iGEM 2019 — Team FAU_Erlangen

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# Team:FAU Erlangen/Notebook

* Team
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* Project
  + [Description](https://2019.igem.org/Team%3AFAU_Erlangen/Description)
  + [Design](https://2019.igem.org/Team%3AFAU_Erlangen/Design)
  + [Experiments](https://2019.igem.org/Team%3AFAU_Erlangen/Experiments)
  + [Notebook](https://2019.igem.org/Team%3AFAU_Erlangen/Notebook)
  + [Contribution](https://2019.igem.org/Team%3AFAU_Erlangen/Contribution)
  + [Results](https://2019.igem.org/Team%3AFAU_Erlangen/Results)
* Parts
  + [Parts Overview](https://2019.igem.org/Team%3AFAU_Erlangen/Parts)
  + [Basic Parts](https://2019.igem.org/Team%3AFAU_Erlangen/Basic_Part)
  + [Composite Parts](https://2019.igem.org/Team%3AFAU_Erlangen/Composite_Part)
  + [Parts Collection](https://2019.igem.org/Team%3AFAU_Erlangen/Part_Collection)
* [Safety](https://2019.igem.org/Team%3AFAU_Erlangen/Safety)
* [Human Practices](https://2019.igem.org/Team%3AFAU_Erlangen/Human_Practices)
* Awards
  + [Model](https://2019.igem.org/Team%3AFAU_Erlangen/Model)
* [Judging Form](https://igem.org/2019_Judging_Form?team=FAU_Erlangen)

* [Team Members](https://2019.igem.org/Team%3AFAU_Erlangen/Team)
* [Collaborations](https://2019.igem.org/Team%3AFAU_Erlangen/Collaborations)
* [Attributions](https://2019.igem.org/Team%3AFAU_Erlangen/Attributions)
* [Safety](https://2019.igem.org/Team%3AFAU_Erlangen/Safety)
* [Human Practices](https://2019.igem.org/Team%3AFAU_Erlangen/Human_Practices)
* [Model](https://2019.igem.org/Team%3AFAU_Erlangen/Model)
* Project
* [Description](https://2019.igem.org/Team%3AFAU_Erlangen/Description)
* [Design](https://2019.igem.org/Team%3AFAU_Erlangen/Design)
* [Experiments](https://2019.igem.org/Team%3AFAU_Erlangen/Experiments)
* [Notebook](https://2019.igem.org/Team%3AFAU_Erlangen/Notebook)
* [Contribution](https://2019.igem.org/Team%3AFAU_Erlangen/Contribution)
* [Results](https://2019.igem.org/Team%3AFAU_Erlangen/Results)
* Parts
* [Parts Overview](https://2019.igem.org/Team%3AFAU_Erlangen/Parts)
* [Basic Parts](https://2019.igem.org/Team%3AFAU_Erlangen/Basic_Part)
* [Composite Parts](https://2019.igem.org/Team%3AFAU_Erlangen/Composite_Part)
* [Parts Collection](https://2019.igem.org/Team%3AFAU_Erlangen/Part_Collection)
* [Judging Form](https://igem.org/2019_Judging_Form?team=FAU_Erlangen)

* Team
  + [Team Members](https://2019.igem.org/Team%3AFAU_Erlangen/Team)
  + [Collaborations](https://2019.igem.org/Team%3AFAU_Erlangen/Collaborations)
  + [Attributions](https://2019.igem.org/Team%3AFAU_Erlangen/Attributions)
* Project
  + [Description](https://2019.igem.org/Team%3AFAU_Erlangen/Description)
  + [Design](https://2019.igem.org/Team%3AFAU_Erlangen/Design)
  + [Experiments](https://2019.igem.org/Team%3AFAU_Erlangen/Experiments)
  + [Notebook](https://2019.igem.org/Team%3AFAU_Erlangen/Notebook)
  + [Contribution](https://2019.igem.org/Team%3AFAU_Erlangen/Contribution)
  + [Results](https://2019.igem.org/Team%3AFAU_Erlangen/Results)
* Parts
  + [Parts Overview](https://2019.igem.org/Team%3AFAU_Erlangen/Parts)
  + [Basic Parts](https://2019.igem.org/Team%3AFAU_Erlangen/Basic_Part)
  + [Composite Parts](https://2019.igem.org/Team%3AFAU_Erlangen/Composite_Part)
  + [Parts Collection](https://2019.igem.org/Team%3AFAU_Erlangen/Part_Collection)
* Safety
* [Human Practices](https://2019.igem.org/Team%3AFAU_Erlangen/Human_Practices)
* Awards
  + [Model](https://2019.igem.org/Team%3AFAU_Erlangen/Model)
* [Judging Form](https://igem.org/2019_Judging_Form?team=FAU_Erlangen)

# Notebook

## May

[ ]

21

Generation of pACYC184 plasmid

|  |  |
| --- | --- |
| Aim / Task | Generation of pACYC184 plasmid |
| Protocol |  |
| Participants | Eva Neugebauer |

* bacteria from -80°C: HVS11 pEcoE, HVS11 pEcoF
* inoculation of both samples in 5 mL LB medium with Chloramphenicol
* overnight culture at 37°C with 200 rpm

[ ]

22

Generation of pACYC184 plasmid

|  |  |
| --- | --- |
| Aim / Task | Generation of pACYC184 plasmid |
| Protocol |  |
| Participants | Eva Neugebauer |

* no turbidity in the ON culture visible
* new bacteria from -80°C: HVS11 pEcoE, HVS11 pEcoF, HVS11 pEcoD
* inoculation of the three samples in 5 mL LB medium with Tetracyclin (2 µg/mL)
* ON culture at 37°C with 200 rpm

[ ]

23

Generation of pACYC184 plasmid

|  |  |
| --- | --- |
| Aim / Task | Generation of pACYC184 plasmid |
| Protocol | Zymo research Miniprep - Classic (Cat.No. D4015, Lot.No. 202653) |
| Participants | Eva Neugebauer |

Turbidity in the ON culture visible

*Miniprep:*

1. Centrifugate the ON culture at 4000 rpm at room temperature
2. Discard the supernatant
3. Add 200 µL P1 buffer
4. Add 200 µL P2 buffer and mix by inverting two times
5. Add 400 µL P3 buffer and mix by inverting three times
6. Centrifugate the samples for 2 min in a microcentrifuge
7. Place the column in a collection tube and transfer the supernatant
8. Centrifugate for 30s
9. Discard the flow-through and return of the column to the collection tube
10. Add 200 µL Endo-Wash buffer and centrifugate for 30 s
11. Add 400 µL Plasmid Wash buffer and centrifugate for 1 min
12. Place the column in a 1.5 mL tube, add 30 µL DNA Elution Buffer and centrifugate for 30 s

Storage of the purified plasmid DNA at 4°C

[ ]

27

Generation of pACYC184 plasmid

|  |  |
| --- | --- |
| Aim / Task | Generation of pACYC184 plasmid |
| Protocol | - |
| Participants | Eva Neugebauer |

inoculation of 6 clones in 5 mL LB medium with Chloramphenicol for the ON culture

[ ]

27
**(2)**

Generation of pACYC184 plasmid

|  |  |
| --- | --- |
| Aim / Task | Generation of pACYC184 plasmid |
| Protocol | - |
| Participants | Eva Neugebauer |

Nanodrop measurement of the samples from 2019-05-23
\* pEcoD: 46.1 ng/µL
\* pEcoE: 70.9 ng/µL
\* pEcoF: 27.6 ng/µL

restriction enzyme digest:

| setpoint | EcoD | EcoE | EcoF |  |
| --- | --- | --- | --- | --- |
| DNA | 500 ng | 10.85 µL | 7.05 µL | 17.5 µL |
| H20 | 20 µL | 6.65 µL | 10.45 µL | 0 µL |
| CutSmart Buffer | 2 µL | 2 µl | 2 µL | 2 µL |
| EcoRI | 0.5 µL | 0.5 µ | 0.5 µL | 0.5 µL |

Digestion 1h at 37°C and 350 rpm, afterwards adding of 4 µL BPB

agarose gel 1%:
1x TBE |60 mL
agarose |0.6 g
ethidiumbromid |2 µL

* load the gel with 20 µL of the samples and 3 µL Gene Ruler Mix
* run the gel at 130 V for 1 h

[ ]

28

Generation of pACYC184 plasmid

|  |  |
| --- | --- |
| Aim / Task | Generation of pACYC184 plasmid |
| Protocol | - |
| Participants | Eva Neugebauer |

*Miniprep:*

1. Centrifugate the ON culture at 4000 rpm at room temperature
2. Discard the supernatant
3. Add 200 µL P1 buffer
4. Add 200 µL P2 buffer and mix by inverting two times
5. Add 400 µL P3 buffer and mix by inverting three times
6. Centrifugate the samples for 2 min in a microcentrifuge
7. Place the column in a collection tube and transfer the supernatant
8. Centrifugate for 30s
9. Discard the flow-through and return of the column to the collection tube
10. Add 200 µL Endo-Wash buffer and centrifugate for 30 s
11. Add 400 µL Plasmid Wash buffer and centrifugate for 1 min
12. Place the column in a 1.5 mL tube, add 30 µL DNA Elution Buffer and centrifugate for 30 s

store the purified DNA at 4°C

*DNA concentration:*
measurement at the NanoDrop

| Clone | Clone 1 | Clone 2 | Clone 3 | Clone 4 | Clone 5 | Clone 6 |
| --- | --- | --- | --- | --- | --- | --- |
| concentration [ng/µL] | 30.6 | 21.3 | 26.1 | 23.6 | 28.1 | 25.4 |

*Digestion:*

digestion for 1.5 h at 37°C

| Clone | 1 | 2 | 3 | 4 | 5 | 6 |
| --- | --- | --- | --- | --- | --- | --- |
| Buffer | 2 µL | 2 µL | 2 µL | 2 µL | 2 µL | 2 µL |
| Enzyme (EcoRV) | 0.5 µL | 0.5 µL | 0.5 µL | 0.5 µL | 0.5 µL | 0.5 µL |
| DNA | 16.3 µL | 17.5 µL | 17.5 µL | 17.5 µL | 17.5 µL | 17.5 µL |
| H20 | 1.2 | - | - | - | - | - |

* load 20 µL of each sample and 3.5 µL of the marker on a 1 % agarose gel
* run 1.5 h at 130 V

*DNA extraction from agarose gel:*

1. add 200 µL NTI per 100 mg gel piece
2. incubate for 5 min at 50°C
3. place the column in a collection tube and transfer the sample
4. centrifugate at 11000 rpm for 30 s
5. add 700 µL NT3 twice adn centrifugate each time at 11000 rpm for 30 s
6. dry the silica membrane for 1 min at 11000 rpm
7. place the column on a sterile 1.5 mL reaction tube
8. add 20 µL NE buffer and centrifugate for 1 min at 11000 rpm

*DNA concentration*:
measurement at the NanoDrop
\* clone 1: 11.2 ng/µL
\* clone 2: 22.3 ng/µl

[ ]

28
**(2)**

Generation of pACYC184 plasmid

|  |  |
| --- | --- |
| Aim / Task | Generation of pACYC184 plasmid |
| Protocol | DNA extraction from agarose gel |
| Participants | Eva Neugebauer |

* run the gel from 2019-05-27 for 45 min at 130 V
* cut out the vector DNA from the gel
* place the gel pieces into reaction tubes

*DNA extraction from agarose gel:*

1. add 200 µL NTI per 100 mg gel piece
2. incubate for 5 min at 50°C
3. place the column in a collection tube and transfer the sample
4. centrifugate at 11000 rpm for 30 s
5. add 700 µL NT3 twice adn centrifugate each time at 11000 rpm for 30 s
6. dry the silica membrane for 1 min at 11000 rpm
7. place the column on a sterile 1.5 mL reaction tube
8. add 20 µL NE buffer and centrifugate for 1 min at 11000 rpm

*Ligation:*
DNA |9 µL
Quick Ligation buffer |10 µL
Quick Ligase |1 µL

Incubate for 10 min at room temperature

*Transformation into competent DH10B e.coli via heat shock:*

1. thaw competent bacteria on ice
2. add 10 µL of the previously ligated DNA, storage of the rest at -20°C
3. incubate 30 min on ice
4. incubate 1 min at 42°C
5. incubate 2 min on ice
6. add 500 µL Soc medium
7. 30 min on 37°C

plate the bacteria on Cam plates, incubate at 37°C over night

*Control gel for the ligation:*

* loading of 2 µL of each sample with 2 µL running buffer
* run the gel for 1 h at 130 V
* for each sample only one band visible

[ ]

29

Generation of pACYC184 plasmid

|  |  |
| --- | --- |
| Aim / Task | Generation of pACYC184 plasmid |
| Protocol | - |
| Participants | Eva Neugebauer |

* some colonies visible on the plates
* store at 4°C

## June

[ ]

13

Generation of more open pACYC184 with EcoRV

|  |  |
| --- | --- |
| Aim / Task | Generation of more open pACYC184 with EcoRV |
| Protocol | Miniprep |
| Participants | Eva Neugebauer |

## Protocol

| Substance | concentration |  |  |
| --- | --- | --- | --- |
| Cut Smart buffer | 2 µL | 2 µg (= 1 µl) | 1 µg (= µl) |
| DNA | 7,98 µL |  |  |
| Enzyme | 1,5 µL |  |  |
| Water | 44,52 µL |  |  |

Incoulation of 200 µL sample 2 from the 27.05.2019

**Miniprep**

* Adding 10 mL Equilibration buffer at the collum
* Centrifugation of overnightculture for 10 min at 4000 x g and removement of all the medium
* Resuspension of the pellet in 4 mL resuspension buffer (R3)
* Adding of 4 mL Lysis buffer and mixing by capping the tube for 5 times
* Incubation for 5 min at RT
* Adding 4 mL precipitation buffer (N3) and inverting till it is homogenous
* Centrifugation for 10 min at 5000 x g at RT
* Loadng supernatant on the collum, drain by gravity
* Adding of 10 mL washing buffer to the collum twice
* Placing collum on a 15 mL tube and adding of 5 mL elution buffer (E4) and letting it drain by gravity
* Adding 3,5 mL isopropanol and mixing it well
* Centrifugation for 30 min at 5000 x g at RT
* Discarding of the supernatant
* Adding 3 mL 70% ethanol
* Centrifugation for 5 min, 5000 x g at RT
* Remove supernatant
* Airdry pellet for 10 min
* Resuspension of pellet in 200 µL
* Storage at -20°C (still in virology lab)

**Nanodrop Measurement**

188,8 ng/ µL

**Digest**

500 ng for 0,5 µL enzyme

Digest of following components at 37°C foe 1 h:

**Digest**

500 ng for 0,5 µL enzyme

Digest of following components at 37°C foe 1 h:

\*see table above

**Gel**

Mixing of 60 µL digest with 12 µL loading buffer

Loading a agarose gel

130 V for 1 h

**Gel extraction**

Cutting the digest out of the gel.

1. Adding 200 µL /per 100 mg gel piece of NTI.
2. 5 min 50°C
3. Placing of the collum in a collection tube and loading of the sample.
4. Centrifugation at 11000 rpm for 30 s.
5. Adding twice 700 µL NT3 and centrifugation each time at 11000 rpm for 30 s.
6. Drying of the silica membrane for 1 min at 11000 rpm.
7. Placing collumn on a 1,5 mL sterile Eppi.
8. Adding 20 µL NE buffer and centrifugation for 1 min at 11000 rpm.
9. Storage at -20°C.

**Nanodrop**

Different results with a wide range

[ ]

17

Concentration of EcoRV opend pACYC184

|  |  |
| --- | --- |
| Aim / Task | Concentration of EcoRV opend pACYC184 |
| Protocol | Nanodrop |
| Participants | Eva Neugebauer |

## Protocol

Nanodrop measurement: concentration still varies

**Concentration determination with agarose gel**

Loading of the sample (2µL and 2µL marker on a agarose gel)

sample: 0,5 µL loading buffer on 2,5 µL digest

Running gel for 1 h at 130 V

Marker 4000 bp ~ 50 ng/µL

![<agarose gel of EcoRV opend pACYC184>](https://static.igem.org/mediawiki/parts/d/d3/T--FAU_ERLANGEN--img_1300.jpg)

[ ]

20

Material and Devices

|  |  |
| --- | --- |
| Aim / Task | Material and Devices |
| Protocol | Material check |
| Participants | Daniel Schreyer |

## Protocol

**Material and Devices**

* E.coli are in the -80°C Refrigerator in our lab - Box 12, reserved for iGEM
  + BL21 = Blank/no name, Star = S, Tuner = T
* 4°C Refrigerator is in our lab - top shelf
* PCR machine is on the other site of the kitchen
* Material for different buffers is on the left side next of the Winkler lab
* Winkler lab is on the opposite site of our lab
* Elektroporator in Jäck AG - Contact Felix Pfister, iGEM for more information
* Shaker Incubator in our Lab - Connect to water flow - Temperature adjustable

[ ]

24

Production of LB medium

|  |  |
| --- | --- |
| Aim / Task | Production of LB medium |
| Protocol | LB-Medium Production Protocol |
| Participants | Daniel Schreyer, Felix Fister |

## Protocol

**Notes**

* Autoclave machine in room 02.052
* Big incubation shaker device in 02.053
* Create medium and label it — “Content, Name-iGEM, room of our lab, date” e.g LB-medium, DS-iGEM, 02.081, 24.06.19
* Created 4L LB-Medium

**Calculations and Results**

20g of LB-Broth dissolved in 1L milli-Q H2O

**Materials used**

* 80g LB-Broth

**Problems faced / possible error causes**

* Medium-bottle do not close 100%
* not 100% contamination proof -use fast
* Our water shaker has the wrong size. Winkler is ordering new sizes for our shaker
  + Look for right size. Send product link to prof. winkler. He is going to order them for us
* Get right flask holder for our shaker?

**Pending tasks, next steps, comments for next people in the Lab**

* Cleaning ladies place the autoclaved medium into our lab. Store at 8°C. Should be finished around 2pm.
* Adding of antibiotics for selection after transformation.
* Use for cultivation and transformation
* Tape and pens are necessary

[ ]

25

E.coli Cultivation

|  |  |
| --- | --- |
| Aim / Task | E.coli Cultivation |
| Protocol | Cultivation with LB-medium |
| Participants | Daniel Schreyer, Lisa Vogg |

## Protocol

**Notes**

* Stored LB-medium in fridge
* Used 3 E.coli strains: Tuner, BL21 Star, BL21
* Pipetting under bunsen burner in Winkler lab
* For each strain: 10 mL LB-medium combined with 1 sterile pipet tip E.coli in 12 ml not fully closed reaction tubes
* Cultivation in top incubation shaker (left side, 3 12 mL reaction tubes) in room 02.053 at 37°C with 130 rpm
* Autoclave 100 mL flasks for tomorrow 26.06.19
* **If we are using stuff from the Winkler lab, bring it back**
* **If we are using workspace in the Winkler lab - clean the bench with 70% Ethanol**

**Protocol:**

1. Unfreeze E.coli
2. Centrifuge tube with Mini-Centrifuge –Solution at the bottom of the reaction tube

**Next Steps are performed next to the bunsen burner**

1. Fill 10 ml LB-medium into 12 ml reaction tube under
2. Dip 1 sterile pipet tip into E.coli reaction tube
3. Throw pipet tip into 12 ml reaction tube and close it
4. Cultivation in incubation shaker for 20 hours at 37°C with 130 rpm

* Store remaining E.coli at -80°C

**Problems faced / possible error causes**

* Incubation flasks are needed for further cultivation. Autoclave 100 mL flasks for tomorrow 26.06.19
* Need lab briefing from some of winklers lab

**Pending tasks, next steps, comments for next people in the Lab**

* OD measurement –further cultivation
* Cultivation in 100mL flasks with the incubation shaker in our lab
* Freeze 1mL from each E.coli strain at -80°C
* Wait for autoclaved flasks. Use them to cultivate our bacteria in our shaker
  …

[ ]

26

E. coli cultivation

|  |  |
| --- | --- |
| Aim / Task | E. coli cultivation |
| Protocol | Cultivation with LB-medium |
| Participants | Lisa Vogg, Martin Borgmann |

## Protocol

**Notes**

* Stored LB-medium in fridge
* Used 3 E.coli strains: Tuner, BL21 Star, BL21
* Pipetting under bunsen burner in Winkler lab
* For each strain: 155 mL LB-medium combined the 10 ml of the previous bacteria culture (25.06.19)
* Cultivation in top incubation shaker in room 02.053 at 37°C with 130 rpm
* stuff flask holder with paper towels to keep the flasks in place
* autoclaved flasks from the previous day not yet usable

**Protocol:**

(Performed next to a bunsen burner)

1. Fill flask with desired volume of LB medium
2. sterilize tip of the pipet, the lid of the bottle with medium and the edge of the flask in the flame to prevent contamination
3. add content of the previous culture tubes to the new flasks
4. Cultivation in incubation shaker for 20 hours at 37°C with 130 rpm

**Problems faced / possible error causes**

* briefing for the lab desperately needed!
* Which kuvettes for OD measurement?
* flasks from the previous day not yet usable

**Pending tasks, next steps, comments for next people in the Lab**

* measure OD
* make cells competent
* freeze a batch

  + 3x 25 ml pipette
* gloves, paper towels, 70% ethanol
* 490 ml LB medium

[ ]

28

Overnight Cultivation of E.coli strains

|  |  |
| --- | --- |
| Aim / Task | Overnight Cultivation of E.coli strains |
| Protocol | E.coli Cultivation |
| Participants | Daniel Schreyer, Leon Altmann, Lisa Vogg, Jannik Wagner |

## Protocol

**Notes**

* Measure OD(600nm) (1:10 dilution) after 2 days incubation in incubation shaker of the E.coli strains BL21, BL21 Star, BL21 Tuner
* OD of 0.71 =7.1

**Protocol:**

**Inoculate overnight culture:**

1. inoculate 100 ml LB-medium with 100µl of the E.coli cultures
2. Fill water shaker with water
3. Place into water shaker at 37°C and 120 RPM
4. Incubate overnight: Start 5pm –19h incubation time

**Materials used**

* OD cuvette
* 25 ml plastic tip
* 3 yellow tips

**Problems faced / possible error causes**

* **NEB Kit was stored at -20°C: competent E.colis should be stored at -80°C, other lab material should be stored at 4°C (DNA Ladder, buffer, …) –Damage is possible**

**Pending tasks, next steps, comments for next people in the Lab**

* Pour agar plates for heat shock test: Test transformation by heat shock with NEB control vector PUC19
* Pour agar plates for first transformations: Agar plates with chloramphenicol
* freeze E.coli from overnight culture
* Heat shock test

[ ]

29

Pour agar plates

|  |  |
| --- | --- |
| Aim / Task | Pour agar plates |
| Protocol |  |
| Participants | Daniel Schreyer, Leon Altmann, Lisa Vogg |

## Protocol

**Notes**

* Pour agar plates:
  + 3 plates with ampicillin resistance
  + 21 plates with chloramphenicol resistance
  + 3 OD measurements of overnight culture. Each around 45 minutes apart
    **Materials used**

agar for 700 mL agar plates

## July

[ ]

1

Preparation Sequences

|  |  |
| --- | --- |
| Aim / Task | Preparation Sequences |
| Protocol | - |
| Participants | Lena Schorr, Ruben Caspary, Eva Neugebauer |

## Protocol

Resuspension of the IDT sequences in 20 µL TE buffer.

Resulting concentration 50 ng/ µL.

Vortexing and incubation for 15 min at 50°C.

Vortexing and storage at -20°C.

Biotwist sequences and primer still not resuspended!
10 µL Filtertip-Spitzen

[ ]

1
**(2)**

Growth kinetics

|  |  |
| --- | --- |
| Aim / Task | Growth kinetics |
| Protocol | OD |
| Participants | Martin Borgmann |

## Protocol

**Freezing of E.coli**

**Goal**: storing the 3 E.coli strains BL21, BL21 Tuner and BL21 Star to have them ready for cultivation for transformation

-Spinned for 10 minutes at 2,500 rpm

-Discarded supernatant without disturbing the pellet, while leaving ~1ml for every sample

-Resuspend the pellet in the remaining supernatant.

-Added 900μl cell culture and 300μl 99% glycerol, to a final glycerol concentration of ~25%. 6.

-Store in -80 °C freezer

**Growth kinetics**

**Goal**: get **Growth kinetics** of the 3 E.coli strains BL21, BL21 Tuner and BL21 Star to estimate the optimal time point for the transformation!

