (make it fresh and store at 4 °C up to a month)
6. 2.5 μl TEMED (add it last)
7. Pipet the separation gel into the casting chamber and fill up to ~ 2.5 cm from the
   top. Add a small layer of isopropanol to the top of the gel prior to polymerization
   to straighten the level of the gel.

###### Stacking gel

1. 2.08 ml dH2O
2. 0.506 ml 30% acrylamide/Bis
3. 0.375 ml 1 M Tris (pH 6.8)
4. 15 μl 20% (w/v) SDS
5. 15 μl 10% ammonium persulfate
6. Remove the isopropanol layer by using a filter paper. Rinse the top layer of the gel
   with ddH2O and dry off as much of the water as possible by using a filter paper. Add
   1.5 µl of TEMED into the stacking gel and mix the solution well. Add the stacking
   gel and insert the comb.

###### Sample buffer (10 mL, 4x)

1. 2.5 ml 1 M Tris-HCl pH 6.8
2. 0.5 ml of ddH20
3. 1.0 g SDS
4. 0.8 ml 0.1% Bromophenol Blue
5. 4 ml 100% glycerol
6. 2 ml 14.3 M β-mercaptoethanol (100% stock)
7. Adjust the final volume to 10 ml with ddH20

more detailed recipes and instructions for preparing the protein samples can be
found at:
<https://bio-protocol.org/bio101/e80>
and at:
 [https://openwetware.org/wiki/SDS-PAGE\_sample\_buffer\_(Morris\_formulation)](https://openwetware.org/wiki/SDS-PAGE_sample_buffer_%28Morris_formulation%29)

[#### Fab acitivity determination on Goat Anti-Human plates with Europium-labeled tracer antibodies](#collapse206)

1. Dilute cell lysates e.g. 1/100, 1/500. 1/5000 ETC.

2. Incubate 200 µL of lysates in Goat Anti-Human plate's wells for 30 min in shaking.

3. Wash the wells twice to remove unbound Ab's.

4. Add 50 ng of Eu-2A11 mAb dilution to each well in the volume of 200 µL. Filter
the Eu-2A11 mAb solution before use.

5. Incubate 30 minutes on shaking.

6. Wash the wells four times to remove unbound Ab's.

7. Add 200 µL of DELFIA Enhancement solution to each well.

8. Incubate for 10 minutes in shaking.

9. Measure time-resolved fluorescence with Hidex or Victor Multilabel Counter

## Testing Site-specific immobilization

To test the function of the antibodies and the effect of site-specific immobilization, the antibodies
were
conjugated to DBCO-coated magnetic beads (Jena Biosciences). Before conjugating the antibodies to
the beads, an azide group was added to the control antibody (Fab0) with NHS-Azide (ThermoFisher)
to simulate unspecific conjugation with biotin. Amount of succesfully coated antibodies
were measured with Eu labeled 226A2 antibodies.

Site-specifically and unspecifically coated beads were saturated with Cy5 labeled
Digoxigenin (University of Turku). Cy5 signal intensity and variation was measured
with flow-cytometer.

Protocols are described bellow.

[#### Coating DBCO magnetic beads with Fabs and Fab activity determination with Euoropium-labeled tracer antibodies](#collapse301)

1. Prepare a standard series (e.g. 10, 30, 60 and 100 ng/per reaction) of azide-reactive-group-containing
Fabs in the volume of 100 µL.

2. Add 1.08 pmol of DBCO beads to the reactions to coat the beads with Fabs. Incubate
overnight at + 4 °C.

**Note: If Fab-coated beads are stored for longer, a preservative, such as DMSO, is recommended.**

3. Wash the beads twice on a magnetic rack.

4. Add 50 ng of Eu-226A2 mAb in the volume of 100 µL. Filter the Eu-226A2 solution
before use.

**Note: A non-Fab-coated bead control should be used.**

5. Incubate for 1 hour at RT.

6. Wash the beads twice on a magnetic rack.

7. Add 200 µl of DELFIA Enhancement solution into each well.

8. Incubate 10 minutes in shaking.

9. Measure time-resolved fluorescence with Hidex or Victor Multilabel Counter.

[#### Comparing site-specific and non-specific immobilization.](#collapse302)