-**Measuring OD600 of over night culture (RT)**

OD600 = 1 equals 8\*108 cells/mL

| Stem | OD | Cell count |
| --- | --- | --- |
| B21: | 1 µg (=0,5 µl) | –5,5\*108 cells / mL |
| B21 Tuner: | OD600 (1) = 0,1966 and OD600 (2) = 0,1402 (double determination!) -average: 0,1684 | –1,35\*109 cells / mL |
| B21 Star: | OD600 = 0,0779 | –6,29\*108 cells / mL |

-**Seeding with same cell coun**t -106 cells / mL in 25 mL LB medium –2,5\*107 cells

| Stem | Calculation |  |
| --- | --- | --- |
| B21: | 5,5\*108 cells / mL -2,5\*107 cells / 5,5\*108 cells / mL = 0,045 mL |  |
| B21 Tuner: | 1,35\*109 cells / mL - 2,5\*107 cells / 1,35\*109 cells / mL = 0,019 mL |  |
| B21 Star: | 6,29\*108 cells / mL - 2,5\*107 cells / 6,29\*108 cells / mL = 0,040 mL |  |

| Time in h | OD | dilution factor | real OD | µ(t) in 1/h |  | OD | dilution factor | real OD | µ(t) in 1/h |  | OD | dilution factor | real OD | µ(t) in 1/h |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| *Stem:* | BL12 |  |  |  |  | BL12 Tuner |  |  |  |  | BL12 Star |  |  |  |
| 0 | 0,0192 | 1 | 0,0192 |  |  | 0,0262 | 1 | 0,0262 |  |  | 0,0199 | 1 | 0,0199 | . |
| 1 | 0,0324 | 1 | 0,0324 | 0,523 |  | 0,0237 | 1 | 0,0237 | -0,100 |  | 0,0283 | 1 | 0,0283 | 0,352 |
| 2 | 0,0674 | 1 | 0,0674 | 0,732 |  | 0,027 | 1 | 0,027 | 0,130 |  | 0,0557 | 1 | 0,0557 | 0,677 |
| 2,5 | 0,1265 | 1 | 0,1265 | 1,259 |  | 0,0405 | 1 | 0,0405 | 0,811 |  | 0,092 | 1 | 0,092 | 1,004 |
| 3 | 0,2873 | 1 | 0,2873 | 1,641 |  | 0,0724 | 1 | 0,0724 | 1,162 |  | 0,217 | 1 | 0,217 | 1,716 |
| 3,5 | 0,2609 | 2 | 0,5218 | 1,194 |  | 0,069 | 2 | 0,138 | 1,290 |  | 0,1933 | 2 | 0,3866 | 1,155 |
| 4 | 0,1663 | 5 | 0,8315 | 0,932 |  | 0,067 | 5 | 0,335 | 1,774 |  | 0,1384 | 5 | 0,692 | 1,164 |
| 4,5 | 0,2182 | 5 | 1,091 | 0,543 |  | 0,142 | 5 | 0,71 | 1,502 |  | 0,1759 | 5 | 0,8795 | 0,480 |
| 5 | 0,1992 | 6 | 1,1952 | 0,182 |  | 0,1605 | 6 | 0,963 | 0,610 |  | 0,1843 | 6 | 1,1058 | 0,458 |

-all strains in log phase at roughly 3-3,5h –**Transformation should be done after 3-3,5h of incubation time after staring a culture!**

![<Beschreibung>](/labjournal-entries/images/Curves.JPG)

[ ]

1
**(3)**

Preparation Sequences

|  |  |
| --- | --- |
| Aim / Task | Preparation Sequences |
| Protocol |  |
| Participants | Lena Schorr, Ruben Caspary, Eva Neugebauer |

## Protocol

Resuspension of the IDT sequences in 20 µL TE buffer.

Resulting concentration 50 ng/ µL.

Vortexing and incubation for 15 min at 50°C.

Vortexing and storage at -20°C.

Biotwist sequences and primer still not resuspended!

10 µL Filtertip-Spitzen

[ ]

2

Amplify construct DNA

|  |  |
| --- | --- |
| Aim / Task | Amplify construct DNA |
| Protocol | - |
| Participants | Lena Schorr, Lisa Vogg |

| Name | Attempt 1 | Attempt 2 | Attempt 3 |
| --- | --- | --- | --- |
| DNA | 1 µg (=0,5 µl) | 2 µg (= 1 µl) | 1 µg (= µl) |
| NEB buffer 3.1 (x10 ) | 5 | 5 | - |

## Protocol

**Notes**

* dissolved primers for amplification in TE buffer to a concentration of 100mM
* froze each primer diluted in TE with a concentration of 1mM (labeled according to construct)
* dissolved all remaining DNA of the constructs in 20 µl TE buffer
* prepared 2 1:100 pre-dilutions of the primer for the PCR, first in TE buffer, second in RNase-free water
* performed PCR according to NEB protocol
* stored PCR products in 4 °C fridge
* prepared agarose gel for electrophresis (needs to be repeated)

**Protocol**

**Dissolving freeze-dried DNA**

* spin down aliquots
* add required volume of TE buffer (20 µl)
* vortex thoroughly
* incubate on the heating block at 50°C for 20 min

**PCR (according to the NEB protocol)**

| template DNA | 5 µl (250 ng) |
| --- | --- |
| forward primer (10 µM) | 1.25 µl (end concentration 0.5 µM) |
| reverse primer (10 µM) | 1.25 µl (end concentration 0.5 µM) |
| Master Mix | 12.5 µl |
| RNase-free water | 5 µlpr |
|  | 25 µl |

* programme is saved under Thomas Winkler >Nina >iGEM
* can otherwise be found in the NEB protocol
  + 30 cycles at 60°C

**Agarose gel**

* 0.8 g Agarose
* 100 ml TAE buffer
* 10 µl SYBR Safe
* medium chamber
* slot chamber onto its sled with the open ends aligning with the edge of the sled
* cautiously pour the liquid agarose into the chamber, avoid air bubbles
* insert the comb
* **DO NOT MOVE AFTER AGAROSE HAS BEEN ADDED!** Otherwise liquid will slosh out and gel will be too thin
* gels can be stored in a plastic dish with a little bit of TAE buffer at 4°C or wrapped in saran film

**Calculations and Results**
- calculated volume for dissolving the primers: **V=m/(M\*c)** with m and M found on the aliquots and c set as **100 mM**

| Primer | m [mg] | M [g/mol] | V [µl] |
| --- | --- | --- | --- |
| Tac Promotor Primer fw 2.0 | 0.97 | 6.38 | 1520 |
| K1 HisStop Primer re 2.0 | 0.61 | 6.467 | 943 |
| K2A HisStop re 2.0 | 0.21 | 7.084 | 296 |
| K3a HisStop Primer re 2.0 | 0.54 | 6.194 | 886 |
| K4a StrepStop re 2.0 | 0.22 | 6.362 | 346 |
| K4b StrepStop re 2.0 | 0.2 | 6.924 | 314 |

**Pending tasks, next steps, comments for next people in the Lab**
- run electrophoresis with PCR products

[ ]

3

Primer Dilution

|  |  |
| --- | --- |
| Aim / Task | Primer Dilution |
| Protocol | - |
| Participants | Lena Schorr |

| Name | Attempt 1 | Attempt 2 | Attempt 3 |
| --- | --- | --- | --- |
| DNA | 1 µg (=0,5 µl) | 2 µg (= 1 µl) | 1 µg (= µl) |
| NEB buffer 3.1 (x10 ) | 5 | 5 | - |

## Protocol

**Notes**

**Prepare Primer Solution:**

* centrifuge primers
* add TE-buffer to dilute primers to a concentration of 100nM
* Vortex
* heat and shake at 50°C in thermomixer
* **freeze and store at -20°C**

**Protocol**

* **Ran gelelectrophoresis with PCR products 02.07.2019 with 100x**

| DNA | 2µl |
| --- | --- |
| Loading Dye(6x) | 2µl |
| H20 | 16µl |

* Put 20µl in each pocket
* put 6µl DNA Ladder in one pocket

**Gibson - PCR:**

| template DNA | 5 µl (250 ng) |
| --- | --- |
| forward primer (10 µM) | 1.25 µl (end concentration 0.5 µM) |
| reverse primer (10 µM) | 1.25 µl (end concentration 0.5 µM) |
| Master Mix | 12.5 µl |
| Autoclaved H20 | 5 µlpr |
|  | 25 µl |

* NEW PCR PROGRAM: igem gibson anhang
* PCR is running over night. Finishes around 9pm and is then stored at 4°C in the PCR cycler

**Calculations and Results**

**Problems faced / possible error causes**

* No Protocol! People should make clear which protocol they want to use
* Tested PCR Products on quantity with biophotometer: No clear results. Results are in our lab notebook

**Materials used**

* 16 PCR Tubes and caps

**Files used / generated**

**Pending tasks, next steps, comments for next people in the Lab**

* Run Gel electrophoresis. See day before
* Gibson Assembly of Fab Fragments
* Gibson Assembly of different constructs into vector
* Loading Dye 6x is stored in the fridge in Winkler’s Lab

[ ]

3
**(2)**

Create E.coli Starter Glycerol Stock

|  |  |
| --- | --- |
| Aim / Task | Create E.coli Starter Glycerol Stock |
| Protocol | see link |
| Participants | Daniel Schreyer, Jannik Wagner |

## Protocol

**Notes**

* Autoclaved 2x 500ml agar plate medium
* Pour antibiotica-free plates
* E.coli strains BL21, Star, Tuner, DH5alpha are cultivated on plates in 3 different dilutions: 1:10, 1:100
* work very sterile
  <http://www.molbi.de/protocols/competent_cells_starter_cultures_v1_0.htm>

**Problems faced / possible error causes**

* Agar dissolves in autoclave
* **work very sterile!!! No antibiotica**

**Materials used**

* 35g LB-Broth with agar

**Pending tasks, next steps, comments for next people in the Lab**

* Cultivate 2 colonies each in 50mL flasks. See protocol day 2 morning!

[ ]

4

Gibson PCR Control Gel-Electrophoresis

|  |  |
| --- | --- |
| Aim / Task | Gibson PCR Control Gel-Electrophoresis |
| Protocol | see link |
| Participants | Felix Pfister |

## Protocol

**Notes**

* 0,8% Agarose gel
* DNA-Mix of Gibson PCR
  + 2 µl DNA
  + 2 µl Loading Dye (6x)
  + 16 µl dH2O
* Gelelectrophoresis @130V for 60 min
* Used only 4µl of 1kb DNA Ladder

**Protocol**

<https://www.addgene.org/protocols/gel-electrophoresis/>

**Calculations and Results**

**Problems faced / possible error causes**

* Gel seems to run a bit crooked
* Amplification of unknown DNA
* Only little desired DNA amplified

**Materials used**

* Gelectrophoresis
  + 0.8g Agarose
  + 100ml TAE-Buffer (1x)
* DNA-Mix
  + 2µl DNA
  + 2µl Loading Dye (6x)
  + 16µl dH2O
* 4µl DNA Ladder (1kb)
* 8 PCR tubes

**Files used / generated**

**Pending tasks, next steps, comments for next people in the Lab**

* Gibson Assembly if correct bases are seen in the gelphoto

![<Beschreibung>](https://static.igem.org/mediawiki/parts/4/4a/T--FAU_ERLANGEN--Gelphoto_Gibson_PCR_04_07_19.jpg)

[ ]

4
**(2)**

Cultivation of E.coli in 15ml flasks with 10ml LB-medium

|  |  |
| --- | --- |
| Aim / Task | Cultivation of E.coli in 15ml flasks with 10ml LB-medium |
| Protocol | see links |
| Participants | Daniel Schreyer, Jannik Wagner |

## Protocol

**Notes**

* Too many colonies on plates
* used 2 µl of each strain and put the whole tip into 10ml LB-medium
* shake at 37°C and 200rpm. Incubate until the evening
* autoclave 1L erlenmeyer flask until the evening

**Protocol**

<http://www.molbi.de/protocols/competent_cells_starter_cultures_v1_0.htm> Day 2: Morning

**Problems faced / possible error causes**

* Too many colonies. Use less amount for cultivation on agar plates

**Materials used**

* 4 Falcons 15mL
* 1 10ml plastic tip

**Pending tasks, next steps, comments for next people in the Lab**

* Cultivate in 1L erlenmeyer flask with 250ml LB until the morning
* OD measurement of cultivated E.coli

[ ]

4
**(3)**

Gibson PCR (Overhang) - New 57°C

|  |  |
| --- | --- |
| Aim / Task | Gibson PCR (Overhang) - New 57°C |
| Protocol | PCR (according to NEB protocol) |
| Participants | Felix Pfister, Daniel Schreyer |

## Protocol

**Notes**

* Defrost Primer on Ice

**Protocol**

Gibson - PCR:

| Template DNA | 5 µl (250 ng) |
| --- | --- |
| Primer fw (10µM) | 1.25 µl (end concentration 0.5 µM) |
| Primer rev (10µM) | 1.25 µl (end concentration 0.5 µM) |
| Master Mix | 12.5 µl |

* NEW PCR PROGRAM: igem gibson anhang —-now with 57°C instead of 65°C
  + Also 35 cycles instead of 30 cycles
* PCR is running over night

**Calculations and Results**

-

**Problems faced / possible error causes**

* Gib pAC Promotor fw almost empty

**Materials used**

* 8 PCR tubes

**Files used / generated**

**Pending tasks, next steps, comments for next people in the Lab**

* Run Gel electrophoresis. See day before
* Gibson Assembly of Fab Fragments
* Gibson Assembly of different constructs into vector
* **Get new pAC fw primer!!!**

[ ]

4
**(4)**

Gibson Assembly - cancelled!

|  |  |
| --- | --- |
| Aim / Task | Gibson Assembly - cancelled! |
| Protocol | see link |
| Participants | Felix Pfister, Antonio Maccataio, Daniel Schreyer |

**Notes**

* Checked Gel electrophoresis from Gibson PCR
  + Didnt work/not enough DNA!!!!

**Protocol**

<https://international.neb.com/protocols/2012/09/25/gibson-assembly-master-mix-assembly>

**Calculations and Results**

**Problems faced / possible error causes**

**Materials used**

**Files used / generated**

**Pending tasks, next steps, comments for next people in the Lab**

* Repeat the Gibson Overhang PCR

[ ]

5

Aliquot E. coli

|  |  |
| --- | --- |
| Aim / Task | Aliquot E. coli |
| Protocol | http://www.molbi.de/protocols/competent\_cells\_starter\_cultures\_v1\_0.htm Day 3: Morning |
| Participants | Daniel Schreyer |

* PCR products from 2019-07-04 are stored at 4 °C
* aliquots are stored at -80 °C, cells are not competent

**Protocol:**

* transfer 200 mL E.coli suspension in four 50 mL flasks –Centrifuge at 4000 rpm for 10 mins with ultra centrifuge
* dilute centrifuged E. coli in 2.5 ml glycerol and 2.5 mL LB-medium
* use 4 mL of each flask to make 1 mL aliquots
* making around 16x 1 mL aliquots of each strain: DH5alpha, Star, Tuner, BL21

[ ]

5
**(2)**

Control electrophoresis Gibson Overhnag PCR from 07-04

|  |  |
| --- | --- |
| Aim / Task | Control electrophoresis Gibson Overhnag PCR from 07-04 |
| Protocol |  |
| Participants | Felix Pfister |

* checked Gibson Overhang
* ran agarose gel at 130 V

[ ]

9

Purify Konstruct DNA from Agarose Gel

|  |  |
| --- | --- |
| Aim / Task | Purify Konstruct DNA from Agarose Gel |
| Protocol | - |
| Participants | Eva Neugebauer, Leon Altmann |

## Protocol

***Notes***

* Before the purification the according DNA bands were cut out of the gel
* The gel pieces were transferred into Eppendorf Reaction Tubes, which were weighed beforehand;
* The mass of the gel pieces was determined by repeated weighing, to determine the necessary amounts of reagents

**Protocol**

Protocol of the NucleoSpin Extract Kit

**Calculations and Results**

* Reaction Tube weight (empty): K2b 1,013g K3b 1,014g K4a 1,013g K4b 1,013g
* Weight of Gelfragments: K2b 0,287g K3b 0,356g K4a 0,288g K4b 0,287g
* For each 100mg gel 300ul of NT1 buffer should be used
* Amount of NT1 buffer: K2b 861ul K3b 1068ul K4a 863ul K4b 861ul

**Problems faced / possible error causes**

The gel fragments were quite large; This might have a negative impact on the efficiency of the purification;

As the lab with the nanodrop was already closed the Concentration of the processed DNA could not be assesed at this day;

**Materials used**

* NucleoSpin Extract Kit

**Files used / generated**

**Pending tasks, next steps, comments for next people in the Lab**

* Measure DNA concentrations

[ ]

9
**(2)**

Production of electrocompetent Bacteria (BL21, BL21 Star, Tuner)

|  |  |
| --- | --- |
| Aim / Task | Production of electrocompetent Bacteria (BL21, BL21 Star, Tuner) |
| Protocol | http://www.molbi.de/protocols/competent\_cells\_electro\_v1\_0.htm |
| Participants | Felix Pfister, Jannik Wagner |

* used only 100 mL of LB medium for 3 flasks
* Problems faced:
  + no ON culture, directly from -80 °C into 100 mL LB medium
  + lost Tuner completely –no competent Tuner bacteria on -80 °C
  + BL21 Star had a smeary pellet in the last washing step –could be problematic

[ ]

10

PCR with K1, K2a, K3a 3% DMSO and gel-electrophoresis

|  |  |
| --- | --- |
| Aim / Task | PCR with K1, K2a, K3a 3% DMSO and gel-electrophoresis |
| Protocol | - |
| Participants | Antonio Maccataio, Lisa Vogg, Marie Wiedemann |

## Protocol

**Notes**

* 5 yL DNA
* 1,25 ym primer fw
* 1,25 yL primer re
* 5 yL DMSO 15% Stock ->3% diluted
* Mastermix 12,5 yL

2x 1:100 Dilution

1mM TE (DNA storage at -20°)

Gibson

gel-electrophoresis

![<Beschreibung>](https://static.igem.org/mediawiki/parts/d/d3/T--FAU_ERLANGEN--C374518F-2355-4212-A19A-9E432DE22E42.jpeg)

[ ]

10
**(2)**

Repeat failed PCR for Gibson overhangs with added DMSO

|  |  |
| --- | --- |
| Aim / Task | Repeat failed PCR for Gibson overhangs with added DMSO |
| Protocol | NEB kit instructions, NEB troubleshooting |
| Participants | Lisa Vogg, Antonio Maccataio |

* aliquoted 15% DMSO
* used original amplified DNA of K1, K2a, K3a
* added DMSO to a final concentration of 3%
* lowered running temperature of the PCR from 57 °C to 55 °C due to DMSO
* prepared 1:100 dilution of the respective primers (–1mM) in TE buffer and stored them at -20 °C
* prepared a further 1:100 dilution (–10 µM) in dH2O for the PCR

| DNA | 5 µL |
| --- | --- |
| Primer fw | 1.25 µL |
| Primer re | 1.25 µL |
| Master Mix | 12.5 µL |
| DMSO (15%) | 5 µL |
|  | 25 µL |

[ ]

11

Electroporation Control

|  |  |
| --- | --- |
| Aim / Task | Electroporation Control |
| Protocol | NEB electroporation protocol |
| Participants | Felix Pfister |

* electroporation of pUC19 into BL21, BL21 Star
* one H2O control in BL21 Star
* generated own electroporation protocol in the GeneUser (>Users>igem>igem1)
* used 25 µL bacteria with 1 µL 1:5 diluted pUC19
  + should be 50 µL next time instead of 25 µL

**Protocol:**
1. incubate labelled agar plates in the incubator at 37 °C
2. incubate medium at 37 °C
3. thaw bacteria from -80 °C on ice
4. when bacteria are thawed, add DNA
5. keep 1 mm cuvettes on ice
6. electroporation of the bacteria with 1 µL DNA –immediately add warm medium to cuvette and mix by pipetting up and down
7. put bacteria on shaker at 37 °C for 30-60 mi at 300 rpm
8. plate them on the 37 °C warm agar plates
9. incubate the bacteria on plates ON –not too long or they will grow too much

[ ]

11
**(2)**

repeat failed PCR with correct amount of primer at 57 °C with and without DMSO

|  |  |
| --- | --- |
| Aim / Task | repeat failed PCR with correct amount of primer at 57 °C with and without DMSO |
| Protocol | NEB PCR kit |
| Participants | Daniel Schreyer, Lena Schorr, Lisa Vogg |

* set up two PCR reactions:

| DNA | 5 µL |
| --- | --- |
| Primer fw (10 µM) | 1.25 µL |
| Primer re (10 µM) | 1.25 µL |
| Master Mix | 12.5 µL |
| dH2O or DMSO (15 %) | 5 µL |
|  | 25 µL |

* run PCR at 57 °C
  + last PCR might have failed due to too low temperatures
  + last PCRs for overhang generation might have failed due to much too high primer concentrations, would explain very bright bands in the previous gel
* amplified constructs form the very first PCR were not sufficient fpr the reaction
* set up another PCR to amplify the constructs K1, K2a and K3a in a 25 µL reaction at 60 °C
* run 0.8 % agarose gel (small) for the amplified constructs
  + 2 µL DNA, 18 µL total volume in each pocket
  + run at 130 V
  + 3 µL 1 kb DNA ladder
* run 0.8 % gel (medium) for the overhang PCR
  + used 2 µL DNA and 6 µL of test vector from the day before
* autoclaved 1 L LB medium

[ ]

12

Check electroporated E. coli on plates

|  |  |
| --- | --- |
| Aim / Task | Check electroporated E. coli on plates |
| Protocol |  |
| Participants | Daniel SChreyer |

* no growth on plates –incubate longer
* E. coli were possibily not transformed
* use bigger amount of eletrocompetent bacteria

[ ]

13

make E. coli strains Tuner and DH5alpha electrocompetent

|  |  |
| --- | --- |
| Aim / Task | make E. coli strains Tuner and DH5alpha electrocompetent |
| Protocol | http://www.molbi.de/protocols/competent\_cells\_electro\_v1\_0.htm |
| Participants | Martin Borgmann |

* thawed stock bacteria on ice
* inocculated 2 25 µL of each strain (Tuner and DH5alpha) in 100 mL LB medium and incubated for 1:45 h until OD600 was 0.4-0.6
* not much 10 % glycerol available so the two cultures of each strain have been pooled and washed together
* frozen in 50 µL aliquots
* modification of used protocol: 8.7 % glycerol used instead of 10 %

[ ]

15

amplification of original constructs

|  |  |
| --- | --- |
| Aim / Task | amplification of original constructs |
| Protocol | NEB kit instructions |
| Participants | Lisa Vogg |

* used 1:10 dilution of 100 µM stock of the primers
* amplified all constructs
* PCR at 60 °C, 25 µL reaction with 5 µL DNA

[ ]

15
**(2)**

produce SOB medium

|  |  |
| --- | --- |
| Aim / Task | produce SOB medium |
| Protocol | - |
| Participants | Antonio Maccataio |

**Protocol:**

Per liter: To 950 mL of deionized H2O, add:

| Tryptone | 20 g |
| --- | --- |
| Yeast extract | 5 g |
| NaCl | 0.5 g |

SOC medium is identical to SOB medium, except that it contains 20 mM glucose. To prepare SOB medium, combine the above ingredients and shake until the solutes have dissolved. Add 10 mL of a 250 mM solution of KCl. (This solution is made by dissolving 1.86 g of KCl in 100 mL of deionized H2O.)
Adjust the pH of the medium to 7.0 with 5 N NaOH (∼0.2 mL). Adjust the volume of the solution to 1 L with deionized H2O. Sterilize by autoclaving for 20 min at 15 psi (1.05 kg/cm2) on liquid cycle.

Just before use, add 5 mL of a sterile solution of 2 M MgCl2 and 5ml of MGSO4. (This solution is made by dissolving 40,66 g of MgCl2 (Hydrate) and 49,3g MgSO4 (Hydrate) respectively in 90 mL of deionized H2O. Adjust the volume of the solution to 100 mL with deionized H2O and sterilize by autoclaving for 20 min at 15 psi [1.05 kg/cm2] on liquid cycle.)
After the SOB medium has been autoclaved, allow it to cool to 60°C or less.
Add 20 mL of a sterile 1 M solution of glucose. (This solution is made by dissolving 18 g of glucose in 90 mL of deionized H2O. After the sugar has dissolved, adjust the volume of the solution to 100 mL with deionized H2O and sterilize by passing it through a 0.22-µm filter.)

* only produced 100 mL

[ ]

16

electroporation control

|  |  |
| --- | --- |
| Aim / Task | electroporation control |
| Protocol | https://international.neb.com/Protocols/0001/01/01/electroporation-protocol-c2986 |
| Participants | Felix Pfister |

* control if BL21 blank/star/tuner and DH5alpha are electrocompetent
* added 2 µL of pUC19 1:5 –electroporation with iGEM protocol
* bacteria plated on 37 °C LB-Agar plates with ampicillin

[ ]

17

Heat shock of DH5alpha and BL21

|  |  |
| --- | --- |
| Aim / Task | Heat shock of DH5alpha and BL21 |
| Protocol |  |
| Participants | Eva Neugebauer, Daniel Schreyer |

1. Thaw chemical competent bacteria on ice (200 µL each)
2. Add 5 µL of pUC vector
3. 30 min incubation on ice
4. 1 min 42°C (450 rmp)
5. 2 min on ice
6. Add 500 µL SOC medium
7. 30 min 37°C (450 rpm)
8. Plate 200 µL bacs on Amp plates
9. Incubation over night at 37°C

[ ]

17
**(2)**

Preparation of chemically competent E. coli

|  |  |
| --- | --- |
| Aim / Task | Preparation of chemically competent E. coli |
| Protocol | https://www.helmholtz-muenchen.de/fileadmin/PEPF/Protocols/Chemically-competent-cells.pdf |
| Participants | Eva Neugebauer, Daniel Schreyer, Ruben Caspary |

* prepared chemically competent E. coli: DH5alpha, BL21 Star, Tuner, BL21
* used E. coli with the following OD:

| Strain | BL21 | BL21 Star | Tuner | DH5alpha |
| --- | --- | --- | --- | --- |
| OD | 0.55 | 0.499 | 0.43 | 0.42 |

* 20x 200 µL aliquots stored at -80 °C of each strain
* CC buffer is stored at 4 °C and is sterilized through 0.22 µm filtration
* used 1⁄5 of each medium –cultivated in 100 mL LB instead of 500 mL
* used stock E. coli, not single colony –lower transformation rate possible

[ ]

19

Check plates of transformation of 2019-07-18

|  |  |
| --- | --- |
| Aim / Task | Check plates of transformation of 2019-07-18 |
| Protocol |  |
| Participants | Daniel Schreyer |

* checked LB agar plates with ampicillin oand our transformed bacteria of 2019-07-18
* growth on both plates –heat shock success
* pick on DH5alpha colony from the agar plate
* cultivated in 250 mL LB-medium with 250 µL (50 mg/ml) Ampicillin
* cultivate at 30 °C and 130 rpm for around 18 h

[ ]

21

Cultivation of transformed DH5alpha and BL21, PCR with new settings

|  |  |
| --- | --- |
| Aim / Task | Cultivation of transformed DH5alpha and BL21, PCR with new settings |
| Protocol |  |
| Participants | Eva Neugebauer, Daniel Schreyer |

**Cultivation for miniprep of pUC19:**

* no bacteria grew in the cultivation of 2019-07-20
* incubator did not heat up
* new clones picked: 5x DH5alpha, 3x BL21
* in 5 mL LB medium with Amp each
* incubation at 37 °C

**PCR overhang K1, K2a, K3a with DMSO:**

* primers used:
  + K1: fw Gib pAC Promotor fw 2.0, re Gib pACK1HisStop prim
  + K2a: fw Gib pAC Promotor fw 2.0, re Gib pAC K2a HisStop
  + K3a: fw Gib pAC Promotor fw 2.0, re Gib pAc K3a fusion re
* PCR program:
  + 98 °C 3 min
  + 98 °C 10 s
  + 53.7 °C 30 s
  + 72 °C 1 min
  + 72 °C 10 min
  + 35 cycles
* PCR products stored at 4 °C

[ ]

22

Overnight culture of transformed E. coli for control

|  |  |
| --- | --- |
| Aim / Task | Overnight culture of transformed E. coli for control |
| Protocol |  |
| Participants | Daniel Schreyer, Lisa Vogg |

* Picked 3 DH5alpha and 1 BL21 Heat shock puC19 transformed E.coli and incubated them over night in 2 ml LB+Amp at 37°C with 220 rpm
* OD measurements in the morning to validate transformation success

[ ]

22
**(2)**

Run test gel for new overhang PCR, mini prep of test transformation

|  |  |
| --- | --- |
| Aim / Task | Run test gel for new overhang PCR, mini prep of test transformation |
| Protocol | Extract me! Mini kit instructions |
| Participants | Felix Pfister, Lisa Vogg |

* 0.8 % gel with 2 µL DNA, 3 µL loading dye, 13 µL dH2o
* out of 8 cultivated colonies, only 2 showed bacterial growth
* performed mini prep for those two cultures (BL21 colonies 2 and 3)
* ran test gel for mini prep

**Protocol:**

1. Pellet cell from bacterial culture by centrifugation for 5 min at 3500g
2. discard supernatant and resuspend cell pellet thoroughly in 250 µl resuspension buffer. Transfer resuspended cell pellet to sterile 1.5 ml tube.
3. add 250 µl lysis buffer and invert tube 4-6 times. DO NOT VORTEX. If sample is not clear immediately, incubate for 1-2 min.
4. add 350 µl neutralization buffer and invert tube several times. DO NOT VORTEX.
5. centrifuge for 5 min at 11000 g. Repeat if supernatant is not clear.
6. Carefully transfer supernatant into a purification microcolumn placed in a collection tube. Do not contaminate sample with pellet! Centrifuge for 60 s at 11000 g.
7. Optional: add 500 µl NW buffer and centrifuge 60 s at 11000g.
8. add 750 µl wash buffer and centrifuge for 60 s at 11000g. Discard filtrate and reuse collection tube.
9. centrifuge for 2 min at 11000g. Discard collection tube and carefully transfer column to a sterile 1.5 ml tube.
10. add 50-100 µl elution buffer directly onto the column. Incubate at room temperature for 60 s.
11. centrifuge for 60 s at 11000g. Remove microcolumn. Isolated DNA should be stored at 4 or -20°C.

[ ]

23

OD of over night culture DH5alpa with pUC19 and BL21 with pUC19

|  |  |
| --- | --- |
| Aim / Task | OD of over night culture DH5alpa with pUC19 and BL21 with pUC19 |
| Protocol | - |
| Participants | Marie, Jannik |

## Protocol

**Notes**

* OD: DH5alpha with pUC19 = 2,79
* OD: BL21 with pUC19= 4,4
* Argarose gel for vector extraction for BL21
* 5 yL Eluate
* 3 yL dye
* 12 yL H2O
* 4 yL Ladder
* Cut lower band out
* Gel volume 230 mg
* Used Qiagen QiAquick gel-extraction kit50 (see Protocol)
* Used 80 yL EB-buffer to eluate
* PCR amplification of K1 with
  + 5 yL DNA
  + 1,25 yL primer re & fw
  + 12 yL Master-mix
  + 5 yL H2O

> BL21 pUC19 concentration: 6,53 ng/µL

**Protocol**

![<Beschreibung>](https://static.igem.org/mediawiki/parts/d/d3/T--FAU_ERLANGEN--45CC5796-285C-4552-9229-78437520F6DF.jpeg)

[ ]

24

Gibson assembly and transformation of K2a, K2b, K3a and K4a in pACYC in BL21

|  |  |
| --- | --- |
| Aim / Task | Gibson assembly and transformation of K2a, K2b, K3a and K4a in pACYC in BL21 |
| Protocol | Gibson assembly instructions, heat shock protocol |
| Participants | Jannik Wagner, Lena Schorr |

* measured DNA concentrations of all constructs
* calculated the amount of DNA and vector for the Gibson assembly
* Gibson assembly of 2a, 2b, 3a+b, 4a+b
* transformation of the constructs into BL21 via heat shock

  + 200 µL bacteria with 2 µL Gibson reagent
* preparation of chloramphenicol plates
* plated transformed bacteria –incubator at 37 °C

**Concentrations of DNA of the constructs:**

* K1: 808.9 ng/µL
* K2a: 868.7 ng/µL
* K2b: 847.2 ng/µL
* K3a: 791 ng/µL
* K3b: 819.1 ng/µL
* K4a: 776.1 ng/µL
* K4b: 805.9 ng/µL

**Lengths of the DNA constructs:**

* K1: 1629 bp
* K2a: 1278 bp
* K2b: 1218 bp
* K3a: 801 bp
* K3b: 753 bp
* K4a: 801 bp
* K4b: 759 bp

**Calculation of the amount of contruct and vector needed for the Assembly:**

* our constructs and vector in pmol/µl (with the formula: amount of DNA \* 1000 / bp of the constructs + 650 Da)
* K1: 1.04 pmol/µL
* K2a: 1.097 pmol/µL
* K2b: 1.46 pmol/µL
* K3a: 1.36 pmol/µL
* K3b: 1.49 pmol/µL
* K4a: 1.34 pmol/µL
* K4b: 1.46 pmol/µL
  –1:3 dilution with H2O –1 µL was taken for the Gibson Assembly (=0.37 pmol/µl for construct 2a)
  –>Vector (pACYC): same amount: ~0.4 pmol/µL

**Gibson Protocol:**

* 0.02-0.5 pmol/µL DNA
* 10 µL Gibson assembly master mix
* 10 µL dH2O

[ ]

25

Transformation of E.coli BL21 with Test Vector pUC

|  |  |
| --- | --- |
| Aim / Task | Transformation of E.coli BL21 with Test Vector pUC |
| Protocol | Transformation mit Ca2+ - kompetente E.coli - Zellen |
| Participants | Martin Borgmann, Marie Wiedemann |

| Name | Attempt 1 | Attempt 2 | Attempt 3 |
| --- | --- | --- | --- |
| DNA | 1 µg (=0,5 µl) | 2 µg (= 1 µl) | 1 µg (= µl) |
| NEB buffer 3.1 (x10 ) | 5 | 5 | - |

## Protocol

**Transformation of BL21 with Test vector pUC (Amp res.):**

* Protocol used is in folder page 1: Transformation mit Ca2+ - kompetente E.coli - Zellen

  + Nanodrop: 6,53ng/µL
  + 50ng DNA ( = 7,7µL) and 200 µL chemocompetent cells
  + heatshock 45s, 42 °C
* plate on Amp plates –test vector with Amp resitance
* incubate at 37°C overnight

**Result**: succesfull transfortmation since the bacteria grew on the Amp plate

Agarose gel electropheres of PCR products from the morning,

**Amplification PCR of Construct 1**

* Protocol from the PCR kit
* Q5 polymerase
* 4µL template
* Procedure:
  1. letting the cycler heat up and wait untilll actuall programm starts
  2. then put in the reaction tube

**Result:** negative

* either bad primers
* or because no DSMO was used

[ ]

26

Preparation of Media LB and SOC

|  |  |
| --- | --- |
| Aim / Task | Preparation of Media LB and SOC |
| Protocol |  |
| Participants | Martin Borgmann |

## Protocol

**Notes**

* Repeated PCR from this morning (26.07.19., entry 56) –Attempt to amplify K1 –not successful
  + 2µL Template
  + 25 µL x2 Q5 MasterMix
  + 2,5 µL FW Primer
  + 2,5 µL RV Primer
  + 2µL H2O
  + 16 µL 15% DSSO –~5% final conc.

**Prepartion of Media SOC and LB**

1L medium -25 g/L LB broth powder dissolved in dH2O

0,2 L SOB Medium for SOC Medium to make E.coli electrocompetent

* 1,25 g Trypton
* 5 g yeast extract
* 0,117 g NaCl

–autoclave 30 min 121 °C

Added the rest of the ingredients for SOC by Eva & Lisa the next day after the Medium cooled off (entry 63)

[ ]

26
**(2)**

Transformation of BL21 with Construct 2a, 2b, 3a + 3b, 4a + 4b in pACYC184 vector, Amplification of Construct 1 with original DNA

|  |  |
| --- | --- |
| Aim / Task | Transformation of BL21 with Construct 2a, 2b, 3a + 3b, 4a + 4b in pACYC184 vector, Amplification of Construct 1 with original DNA |
| Protocol | Transformation mit Ca2+ - kompetente E.coli Zellen (Ordner Seite 1) |
| Participants | Daniel Schreyer, Ruben Caspary |

**Notes**

* amplified 2µl original K1 DNA
* H20 Control

## Protocol

| DNA | 2µl |
| --- | --- |
| 2x MasterMix | 10µl |
| Primer fw 10µM | 1µl |
| Primer re 10µM | 1µl |
| DMSO 3% in dH20 | 6µl |

Program used **Construct Amplification (30 cycles):**

| Denaturation | 98°C | 10s |
| --- | --- | --- |
| Annealing | 51°C | 30s |
| Extension | 72°C | 60s |

* Control with 0.8% Agarose-Gelelectrophoresis
* 17 **µl K1 Amp remaining**

**Transformation of BL21 with Construct 2a, 2b, 3a + 3b, 4a + 4b in pACYC184 vector:**

* Protocol used is in folder page 1: Transformation mit Ca2+ - kompetente E.coli - Zellen
* Contructs used: 2a, 2b, 3a+b, 4a+b **2µl each**
* positive Control with **NEB pUC Control vector: used 10µl -500pg + purified pUC Vector from colony 3 –4µl: around 25ng DNA**
* plate on Amp plates: positive Controls
* plate on Chloramphenicol plates from **29.06.2019 Construc 2a, 2b, 3a + 3b, 4a + 4b**
* **Incubate in the afternoon or until tomorrow morning at 37°C**

**Problems faced / possible error causes**

* **Used old plates 29.06.2019**
* used SOC-Medium, which was opened on the 11.07.2019 –might be contaminated, even though medium was clear
* used maybe too much DNA Ladder

**Materials used**

* 3mL SOC-Medium
* NEB pUC Vector: 10µl
* 0,4 Agarose
* 10µl Quick 1kb DNA Ladder

**Pending tasks, next steps, comments for next people in the Lab**

* Control of Gelelectrophoresis under UV-Light
* autoclave SOC-Medium
* Pour new plates: Chloramphenicol and Ampicillin plates
* Check transformed bacteria on plates in the next morning 27.07.2019

[ ]

27

Preparation of Media

|  |  |
| --- | --- |
| Aim / Task | Preparation of Media |
| Protocol |  |
| Participants | Lisa Vogg, Eva Neugebauer |

## Protocol

**Notes**

1. 1% Gel

Sample from 26.07.2019 for the gel (2µL DNA, 3µL Stain, 13µL H2O)

**No bands visable!**

1. Each 2 colonies of K2a, K2b, K4ab and one colonie of K3ab

Incoluation overnight at 37°C in 5 mL LB with Camp

1. Finishing the SOC medium from th 26.06.2019 by adding 1 M MgSo4 and 0,72 g Glucose

**Pending tasks, next steps, comments for next people in the Lab**

Miniprep of clones

[ ]

28

Store transformed E.coli, K1 Gel Extraction

|  |  |
| --- | --- |
| Aim / Task | Store transformed E.coli, K1 Gel Extraction |
| Protocol |  |
| Participants | Eva Neugebauer, Daniel Schreyer |

## Protocol

**Notes**

* **No mini-prep columns left!! –Wait for new order.**
* Transformed E.coli are stored at 4°C and are waiting for their mini-prep **(4**.5 ml left)\*\*
* **Aliquoted 50% Glycerol in LB-medium in 15 ml Falcon**

**Storage of 500µl transformed E.coli at -80°C**

**Protocol:**

1. Mix 500µl vortexed transformed E.coli with 500µl 100% Glycerol solution
2. Different colonies are labeled 1 and 2. Some Construct transformed E.colis have 2 aliquots, because of 2 colonies picked
3. Store at -80°C

**K1 Gel Extraction:**

* Preparation for Blunt-End Cloning
* Extract K1 amplificat (11.07.2019) out of a 0.8% agarose gel
* Use 6µl of K1 and 15µl of H20 control –2µl of K1 are left for further PCRs
* Run at 110V for 90 minutes
* Plot: 4µl Ladder - empty - K1 - empty - H20

**Plated 200µl K3a+b on Agar-Chloramphenicol plates**

**Problems faced / possible error causes**

* Amplification PCR are not perfectly labeled. Unsure if it was K1 or Water control –Used both in our gel electrophoresis
* **NO K1 Amplificat left in tube! Gel Extraction was not possible**

**Materials used**

* 4µl DNA Ladder
* Gel extraction Kit (1 prep)
* 10ml Glycerol

**Pending tasks, next steps, comments for next people in the Lab**

* Blunt-End cloning of purified K1 in pACYC184
* Mini-prep of all our transformed E.coli
  + PCR to validate Gibson Assembly and transformation success.
* Pick colony of K3a+b and Incubate in Lb - Chloramphenicol medium for mini-prep

[ ]

30

Cultivation of K3ab for Mini-Prep

|  |  |
| --- | --- |
| Aim / Task | Cultivation of K3ab for Mini-Prep |
| Protocol |  |
| Participants | Daniel Schreyer |

## Protocol

**Notes**

* **Picked 2 colonies of K3ab plate (overnight plate from the 29.07.19)**
* Cultivate in Lb-Chloramphenicol medium

**Pending tasks, next steps, comments for next people in the Lab**

* Check growth on the 30.07.19
* Perform mini-prep
* Validate Insertion success of our Gibson Assembly

[ ]

31

Mini-Prep of the reagenz K2a (2x), K2b (2x), K3ab (3x), K4ab (2x) = 9 samples

|  |  |
| --- | --- |
| Aim / Task | Mini-Prep of the reagenz K2a (2x), K2b (2x), K3ab (3x), K4ab (2x) = 9 samples |
| Protocol | Protocol of the Kit "ExtractMe" |
| Participants | Martin Borgmann, Lena Schorr |

## Protocol

**Notes**

* first we down-centrifuged the over-night culture from K3ab
* We then put 500µl of K3ab together with 500µl of Glycerol in the freezer at -80°C (box at the bottom of rang 12)
* Then the Mini-Prep with the Extract gel was performed
* The DNA was then stored at 4°C

**Protocol**

Protocol of the Extract me Kit

**Calculations and Results**

**Problems faced / possible error causes**

We used 14ml overnight-culture, which was a little bit too much, because after the lysis there was a lot protein waste so we had to repeat this step several times

**Materials used**

* Glycerol
* Extract Me Kit

**Files used / generated**

**Pending tasks, next steps, comments for next people in the Lab**

* Measure DNA concentrations
* Performing a gel-electrophoresis with the isolated DNA (first cleave the DNA with restriction-enzymes)
* Sent a part of the isolated DNA to sequencing-company

[ ]

31
**(2)**

Concentration of Minipreps (K2a (2x), K2b (2x), K3ab (3x), K4ab (2x) = 9 samples)

|  |  |
| --- | --- |
| Aim / Task | Concentration of Minipreps (K2a (2x), K2b (2x), K3ab (3x), K4ab (2x) = 9 samples) |
| Protocol | Eva’s Digest Protokoll |
| Participants | Marie Wiedemann, Ruben Caspary |

## Protocol

**Notes**

* Nanodrop of all Mini-Preps
* Digest at 37° 350 rpm heat-block
  + 12 yL H2O
  + 5 yL Probe
  + 2 yL CutSmart Buffer
  + 0,5 yL Hind\_III
  + 0,5 yL BamHI-HF
* Concentrations similar to Eva’s vector preperation
* Gel, 14 lanes for vector-insert digest of:
  + K2a (2x colonie 1 & 2),
  + K2b (2x colonie 1 & 2),
  + K3ab (3x colonie 1,2,3),
  + K4ab (2x colonie 1 & 2 no Kol 3) = 9 samples

**Protocol**

**Calculations and Results**

| Construct |  | Colonie 1 | [c] ng/yL |  | Colonie 2 |  | Colonie 3 |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2a |  |  | 40,22 |  | 46,00 |  |  |
| 2b |  |  | 46,08 |  | 41,48 |  |  |
| 3ab |  |  | 62,29 |  | 36,44 |  | 32,29 |
| 4ab |  |  | 33,01 |  |  |  | 3,01 |

**Problems faced / possible error causes**

**Materials used**

CutSmart Buffer

Hind\_III

BamHI\_HF

TAE-Buffer

Agarose

**Files used / generated**

eluate gel, send Insert

**Pending tasks, next steps, comments for next people in the Lab**

## August

[ ]

1

Transformation colony screening with PCR

|  |  |
| --- | --- |
| Aim / Task | Transformation colony screening with PCR |
| Protocol | Standard PCR protocol |
| Participants | Daniel Schreyer |

* used PCR primers for amplification to screen for our inserted sequence
* forward primer the same for all the samples, used the respective reverse primers of each sample. For the ligated samples, the “b” primers were used.

| Template DNA | 5 µL |
| --- | --- |
| Primer fw (10 µM) | 1.25 µL |
| Primer rev (10 µM) | 1.25 µL |
| Master Mix | 12.5 µL |
| autoclaved H2O | 5 µL |
|  | 25 µL |

* checked success with a 1% agarose gel, run at 150 V for 1 h
* PCR was negative
  + too little amplification primers
  + wrong primers used
  + or no insert in our vector

[ ]

1
**(2)**

Transformation Colony Screening with PCR

|  |  |
| --- | --- |
| Aim / Task | Transformation Colony Screening with PCR |
| Protocol | Standard PCR Protocol |
| Participants | Daniel Schreyer |

| Name | Attempt 1 | Attempt 2 | Attempt 3 |
| --- | --- | --- | --- |
| DNA | 1 µg (=0,5 µl) | 2 µg (= 1 µl) | 1 µg (= µl) |
| NEB buffer 3.1 (x10 ) | 5 | 5 | - |

## Protocol

**Notes**

* Use PCR Primers for Amplification to screen for our inserted sequence
* Only if Primers can bind to our vector and PCR produces the right Insert size our sequence is in our vector and the colony can be used for dowstream experiments
* Use undiluted amplification primers, because we don’t trust the 100µM note.
  + We have enough Primers anyway
* used always the same forward Primer, but the reverse primer differed from construct to construct. Used always b re primers, if we assembled multiple constructs together
* **Used 2 x 2a, 2 x 2b, 3 x 3ab, 2 x 4ab –labeled them respectively**

**Gel Electrophoresis:**

* 1% Agarose gel. Run with 150 V for 1 hour

**Protocol**

**PCR:**

Primers:

fw: Tac Promoter fw 2.0 Primer

re: 2a: 2a re Primer, 2b: 2b re Primer, 3ab: 3b re Primer, 4ab: 4b re Primer

| Name | Volume |
| --- | --- |
| Template DNA | 5 µl |
| Primer fw (10µM) | 1.25 µl |
| Primer rev (10µM) | 1.25 µl |
| Master Mix | 12.5 µl |
| Autoclaved H2O | 5 µl |
| Total Volume | 25 µl |

**Problems faced / possible error causes**

* Too little amplification primers
* Wrong amplification primers used
* No Sequence in our vector

**Materials used**

* 100 µl 2x MasterMix
* 12 PCR Tubes and lid

[ ]

1
**(3)**

Control of the Gibson assembly

|  |  |
| --- | --- |
| Aim / Task | Control of the Gibson assembly |
| Protocol |  |
| Participants | Martin Borgmann, Jannik Wagner |

* restriction digest with Ase1 and Pvu1: Ase1 cuts once in the vector and once in the promoter region of the insert, Pvu1 was used to cut the insert, however it only cuts in construct 2a
  + 12 µL H2O
  + 5 µL DNA
  + 2 µL CutSmart Buffer 3.1
  + 0.5 µL Ase1
  + 0.5 µL Pvu1
* incubation for 1 h at 37 °C
* gel electrophoresis in 1% agarose in TAE with SYBR Safe Gel Stain

[ ]

1
**(4)**

Control if the Assemblys worked

|  |  |
| --- | --- |
| Aim / Task | Control if the Assemblys worked |
| Protocol | - |
| Participants | Martin Borgmann, Jannik Wagner |

## Protocol

**Notes**

* Restriction digest with Ase1 and Pcu1.

  + 12 µL H2O
  + 5 µL DNA
  + 2 µL CutSmart Buffer 3.1
  + 0,5 µL Ase1
  + 0,5 µL Pvu1

  -incubation 1h 37 °C

Gel Electrophoresis in 1% Agarose in TAE + SYBR green
**Calculations and Results**

Ase1 part of the vector and also part of each construct

-Ase1 cuts sticky ends -occurence of multimers

-Smallest piece is 329 kb, additinal bands due to multimers

-These bands can be found for every construct

Pvu1 cuts only 2a in two site

-many fragments from construct 2a

**Result**: The Construct had been inserted into the vector successfully!

**Problems faced / possible error causes**

Pvu1 was mistakingly used in an attempt to cut the insert. However, cutting site is only in 2a. Ase1 sufficient for the intepretation of the results.