1. Vortex all bead samples
2. Transfer similar amout of coated beads to reaction (we used 3 million beads/reaction).
3. Add PBS + 0,5% Tween -buffer to final volume of 496 µL
4. Add 4 µl 25µM Cy5 labeled Digoxigeninto each reaction
5. Incubate in orbital shaker for 1h
6. Wash beads 1x with PBS + 0,5% Tween(remember to immobilize beads with a magnet!)
7. Add PBS 0,5% Tween to a final volume on 500 µL
8. Analyze beads with flow cytometer, settings as follows:
   * Number of counts: 100 000
   * Flow rate 14 µL/min
   * Core size 10 µm
   * Excitation: 650 nm
   * Emission: 670 nm

[#### Capacity determination of streptavidin plates with Fabs biotinylated with different strategies](#collapse303)

1. Prepare a standard series (e.g. 0, 10, 30, 60 and 100 ng/per reaction) of biotinylated
Fabs and add 200 µL of each dilution to wells of a streptavidin-coated plate.

2. Incubate the biotin-streptavidin reaction for one hour in shaking.

3. Wash the wells four times with Kaivogen Wash Buffer.

4. Add 50 ng of Eu-226A2 mAb in the volume of 200 µL. Filter the Eu-226A2 solution
before use.

5. Incubate for one hour in shaking.

6. Wash the wells four times with Kaivogen Wash Buffer.

7. Add 200 µl of DELFIA Enhancement soltuion into each well.

8. Incubate 10 minutes in shaking.

9. Measure Time-resolved fluorescence with Hidex or Victor Multilabel Counter.

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Aboa/Results

Aboa Navbar

![](https://static.igem.org/mediawiki/2019/a/a2/T--Aboa--Aboa_banner.png)

[![logo](https://static.igem.org/mediawiki/2019/3/32/T--Aboa--LogoVaaka.png)](https://2019.igem.org/Team%3AAboa)

* [Home](https://2019.igem.org/Team%3AAboa)
* Team
  + [Team Members](https://2019.igem.org/Team%3AAboa/Team)
  + [Collaborations](https://2019.igem.org/Team%3AAboa/Collaborations)
* Project
  + [Description](https://2019.igem.org/Team%3AAboa/Description)
  + [Design](https://2019.igem.org/Team%3AAboa/Design)
  + [Experiments](https://2019.igem.org/Team%3AAboa/Experiments)
  + [Notebook](https://2019.igem.org/Team%3AAboa/Notebook)
  + [Contribution](https://2019.igem.org/Team%3AAboa/Contribution)
  + [Results](https://2019.igem.org/Team%3AAboa/Results)
  + [Demonstrate](https://2019.igem.org/Team%3AAboa/Demonstrate)
  + [Improve](https://2019.igem.org/Team%3AAboa/Improve)
  + [Attributions](https://2019.igem.org/Team%3AAboa/Attributions)
* [Parts](https://2019.igem.org/Team%3AAboa/Parts)
* [Safety](https://2019.igem.org/Team%3AAboa/Safety)
* Human practices
  + [Human practices](https://2019.igem.org/Team%3AAboa/Human_Practices)
  + [Education & Engagement](https://2019.igem.org/Team%3AAboa/Public_Engagement)
* Awards
  + [Measurement](https://2019.igem.org/Team%3AAboa/Measurement)
* [Judging form](https://igem.org/2019_Judging_Form?team=Aboa)

# Results

Our project aimed for the creation of a novel immunoassay that uses expanded genetic
code to introduce a reactive azide moiety site specifically to Fabs that can then
be forced to adopt a correct orientation on a biosensor surface. This would improve
the repeatability and sensitivity of any assay that currently uses a random immobilization
of antibodies.

## Contents

* [Characterization of E.coli 321.A](#Characterization)
* [Producing Anti-Digoxigenin Fab](#Producing_digox_fab)
* [Producing Anti-Dengue Fab](#Producing_dengue_fab)
* [Coating and Testing Anti-Digoxigenin Fabs](#Coating_and_testing_anti_digox)
* [Temperature stability measurements of Fab fragments](#Thermal_stability)

## Characterization of E.coli 321.A

As C321.ΔA.exp is the first genomically recoded organism (GRO) with interesting
properties such as the inability to terminate TAG codons as it has had Release Factor
1 (RF1) completely removed (Lajoie et al. 2013). This is why we wanted to characterize
this strain and see what sort of properties it has together with plasmid pEVOL-pAzF
in producing a protein that has a site for incorporation of unnatural amino acids.