**Pending tasks, next steps, comments for next people in the Lab**

confirm results with PCR of the insert

![<Control if the Assemblys worked>](https://static.igem.org/mediawiki/parts/d/d3/T--FAU_ERLANGEN--68ed1d2a-107d-4b25-a930-ffcaa0a7ade4.jpg)

[ ]

2

Check RE digestion of the miniprep, dissolved new DNA samples

|  |  |
| --- | --- |
| Aim / Task | Check RE digestion of the miniprep, dissolved new DNA samples |
| Protocol | - |
| Participants | Lisa Vogg, Leon Altmann |

## Protocol

**Notes**

* ran gel (2 µl plasmid DNA, 3 µl loading dye, 13 µl dH20)
* dissolved new DNA (CHO expression and measured part) in 20 µl TE buffer and stored at -20 °C
* DNA should be thoroughly resuspended before usage just in the case it is not already

[ ]

2
**(2)**

Check RE digestion of the miniprep

|  |  |
| --- | --- |
| Aim / Task | Check RE digestion of the miniprep |
| Protocol |  |
| Participants | Leon Altmann, Lisa Vogg |

* ran 1% agarose gel: 2 µL plasmid DNA, 3 µL loading dye, 13 µL dH20 at 130 V for 1 h
* dissolved newly arrived DNA for CHO expression and measurement part in 20 µL TE buffer, stored at -20 °C

[ ]

5

Gibson overhang PCR, pACYC184 EcoRV digest

|  |  |
| --- | --- |
| Aim / Task | Gibson overhang PCR, pACYC184 EcoRV digest |
| Protocol |  |
| Participants | Lena Schorr, Daniel Schreyer |

**Digest:**

* EcoRV digest with Eva’s protocol
* digest mini-prep from 2019-08-02
* concentration of pACYC184: 154 ng/µL
* digest 1.5 µg plasmid: 9.74 µL at 37 °C for 60 min

| CutSmart buffer | 5 µL |
| --- | --- |
| DNA | 9.74 µL |
| EcoRV-HF enzyme | 1.5 µL |
| Water | 33.76 µL |
|  | 50 µL |

* inactivate for 20 min at 65 °C
* run 0.8% agarose gel

**PCR:**

* PCR of constructs from 07-16 K1, K2a, K3a + DMSO control
* PCR of constructs from 07-04 K1, K2a, K2b, K3a, K3b, K4a, K4b + DMSO control
* used residual DNA of 2b, 3b, 4a, 4b from 07-16
* gel electrophoresis with 0.8% agarose gel

| DMSO (15%) | 5 µL |
| --- | --- |
| DNA | 5 µL |
| fw Primer | 1.25 µL |
| re Primer | 1.25 µL |
| Master Mix (2x) | 12.5 µL |
|  | 25 µL |

**Second PCR with 3% DMSO:**

* used overhang PCR products from 07-16 of K1, K2a, K3a

**Problems faced:**

* used wrong plasmid concentration! after mini-prep concentration of 50 ng/µL
* used 100% DMSO instead of 15% as H2O replacement in the first PCR

[ ]

6

Control of overhang PCR, purification of the cut pACYC184

|  |  |
| --- | --- |
| Aim / Task | Control of overhang PCR, purification of the cut pACYC184 |
| Protocol | Monarch DNA Gel Extraction Kit |
| Participants | Martin Borgmann, Lena Schorr |

* Control gel:
  + used 1% agarose gel for samples of the overhang PCR and the cut vector
  + run gel at 120 V for 1 h
* purification of the cut vector with **Monarch DNA Gel Extraction Kit:**

1. Excise the DNA fragment from the agarose gel, taking care to trim excess agarose.Transfer to a 1.5 ml microfuge tube, and weigh the gel slice. Minimize exposure to UV light.
2. Add 4 volumes of Gel Dissolving Buffer to the gel slice (e.g., 400 μl buffer per 100 μl or 100 mg agarose).
3. Incubate the sample between 37-55°C (typically 50°C), vortexing periodically until the gel slice is completely dissolved (generally 5-10 minutes). For DNA fragments 8 kb, an additional 1.5 volumes of water should be added after the slice is dissolved to mitigate the tighter binding of larger pieces of DNA (e.g., 100 μl gel slice: 400 μl Gel Dissolving Buffer: 150 μl water).
4. �Insert column into collection tube and load sample onto the column. Spin for 1 minute, then discard flow-through.
5. Re-insert column into collection tube. Add 200 μl DNA Wash Buffer and spin for 1 minute. Discarding flow-through is optional.
6. Repeat step 5.
7. Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column does not come into contact with the flow-through. If in doubt, re-spin for 1 minute.
8. Add ≥ 6 μl of DNA Elution Buffer to the center of the matrix. Wait for 1 minute, and spin for 1 minute to elute DNA. Typical elution volumes are 6-20 μl. Nuclease-free water (pH 7-8.5) can also be used to elute the DNA. Yield may slightly increase if a larger volume of DNA Elution Buffer is used, but the DNA will be less concentrated. For larger size DNA (≥ 10 kb), heating the elution buffer to 50°C prior to use can improve yield.

* measurement of purified vector: 4.9 ng/µL
* storage of purified cut vector at -20 °C
* digestion of purified cut vector with EcoRV-HF enzyme
* run gel with samples of the overhang PCR of K1, K2a, K3a from the previous day, the digested vector and uncut vector as control
* extraction of the digested vector of the gel, store at 4 °C

**Problems faced:**

* Control gel was negative, only K3a was visible in the overhang PCR
* Gel for purification was empty (broken slide), had to be repeated
* Second gel also negative except for K3a

[ ]

8

PCR of CFP part for measurement

|  |  |
| --- | --- |
| Aim / Task | PCR of CFP part for measurement |
| Protocol |  |
| Participants | Felix Pfister, Leon Altmann |

* eluted the Measurement Primers to a concentration of 100 µM
* used for amplification via PCR: annealing temperature 57 °C, elongation time 45 s

| Name | Volume |
| --- | --- |
| Primer fw (10 µM) | 1.25 µL |
| Primer rev (10 µM) | 1.25 µL |
| template DNA | 1 µL |
| Q5 2x Master Mix | 12.5 µL |
| Nuclease free H2O | 9 µL |

[ ]

8
**(2)**

Create good Gibson overhangs and perform Gibson Assembly

|  |  |
| --- | --- |
| Aim / Task | Create good Gibson overhangs and perform Gibson Assembly |
| Protocol | Q5 HiFi DNA-Pol NEB Protocol// Taq DNA-Pol NEB Protocol // NEBuilder HiFi DNA Assembly Kit Protocol |
| Participants | Antonio Maccataio, Jannik Wagner |

**PCR:**

* annealing temperature: 53.7 °C
* polymerase: Taq and Q5
* DNA: 2 µL
* nuclease-free water

**Gel:**

* 1% agarose gel in TAE buffer
* SYBR Safe Gel Stain: 7:100,000
* run at 120 V

**Analysis:**

* Taq PCR: total failure
* Q5: good amplification of 3a, 3b and 4b, rest: failure
* 3a and 3b measured by photometer
  + 500 ng 3a = 1,66µl (500ng/µl)
  + 500 ng 3b = 1,00µl (300ng/µl)
  + 370 ng pACYC = 2µl

[ ]

8
**(3)**

gel extraction of pSec Hygro Tag, CHO constructs K1, K2a, K2b

|  |  |
| --- | --- |
| Aim / Task | gel extraction of pSec Hygro Tag, CHO constructs K1, K2a, K2b |
| Protocol | Qiaquick Gel Extraction Kit |
| Participants | Felix Pfister, Leon Altmann |

* gel extraction of pSec Hygro Tag, and the CHO constructs K1, K2a, K2b
* Qiaquick Gel Extraction Kit:

1. Add 5 volumes of Buffer PB to 1 volume of the PCR sample, and then mix. It is not necessary to remove mineral oil or kerosene. For example, add 500 μl of Buffer PB to 100 μl PCR sample (not including oil).
2. If pH Indicator I has been added to Buffer PB, check that the mixture’s color is yellow. If the color of the mixture is orange or violet, add 10 μl of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn yellow.
3. Place a QIAquick spin column in a provided 2 ml collection tube.
4. To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s.
5. Discard flow-through. Place the QIAquick column back into the same tube. Collection tubes are reused to reduce plastic waste.
6. To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30–60 s.
7. Discard flow-through and place the QIAquick column back into the same tube. Centrifuge the column for an additional 1 min. IMPORTANT: Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation.
8. Place QIAquick column in a clean 1.5 ml microcentrifuge tube.
9. To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water (pH 7.0–8.5) to the center of the QIAquick membrane and centrifuge the column for 1 min. Alternatively, for increased DNA concentration, add 30 μl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge. IMPORTANT: Ensure that the elution buffer is dispensed directly onto the QIAquick membrane for complete elution of bound DNA. The average eluate volumes are 48 μl from 50 μl elution buffer volume and 28 μl from 30 μl elution buffer. Elution efficiency is dependent on pH. Maximum elution efficiency is achieved between pH 7.0 and 8.5. When using water, make sure that the pH value is within this range, and store DNA at –20°C because DNA may degrade in the absence of a buffering agent. The purified DNA can also be eluted in TE buffer (10 mM Tris·Cl, 1 mM EDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic reactions.
10. If the purified DNA is to be analyzed on a gel, add 1 volume Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting it up and down before loading the gel. Loading Dye contains 3 marker dyes – bromophenol blue, xylene cyanol and orange G – that facilitate estimation of DNA-migration distance and optimization of the agarose gel run time. Refer to Table 2 (page 17) to identify the dyes according to migration distance and agarose gel percentage and type.

[ ]

8
**(4)**

PCR of CHO constructs

|  |  |
| --- | --- |
| Aim / Task | PCR of CHO constructs |
| Protocol |  |
| Participants | Felix Pfister, Leon Altmann |

* eluted the CHO Primers to a concentration of 100 µM
* used for amplification via PCR: annealing temperature 57 °C, elongation time 45 s

| Name | Volume |
| --- | --- |
| Primer fw (10 µM) | 1.25 µL |
| Primer rev (10 µM) | 1.25 µL |
| template DNA | 1 µL |
| Q5 2x Master Mix | 12.5 µL |
| Nuclease free H2O | 9 µL |

* many unspecific bands, especially K1
* new agarose gel with 10 µL PCR product and gel extraction

[ ]

8
**(5)**

Digestion of pSec Hygro Tag

|  |  |
| --- | --- |
| Aim / Task | Digestion of pSec Hygro Tag |
| Protocol | Digest protocol of NEB |
| Participants | Felix Pfister, Leon Altmann |

* digested pSec Hygro Tag with NheI
* 15 min at 37 °C, then 20 min at 80 °C

[ ]

10

Heatshock of Gibson CHO-S (K1, K2a, K2b)

|  |  |
| --- | --- |
| Aim / Task | Heatshock of Gibson CHO-S (K1, K2a, K2b) |
| Protocol | NEB Heatshock |
| Participants | Felix Pfister, Jannik Wagner |

* made 15 LB-Agar plates with Ampicilin
* heatshock of the constructs of CHO constructs K1, K2a, K2b in BL21, BL21 Star, Tuner and DH5alpha, plus a positive and negative control in BL21 Star (=14 different transformations)
  + used 2 µL of the Gibson assembly
  + used SOC medium for the bacteria after the heatshock
* plated bacteria afterwards on the LB-Agar plates
* incubation at 37 °C

[ ]

12

Production of single colonies (Star; K1, K2a,K2b of 11.08); Preparation of new amp-plates

|  |  |
| --- | --- |
| Aim / Task | Production of single colonies (Star; K1, K2a,K2b of 11.08); Preparation of new amp-plates |
| Protocol |  |
| Participants | Felix Pfister, Lena Schorr |

[ ]

13

Miniprep of ON Culture & digestion; PCR clean-up

|  |  |
| --- | --- |
| Aim / Task | Miniprep of ON Culture & digestion; PCR clean-up |
| Protocol |  |
| Participants | Eva Neugebauer, Jannik Wagner |

* Miniprep of the samples:

  + K1-1 Tuner, K1-2 Tuner, K1-3 Tuner
  + K2a-1 Tuner, K2a-2 Tuner, K2a-3 Tuner
  + K2b-1 Tuner, K2b-2 Tuner, K2b-3 Tuner
  + K1 Star
  + negative control
* digestion of isolated DNA from Miniprep:

| Name | Volume |
| --- | --- |
| EcoRV | 1 µL |
| NheI | 1 µL |
| CutSmart Buffer | 5 µL |
| DNA | 1 µL |
| dH2O | ad 50 µL |

* incubation for 15 min at 37 °C
* PCR clean up of the samples:

  + K2a, K2b, K3a from 2nd amplification from 15.7 !! no clear description on tubes of 2nd amplification and no picture of gel ==possible error source !!
  + K3b, K4a, K4b from 1st amplification
  + end volume 50 µL

[ ]

14

PCR - Amplification, Overhang / Puc19 digest with Sma1 / Digested vector gel extraction / Mini-Prep CHO-S Parts

|  |  |
| --- | --- |
| Aim / Task | PCR - Amplification, Overhang / Puc19 digest with Sma1 / Digested vector gel extraction / Mini-Prep CHO-S Parts |
| Protocol |  |
| Participants | Eva Neugebauer, Daniel Schreyer |

**PCR Amplification and overhang:**

* used every construct besides K1 from clean up

**pUC19 digest:**

* digest for 1 h with Sma1 at 25 °C in CutSmart Buffer
* 5 µL pUC19 (Bl21 K3 from gel 07-23)
* 2 µL restriction enzyme
* pUC19 gel extraction: 0.8% agarose gel electrophoresis with undigested pUC19 as control
* no pUC19 was digested! BL21 K3 is empty!

**Miniprep from the ON cultures 2019-08-13:**

* extract-me miniprep kit used
* nanodrop measurements are on the respective tubes
* control digestion with Pst1
  + used 500 ng of each miniprep
  + used 1 µL Pst1-HF and 5 µL CutSmart Buffer
  + stopped reaction with 10 µL 6x loading dye and used 20 µL for gel electrophoresis

[ ]

15

Control Digestion of pSec/Tag2/Hygro

|  |  |
| --- | --- |
| Aim / Task | Control Digestion of pSec/Tag2/Hygro |
| Protocol | - |
| Participants | Jannik Pfister, Felix Wagner |

* Did single enzyme digestion of pSec
* EcoRI –2 sites
* EcoRV –1 site
* NheI –1 site
  \*See picture attachment for estimation and results

|DNA |pSec + EcorI |Laddder|pSec + EcorV|pSec + NheI|
|—|—|—|
|

![<Hygro digestion with EcoRV,EcoRI,NheI>](https://static.igem.org/mediawiki/parts/d/d3/T--FAU_ERLANGEN--WhatsApp_Image_2019-08-15_at_14.53.19.jpeg)

[ ]

15
**(2)**

Gel extraction of Gibson Overhang PCR (K2a -K4b) (from 14.08)

|  |  |
| --- | --- |
| Aim / Task | Gel extraction of Gibson Overhang PCR (K2a -K4b) (from 14.08) |
| Protocol |  |
| Participants | Felix Pfister, Lena Schorr |

* no bands in K2b and K3a
* many different bands in K2a
* K3b - K4b look good
  –gel extraction only of K2a, K3b, K4a, K4b

[ ]

15
**(3)**

pSec+CHO (K1, K2a K2b) / Gel extraction

|  |  |
| --- | --- |
| Aim / Task | pSec+CHO (K1, K2a K2b) / Gel extraction |
| Protocol |  |
| Participants | Felix Pfister, Lena Schorr |

* used 2x 20 µL of the miniprep of the vector+construct
* did not start the gel extraction
* ran agarose gel
* gel extraction only of K1E1-1, K1E1-2, K2aE1-3
* gel weight for the extraction:
  + K1E1-1: 0.17 g
  + K1E1-2: 0.16 g
  + K2aE1-3: 0.12 g

[ ]

15
**(4)**

pUC19 / MiniPrep / Nanodrop / Gel-Extraction / Dephosphorylation Test / Heat-Shock

|  |  |
| --- | --- |
| Aim / Task | pUC19 / MiniPrep / Nanodrop / Gel-Extraction / Dephosphorylation Test / Heat-Shock |
| Protocol |  |
| Participants | Felix Pfister |

* Miniprep: used 30 µL MiliQ for eluation
* Nanodrop:
  + colony 1: 286 ng/µL
  + colony 2: 283 ng/µL
  + colony 3: 306 ng/µL
* gel weight for the extraction:
  + pUC19 1: 0.31 g
  + pUC19 2: 0.23 g
  + pUC19 3: 0.25 g

[ ]

17

Produce new Amp+ Agar Plates/ Measure Miniprep of pUC19/ Digest,De-phosphorylate pUC19 with SmaI/ Seed new single colonies from Gibson CHO-S K1, K2a, K2b

|  |  |
| --- | --- |
| Aim / Task | Produce new Amp+ Agar Plates/ Measure Miniprep of pUC19/ Digest,De-phosphorylate pUC19 with SmaI/ Seed new single colonies from Gibson CHO-S K1, K2a, K2b |
| Protocol |  |
| Participants | Marie Wiedemann, Leon Altmann |

## Protocol

***Notes***

**Prepare new Amp plates:**

Prepare 500ml LB-Broth with Agar (17,5 g Agar) for 19 new colony plates;

Autoclave the Mixture to make it sterile;

After a cooling phase Amp got added (500 µl –concentration: 50 µg/ml);

Pour the plates under sterile conditions and put them in the refrigerator @4 °C;

For the digestion of pUC19 with SmaI the miniprep samples were measured in the nanodrop;

| Sample Name | 260 | 280 | 260⁄280 | ng/µl |
| --- | --- | --- | --- | --- |
| pUC19 c1 | 1,143 | 0,606 | 1,89 | 57,15 |
| pUC19 c2 | 1,426 | 0,873 | 1,63 | 71,30 |
| pUC19 c3 | 1,073 | 0,533 | 2,01 | 53,65 |

As the measurement diverged greatly from the measurement the day before that was done right after the miniprep, but with out further purification two approaches were prepared. One with the old nanodrop results from the day before and one with the new results. It is thought that the approach from the day before won’t have the right amount of Vector inside but it is prepared either way to be on the safe side.

Approach one:

| Name | Amount [µl] |
| --- | --- |
| DNA | 3,3 |
| CutSmart x10 NEB | 5 |
| Arctic Buffer x10 NEB | 5 |
| Arctic Phosphatase NEB | 1 |
| SmaI NEB | 0,5 |
| H2O to 50µl | 35,2 |

Approach two:

| Name | Amount [µl] |
| --- | --- |
| DNA | 14 |
| CutSmart x10 NEB | 5 |
| Arctic Buffer x10 NEB | 5 |
| Arctic Phosphatase NEB | 1 |
| SmaI NEB | 0,5 |
| H2O to 50µl | 24,5 |

Incubate at 37°C for 1 h;

Inactivate at 80 °C for 20 min (80 °C necessary to inactivate the phosphatase, SmaI itself 65 °C for 20 min would be sufficient);

**Preparation of 0,8 % Agarose Gel:**

Weigh in 0,8g Agarose and solve in 100ml TAE-buffer –solve Agarose by microwaving;

After cooling adding of SybrGreen 7 µl;

**Preparation of Single Colonies from CHO-S K1, K2a, K2b**

For each of the three constructs three colonies were picked and carefully plated under sterile conditions onto the Agar (Amp+) Plates that were earlier prepared;

**Problems faced / possible error causes**

The Gel did not show a clear enough band to extract the digested vector. This is due incubation temperature (37 °C);
SmaI does only require 25 °C to cut properly and shows only 50 % performance at 37 °C;

**Pending tasks, next steps, comments for next people in the Lab**

* **Measurement part!: Repeat digestion (SmaI) and dephosphorylation of puc19 at 25 °C (Stored at 4 °C);**
* **Control: Gel electrophoresis after digest;**
* **Perform Gibson Assembly puc19 + CFP;**
* **Check Plates seeded today (Incubator at 37 °C);**
* **New Gibson Assembly with K1, K2a, K2b (important)**

![<pUC19>](https://static.igem.org/mediawiki/parts/d/d3/T--FAU_ERLANGEN--pUC19_SmaI_-_Benchling.png)

[ ]

19

Miniprep of Gibson CHO Constructs (ON Cultures 18.08) / Preperation for MidiPrep of pUC19 /Control digestion of Gibson CHO Constructs

|  |  |
| --- | --- |
| Aim / Task | Miniprep of Gibson CHO Constructs (ON Cultures 18.08) / Preperation for MidiPrep of pUC19 /Control digestion of Gibson CHO Constructs |
| Protocol | - |
| Participants | Felix Pfister |

## Protocol

**Notes**

* Did a MidiPrep of ON Cultures from 18.08
  + Concentration:

Agarose-Gel electrophoresis for validation

| Name | K1-1 | K1-2 | K1-3 | K2a-1 | K2a-2 | K2a-3 | K2b-1 | K2b-2 | K2b-3 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Conc [ng/µl] | 33,9 | 31,9 | 27,4 | 38,7 | 31,7 | 27,4 | 28,5 | 44,4 | 26,1 |

| Component | Amount |
| --- | --- |
| DNA | 3 µl |
| 6X Loading Dye | 2 µl |
| dH2O | 1 µl |

-Same Loading order as NanoDrop concentration measurment

* Did an ON Culture of a single conlony of pUC19 (18.08)

  + Picked one colony and incubated it in 5 ml LB+Amp in the shaker @37°C
  + In the late afternoon –1:500 in 85ml LB+Amp –shake for 12-16h @37°C
* Control digestion of CHO Constructs

  + Restriction enzymes: ApaI and HindIII-HF

| Component | Amount |
| --- | --- |
| DNA | 300 ng |
| ApaI | 1 µl |
| HindIII-HF | 1 µl |

| CutSmart Buffer | 5 |
| --- | --- |
| dH2O | ad 50 µl |

* - Protocol
  - Incubate ApaI at 25°C for 5-15 minutes
  - Add 1.0 µl (or 10 units) of HindIII-HF and Incubate at 37°C for 5-15 minutes
  - Inactivate both at 80°C for 20 minutes

**Problems faced / possible error causes**

* Low concentration of construc –more LB+amp Medium next time with longer incubation time in the shaker?

**Pending tasks, next steps, comments for next people in the Lab**

* MidiPrep of ON Culture of pUC19
* Empty constructs
* repeat gibson

![<Beschreibung>](https://static.igem.org/mediawiki/parts/c/c4/T--FAU_ERLANGEN--Unbenannt.png-with-annotations.png)
![<Beschreibung>](https://static.igem.org/mediawiki/parts/5/5d/T--FAU_ERLANGEN--TS05376Aug192019.jpg)
![<Beschreibung>](https://static.igem.org/mediawiki/parts/6/6e/T--FAU_ERLANGEN--TS05375Aug192019.jpg)

[ ]

19
**(2)**

New Attempts for pUC19 Digest/ Heatshock pSEC and ON culture/ ON cultures from K1,K2a,K2b

|  |  |
| --- | --- |
| Aim / Task | New Attempts for pUC19 Digest/ Heatshock pSEC and ON culture/ ON cultures from K1,K2a,K2b |
| Protocol | - |
| Participants | Marie Wiedemann, Leon Altmann |

## Protocol

**Notes**

**Check the Plates from 17.8.19 CHO-S Constructs K1, K2a, K2b:**

On all of the plates the seeding worked fine and clones can be picked later on for ON cultures;

**New Attempt to digest pUC19:**

| Name | Attempt 1 [µl] | Attempt 2 [µl] |
| --- | --- | --- |
| DNA | 17,5 (pUC19 c1) | 18,6 (pUC19 c3) |
| CutSmart x10 | 5 | 5 |
| AntarcticBuffer x10 | 5 | 5 |
| Antarctic Phosphatase | 1 | 1 |
| SmaI | 0,5 | 1 |
| H2O | 21 | 19,4 |

The first attempt was @25 °C for 1 hour;

Inactivation @ 80 °C for 20 min;

Second attempt incubation @37 °C for 1 hour;

Inactivation @ 80 °C for 20 min;

Both of the samples were to find better conditions as the digest from 17.8.19 were not optimal;

25 °C is the optimal temperature for SmaI, whereas 37 °C is the optimum for the phosphatase;

With 1 hour incubation time also the 25 °C should be suitable for the phosphatase and likewise the 37 °C are acceptable for SmaI, given that the efficacy of the digest is lowered by approx 50% (information on the NEB site);

None of the Digests showed optimal results;

Both were looked at in an 0,8% agarose gel, each for their own.