This experiment was performed using a 96-well plate and Cytation 5 multimode fluorescence
imager. First the C321.ΔA.exp containing pEVOL-pAzF and pUC19[GFP-TAG-RFP] were precultured
in the wells at 37°C in vigorous shaking and their OD600 was measured until it reached
an average of 0.5. After that the wells were induced with varying amounts of Arabinose,
IPTG and pAzF (Figure 1). The fluorescence was measured every 30 minutes at manufacturer
preset GFP and mCherry channels. The results were normalized by reporting a relative
change of RFP and GFP ratio.

![Important results](https://static.igem.org/mediawiki/2019/3/38/T--Aboa--GFP-TAG-RFP--chart.png)

***Figure 1.**
The relative change of fluorescence measured in preset mCherry and GFP channels.
pAzF has the strongest effect on the expression of RFP when the synthetase is present.
This also shows that when the synthetase is not present, there still is some misincorporation
of other amino acids to the TAG site as it cannot be terminated either. IPTG does
not have an effect on the reading of TAG stop codon as expected.*

## Producing Anti-Digoxigenin Fab

We used a digoxigenin binding Fab fragment as our model antibody in this project.
First of all this Fab has been vigorously optimized by codon harmonization to reduce
its toxicity and to increase its yields as we were not sure how well this new strain
could tolerate these humanized and toxic proteins (Kulmala et al. 2017). The second
reason for using this was that the analyte, digoxigenin was readily available for
us at the Department of Biotechnology as a labeled and non-labeled version. Although
we later found out thet the Eu3+ labeling by the department chemists was not successful
so we had to use other means to complete our measurements.

We started the production phase with an optimization task to find out what were
effects of Fab variants to our cell viability and also to optimize the amount of
IPTG, Arabinose and pAzF to use in an attempt to conserve expensive reagents. We
were successful in producing three variants of the Fab fragment: Fab0 (unmodified
Fab), Lys 108 (Lys->TAG, 108 representing the site) and Lys 191. We also made vector
that contained a C-terminal TAGTAA modification after a 6xHis tag that we used together
with the unmodified Fab. The histidine tag is directly opposite to the binding part
so we used this as the binder that forces a correct orientation for the antibodies.

![More important results](https://static.igem.org/mediawiki/2019/0/08/T--Aboa--DigoxEndOD600.png)

***Figure 2.**
The end densities of the cells after a test production round. A small effect
is already observed with these seemingly similar versions of the same protein.*

![Very important results, shame that you don't see them](https://static.igem.org/mediawiki/2019/2/20/T--Aboa--DigoxOptimization.png)

***Figure 3.**
Production optimization of Fab variants. The cells were tested (A and B) without pAzF present (C and D) and 1 mM pAzF present. (A and C) Concentration of Arabinose was tested and (B and D) concentration of IPTG was tested. From here we can observe that pAzF has
the most dramatic effect in expression of the Fab when the synthetase is present.
IPTG has a relative small, if any effect on the expression which is somewhat surpricing.
We chose to use 1mM pAzF, 0,5 mM IPTG and 0,5 % Arabinose in our work. This also
proved that we can produce Fabs using our GRO strain.*

![Very interesting results! Get better interwebs.](https://static.igem.org/mediawiki/2019/a/a9/T--Aboa--ProskutGeelilla3.png)

***Figure 4.**
(A) 1µg of Ni-NTA purified pAzF modified and unmodified Fabs. (B) SEC purified unmodified Fab. (C) The labeling of our produced Fab with DBCO-Cy5.5 visualized using Li-COR Odyssey at 700 nm bandwidth. A clear labeling of the C-terminal pAzF (amino acid residue 460) can be observed from this picture. No unspecific labeling of Fab0 can be observed. Copper free click-chemistry displays a very specific way of labeling irrespective of the purity of the sample. The other variants (191, 108) were not labeled very efficiently. This could be due to poor solvent accessibility of the pAzF residue or degradation of the protein. Unbound label can be seen at 1 kDa.*

## Producing Anti-Dengue Fab

We were not able to successfully produce any dengue binding Fab fragments. This
Fab sequence proved out to very toxic for our cell lines even in XL-1 Blue. Using
IDT gBlocks as a synthesis method for these Fabs is not recommended as most of the
genes we were able to sequence, proved out to be either truncated proteins or frameshifted.
This also shows that the viability of those cells that harbor the correct gene was
very low. After a huge sequencing effort we were able to find one unmodified fragment
that we produced but we could not measure any specific Fab activity using Eu labeled
2A11 anti Fab antibodies.

## Coating and testing Anti-Digox Fab

This part was what we were aiming for during the whole project. Because there wasn’t
any DBCO modified 96-well plates available anywhere, we decided to use DBCO-coated
magnetic beads (Jena, Biosciences, Germany). These beads could be coated with our
pAzF modified Fabs. We first had to prove that the Fabs are able to bind to the beads
specifically via the pAzF and not passively or in any other way.