The order looked like the following:

|1kb DNA Ladder (5µl)|Half Sample (30µl):5 µl Staining|25 µl Sample|Half Sample (30 µl):5 µl Staining25 µl Sample|

Attempt 1 of todays pUC19 digest did show a similar result to yesterdays digestion

Expected was one band of 2,7 kb length (see picture from virtual digest 17.8.19);

The suspected bands were cut out and extracted from the gel (QiaQuick Gel Extraction Kit);

Sample 1: 0,36 g; Sample2: 0,3 g;

–1060 µl QG; –900 µl QG

Protocoll of the Kit was used, the DNA was solved in 50 µl H2O;

The resulting DNA was measured in the Nanodrop;

| Sample Name | 260 | 280 | 260⁄280 | ng/µl |
| --- | --- | --- | --- | --- |
| Attempt 1 first half | 0,026 | 0,030 | 0,87 | 1,29 |
| Attempt 1 second half | 0,033 | 0,041 | 0,81 | 1,66 |

With these low DNA concentrations Gibson Assembly is not feasible without increasing the reaction volume substantially;
Therefor one should ask one of the TAs for advise to increase the DNA concentrationin a reliable way;

**ON culture of Single Colonies from CHO-S K1, K2a, K2b**

For each of the three constructs three colonies were picked and carefully seeded in 5 ml LB-Medium (Amp+) under sterile conditions;

Placed in the incubator @ 37 °C 130 rpm (time approx. 18:30);

**Heatshock pSec:**

Stored pSec has a concentration of 2 µg/µl –a 1:10 dilution was performed to pipette 100 ng of vector DNA for the heatshock procedure;

The 100 ng of pSec vector DNA were used for 200 µl DH5a;

Evas’ protocoll was used;

A plate of LB-Agar (Amp+) was seeded and put in the incubator @ 37 °C (time approx. 18:30);

**Problems faced / possible error causes**

Gel bands did indicate a non optimal digest –Not suspected bands and maybe also religation??;

The other digestion Attempt worked as bad as the first or the one from 17.8.19;

Not sure which of the bands is the actual cut vector as one bamd is visible at approx. 3kb, one band looks more like 2 kb and maybe there is a very thin band at 2,7 kb which would be the expected position;

Ask TA with the gel photo for advise and maybe also advise for a proper digestion –Might be that it was not to few but rather to much enzyme??

Also DNA purification from the Gel yielded only an extremly low amount of DNA –Gibson not performable;

**Pending tasks, next steps, comments for next people in the Lab**

Take the overnight cultures of Gibson CHO K1, K2a, K2b out of the incubator

Take the plate from pSec out of the incubator

Mini-Prep of pSec & freeze

Speak to the TA or PhD: Discuss the gel bands of the digests & how to increase the concentration of pUC19 digest 2 to continue with gibson assembly

Gibson assembly of pUC19 and measurement part (has conc of 1000 ng in 20 µl according to TAs advice

Heatshock of pUC19 and measurement part in competent E.coli

Generate more pUC19 vector

![<pUC19 Digest>](https://static.igem.org/mediawiki/parts/b/bb/T--FAU_ERLANGEN--Verdau_2_18.8_pUC19_SmaI_Dephos.jpeg)
![<pUC19 Digest>](https://static.igem.org/mediawiki/parts/e/eb/T--FAU_ERLANGEN--Verdau_3_18.8_pUC19_SmaI_Dephos.jpeg)

[ ]

20

Midi-prep of overnight-culture from DH5a with puc19; Digestion of puc19 with SmaI at 37°C

|  |  |
| --- | --- |
| Aim / Task | Midi-prep of overnight-culture from DH5a with puc19; Digestion of puc19 with SmaI at 37°C |
| Protocol | Midi prep protocol |
| Participants | Lena Schorr |

## Protocol

**Notes**

* **performance of a midi prep of the overnight culture of DH5a with puC19**

  + 2 x 25ml from overnight culture
  + See procotol below
  + 467,5 ng/µl (total: 400µl)
  + Storage in -20°C in 15ml falcon
* **Digestion of the puC19 vector with SmaI under new conditions**

  + all materials in the fridge of TW (-20°C)
  + digestion at 37°C for 1 h
  + Inactivation at 80°C for 20 minutes
  + Storage at -20°C (annotated with attempt 1; attempt 2)

| Name | Attempt 1 [µl] | Attempt 2 [µl] |
| --- | --- | --- |
| DNA | 10µl (=6µg) | 20µl (=12µg) |
| CutSmart x10 | 5 | 5 |
| AntarcticBuffer x10 | 5 | 5 |
| Antarctic Phosphatase | 1 | 1 |
| SmaI | 0,5 | 0,5 |
| H2O | 28,5 | 18,5 |

**Protocol Midi:**

1. Harvest overnight bacterial culture by centrifuging at 6000 x g for 15 min at 4°C. (at the fuge in the Behrensen Lab!!!)
2. Resuspend the bacterial pellet in 4 ml Buffer P1.
3. Add 4 ml Buffer P2, mix thoroughly by vigorously inverting 4–6 times and incubate at room temperature (15–25°C) for 5 min. If using LyseBlue reagent, the solution will turn blue.
4. Add 4 ml Buffer P3, mix thoroughly by vigorously inverting 4–6 times. Incubate on ice for 15 min. If using LyseBlue reagent, mix the solution until it is colorless.
5. Centrifuge at ≥20,000 x g for 30 min at 4°C. Re-centrifuge the supernatant at ≥20,000 x g for 15 min at 4°C.
6. Equilibrate a QIAGEN-tip 100 by applying 4 ml Buffer QBT, and allow column to empty by gravity flow.
7. Apply the supernatant from step 5 to the QIAGEN-tip and allow it to enter the resin by gravity flow.
8. Wash the QIAGEN-tip with 2 x 10 ml Buffer QC. Allow Buffer QC to move through the QIAGEN-tip by gravity flow.
9. Elute DNA with 5 ml Buffer QF into a clean 15 ml vessel. For constructs larger than 45 kb, prewarming the elution buffer to 65°C may help to increase the yield.
10. Precipitate DNA by adding 3.5 ml roomtemperature isopropanol to the eluted DNA and mix. Centrifuge at ≥15,000 x g for 30 min at 4°C. Carefully decant the supernatant.
11. Wash the DNA pellet with 2 ml room-temperature 70% ethanol and centrifuge at ≥15,000 x g for 10 min. Carefully decant supernatant.
12. Air-dry pellet for 5–10 min and redissolve DNA in a suitable volume of appropriate buffer (e.g., TE buffer, pH 8.0, or 10 mM Tris·Cl, pH 8.5).

**Pending tasks, next steps, comments for next people in the Lab**

* 0,8% agarose gel of the digested puc vector (attempt 1 and 2)
* Performing gibson assembly with construct 1, and 2a+b in psec for CHOs

[ ]

21

Digestion of pSEC with EcoR5 and NHeI for Gibson Assembly, agarose gel (0,8%) for control of digestion of pSec and pUC19, Extraction of digested pSEC vector from gel

|  |  |
| --- | --- |
| Aim / Task | Digestion of pSEC with EcoR5 and NHeI for Gibson Assembly, agarose gel (0,8%) for control of digestion of pSec and pUC19, Extraction of digested pSEC vector from gel |
| Protocol | Extraction protocol from Qiagen |
| Participants | Lena Schorr |

## Protocol

**Notes**

* Digestion of pSEC with EcoR5 and NHE1

| Name | Attempt 1 [µl] | Attempt 2 [µl] | Attempt 3 [µl] |
| --- | --- | --- | --- |
| DNA | 1 µg (=0,5µl) | 2 µg (=1µl) | 1 µg (=0,5µl) |
| NEB buffer 3.1 (x10) | 5 | 5 | - |
| Cut-Smart (10x) | - | - | 3 |
| EcoR5 | 1µl | 1µl | 1µl |
| NHE1 | 1µl | 1µl | 1µl |
| H2O | 42,5µl | 42µl | 42µl |

* attempt 1 and 2:
  + 15 minutes on 37°C and stop reaction by adding 10 µl 6 x loading dye
* attempt 3:
  + 60 minutes on 37°C and inactivation at 80°C

Preparation of a 0,8% agarose gel

* scheme see below
* 25 µl of the samples and 5µl ladder were used
* gel run for 45 minutes at 120 V

Extraction of line 7 (digested pSEC vector with EcoRV and NheI)

* Extraction kit was used
* sample stored at -20°C

**Pending tasks, next steps, comments for next people in the Lab**

* new digestion of puC19 and maybe pSEC
* Gibson Assembly of CHO constructs in pSEC and iGEM Part in Puc18
* Heat Shock

![<Beschreibung>](https://static.igem.org/mediawiki/parts/d/d3/T--FAU_ERLANGEN--Puc_and_pSEC_digestion_prediction.PNG)
![<Beschreibung>](https://static.igem.org/mediawiki/parts/d/d3/T--FAU_ERLANGEN--digestion_puc19_and_pSEC.png)

[ ]

22

Digestion of pSEC with EcoR5 and NHeI for Gibson Assembly, agarose gel (0,8%) for control of digestion of pSec and pUC19, Extraction of digested pSEC vector from gel

|  |  |
| --- | --- |
| Aim / Task | Digestion of pSEC with EcoR5 and NHeI for Gibson Assembly, agarose gel (0,8%) for control of digestion of pSec and pUC19, Extraction of digested pSEC vector from gel |
| Protocol | Extraction protocol from Qiagen |
| Participants | Leon Altmann |

## Protocol

**Notes**

Digestion of pSEC with EcoR5 and NHE1

| Name | Amount [µl] |
| --- | --- |
| DNA | 1 µl (=2 µg) |
| Cut-Smart (10x) | 5 |
| EcoR5 | 0,5 |
| NHE1 | 0,5 |
| H2O | 43 |

* 60 minutes @ 37°C
* 20 minutes inactivation @ 80°C

Digestion of pUC19 with SmaI + Antarctic Phosphatase

| Name | Amount [µl] |
| --- | --- |
| DNA | 10,6 µl (of Midiprep) |
| Cut-Smart (10x) | 5 |
| Antarctic Buffer x10 | 5 |
| Antarctic Phosphatase | 1 |
| EcoR5 | 0,5 |
| H2O | 27,9 |

* 60 minutes @ 37°C
* 20 minutes inactivation @ 80°C

Preparation of a 0,8% agarose gel

* scheme see below
* 25 µl of the samples + 5 µl of 6x loading dye
* 5µl ladder
* 1h @130V
* Extraction of line 5, 6 (digested pSEC vector with EcoRV and NheI)
* QiaGen Gelextraction kit was used
* sample stored at -20°C

**Pending tasks, next steps, comments for next people in the Lab**

* new digestion of pUC19 better would be to ask wether they have pUC19 stored somewhere as ours is out of bounds
* Gibson Assembly of CHO constructs in pSEC and potentially pUC19
* Heat Shock

[ ]

23

Gibson Assembly of CHO K1, K2a, K2b and measurement part in pSec; Heatshock in DH5a bacs; Gel electrophoresis of Gibson Assembly Samples

|  |  |
| --- | --- |
| Aim / Task | Gibson Assembly of CHO K1, K2a, K2b and measurement part in pSec; Heatshock in DH5a bacs; Gel electrophoresis of Gibson Assembly Samples |
| Protocol | Gibson Assembly; Heatshock; Gel electrophoresis |
| Participants | Marie Wiedemann |

## Protocol

**Notes**

**Gibson Assembly**

According to 2.8.19: CHO constructs and measurement part were taken up in 20 µl TE buffer. –concentration: 50ng/µl

concentration pSec: 26,8 ng/µl

using 50 ng pSec 1,86 µl

using insert three-fold: 150 ng ~3 µl ; used inserts: CHO K1, CHO K2a, CHO K2b, measurement part

also performed a positive control with the provided kit.

| Amount in µl | Name |
| --- | --- |
| 1,86 | Vector pSec |
| 3 | Insert |
| 10 | Gibson Assembly Master Mix |
| 5,14 | dH2O |

Incubated at 50 °C in thermocycler for 30mins.

**Heatshock**

performed a heatshock with the Gibson Assembly with DH5a bacteria, following Evas protocoll. Used 18 µl of the assembly samples.

Plated the bacteria on Amp plates and put in incubator to be continued with tomorrow.

**Gel electrophoresis**

prepared a 0.8% agarose gel with 0.8 g agarose in 100 ml TE buffer. microwaved and added SybrGreen after short cooling period.

Mixed 2 µl of DNA sample with 2 µl of DNA Purple Dye and 16 µl of water.

Added 7 µl DNA Ladder

Order: wayward DNA Ladder - empty - DNA Ladder - CHO K1 - CHO K2a - CHO K2b - Measurement Part - Positive control

**Problems faced / possible error causes**

Nothing just DNA ladder visible on gel. Might have used not enough DNA sample?

**Pending tasks, next steps, comments for next people in the Lab**

* take plates out of incubator, pick colonies
* find a new pUC19 vector to try

[ ]

27

Isolation of DNA (CHO constructs) from overnight culture

|  |  |
| --- | --- |
| Aim / Task | Isolation of DNA (CHO constructs) from overnight culture |
| Protocol | Extract Me - for mini prep |
| Participants | Lena Schorr |

## Protocol

**Notes**

* BACS stock of
  + CHO K1, clone 2
  + CHO K1, clone 1
  + CHO K2a, clone 4
  + DH5a positive control
    - storage at -80°C (in box of electrocompetent tuner and DH5a)
* Mini-prep of overnight culture
  + eluation with 50µl MiliQ water

**Pending tasks, next steps, comments for next people in the Lab**

* Measerment of the DNA concentrations
* gel of Mini-Prep (did Gibson work?)
* Digestion of puC19 with ecoRV
* Gibson Assembly
* sent CHOs to Kiel

[ ]

28

Mini Prep from ON cultures (K1,K2a, K2b), Gel electrophoresis, PCR

|  |  |
| --- | --- |
| Aim / Task | Mini Prep from ON cultures (K1,K2a, K2b), Gel electrophoresis, PCR |
| Protocol | Extract Me - Plasmid Preparation Kit |
| Participants | Leon Altmann |

## Protocol

**Notes**

* Mini-prep of overnight cultures (K1, K2a, K2b) of each 5 clones
  + eluation with 50µl MiliQ water
* Ran a 0,8% Gel
* Measured Plasmid DNA at Nanodrop

| Sample Name | 260 | 280 | 260⁄280 | ng/µl |
| --- | --- | --- | --- | --- |
| K1 c1 | 0,525 | 0,293 | 1,79 | 26,25 |
| K1 c2 | 1,408 | 0,748 | 1,88 | 70,42 |
| K1 c3 | 1,088 | 0,619 | 1,76 | 54,39 |
| K1 c4 | 1,275 | 0,694 | 1,84 | 63,77 |
| K1 c5 | 1,404 | 0,760 | 1,85 | 70,21 |
| K2a c1 | 1,809 | 0,983 | 1,84 | 90,45 |
| K2a c2 | 1,503 | 0,830 | 1,81 | 75,14 |
| K2a c3 | 1,214 | 0,665 | 1,83 | 60,72 |
| K2a c4 | 1,241 | 0,649 | 1,91 | 62,07 |
| K2a c5 | 1,227 | 0,658 | 1,86 | 61,37 |
| K2b c1 | 0,409 | 0,239 | 1,71 | 20,45 |
| K2b c2 | 1,024 | 0,562 | 1,82 | 51,18 |
| K2b c3 |  |  |  |  |
| K2b c4 | 1,222 | 0,649 | 1,88 | 61,12 |
| K2b c5 | 0,960 | 0,476 | 2,02 | 47,99 |

Performed a PCR with all of the Minis to confirm bands that are potentially depicting vector + insert

| Name | K1 c1 | K1 c2 | K1 c3 | K1 c4 | K1 c5 | K2a c1 | K2a c2 | K2a c3 | K2a c4 | K2a c5 | K2b c1 | K2b c2 | K2b c3 | K2b c4 | K2b c5 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| CHO-S fw | 1,25 | 1,25 | 1,25 | 1,25 | 1,25 | 1,25 | 1,25 | 1,25 | 1,25 | 1,25 | 1,25 | 1,25 | 1,25 | 1,25 | 1,25 |
| CHO-S Primer re | 1,25 | 1,25 | 1,25 | 1,25 | 1,25 | 1,25 | 1,25 | 1,25 | 1,25 | 1,25 | 1,25 | 1,25 | 1,25 | 1,25 | 1,25 |
| DNA | 3,8 | 1,4 | 1,8 | 1,6 | 1,4 | 1,1 | 1,3 | 1,6 | 1,6 | 1,6 | 4,9 | 2 |  | 1,6 | 2,1 |
| Water | 6,2 | 8,6 | 8,2 | 8,4 | 8,6 | 8,9 | 8,7 | 8,4 | 8,4 | 8,4 | 5,1 | 8 |  | 8,4 | 7,9 |
| Q5 Hifi MM 2x | 12,5 | 12,5 | 12,5 | 12,5 | 12,5 | 12,5 | 12,5 | 12,5 | 12,5 | 12,5 | 12,5 | 12,5 | 12,5 | 12,5 | 12,5 |
| Total | 50 | 50 | 50 | 50 | 50 | 50 | 50 | 50 | 50 | 50 | 50 | 50 | 50 | 50 | 50 |

Primer for this PCR are stored at -20°C the original primer tubes have a concentration of 100µM. Besides them there are also Eppis with the ready to pcr concentration of 10µM;

**Problems faced / possible error causes**

Not a real problem but teh bands of corerct lenght were quite weak in comparison to what is probably just oure vector at 5,7kb;
To tackle this issue PCR was performed. If the PCR is successfull, then Samples with vector + Insert will show a band of the corresponding inserts length in a gel;

**Materials used**

Q5 Hifi Polymerase **empty!**

**Pending tasks, next steps, comments for next people in the Lab**

* Perform Gel with PCR Products;
* Send positive ones to Kiel (call Matthias Peipp beforehand)
* Furhter try to make the Measurement Part work –Puc digest or also possible to open any other vector blunt and clone the part behind a promotor
* If successfull heatshock
* Plate out

[ ]

29

Gel of PCR, Digest of pUC19 (EcoRI, HindIII), ON cultures, Gel electrophoresis of unclear samples

|  |  |
| --- | --- |
| Aim / Task | Gel of PCR, Digest of pUC19 (EcoRI, HindIII), ON cultures, Gel electrophoresis of unclear samples |
| Protocol | - |
| Participants | Felix Pfister, Leon Altmann |

## Protocol

**Notes**

The PCR samples from 28.8 were applied to two 0,8% Agarose Gel and ran at 130V for 1 hour.

Gel 1:

Ladder K1 c1 K1 c2 K1 c3 K1c4 K1 c5 K2a c1 K2a c2 K2a c3 K2a c4 K2a c5

Gel 2:

Ladder K2b c1 K2b c2 K2b c3 K2b c4 K2b c5 Control

The PCR was performed to see whether the desired Insert was implemented into the vector, as the Gel from 28.8 allowed no precise determination.

The PCR Water-control showed a clear band at approximately 1,2 kb. This band was also seen in every other samples, hinting towards contamination of the water or PCR mix.

Other than that, the PCR samples did not show bands that would allow the assumption of successful Gibson assembly. Probably all the vectors are empty.

Another Digestion of pUC19 was performed to exclude potential problems with the blunt cutting enzymes due to re-ligation.

EcoRI and HindIII were used and were expected to yield a 2,6 kb and a 0,1 kb fragment.
Of EcoRI and HindIII a 1:10 Dilution was performed for easier pipetting.

Digestion Mix:

| Name | Amount [µl] |
| --- | --- |
| DNA (pUC Midi 467,5 ng/µl) | 4,27 |
| CutSmart x10 | 5 |
| EcoRI | 1 |
| HindIII | 2 |
| Water | 37,73 |
| Total Amount | 50 |

Digestion @ 37°C for 1h;

Inactivation @ 80°C for 20min;

Samples applied to a 0,8% Agarose Gel: Ladder pUC19

The digest gave the same result as every other digest with blunt end enzymes, indicating that the pUC19 sample we use is in fact contaminated, or no pUC19 at all!

To clarify which of the stored samples in the freezer @ -20°C and in the refrigerator @ 4°C are still of use Gel electrophoresis was performed.

| Gel 1 part 1 |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| DNA Ladder | Gelex K2a 16.8 | Gelex K3b 16.8 | Gelex K4a 16.8 | Gelex K4b 16.8 | Gelex K2b 09.7 | Gelex K3b 09.7 | Gelex K4b 09.7 | Gelex K4b 09.7 | PC | Vector 4b/a | 3ab pACYC184 p3 | Psec digested Gelex 21.08 | Attempt 3 pSec |
| Gel 1 part 2 |  |  |  |  |  |  |  |  |  |  |  |  |  |
| DNA Ladder | K2ab 2 30.07 | K2a 1 30.07 | K2a 2 30.07 | K2b 2 30.07 | K2b 1 30.07 | Gelex K1 CHO-S 8.8 | Gelex K2a CHO-S 8.8 | Gelex K2b CHO-S 8.8 | Purified CFP 9.8 | / | 3ab 1 | 3ab 2 | 3ab 3 |
| Gel 2 |  |  |  |  |  |  |  |  |  |  |  |  |  |
| DNA Ladder | 25.8 K1 c1 | 25.8 K1 c2 | 25.8 K1 c3 | 25.8 K1 c4 | 25.8 K2a c1 | 25.8 K2a c3 | 25.8 K2a c4 | 25.8 K2a c6 | DNA CHO K2b c1 25.8 | DNA CHO K2b c2 25.8 | DNA CHO K2b c3 25.8 | DNA CHO K2b c4 25.8 |  |
| Gel 3 |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Ladder | 2a | 2a 1 | 2a 2 | 2b | 2b 1 | 4ab | 3ab |  |  |  |  |  |  |

**Pending tasks, next steps, comments for next people in the Lab**

* Decision which of the tested samples remains usefull and should therefore be kept in storage;
* Find alternative to pUC19 for the measurement part
  + One can **not** perform blunt end cloning with the ordered gBlocks
  + However it should be possible with the PCr products of the gBlocks as the have 5’ phosphate groups

![<Beschreibung>](https://static.igem.org/mediawiki/parts/d/d3/T--FAU_ERLANGEN--Gel_1_doppel.png)
![<Beschreibung>](https://static.igem.org/mediawiki/parts/d/d3/T--FAU_ERLANGEN--Gel_2_einzel_heller.png)
![<Beschreibung>](https://static.igem.org/mediawiki/parts/d/d3/T--FAU_ERLANGEN--Gel_2_einzel.png)
![<Beschreibung>](https://static.igem.org/mediawiki/parts/d/d3/T--FAU_ERLANGEN--Gel_3_einzel_klein.png)

[ ]

30

MiniPrep of Gibson Assembly Colonies from 23.08 / MiniPrep of sequenced CHO K1 E1-2 and CHO K2a E1-3 (16.08)

|  |  |
| --- | --- |
| Aim / Task | MiniPrep of Gibson Assembly Colonies from 23.08 / MiniPrep of sequenced CHO K1 E1-2 and CHO K2a E1-3 (16.08) |
| Protocol | - |
| Participants | Felix Pfister, Jannik Wagner |

## Protocol

**Notes**

* MiniPrep of Gibson Assembly Colonies K1, K2a, K2b (5 each)
  + Eluated in 50 µl dH2O
* MiniPrep of CHO K1 E1-2 and K2a E1-3
  + Lost K1 CHO K1 E1-2
  + Eluated in 50 µl dH2O
* Control per Electrophoresis

  + Loading
    - DNA Ladder/K1-1/K1-2/K1-3/K1-4/K1-5/empty/K2a-1/K2a-2/K2a-3/K2a-4/K2a-5/
    - DNA Ladder/K2b-1/K2b-2/K2b-3/K2b-4/K2b-5/empty/CHO K2a E1-3
* Control digestion of both MP

![<Beschreibung>](https://static.igem.org/mediawiki/parts/2/22/T--FAU_ERLANGEN--Unbenannt.PNG)
![<Beschreibung>](https://static.igem.org/mediawiki/parts/d/d3/T--FAU_ERLANGEN--TS05396Aug302019.jpg)

[ ]

30
**(2)**

MiniPrep of Gibson Assembly Colonies from 23.08 / MiniPrep of sequenced CHO K1 E1-2 and CHO K2a E1-3 (16.08)

|  |  |
| --- | --- |
| Aim / Task | MiniPrep of Gibson Assembly Colonies from 23.08 / MiniPrep of sequenced CHO K1 E1-2 and CHO K2a E1-3 (16.08) |
| Protocol | - |
| Participants | Felix Pfister, Jannik Wagner |

## Protocol

**Notes**

* MiniPrep of Gibson Assembly Colonies K1, K2a, K2b (5 each)
  + Eluated in 50 µl dH2O
* MiniPrep of CHO K1 E1-2 and K2a E1-3
  + Lost K1 CHO K1 E1-2
  + Eluated in 50 µl dH2O
* Control per Electrophoresis

  + Loading
    - DNA Ladder/K1-1/K1-2/K1-3/K1-4/K1-5/empty/K2a-1/K2a-2/K2a-3/K2a-4/K2a-5/
    - DNA Ladder/K2b-1/K2b-2/K2b-3/K2b-4/K2b-5/empty/CHO K2a E1-3
* Control digestion of both MP

![<Beschreibung>](https://static.igem.org/mediawiki/parts/d/d3/T--FAU_ERLANGEN--TS05396Aug302019.jpg)
![<Beschreibung>](https://static.igem.org/mediawiki/parts/2/22/T--FAU_ERLANGEN--Unbenannt.PNG)

[ ]

31

PCR of measurment part, gel-electrophoresis of measurment part, gel extraction of measurment part

|  |  |
| --- | --- |
| Aim / Task | PCR of measurment part, gel-electrophoresis of measurment part, gel extraction of measurment part |
| Protocol | Q5 high fidelity PCR Kit, qiagen gel extraction kit |
| Participants | Lena Schorr |

## Protocol

**Notes**

* PCR of the iGEM measurment part CFP
  + 50µl reaction

|  | 1x | 2,5x |
| --- | --- | --- |
| Q5 high fidelity master mix | 25µl | 62,5µl |
| 10µM forward primer | 1,25 µl | 3,125µl |
| 10µM reverse primer | 1,25µl | 3,125µl |
| template DNA (CFP) | 0,5µl (=25ng) | - |
| nuclease free water | 22µl | 55µl |

* with melting temperature of 56°C
* program from the protocol
* PCR products are stored at -20°C
  + M = measurement part
  + C = control with water
* gel electrophoresis
  + 1% gel
  + run for 1h, 120V

**Scheme:**

ladder (7µl) - measurment part (25µl –>10 µl PCR product + 10µl MilliQ + 5µl loading Dye) - water control (“”)

–>see below

* gel extraction via qiagen gel extraction kit
  + band of CFP at 723 bp (see picture below)
  + elution with 50µl MiliQ water
  + storage at -20°C

**Protocol**

Q5 high fidelity PCR Kit, qiagen gel extraction kit

**Pending tasks, next steps, comments for next people in the Lab**

Measurement of DNA concentration of extracted PCR product (CFP –stored at -20°C)

![<Beschreibung>](https://static.igem.org/mediawiki/parts/5/55/T--FAU_ERLANGEN--gel_picture_PCR_CFP_new.jpg)

## September

[ ]

3

CHO parts of 30.07. sequencing + ampicilin aliquots

|  |  |
| --- | --- |
| Aim / Task | CHO parts of 30.07. sequencing + ampicilin aliquots |
| Protocol | Lsg Sequenzing Anleitung |
| Participants | Lena Schorr, Leon Altmann |

**Notes**

Total volume should be 15 µL with a concentration of 100 µg/µL

Accodring to the nanodrop results of 30.08. following DNA volumes were used:

K 1-1: 5,9 µL (C159961)

K 1-2: 6,2 µL (C159960)

K 1-3: 2,96 µL (C159959)

K 1-4: 2,8 µL (C159958)

K 1-5: 4,2 µL (C159957)

K 2a-1: 2,44 µL (C159956)

K 2a-2: 5,3 µL (C159955)

K 2a-3: 3,2 µL (C159954)

K 2a-4: 2,9 µL (C159953)

K 2a-5: 2,66 µL (C159952)

K 2b-1: 6,82 µL (C159951)

K 2b-2: 3,5 µL (C159950)

K 2b-3: 4,5 µL (C159949)

K 2b-4: 4,52 µL (C159948)

K 2b-5: 4,1 µL (C159947)

ad 15 µL with milliQ water

Amp aliqouts:

50 mg/ mL

| **Aim/Task:** | Ligation of pUC19 with Measurement Part, Heatshock into DH5a, PCR of Measurement Part |
| --- | --- |
| **Protocoll used:** | Heat Shock Protocol, NEB Ligation Protocol (M0202), Q5 Hifi PCR Protocol |
| **Date, Site:** | 03.09.19 , nfz |
| **Name:** | Felix, Eva |

***Notes***

Ligation of Measurement Part with cut pUC19 from earlier of the day;

Relative amounts of vector 1 : Insert 4;

10 ng of Vector and approx. 10 ng of Insert as the Insert is only 700 bp long;

Ligation with T4 DNA Ligase Protocol (M020) from NEB;

Reaction set up on ice;

| **Name** | **Amount [µl]** |
| --- | --- |
| T4 DNA Ligase Buffer x10 | 3 |
| Vector DNA | 20 |
| Insert DNA | 0,5 |
| Water | 5,5, |
| T4 Ligase | 1 |
| **Total amount** | **30 µl** |

Incubate @RT for 10 min;

Heat inactivate @65 for 10 min;

With the sample **Heat shock** was performed after Evas Protocol;

The transformed cells were seeded on an Amp+ plate and placed into the incubator @ 37 °C;

For the case of a failed ligation a PCR with the measurement part was set up;

||
| — || — |
| **Name** | **Amount [µl]** |
| ——————————- | ————— |
| DNA | 0,5 |
| Q5 Hifi Polymerase MasterMix x2 | 12,5 |
| Measurement Primer fw | 1,25 |
| Measurement Primer re | 1,25 |
| Nuclease free water | 9,5 |
| **Total amount** | **30 µl** |

**Problems faced / possible error causes**

Q5 Hifi Polymerase is stored in the topmost drawer in the -20 °C fridge in the winkler lab and no longer in the Plastik Box in drawer 2;

**Pending tasks, next steps, comments for next people in the Lab**

- Check Plate in the Incubator and pick colonies if transformation was successful;

- IF not new digestion with the ordered pUC19 (XbaI/EcoRI)

[ ]

3
**(2)**

Digestion of pUC19 and CFP with Xbal and EcoRI

|  |  |
| --- | --- |
| Aim / Task | Digestion of pUC19 and CFP with Xbal and EcoRI |
| Protocol | Heat Shock Protocol, NEB Ligation Protocol (M0202), Q5 Hifi PCR Protocol |
| Participants | Eva Neugebauer, Felix Pfister |

**Notes**

Nanodrop measurement of gelextraction from 31.07. and and 01.08.

CFP PCR gel extr. 31.07.: 13,6

CFP PCR gel extr. 01.08. 1: 21,82

CFP PCR gel extr. 01.08. 2: 16,96

Digest of gelextr. (31.08.) and pUC19 with EcoRI and XbaI:

43µL DNA + 5 µL cutsmart + 1 µL of each enzyme (HF)

15 min 37°C

20 min 65°C heat inactivation

**Pending tasks, next steps, comments for next people in the Lab**

Ligation of puc19 and CFP

New PCR of CFP

[ ]

3
**(3)**

Ligation of pUC19 with Measurement Part, Heatshock into DH5a, PCR of Measurement Part

|  |  |
| --- | --- |
| Aim / Task | Ligation of pUC19 with Measurement Part, Heatshock into DH5a, PCR of Measurement Part |
| Protocol | Heat Shock Protocol, NEB Ligation Protocol (M0202), Q5 Hifi PCR Protocol |
| Participants | Eva Neugebauer, Felix Pfister |

**Notes**

Ligation of Measurement Part with cut pUC19 from earlier of the day;

Relative amounts of vector 1 : Insert 4;

10 ng of Vector and approx. 10 ng of Insert as the Insert is only 700 bp long;

Ligation with T4 DNA Ligase Protocol (M020) from NEB;

Reaction set up on ice;

| Name | Amount [µl] |  |
| --- | --- | --- |
| T4 DNA Ligase Buffer x10 | 3 |  |
| Vector DNA | 20 |  |
| Insert DNA | 0,5 |  |
| Water | 5,5, |  |
| T4 Ligase | 1 |  |
| **Total amount** | **30 µl** |  |

Incubate @RT for 10 min;

Heat inactivate @65 for 10 min;

With the sample **Heat shock** was performed after Evas Protocol;

The transformed cells were seeded on an Amp+ plate and placed into the incubator @ 37 °C;

For the case of a failed ligation a PCR with the measurement part was set up;

| Name | Amount [µl] |
| --- | --- |
| DNA | 0,5 |
| Q5 Hifi Polymerase MasterMix x2 | 12,5 |
| Measurement Primer fw | 1,25 |
| Measurement Primer re | 1,25 |
| Nuclease free water | 9,5 |
| **Total amount** | **30 µl** |

**Problems faced / possible error causes**

Q5 Hifi Polymerase is stored in the topmost drawer in the -20 °C fridge in the winkler lab and no longer in the Plastik Box in drawer 2;

**Pending tasks, next steps, comments for next people in the Lab**

* Check Plate in the Incubator and pick colonies if transformation was successful;
* IF not new digestion with the ordered pUC19 (XbaI/EcoRI)

[ ]

3
**(4)**

Digestion of pUC19 and CFP with Xbal and EcoRI

|  |  |
| --- | --- |
| Aim / Task | Digestion of pUC19 and CFP with Xbal and EcoRI |
| Protocol | Lsg Sequenzing Anleitung |
| Participants | Eva Neugebauer, Felix Pfister |

**Notes**

Nanodrop measurement of gelextraction from 31.07. and and 01.08.

CFP PCR gel extr. 31.07.: 13,6

CFP PCR gel extr. 01.08. 1: 21,82

CFP PCR gel extr. 01.08. 2: 16,96

Digest of gelextr. (31.08.) and pUC19 with EcoRI and XbaI:

43µL DNA + 5 µL cutsmart + 1 µL of each enzyme (HF)

15 min 37°C

20 min 65°C heat inactivation

**Pending tasks, next steps, comments for next people in the Lab**

Ligation of puc19 and CFP

New PCR of CFP

[ ]

3
**(5)**

CHO parts of 30.07. sequencing + ampicilin aliquots

|  |  |
| --- | --- |
| Aim / Task | CHO parts of 30.07. sequencing + ampicilin aliquots |
| Protocol | Lsg Sequenzing Anleitung |
| Participants | Eva neugebauer, Leon Altmann |

**Notes**

Total volume should be 15 µL with a concentration of 100 µg/µL

Accodring to the nanodrop results of 30.08. following DNA volumes were used:

K 1-1: 5,9 µL (C159961)

K 1-2: 6,2 µL (C159960)

K 1-3: 2,96 µL (C159959)

K 1-4: 2,8 µL (C159958)

K 1-5: 4,2 µL (C159957)

K 2a-1: 2,44 µL (C159956)

K 2a-2: 5,3 µL (C159955)

K 2a-3: 3,2 µL (C159954)

K 2a-4: 2,9 µL (C159953)

K 2a-5: 2,66 µL (C159952)

K 2b-1: 6,82 µL (C159951)

K 2b-2: 3,5 µL (C159950)

K 2b-3: 4,5 µL (C159949)

K 2b-4: 4,52 µL (C159948)

K 2b-5: 4,1 µL (C159947)

ad 15 µL with milliQ water

Amp aliqouts:

50 mg/ mL

[ ]

3
**(6)**

Digestion of pUC19 and CFP with Xbal and EcoRI

|  |  |
| --- | --- |
| Aim / Task | Digestion of pUC19 and CFP with Xbal and EcoRI |
| Protocol | - |
| Participants | Felix Pfister, Eva Neugebauer |

**Notes**

Nanodrop measurement of gelextraction from 31.07. and and 01.08.

CFP PCR gel extr. 31.07.: 13,6

CFP PCR gel extr. 01.08. 1: 21,82

CFP PCR gel extr. 01.08. 2: 16,96

Digest of gelextr. (31.08.) and pUC19 with EcoRI and XbaI:

43µL DNA + 5 µL cutsmart + 1 µL of each enzyme (HF)

15 min 37°C

20 min 65°C heat inactivation

**Pending tasks, next steps, comments for next people in the Lab**

Ligation of puc19 and CFP

New PCR of CFP

[ ]

4

Digest of new pUC19 (XbaI/EcoRI), PCR clean up of CFP, Gel extraction of CFP, Digest purified CFP, Ligation of pUC19 with CFP, Heatshock

|  |  |
| --- | --- |
| Aim / Task | Digest of new pUC19 (XbaI/EcoRI), PCR clean up of CFP, Gel extraction of CFP, Digest purified CFP, Ligation of pUC19 with CFP, Heatshock |
| Protocol |  |
| Participants | Leon Altmann |

**Notes**

**Digestion of pUC19 with EcoRI and XbaI:**

| Name | Amount [µl] |
| --- | --- |
| pUC19 DNa | 2 (= 2µg –DNA conc. 100ng/µl) |
| EcoRI-HF | 1 |
| XbaI | 1 |
| Cut Smart Buffer x10 | 2 |
| Water | 14 |
| **Total amount** | **20 µl** |

Incubate @37 °C for 20 min;

Inactivate @ 65 °C for 20 min;

---

**PCR Purification:**

Purified PCR sample from 3.9.19 with the PCR Purification Kit and followed the Kits protocol;

Eluted DNA in 30 µl Buffer EB;

---

**Gel Extraction:**

Extracted DNA from the Gel prepared earlier this DNA to extract the Measurement Part;

Used the QiaQuick Gel Extraction Kit and followed its protocol;

Weight of Gel fragment ~0,02g –60 µl Buffer QG for Gel solving;

Eluted the DNA in 35 µl Buffer EB;

---

**DNA Measuring at Nanodrop:**

| Name | ng/µl | 260 | 280 | 260⁄280 | 260⁄230 |
| --- | --- | --- | --- | --- | --- |
| PCR Clean Up CFP | 45,6 | 0,912 | 0,500 | 1,82 | 1,69 |
| Gel Ex CFP | 8,5 | 0,169 | 0,085 | 2,00 | 0,02 |

---

**Digestion of the purified samples (Gel Ex CFP, PCR Clean Up CFP):**

| Name | Amount [µl] PCR Purified | Amount [µl] Gel Ex CFP |
| --- | --- | --- |
| CFP DNA | 30 (= 1,37 µg) | 35 (= 927,5 ng) |
|  |  |  |
| EcoRI-HF | 1 | 1 |
|  |  |  |
| XbaI | 1 | 1 |
|  |  |  |
| Cut Smart Buffer x10 | 5 | 5 |
|  |  |  |
| Water | 15 | 8 |
|  |  |  |
| **Total amount** | **50 µl** | **50 µl** |
|  |  |  |

Incubate @37 °C for 20 min;

Inactivate @ 65 °C for 20 min;

Ligation of Measurement Part with the digested pUC in different ratios

Ligation with T4 DNA Ligase Protocol (M020) from NEB;

Reaction set up on ice;

| Name | Amount [µl] CFP PCR 1:5 | Amount [µl] CFP PCR 1:7 | Amount [µl] CFP PCR 1:10 | Amount [µl] Gel Ex 1:5 |
| --- | --- | --- | --- | --- |
| T4 DNA Ligase Buffer x10 | 2 | 2 | 2 | 2 |
| Vector DNA | 0,5 | 0,5 | 0,5 | 0,5 |
| Insert DNA | 1,4 | 2 | 2,84 | 11,76 ( = 100 ng) |
| Water | 15,1 | 14,5 | 13,7 | 5,24 |
| T4 Ligase | 1 | 1 | 1 | 1 |
| **Total amount** | **20 µl** | **20 µl** | **20 µl** | **20 µl** |

Incubate @RT for 10 min;

Heat inactivate @65 for 10 min;

With the sample **Heat shock** was performed after Evas Protocol;

The transformed cells and one sample being ony digested vector were seeded on Amp+ plates and placed into the incubator @ 37 °C overnight;

---

Placed the colonies picked earlier by Eva into the Shaker overnight @ 37 °C and 230 rpm;

**Files used / generated**

In parallel designed Primer for the restriction digestion of the Constructs for the *E. coli* expression;

Designed them with the intent to open the pACYC184 vector with ClaI and SalI and accordingly also digest the constructs with the same enzymes to perform classical cloning;

**Pending tasks, next steps, comments for next people in the Lab**

* Mini Prep from the Overnight cultures in the Shaker (one could also look at the cells under the fluorescence microscope as successfull ligation + transformation should produce CFP);
* Gel electrophoresis thereof
* Heatshock into other Strains BL21, Star, Tuner;
* Check Plates in the Incubator, prepare ON cultures;

[ ]

4
**(2)**

CFP ligation with restrictionenzyme digest

|  |  |
| --- | --- |
| Aim / Task | CFP ligation with restrictionenzyme digest |
| Protocol |  |
| Participants | Eva Neugebauer |

**Notes**

**Plates of Heatshock (03.09.2019)**

4 small clonies detactable

Plate back to incubator

Later clone picking

**1% Agarose gel**

M (5µL) | 1 CFP 01. | 2 CFP 01. | puc19 Verdau 3.9. | - | - | M(5µL) | PCR L | PCR R

For: 1 CFP 01., 2 CFP 01., puc19 Verdau 3.9. following composition was used:

5µL DNA, 3,3 µL Loading Dye, 11,7 µL MilliQ water

For: PCR R and PCR L following composition was used:

12µL DNA, 3,3 µL Loading Dye, 4,7 µL MilliQ water

Gel running running at 110V

No band for the vector -probably not enough DNA loaded on the gel

Cutting out the CFP from the PCR (PCR L was the probe and R the control)

**Problems faced / possible error causes**

Plate was the wrong way round in the incubator

No negative control for the heatshock -clones might be just undigested vector

**insert picture from 4.9.19 here; name on labfolder:TS05405Sept42019.jpg**

| **Aim/Task:** | Digest of new pUC19 (XbaI/EcoRI), PCR clean up of CFP, Gel extraction of CFP, Digest purified CFP, Ligation of pUC19 with CFP, Heatshock |
| --- | --- |
| **Protocoll used:** |  |
| **Date:** | 2019-09-04 |
| **Name:** | Leon |

***Notes***

**Digestion of pUC19 with EcoRI and XbaI:**

| **Name** | **Amount [µl]** |
| --- | --- |
| pUC19 DNa | 2 (= 2µg –DNA conc. 100ng/µl) |
| EcoRI-HF | 1 |
| XbaI | 1 |
| Cut Smart Buffer x10 | 2 |
| Water | 14 |
| **Total amount** | **20 µl** |

Incubate @37 °C for 20 min;

Inactivate @ 65 °C for 20 min;

---

**PCR Purification:**

Purified PCR sample from 3.9.19 with the PCR Purification Kit and followed the Kits protocol;

Eluted DNA in 30 µl Buffer EB;

---

**Gel Extraction:**

Extracted DNA from the Gel prepared earlier this DNA to extract the Measurement Part;

Used the QiaQuick Gel Extraction Kit and followed its protocol;

Weight of Gel fragment ~0,02g –60 µl Buffer QG for Gel solving;

Eluted the DNA in 35 µl Buffer EB;

---

**DNA Measuring at Nanodrop:**

| Name | ng/µl | 260 | 280 | 260⁄280 | 260⁄230 |
| --- | --- | --- | --- | --- | --- |
| PCR Clean Up CFP | 45,6 | 0,912 | 0,500 | 1,82 | 1,69 |
| Gel Ex CFP | 8,5 | 0,169 | 0,085 | 2,00 | 0,02 |

---

**Digestion of the purified samples (Gel Ex CFP, PCR Clean Up CFP):**

| **Name** | **Amount [µl] PCR Purified** | **Amount [µl] Gel Ex CFP** |
| --- | --- | --- |
| CFP DNA | 30 (= 1,37 µg) | 35 (= 927,5 ng) |
| EcoRI-HF | 1 | 1 |
| XbaI | 1 | 1 |
| Cut Smart Buffer x10 | 5 | 5 |
| Water | 15 | 8 |
| **Total amount** | **50 µl** | **50 µl** |

Incubate @37 °C for 20 min;

Inactivate @ 65 °C for 20 min;

Ligation of Measurement Part with the digested pUC in different ratios

Ligation with T4 DNA Ligase Protocol (M020) from NEB;

Reaction set up on ice;

| **Name** | **Amount [µl] CFP PCR 1:5** | **Amount [µl] CFP PCR 1:7** | **Amount [µl]** **CFP PCR 1:10** | **Amount [µl] Gel Ex 1:5** |
| --- | --- | --- | --- | --- |
| T4 DNA Ligase Buffer x10 | 2 | 2 | 2 | 2 |
| Vector DNA | 0,5 | 0,5 | 0,5 | 0,5 |
| Insert DNA | 1,4 | 2 | 2,84 | 11,76 ( = 100 ng) |
| Water | 15,1 | 14,5 | 13,7 | 5,24 |
| T4 Ligase | 1 | 1 | 1 | 1 |
| **Total amount** | **20 µl** | **20 µl** | **20 µl** | **20 µl** |

Incubate @RT for 10 min;

Heat inactivate @65 for 10 min;

With the sample **Heat shock** was performed after Evas Protocol;

The transformed cells and one sample being ony digested vector were seeded on Amp+ plates and placed into the incubator @ 37 °C overnight;

---

Placed the colonies picked earlier by Eva into the Shaker overnight @ 37 °C and 230 rpm;

**Files used / generated**

In parallel designed Primer for the restriction digestion of the Constructs for the *E. coli* expression;

Designed them with the intent to open the pACYC184 vector with ClaI and SalI and accordingly also digest the constructs with the same enzymes to perform classical cloning;

**Pending tasks, next steps, comments for next people in the Lab**

- Mini Prep from the Overnight cultures in the Shaker (one could also look at the cells under the fluorescence microscope as successfull ligation + transformation should produce CFP);

- Gel electrophoresis thereof

- Heatshock into other Strains BL21, Star, Tuner;

- Check Plates in the Incubator, prepare ON cultures;

![<CFP_gel>](https://static.igem.org/mediawiki/parts/3/3a/T--FAU_ERLANGEN--TS05405Sept42019.jpg)

[ ]

4
**(3)**

CFP ligation with restrictionenzyme digest

|  |  |
| --- | --- |
| Aim / Task | CFP ligation with restrictionenzyme digest |
| Protocol |  |
| Participants | Eva Neugebauer |

**Notes**

**Plates of Heatshock (03.09.2019)**

4 small clonies detactable

Plate back to incubator

Later clone picking

**1% Agarose gel**

M (5µL) | 1 CFP 01. | 2 CFP 01. | puc19 Verdau 3.9. | - | - | M(5µL) | PCR L | PCR R

For: 1 CFP 01., 2 CFP 01., puc19 Verdau 3.9. following composition was used:

5µL DNA, 3,3 µL Loading Dye, 11,7 µL MilliQ water

For: PCR R and PCR L following composition was used:

12µL DNA, 3,3 µL Loading Dye, 4,7 µL MilliQ water

Gel running running at 110V

No band for the vector -probably not enough DNA loaded on the gel

Cutting out the CFP from the PCR (PCR L was the probe and R the control)

**Problems faced / possible error causes**

Plate was the wrong way round in the incubator

No negative control for the heatshock -clones might be just undigested vector

![<Agarose_gel_cfp>](https://static.igem.org/mediawiki/parts/3/3a/T--FAU_ERLANGEN--TS05405Sept42019.jpg)

[ ]

5

CFP ligation with restrictionenzyme digest

|  |  |
| --- | --- |
| Aim / Task | CFP ligation with restrictionenzyme digest |
| Protocol |  |
| Participants | Leon Altmann |

***Notes***

ON Plates in the incubator were checked. The Control plate with only cut vector had 4 very small colonies on it. Whereas all Plates with Vector + Insert (pUC19 + CFP) had much higher numbers. Comparing the plates, the Gel Ex. Plate 1:5 had the least amount and within the PCR Plates a nice gradient was visible. The higher the Vector: Insert ratio was, the larger was the number of colonies.