We used two different approaches to immobilize the Fabs. In the first one, we added
biotin group to the azide group by using biotin-DBCO, after which the Fabs were immobilized
on a streptavidin-avidin plate (SA-plate) to react with the biotin. In the second
approach, the Fabs were directly immobilized onto DBCO-magnetic beads. With Eu-2A11
in both the approaches we observed, that the azide group in pAzF can be used to immobilize
the Fabs (Figure 5).

![I have no idea what these results mean](https://static.igem.org/mediawiki/2019/thumb/b/b9/T--Aboa--Aboa-Fab_immobilization.png/800px-T--Aboa--Aboa-Fab_immobilization.png)

***Figure 5.**
Determining the immobilization of Fabs via their pAzF onto a SA-plate (A) and onto
DBCO magnetic beads (B). According to the fluorescent measured, there is more randomly
immobilized Fab in the SA-plates compared to c-terminal conjugation. However, on
the magnetic beads the situation is the opposite. Also, the signal from the Fab 191,
Fab 108 and Fab0 are near the same level.*

Because we did not have Eu-labeled digoxigenin available, we resorted in using Cy5-labeled
digoxigenin kindly provided to us by our advisor Tuomas Huovinen. We then proceeded
to measure these coated beads in a flow cytometer.

![These some interesting results](https://static.igem.org/mediawiki/2019/9/9d/T--Aboa--BeaditSytometrissaAB.png)
![These are really important and interesting results!](https://static.igem.org/mediawiki/2019/3/3e/T--Aboa--BeaditSytometrissaC.png)

***Figure 6.**
(A) The size (Forward scatter, FSC-area) and shape (Side scatter, SSC-area) distribution
of randomly and site-specifically immobilized beads. This shows how a site specifically
labeled antibody with only one reactive site does not cross-link the beads in the
same manner as a randomly conjugated antibody. (B) A histogram of fluorescence intensity
of Cy5 at 700nm. the total population 100000 beads. Uncoated beads in red. Passively
adsorbed beads in blue, random conjugation in orange and site specific conjugation
using pAzF in green. A much larger variance is achieved when using random conjugation
because of the crosslinking of beads. Site specifically coated beads are more uniform.
(C) when monomeric beads are measured, a much more uniform coating is once more observed
with virtually no unspecific binding or empty beads with a huge difference in variance.
Variance of the assay is a critical factor for the sensitivity and repeatability of any test.*

![Results](https://static.igem.org/mediawiki/2019/thumb/d/d3/T--Aboa--Results.jpeg/800px-T--Aboa--Results.jpeg)

***Figure 7.** A) and B) show the assay performance as signal to background ratio and the error as a robust coefficent of variation. The test error is already, without any optimization at a clinically relevant level*

## Temperature stability measurements of Fab fragments

Melting temperature of Fabs was performed with Prometheus NanoDSF differential scanning
fluorometer (Munich, Germany). These measurements were kindly provided to us by our
sponsor PerkinElmer Turku.

![Nice thermal stability tests](https://static.igem.org/mediawiki/2019/thumb/9/9b/T--Aboa--TemperatureStability.png/800px-T--Aboa--TemperatureStability.png)

***Figure 8.**
(A) Melting temperatures of Fabs. (B) Shown as the proteins change of fluorescence
at 330 nm. The temperature stability of Fab with a pAzF at the histag does not alter
the stability of Fabs.*

---

[![](https://static.igem.org/mediawiki/2019/e/eb/T--Aboa--hytest.png)](https://www.hytest.fi/home)

[![](https://static.igem.org/mediawiki/2019/2/2c/T--Aboa--syrinx.png)](http://www.syrinxbioanalytics.com/)

[![](https://static.igem.org/mediawiki/2019/a/a8/T--Aboa--biotop.png)](https://www.biotop.fi/content/en/2/10005/Home.html)

[![](https://static.igem.org/mediawiki/2019/b/b5/T--Aboa--edita.png)](https://www.editapublishing.fi/)

[![](https://static.igem.org/mediawiki/2019/3/36/T--Aboa--AboAkademi.png)](https://www.abo.fi/en/)

[![](https://static.igem.org/mediawiki/2019/thumb/4/4f/T--Aboa--UtuLogo.png/800px-T--Aboa--UtuLogo.png)](https://www.utu.fi/en)