With the fluorescence microscope of the AG Behrens the plates were screened for positive clones.

Number of CFp pos. clones was rather low <10 %, positive clones were marked and later on picked to set up ON cultures;

Incubated ON cultures @ 37 °C, 230 rpm;

The ON cultures in the shaker were split. A 500 µl Aliquot was stored at -20 °C after spinning down the cells.
The other 4,5 ml were spinned down in the Megafuge (Mouse Lab), Miniprep not performed due to the low efficycy of the better ligation approach, so it is to be assumed that these clones are all negative;

Sequencing data was analyzed –all the sent in clones seem to be positive

[ ]

6

estriction OH PCR with the E. coli Parts, Mini Prep of the CFP clones, Control Digest

|  |  |
| --- | --- |
| Aim / Task | estriction OH PCR with the E. coli Parts, Mini Prep of the CFP clones, Control Digest |
| Protocol |  |
| Participants | Eva Neugebauer, Leon Altmann |

**Notes**

To generate the necessary restriction sites for the classical cloning PCr was performed with the constructs for the E. coli expression.

| Restriction OH PCR | K1 | K2a | K2b | K3a | K3b | K4a | K4b | Water Control |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Q5 MM x2 | 12,5 | 12,5 | 12,5 | 12,5 | 12,5 | 12,5 | 12,5 | 12,5 |
| fw Primer | 1,25 | 1,25 | 1,25 | 1,25 | 1,25 | 1,25 | 1,25 | 1,25 |
| re Primer | 1,25 | 1,25 | 1,25 | 1,25 | 1,25 | 1,25 | 1,25 | 1,25 |
| DNA | 0,5 | 0,5 | 2,5 | 0,5 | 0,5 | 0,5 | 0,5 | 0 |
| Water | 9,5 | 9,5 | 7,5 | 9,5 | 9,5 | 9,5 | 9,5 | 10 |
| **Total** | **25** | 25 | 25 | 25 | 25 | 25 | 25 | 25 |

The normal PCR Amplification Program was used in the PCR cycler, Samples were stored @ 4 °C;

Miniprep of the ON cultures that were now for 4 more hours in hte Shaker was performed;

Eluted the samples in 50 µl of EB Buffer;

DNA concentration of the samples was measured with the Nanodrop;

| Name | ng/µl | 260 | 280 | 260⁄280 | 260⁄230 |
| --- | --- | --- | --- | --- | --- |
| C1 | 77,25 | 1,545 | 0,706 | 2,19 | 2,28 |
| C2 | 166,47 | 3,329 | 1,552 | 2,14 | 2.25 |
| C3 | 50,54 | 1,011 | 0,519 | 1,95 | 1,82 |
| C4 | 34,62 | 0,692 | 0,356 | 1,95 | 1,91 |
| C5 | 80,80 | 1,616 | 0,782 | 2,07 | 1,99 |
| C6 | 24,83 | 0,497 | 0,227 | 2,19 | 2,04 |
| C7 | 33,04 | 0,661 | 0,308 | 2,14 | 2,08 |
| C8 | 22,01 | 0,440 | 0,217 | 2,02 | 1,88 |

**Test Digest**

Miniprep samples were digested with EcoRI HF and SalI

| Name | C1 | C2 | C3 | C4 | C5 | C6 | C7 | C8 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| SalI | 0,5 | 0,5 | 0,5 | 0,5 | 0,5 | 0,5 | 0,5 | 0,5 |
| EcoRI HF | 0,5 | 0,5 | 0,5 | 0,5 | 0,5 | 0,5 | 0,5 | 0,5 |
| Cut Smart x 10 | 2,5 | 2,5 | 2,5 | 2,5 | 2,5 | 2,5 | 2,5 | 2,5 |
| DNA | 6,5 | 3 | 10 | 14,5 | 6,2 | 20 | 15,2 | 0 |
| Water | 15 | 18,5 | 11,5 | 7 | 15,3 | 1,5 | 6,3 | 21,5 |
| **Total** | **25** | **25** | **25** | **25** | **25** | **25** | **25** | **25** |

Incubation @ 37 °C for 15 min;

Inactivation @ 65 °C for 20 min;

***Problems faced / possible error causes***

During the Miniprep a washing step was forgotten so the eluted DNA presumable also contained genomic DNA;
To assay this the samples were once more transferred onto purifiation säulchen to be washed and finally aeluted again;

***Pending tasks, next steps, comments for next people in the Lab***

* Run a Gel of the digested Miniprep samples
* Run a Gel of the PCR samples
* Prepare new competent Cell ( DH5a, Tuner, BL21, Star)
* Heat Shock with the PCR samples (Tuner, Bl21, Star) and positive Miniprep Samples (Tuner, BL21, Star)

[ ]

6
**(2)**

Control Gel of PCR and Mini, PCR Purification/Gel Extraction, New Amp+ Plates, Heatshock of CFP c2 into Star/BL21/Tuner

|  |  |
| --- | --- |
| Aim / Task | Control Gel of PCR and Mini, PCR Purification/Gel Extraction, New Amp+ Plates, Heatshock of CFP c2 into Star/BL21/Tuner |
| Protocol |  |
| Participants | Leon Altmann, Lena Schorr, Eva Neugebauer |

**Notes**

**Control Gel electrophoresis:**

A 1% Agarose Gel was prepared and the samples from the digested Mini Prep (6.7.) and the E. coli Restriction PCR (6.7.) were applied;

Gel 1:

| Ladder | C1 | C2 | C3 | C4 | C5 | C6 | C7 | C8 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  |  |  |  |

Gel 2:

| Ladder | K1 | K2a | K2b | K3a | H20 | H20 | K4a | K4b | K3b |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  |  |  |  |  |

Of the PCR 3 µl were used to create a 20 µl sample with loading dye and water;

Of the digested Mini Prep 25 µl were used together with 4,2 µl;

To the gel 15 µl of each sample were applied;

**PCR Purification of K2b, K3a, K3b, K4a, K4b:**

Performed PCR Purification according to the QiaQuick Purification Protocol and eluted the samples in 30 µl EB-Buffer;

**Gel extraction K1, K2a:**

For the Gel extraction another Gel of K1 and K2a with a higher sample concentration was prepared;

For K1 the band at approx 1,7 kb was extracted;

Construct 2a with the Sites for Restriction cloning is approx. 1400 bp long, to be safe though also the bad that was seen at the height of roughly 1200 bp was extracted and purified;

The samples were eluted in 50 µl EB Buffer;

**Cast new Amp+ Plates:**

New AMP Plates were necessary so 35g of LB-Agar Powder were mixed with 1l of MilliQ Water and mixed thoroughly.
Mixture was autoclaved and after sufficient cooling Amp (50 mg/ml) was added to reach a concentration of 50 µg/ml;

With the Broth new Plates were casted;

---

**Heatshock of CFP c2 into Star/Bl21/Tuner:**

Heatshock with CFP+Puc19 clone 2 into Bl21, Star and Tuner with 2 µl DNA;

Eva Heatshock Protocol was used plus an extra Step with 200 µl SOC Medium after the 2 min incubation on ice;

Plated 200 µl of each sample onto Amp+ plates and stored the remaining 200 µl @ 4 °C;

**Digestion of pACYC184 and purified samples with ClaI, SalI-HF:**

pACYC184:

| **Name** | **Amount [µl] pACYC184** | **Amount [µl] PCR purified K2b, K3b ,K4a, K4b** | **Amount [µl] Gel Ex. K1, K2a** |
| --- | --- | --- | --- |
| DNA | 5,32 | 15 | 40 |
| ClaI | 1 | 1 | 1 |
| SalI-HF | 1 | 1 | 1 |
| Cut Smart Buffer x10 | 5 | 2,5 | 5 |
| Water | 44,86 | 4,5 | 4 |
| **Total amount** | **50 µl** | **25 µl** | **50 µl** |

Incubate @37 °C for 15 min;

Inactivate @ 65 °C for 20 min;

To avoid uncut vector, that might reduce the efficacy of a following ligation Gel Extraction of the 3,6 kb long fragment was performed;

The sample was eluted in 30 µl Buffer EB;

**Ligation of digested and gel extracted pACYC184 with the digested the digested E. coli constructs (K1-K4b) in a 1:7 ratio (vector:insert):**

Before the Ligation the amount DNA amount was measured in the Nanodrop:

| Name | ng/µl | 260 | 280 | 260⁄280 | 260⁄230 |
| --- | --- | --- | --- | --- | --- |
| pACYC184 | 14,94 | 0,290 | 0,160 | 1,81 | 0,02 |
| K1 dig | 75,94 | 1,519 | 0,867 | 1,75 | 0,45 |
| K2a 1 dig (oben) | 127,04 | 2,541 | 1,624 | 1,56 | 0,44 |
| K2a 2 dig (unten) | 123,97 | 2,479 | 1,584 | 1,56 | 0,43 |
| K2b dig | 71,40 | 1,428 | 0,839 | 1,70 | 1,01 |
| K3a dig | 71,79 | 1,463 | 0,830 | 1,73 | 0,87 |
| K3b dig | 63,78 | 1,276 | 0,776 | 1,64 | 0,81 |
| K4a dig | 56.60 | 1,132 | 0,665 | 1,70 | 0,86 |
| K4b dig | 76,38 | 1,528 | 0,993 | 1,54 | 0,80 |

With the results from the Nanidrop Measurement Ligation with T4 DNA Ligase Protocol (M020) from NEB was performed;

Reaction set up on ice;

| **Name** | **Amount [µl] K1** | **Amount [µl] K2a 1** | **Amount [µl]** **K2a 2** | **Amount [µl] K2b** | **Amount [µl] K3a** | **Amount [µl] K3b** | **Amount [µl] K4a** | **Amount [µl] K4b** |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| T4 DNA Ligase Buffer x10 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 |
| Vector DNA | 3,5 | 3,5 | 3,5 | 3,5 | 3,5 | 2,7 | 2,7 | 2,7 |
| Insert DNA | 4,6 | 2,75 | 2,8 | 3,5 | 3,5 | 5,5 | 6,2 | 4,6 |
| Water | 8,9 | 10,75 | 10,7 | 10 | 10 | 8 | 7,3 | 8,9 |
| T4 Ligase | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
| **Total amount** | **20 µl** | **20 µl** | **20 µl** | **20 µl** | **20 µl** | **20 µl** | **20 µl** | **20 µl** |

Short Spin the Samples and incubate @RT for 10 min;

With the samples **Heat shock** was performed after Evas Protocol + SOC Step;

The 200µl of the ransformed cells were seeded on Amp+ plates and placed into the incubator @ 37 °C overnight;

**Calculations and Results**

**Control Gel electrophoresis:**

The control gel electrophoresis showed that the digestion in most of the samples were successfull. In some (4,6,7,8) uncut DNA was visible;

Sample 5 and 8 do also not show the expected band at approx 0,7 kb;

All PCR samples showed bands at their expected heights;

Just K1 and K2a had a strong unspecific bands. This is why the correct band was extracted out of the Gel instead of PCR Purification like with the other samples;

***Problems faced / possible error causes***

Forgot to inactivate the Ligase @ 65 °C for 10 min;

**pirctures have to be inserted here:**

7.9\_Restriction\_PCR,\_Miniprep\_Gel.jpeg

7.9\_Restriction\_K1,\_K2a\_for\_Gel\_Ex.jpeg

![<Restriction_gel>](/labjournal-entries/images/7.9_Restriction_K1,_K2a_for_Gel_Ex.jpeg-with-annotations.png)
![<Restriction_PCR>](/labjournal-entries/images/7.9_Restriction_PCR,_Miniprep_Gel.jpeg-with-annotations.png)

[ ]

6
**(3)**

CFP ligation with restrictionenzyme digest

|  |  |
| --- | --- |
| Aim / Task | CFP ligation with restrictionenzyme digest |
| Protocol |  |
| Participants | Leon Altmann |

***Notes***

Checked the ON cultures in the Shaker –A 500 ml Aliquot was frozen away after centrifuging and getting rid of the supernatant;

The other 4,5 ml were spinned down in hte Megafuge for 5 min, 4000 rpm –All the pellets were rather small so the Samples were put into the Shaker under the same conditions for a couple more hours;

The ordered Primer for Restriction Overhang PCR were diluted in TE-Buffer, so that the concentration is 100 µM;

| **Name** | **DNA [nmol]** | **TE-Buffer [µl]** |
| --- | --- | --- |
| K1 restriction fw | 31,9 | 319 |
| K1 restriction re | 32,4 | 324 |
| Restriction fw | 99,9 | 999 |
| Restriction re | 106,8 | 1068 |

Incubation @ 50 °C to elute the Primers;

[ ]

8

CFP ligation with restrictionenzyme digest

|  |  |
| --- | --- |
| Aim / Task | CFP ligation with restrictionenzyme digest |
| Protocol |  |
| Participants | Leon Altmann |

**Notes**

Checked the Plates in the Incubator;

The Cells with the E. coli Restriction constructs were seeded on the wrong plates AMp+ Plates instead of CAM plates;

The Cells with the Measurement Part (CFP) have grown nicely; Most colonies grew with the Tuner strain;

2l New LB Medium with 20g LB powder per Liter were prepared and autoclaved;

New Heatshock with the Rest of the Ligation samples (15 µl) into Tuner;

200 µl of the Heatshock samples were plated on CAM+ Plates and Placed in the incubator @ 37 °C; (Rest stored @ 4 °C)

Made 3 ON cultures with LB Amp+ (300 ml) one for each of the Measurement Part Plates (BL21, Tuner, Star);

Put them in the Shaker @ 37 °C, 230 rpm

**Pending tasks, next steps, comments for next people in the Lab**

* Prepare new competent cells (BL21, Star, Tuner, DH5a);
* Check Plates in the Incubator and prepare cultures –Mini Prep;
* Check Cultures in the Shaker and prepare for CFP Measuring;

[ ]

9

colony PCR

|  |  |
| --- | --- |
| Aim / Task | colony PCR |
| Protocol |  |
| Participants | Eva Neugebauer |

**Notes**

Preparation of labled Eppis with 1 mL LB with Chloramphenicol.

Preparation of Mastermixes for the colony PCR with: Q5 MM, Primer, Water like following:

|  | K1 | All other constructs |
| --- | --- | --- |
| Q5 MM | 75 µL | 475 µL |
| fw primer | 7,5 µL | 47,5 µL |
| re primer | 7,5 µL | 47,5 µL |
| H2O | 60 µL | 380 µL |

25 µL per colony master mix in the according PCR tube.

Picking of clones with a pipet. Putting the pipette in the PCR tube and then into the LB.

PCR program: Konstrukt amplification

Running gel on a 1% agarose gel.

5 µL Loading dye was added to the 25µL PCR and then 15µL loaded on the gel.

Running at 130 V.

Loading scheme of the gel:

Gel 1:

Marker | K1.1 | K1.2 | K1.3 | K1.4 | K1.5 | K2a.1 | K2a.2 | K2a.3 | K2a.4 | K2a.5 | K2a.1obere |K2a.2obere| K2a.3obere

Gel 2:

Marker| K2a.4obere | K2a.5obere | K2b.1 | K2b.2 | K2b.3 | K2b.4 | K3a.1 | K3a.2 | K3a.3 | K3a.4 | K3a.5 | K3b.1 | K3b.2

Gel 3:

Marker| K3b.3 | K3b.4 | K3b.5 | K4a.1| K4a.2| K4a.3| K4a.4|K4b.1|K4b.2|K4b.3|K4b.3|K4b.4|h20 PCR|milliq

[ ]

10

colony PCR

|  |  |
| --- | --- |
| Aim / Task | colony PCR |
| Protocol |  |
| Participants | Martin Borgmann, Felix Pfister |

**Notes**

**Colony PCR**

Preparation of labled Eppis with 1 mL LB with Chloramphenicol.

Preparation of Mastermixes for the colony PCR with: Q5 MM, Primers, Water as followed (3 colonies of each construct):

|  | K1 (3 reactions) | All other constructs (21 reactions) |
| --- | --- | --- |
| Q5 MM | 37,5 µL | 262,5 µL |
| fw primer | 3,75 µL | 26,25 µL |
| re primer | 3,75 µL | 26,25 µL |
| H2O | 30 µL | 210 µL |

Primers:

* ​ K1: “K1 restriction fw” and “K1 restriction re”
* ​ all other constructs: “restriction fw” and “restriction re”

25 µL per colony master mix in the according PCR tube.

Picking of clones with a pipet tip

PCR program: Konstrukt amplification

​ modifcation: 1st step at 90°C 10 min

Gel electrophores (1% agarose, 130 V)

2 µL Loading dye was added to the 10 µL PCR and then loaded on the gel.

Loading scheme of the gel (2 x 14 pockets, 1mm wide):

First Row:

1 kb Ladder | K1.1 | K1.2 | K1.3 |K2a1.1 | K2a1.2 | K2a1.3 | K2b.1| K2b.2 | K2b.3 | K3a.1 | K3a.2 | K3a.3

Second Row:

1 kb Ladder K3b.1 | K3b.2 | K3b.3 | K4a.1 | K4a.2 | K4a.3 | K4b.1 | K4b.2 | K4b.3 | K2a2.1 | K2a2.2 | K2a.3

**Calculations and Results**

Weak bands

​ might be repeated with with more cycles!

**Problems faced / possible error causes**

Didn’t prepare 1 replicate in the MM, therefore the last tube had a little less volume than the others.

Switched poles during electrophoresis, therefore lost the first row.

**Mini Prep**

Miniprep of ON cultures of the colonies that were incubated after colony PCR (09.09.19)

Assessment of success using gel electrophoresis (1% gel, 130 V)

[ ]

11

Testing of miniprep from 10.09.

|  |  |
| --- | --- |
| Aim / Task | Testing of miniprep from 10.09. |
| Protocol | https://www.helmholtz-muenchen.de/fileadmin/PEPF/Protocols/Chemically-competent-cells.pdf |
| Participants | Marie Wiedemann |

**Notes**

* Used prepared bacteria from -80 °C (DH5a, Star, BL21, Tuner)
* defrosted them at RT, put in 125 ml LB Medium
* incubated at 30 °C for 1h
* checked oD until between 0.4 and 0.6
* oD: DH5a: 0,564; Star: 0,654; BL21: 0,401; Tuner: 0,82
* diluted Tuner to oD of 0,628
* Following steps consitently on ice
* split in 50ml falcons
* incubate ion ice 10 min
* spin in ultracentrifuge 10 min 4000rpm 4 °C
* resuspend pellet in 25 ml CC-Puffer (already prepared)
* incubate 10 min on ice
* centrifuge 10 min 4000 rpm 4 °C
* resuspend pellet in 4,65 ml CC-Puffer + 0,35 ml 15% DMSO per falcon
* incubate on ice 10 min
* aliquoted in 1,5 ml eppis 200 µl per eppi
* freezed in -80 °C

**Materials used**

CC-Puffer

DMSO

LB-Medium

[ ]

11
**(2)**

Testing of miniprep from 10.09.

|  |  |
| --- | --- |
| Aim / Task | Testing of miniprep from 10.09. |
| Protocol |  |
| Participants | Eva Neugebauer |

**Notes**

Measurement of the miniprep samples from 10.09. at the nanodrop.

Concentration in ng/µL:

K1.1: 64,2; K1.2 168,7; K2a.1.1 61,1; K2a.1.2 179,4; K2a2.1 132,6; K2a2.2 88,9; K2b.1 201,4; K2b.2 152,0; K3a.1 191; K3a.2 173,1; K3b.1 189,5; K3b.2 175; K4a.1 91,9; K4a.2 72,5

K4b.1 73,5; K4b.2 166,2

Testdigestion with 500 ng DNA, 0,5 µL Cla-I and 0,5 µL SalI, 5 µL Cut smart ad 25 µL for 15 min at 37°C

Adding of 5 µL Loading Dye to the digest. Running a 0,8% gel with 5µL Marker and 15µL of the digestion samples at 130V.

Pipetting scheme was this:

Gel1:

M | K1.1 | K1.2 | K2a.1.1 | K2a.1.2 | K2a2.1 | K2a2.2 | K2b.1| K2b.2 | K3a.1| K3a.2 | K3b.1 | K3b.2 | K4a.1

Gel2:

M | K4a.2 | K4b.1 | K4b.2

[ ]

12

Digestion of E.coli constructs, ligation of vector and constructs, Heat-Shock of CHO-constructs in DH5a, overhang PCR with bacterial constructs

|  |  |
| --- | --- |
| Aim / Task | Digestion of E.coli constructs, ligation of vector and constructs, Heat-Shock of CHO-constructs in DH5a, overhang PCR with bacterial constructs |
| Protocol |  |
| Participants | Martin Borgmann, Lena Schorr, Leon Altmann |

**Notes**

* Digestion of paCYC and K1, K2a, K2b, K3a, K3b, K4a, K4b with ClaI and SmaI –wrong enzymes!!! (instead of SmaI, SalI should have been unsed)
* Heat-Shock of constructs (K1-1, K1-2, K2a-1, K2a-2, K2b-1, K2b-2) in DH5a
  + with 0,5µl of Mini samples from 30.8.19
  + plating the constructs on Amp plates
  + storage in the incubator
* Overhang PCR with K1, K2a, K2b, K3a, K3b, K4a, K4b (from initial IDT constructs) and K2a, K2b, K3a, K4b from PCR clean up from 13.08.

|  | 1x | 16x |
| --- | --- | --- |
| Q5 high fidelity | 25µl | 400µl |
| 10µM forward primer | 2,5µl | 40µl |
| 10µM reverse primer | 2,5µl | 40µl |
| DNA template | 1µl | - |
| nuclease free water | 19µl | 304µl |

* gel electrophoresis (0,8% and 1%)

  + 0,8%: ladder - K1 - K2a - K2b - K3a - K3b - K4a - K4b - H2O (from the initiall constructs)
  + 1%: ladder - K2a - K2b - K3a - K3b - K4a - K4b - H20
* Preparation of overnight culture of heat shocked bacteria with LB and Amp

![<Overhang_PCR_restriction>](/labjournal-entries/images/Overhang_PCR_restriction.jpg)
![<Overhang_PCR_restriction2>](https://static.igem.org/mediawiki/parts/9/97/T--FAU_ERLANGEN--overhang_PCR_restriction_2.jpg)

[ ]

14

Midi Prep of ON cultures (K1-1, K1-2, K2a-1, K2a-2, K2b-1, K2b-2); Preparation of new LB Medium, PCR purification of PCR samples from the 12.9.; inoculation of clones from the heatshock from the 13.9. in 5ml LB-Amp

|  |  |
| --- | --- |
| Aim / Task | Midi Prep of ON cultures (K1-1, K1-2, K2a-1, K2a-2, K2b-1, K2b-2); Preparation of new LB Medium, PCR purification of PCR samples from the 12.9.; inoculation of clones from the heatshock from the 13.9. in 5ml LB-Amp |
| Protocol | Midi prep protocol |
| Participants | Martin Borgmann, Lena Schorr |

**Notes**

* PCR purification o

  + PCR samples from the 12.9
  + Elution in 150 µl MiliQ water
  + concentration:
* Preparation of 3 1l bottles LB medium
* Midi Prep of the overnight cultures of

K1-1, K1-2, K2a-1, K2a-2, K2b-1, K2b-2 (CHO constructs) in DH5a

* protocol ion labfolder at the 20.8.
* eluation in 100µl MilliQ water
* Inoculation of clones from the heatshock (13.9.) in 5ml LB-Amb

  + over night in the shaker at 37°

**Pending tasks, next steps, comments for next people in the Lab**

–>Mini Prep of the overnight cultures

–>gel-electrophoresis of the midi prep + from the current mini-prep

[ ]

15

iniprep of ON cultures from 14.09.19 + Gel electrophoresis + Gel electrophoresis from Midiprep from 14.09.19

|  |  |
| --- | --- |
| Aim / Task | iniprep of ON cultures from 14.09.19 + Gel electrophoresis + Gel electrophoresis from Midiprep from 14.09.19 |
| Protocol | ExtractMe Plasmid Mini Kit |
| Participants | Martin Borgmann |

**Notes**

* Miniprep of over night cultures from 14.09.19 using “ExtractMe Plasmid Mini Kit”

  + Elution in 60 µL (50µL left after taking 10 µL for electrophoresis)
* Preparation of Gel electrophoresis

  + Dilution of Midiprep samples from 14.09.19

  1:100

  to avoid extrem bright fluorescence that might disturb the picture of the upcoming gel electrophoresis (samples have a DNA conc. of up to 5700 ng/µL)

  + 10 µL sample + 990 µL H2O
  + Sample “Midi K2-a-2” diluted 1:10 since its DNA conentration was only 337 ng/µL)
  + 10 µL sample + 90 µL H2O
* Gel electrophoresis in 0,8% agarose gel

  + Loading scheme: 1kb ladder | Mini K1-1 | Mini K1-2 | Mini K2a-1 | Mini K2a-2 | Mini K2b-1 | Mini K2b-2 | Midi K1-1 | Midi K1-2 | Midi K2a-1 | Midi K2a-2 | Midi K2b-1 | Midi K2b-2

![<Miniprep_Gel_electrophoresis>](https://static.igem.org/mediawiki/parts/9/98/T--FAU_ERLANGEN--gel_photo_14.09..JPG)

[ ]

18

Control Digest of MidiPrep samples (14.9) and Mini Prep samples (15.9); Prepare samples for Kiel

|  |  |
| --- | --- |
| Aim / Task | Control Digest of MidiPrep samples (14.9) and Mini Prep samples (15.9); Prepare samples for Kiel |
| Protocol |  |
| Participants | Leon Altmann |

***Notes***

Digest of MidiPrep samples (14.9) and Mini Prep samples (15.9) with EcoRI-HF and NHeI-HF to check properly check the samples;

| **Name** | **Mini Prep samples [µl]** | **Midi Prep samples [µl]** |
| --- | --- | --- |
| DNA | 2 | 0,5 |
| EcoRI-HF | 0,5 | 0,5 |
| NheI-HF | 0,5 | 0,5 |
| Cut Smart Buffer x10 | 2 | 2 |
| Water | 15 | 1,5 |
| **Total amount** | **20 µl** | **20 µl** |

Incubate @37 °C for 20 min;

Inactivate @ 65 °C for 20 min;

Prepare 0,8 % Agarose Gel to examine digested samples;

**Loading order:**

| Ladder | Midi K1-1 | Midi K1-2 | Midi K2a- 1 | Midi K2a-2 | Midi K2b-1 | Midi K2b-2 | Mini K1-1 | Mini K1-2 | Mini K2a-1 | Mini K2a-2 | Mini K2b-1 | Mini K2b-2 | Ladder |

Prepared 100 µg of the Midi Prep samples K1-2, K2a-1, K2b-1 for Kiel and sent them there via Post;

[ ]

22

Splitting of the 293T cells

|  |  |
| --- | --- |
| Aim / Task | Splitting of the 293T cells |
| Protocol | - |
| Participants | Martin Borgmann, Eva Neugebauer |

**Notes:**

-293T cells were not properly attached
-Probably most of them died

**Work done:**

-Mediumchange of the cells

**Pending tasks, next steps, comments for next people in the Lab**

Check if cells are growing

Getting new cells and culture them proberly :)

[ ]

23

Splitting 239T cells

|  |  |
| --- | --- |
| Aim / Task | Splitting 239T cells |
| Protocol |  |
| Participants | Martin Borgmann, Eva Neugebauer |

**Notes**

Check HEKs in the Incubator –All of them died

**Pending tasks, next steps, comments for next people in the Lab**

Get new HEKs

Proceed with Measurement Part

[ ]

24

Splitting 239T cells

|  |  |
| --- | --- |
| Aim / Task | Splitting 239T cells |
| Protocol |  |
| Participants | Martin Borgmann, Eva Neugebauer, Leon Altmann |

**Notes**

Repeat the IPTG

**Pending tasks, next steps, comments for next people in the Lab**

Check if cells are growing

Getting new cells and culture them properly

**CFP part:**

Cheking plates in the incubator

Plenty colonies in Tuner but not BL21 and STAR (Those were the “new” competent cells)

=BL21 and STAR not properly competent!!

New heatshock with old stock of competent bacs (STAR and BL21) acording to Evas protocol

HEK:

Check the flasks in the incubator: Live and happy ~ 50% confluent

Seewing of 4 x 10^6 cells in a plate (10 mL) like following.

1. Discard medium
2. Washing with 3 mL Trypsin
3. Incubation with 3 mL trypsin
4. When cells were detached mixing with 7 mL Medium
5. Counting in neubauer chamber (total cell number 10,2 x 10^6 cells)
6. 8 mL cell mix in the plate + 2 mL medium

Rest of the cells back in the flask, ad 20 mL with medium

E. coli:

Digest of pACYC184: 5µL cutsmart, 11µL DNA, 1µL Cla-I, 1µL Sal-I

Water was not added! (WRONG)

Digest of the constructs which were already digested on the 07.09. but not for 1 h just 15 min.

1 h digest with Cla-I and Sal-I (each 0,5µL)

Ligation

[ ]

25

HEK transfer, vector Digestion, Ligation, Heatshock

|  |  |
| --- | --- |
| Aim / Task | HEK transfer, vector Digestion, Ligation, Heatshock |
| Protocol |  |
| Participants | Eva Neugebauer, Leon Altmann |

***Notes***

CFP part:

Checking plates in the incubator

Plenty colonies in Tuner but not BL21 and STAR (Those were the “new” competent cells)

=BL21 and STAR not properly competent!!

New heatshock with old stock of competent bacs (STAR and BL21) acording to Evas protocol

HEK:

Check the flasks in the incubator: Live and happy ~ 50% confluent

Seeding of 4 x 10^6 cells in a cell culture plate (10 ml) like following.

1. Discard medium
2. Washing with 3 ml trypsin
3. Incubation with 3 ml trypsin
4. When cells are detached mixing with 7 ml Medium
5. Counting in neubauer chamber (total cell number 10,2 x 10^6 cells)
6. 8 ml cell mix in the plate + 2 ml medium

Rest of the cells back in the flask, ad 20 ml with medium

E. coli:

Digest of pACYC184: 5µL cutsmart, 11µL DNA, 1µL Cla-I, 1µL Sal-I

Water was not added! (WRONG);

Incubation @37 °C for 1h and following inactivation @65 °C for 20 min;

Proceeded nonetheless and added water aftewards to have a convenient volume of 50 µl

Digest of the constructs (K1 - K4b) which were already digested on the 07.09.19. ClaI is not a time saver qualified enzyme and thus the digestion time of 15 minutes might have not been sufficient.
1 h digest with Cla-I and Sal-I (each 0,5µL) in a volume of 20 µl @37 °C;

Inactivation @65 °C for 20 min;

With the sample concentration determined on 7.9 the new sample concentration was calculated, as the digest altered the volume;

Sample Concentration:

| **K1** | **K2a** | **K2b** | **K3a** | **K3b** | **K4a** | **K4b** |
| --- | --- | --- | --- | --- | --- | --- |
| 67.5 ng | 110 ng | 58,4 ng | 71,8 ng | 63,8 ng | 56,6 ng | 62,9 ng |

Only 6 Chloramphenicol plates were left in the fridge, so ligation was only performed with K1, K2a and K2b;

Amounts necessary for the ligation were calculated for all constructs;

| **Construct** | **1:10 ng** | **1:10 µl** | **1:15 ng** | **1:15 µl** |
| --- | --- | --- | --- | --- |
| **K1** | 207,4 | 3 | 311,1 | 4,5 |
| **K2a** | 151,4 | 1,4 | 227,1 | 2,1 |
| **K2b** | 144,2 | 2,46 | 216,3 | 3,7 |
| **K3a** | 105,6 | 1,8 | 158,4 | 2,7 |
| **K3b** | 100 | 1,9 | 150 | 2,85 |
| **K4a** | 105,6 | 2,3 | 158,4 | 3,45 |
| **K4b** | 100,6 | 1,6 | 150,9 | 2,4 |

Ligation

|  | **K1** |  | **K2a** |  | **K2b** |  |
| --- | --- | --- | --- | --- | --- | --- |
|  | **1:10** | **1:15** | **1:10** | **1:15** | **1:10** | **1:15** |
| **DNA** | 3 | 4,5 | 1,4 | 2,1 | 2,46 | 3,7 |
| **Vector** | 1,25 | 1,25 | 1,25 | 1,25 | 1,25 | 1,25 |
| **Ligase Buffer x10** | 2 | 2 | 2 | 2 | 2 | 2 |
| **Ligase** | 1 | 1 | 1 | 1 | 1 | 1 |
| **H20** | 12,75 | 11,25 | 14,35 | 13,65 | 13,3 | 12,05 |
| **Total** | **20 µl** | **20 µl** | **20 µl** | **20 µl** | **20 µl** | **20 µl** |

Incubation @RT for 20 min;

Inactivation @65 °C for 20 min;

Perform Heatshock after Evas Protocol;

Heatshock samples were not plated on plates, as the remaining Chloramphenicol**+** were found to be contaminated;
Samples were therefor stored @4 °C;

**Pending tasks, next steps, comments for next people in the Lab**

* Produce new Chloramph Plates
* Test Transfection of HEK293T cells with K1

[ ]

26

calciumchoride transfection of heks with K1

|  |  |
| --- | --- |
| Aim / Task | calciumchoride transfection of heks with K1 |
| Protocol |  |
| Participants | Eva Neugebauer, Leon Altmann |

**Notes**

Take Plates with negative control for measurement Part out of the incubator, colonies grew on alle of them!

Picked clones and prepared ON cultures with 8 ml LB-Amp, put them in the fridge until later;

Medium Change 1h prior to Transfection of the HEKs, DMEM without Hepes (cells were already in Medium without Hepes);

Confluence ~ 70%;

Prepare new LB Agar for new Chloramphenicol Plates;
35 g LB Agar were weighed in and mixed thorougly with 1 l Water;

Autoclaved Medium;

Performed Calciumchlorid Transfection:

Perpared two mixes:

A) 1 ml of 2x HBS in a 15 ml Falcon;

B) DNA Mix:

| Water | 860 µl |
| --- | --- |
| Plasmid | 20 µl (1 µg/µl) |
| 2,5 M CaCl2 | 100 µl |
| 2,5 mM Chloroquine | 20 µl |

Carefully apply the DNA mix to the 2x HBS over the time of 1 min, while constantly blowing air into the HBS with a Pipette boy;

Vortex the resulting Mix thorougly and apply it dropwise to the Cells;

After the prepared Medium has cooled sufficiently added Chloamphenicol (35 mg/ml) to reach a concentration of 25 µg/ml –735 µl;

Poured new Chloramph Plates;
Seeded the Heatshock Samples from 25.9 that were stored in the fridge onto Chloramphenicol Plates;

Placed them in the Incubator;

Placed the earlier prepared ON cultures in the shaker;

Performed Medium Change 6 h post transfection with 10 ml Medium;

[ ]

27

measuring measuring part

|  |  |
| --- | --- |
| Aim / Task | measuring measuring part |
| Protocol |  |
| Participants | Eva Neugebauer, Leon Altmann |

**Notes**

**Measurement part:**

Take ON cultures out of shaker

Measure the smaples with spectrometer

Bl21 puc19 neg: 1,098

STAR puC19 neg: 1,17

TUNER pUC19: CFP neg: 1,41

Prepared samples and induced them with IPTG

| Culture [mL] | LB Amp [mL] | IPTG 1mM with LB amp [mL] |  |
| --- | --- | --- | --- |
| BL21 pos | 5 | 0 | 5 |
| STAR pos | 5 | 0 | 5 |
| TUNER pos | 5 | 0 | 5 |
| BL21 negative | 2,5 | 2,5 | 5 |
| STAR negative | 2,5 | 2,5 | 5 |
| TUNER negative | 2 | 3 | 5 |

Incubation for 3 hours at 37°C

Sonification:

Measuring cell desnity at spectrometer

acording to this 10^7 cells/ mL were added for a 10 mL endvolume solution

centrifugation for 10 min 4000 g

Resuspend in 10 mL sonification buffer

3 10s pulses at 30W

Remove cell debris at 10.000g for 10 min

Measuring in ELISA reader:

Not possible ELISA has just OD detection no flourescence

**E. Coli constructs:**

Plates from heatshock out of the incubator

they were all overgrown

K1 293T:

Medium chance

1:5 splitting of the 293T cell flask

[ ]

29

ITPG induction of ON culture

|  |  |
| --- | --- |
| Aim / Task | ITPG induction of ON culture |
| Protocol |  |
| Participants | Eva Neugebauer |

**Notes**

OD measurement of the ON cultures

| Culture | OD | amount ON culture for OD of 0,5 in 5 mL [mL] |
| --- | --- | --- |
| Tuner CFP pos | 2,2075 | 1,13 |
| Star CFP pos | 2,59 | 0,96 |
| BL21 CFP pos | 1,757 | 1,48 |
| Tuner CFP neg | 1,97 | 1,25 |
| Star CFP neg | 1,769 | 1,43 |
| BL21 CFP neg | 1,672 | 1,5 |

5mL induction culture for each ON culture with

1. 0,5 mM IPTG (2,5mL of 1mM IPTG solution in LB Amp)
2. 0,3 mM IPTG (1,5mL of 1mM IPTG solution in LB Amp)

ad 5 mL with LB

ON cultures filled up to ca 5 mL with LB amp

[ ]

30

transfection

|  |  |
| --- | --- |
| Aim / Task | transfection |
| Protocol | - |
| Participants | Eva Neugebauer, Leon Altmann |

## Protocol

***Notes***

Medium changen of the HEK plates (K2a, K2b) 1 h pre-transfection;

Harvesting supernatant from last weeks K1;

Splitting of the 5 flasks with HEK cells (ratio 1:5);

DNA for transfection stems from the Midi-Prep of 14th of September;

20 µg of DNA were brought into a 1 µg/µl concentration for the transfection;

Transfection with K2a, K2b according to Annes’ protocol, time of transfection approx. 11:00;

Changed Medium 8 h post-transfection with 10 ml DMEM;

[ ]

30
**(2)**

transfection

|  |  |
| --- | --- |
| Aim / Task | transfection |
| Protocol | - |
| Participants | Eva Neugebauer, Leon Altmann |

## Protocol

**Notes**

Medium changen of the HEK plates (K2a, K2b) 1 h pre-transfection;

Harvesting supernatant from last weeks K1;

Splitting of the 5 flasks with HEK cells (ratio 1:5);

DNA for transfection stems from the Midi-Prep of 14th of September;

20 µg of DNA were brought into a 1 µg/µl concentration for the transfection;

Transfection with K2a, K2b according to Annes’ protocol, time of transfection approx. 11:00;

Changed Medium 8 h post-transfection with 10 ml DMEM;

## October

[ ]

1

Western blot

|  |  |
| --- | --- |
| Aim / Task | Western blot |
| Protocol | - |
| Participants | Eva Neugebauer |

## Protocol

**Notes**

1. Preperation of a 10% Bis-tris Gel.
2. Mixing of 10 µL harvest of K1 day1, K1 day2, K2a, and K2b with 2x loading buffer
3. 95°C for 5 min
4. Loading of the gel with 5µL marker |K1 day1|K1 day4|K2a|K2b (samples 15µL)
5. Running gel for 1 h at 130V in 1x MOPS buffer
6. Blotting to the membrane at 65mA for 1h30
7. Blocking membrane in 10% milkpowder for 1h30
8. Washing with PBST
9. Adding primary antibody (mouse anti-Histag in 1:2000 dilution) 10mL in 10% milkpowder
10. Shaking overnight at 4°C
11. 3 times washing for 10 min with PBST
12. Adding secondary antibody (HRP anti mouse in 1:2000 dilution) 10 ml in 10% milkpowder
13. Shaking at RT for 1h
14. 3 times washing for 10 min with PBST
15. Adding 2mL of the reaction mix directly before taking the picture of the blot

![<Westernblot_02_10_2019>](/labjournal-entries/images/WB_02.10.png)

[ ]

1
**(2)**

Harvesting K2a and K2b supernatant

|  |  |
| --- | --- |
| Aim / Task | Harvesting K2a and K2b supernatant |
| Protocol | - |
| Participants | Eva Neugebauer, Leon Altmann |

## Protocol

**Notes**

Harvesting K2a and K2b supernatant, reapply 4 ml of Medium to the plates;

Prepared new plates for transfection;

Took 3 flasks with HEk Cells and collected the cells in a 50 ml Falcon after making them loose with trypsin and inactivating it again with Medum;

Cells were counted using a Neubauer Chamber in a 1:1 Dilution with Trypan Blue;
Counted: 115/131/119 –Mean is 121,6 Cells per quadrant –Therefore 2,432\*106 cells/ml;

Seed 4\*106 cells per Plate for the transfection tomorrow (1,64 ml of the cell suspension);

The remaining 5,4 ml (~13\*106 cells) were applied to a new cell culture flask;
Everything was put in the incubator @37 °C, 5% Co2;

[ ]

2

Harvesting supernatant

|  |  |
| --- | --- |
| Aim / Task | Harvesting supernatant |
| Protocol | - |
| Participants | Eva Neugebauer, Leon Altmann |

## Protocol

**Notes**

Change Medium 1h pre-transfection on the 6 prepared plates;

Harvesting supernatant from yesterdays transformation (K2a, K2b);

DNA for transfection stems from the Midi-Prep of 14th of September (K1-2, K2a-1, K2b-1);

20 µg of each DNA were brought into a 1 µg/µl concentration for the transfection;

With each Construct two plates were transfected (K1, K2a, K2b) according to Annes’ protocol, time of transfection approx. 11:30;

[ ]

3

Test Miniprep of used strains for Measurement Part

|  |  |
| --- | --- |
| Aim / Task | Test Miniprep of used strains for Measurement Part |
| Protocol | Miniprep |
| Participants | Leon Altmann |

## Protocol

Notes

Check XGal/IPTG/Amp+ Plates –overgrown, all colonies blue (on negative and positive plates!)
Induced the ON cultures with 0 mM, 0,33 mM and 0,5 mM IPTG;
Placed them in the incubator
Meanwhile Miniprep with 1 ml of each of the uninduced CFP pos cultures was performed.
Applied 10 µl sample + 3 µl 6xDye + 5 µl H2O to a 0.8% Agarose Gel

Gel: Ladder/Bl21/Star/Tuner

–Tuner does not contain the desired insert. This notion is corraborated by the pellets colour.

[ ]

5

Harvesting

|  |  |
| --- | --- |
| Aim / Task | Harvesting |
| Protocol | - |
| Participants | Eva Neugebauer |

## Protocol

**Notes**

Harvesting of all transfected cells:

collection of the medium and centrifugation at max for 10 minutes.

The supernatant still needs to be filtrated.

[ ]

8

Westernblot

|  |  |
| --- | --- |
| Aim / Task | Westernblot |
| Protocol | - |
| Participants | Eva Neugebauer, Leon Altmann |

## Protocol

***Notes***

1. Removal of of the storage buffer by centrifugation with 100 g for 1 min
2. Adding 600 µL binding buffer and centrifugation with 100 g for 1 min
3. Adding the whole sample in multiple steps to the colume with each time centrifugation for 30s at 100g (Keeping the flow through)
4. Washing once with 600 µL of binding buffer
5. First Elution step: Adding 200µL Elution buffer to the colum
6. Second Elution step: Adding 200 µL Elution buffer to the colum

Running 15% SDS Gel at 300V for 30 min

10 µL of the samples were applied and 7 µL of the ladder

Pipetting scheme:

Ladder | Wash K1 | Wash K2a | Wash K2b | Elution1 K1 | Elution 1 K2a | Elution 1 K2b | Elution 2 K1 | Elution 2 K2a | Elution 2 K2b

Stain the finished Gel with Coomasie and unstain it sufficiently to see clear bands;

Measure the samples in the Nanodrop A280:

Determine the Extinction Coefficient for each of the constructs with ProtParam (OnlineTool);

|  |  | 1 | 2 | 3 | Mean |
| --- | --- | --- | --- | --- | --- |
| Elution 1 | K1 | 25,54 | 25,08 | 27,04 | 25,9 |
|  | K2a | 75,6 | 72,57 | 72,02 | 73,22 |
|  | K2b | 47,28 | 48,16 | 48,99 | 48,14 |
| Elution 2 | K1 | 13,0 | 12,02 | 12,41 | 12,47 |
|  | K2a | 11,74 | 11,58 | 12,53 | 11,95 |
|  | K2b | 25,09 | 24,75 | 24,78 | 24,87 |

For a 1:1 ratio of K2a and b following volumes were added together:

E1: K2b 100µL with K2a 65,9µL

E2: K2a 100µL with K2b 48µL

Samples were sended to Kiel for killing assay.

10µL of each sample was kept for a westernblot to verify that the tag and catcherreaction happend.

[ ]

9

Validate data from 3.10.19

|  |  |
| --- | --- |
| Aim / Task | Validate data from 3.10.19 |
| Protocol | - |
| Participants | Leon Altmann |

## Protocol

**Notes**

Notes

Performed restriction digest with Tuner and BL21 (control) to check the result from 3rd October.
Took Miniprep samples from 3.10.19 and digested the samples with SalI and EcoRI;
Incubate @ 37 °C for 45 min;
Inactivation fro 20 min @ 65 °C;

Applied digested samples to 0.8% Agarose Gel:
Ladder/Bl21/Tuner

[ ]

10

Westernblot of the purified proteins

|  |  |
| --- | --- |
| Aim / Task | Westernblot of the purified proteins |
| Protocol | - |
| Participants | Eva Neugebauer |

## Protocol

10% BisTris Gel

10µL purified samples were mixed with 2x loading dye.

95°C for 5 min

Running gel for 1 h 130V:

Pipetting scheme of the gel: 5µL each

Ladder | E1 K1 | E1 K2a | K1 K2b | K1 K2ab | E2 K1 | E2 K2a | E2 K2b | E2 K2ab

Blotting for 1:30 h at 65mA

Blocking the membrane with 10% milkpowdersolution at 4°C for 5h

1x washing with PBST

Adding his-Ab (1:5000 in milkpowdersolution) overnight at 4°C

[ ]

15

New Transfection

|  |  |
| --- | --- |
| Aim / Task | New Transfection |
| Protocol | - |
| Participants | Eva Neugebauer |

## Protocol

**Notes**

Splitting HEK293T cells in 3 wells of a 6 well plate

[ ]

16

New Transfection, Purification, Western blot

|  |  |
| --- | --- |
| Aim / Task | New Transfection, Purification, Western blot |
| Protocol | - |
| Participants | Eva Neugebauer |

## Protocol

* Medium change 1 h before transfection
* For transfection 2 µg DNA were used of K1-1, K2a-1 and K2b-1: Diluted 500 µL medium with nothing added
* Mixed with 500µL Medium with nothing added with 4µL genjet
* Incubation for 10 min at RT

[ ]

18

New Transfection, Purification, Western blot

|  |  |
| --- | --- |
| Aim / Task | New Transfection, Purification, Western blot |
| Protocol | - |
| Participants | Eva Neugebauer |

## Protocol

Harvesting of the cells
Centrifuagtion at 2500 rpm for 5 min
600µL of supernatant were used for the purification via Histrap

Histrap Purification:
centrifuge at 4°C
open columns a quater and placing it in a 2 mL eppi
Centrifuation at 500 rpm for 1 min
Adding 600 µL Binding buffer
Centrifugation at 500 rpm for 1 min
Adding 600µL sample
Centrifugation at 500 rpm for 1 min
Washing with 600µL binding buffer
Centrifugation at 500 rpm for 1 min
3x Elution step: Adding 100µL Elution buffer and centrifugation at 500 rpm for 1 min

Tag-Catcher reaction:
Adding same colume K2a and K2b and incubate them 20 min at RT
For this samples: Supernatant (SN) harvest, Elution (E) 1-3

Gel running :

at 130V for 1 h with following pipetting scheme

Gel1
M, SN K1, SN K2a, SN K2b, SN K2ab, E1 K1, E1 K2a, E1 K2b, E1 K2ab, E2 K1, E2 K2a, E2 K2b, E2 K2ab

Gel 2
M, SN K1, SN K2a, SN K2b, SN K2ab, E3 K1, E3 K2a, E3 K2b, E3 K2ab

Marker 5µL, Samples 20µL (10µL sample+10µL loading dye)
After loading dye adding samples were cooked for 2 min at 95°C except the SN samples of the gel 2

Blotting:
Both gels were blotted semidry for 1:23 h at 130mA

Blocking:
Membranes were blocked for 1h 45 with 10% milkpowdersolution

Primary Ab:
2 washing steps with PBST
anti-Histag ab was diluted 1:2000 in 10mL 10% milkpowdersolution and added to the membrane
Incubation overnight at 4°C

[ ]

19

Westernblot

|  |  |
| --- | --- |
| Aim / Task | Westernblot |
| Protocol | - |
| Participants | Eva Neugebauer |

## Protocol

**Notes**

3x washing with PBST for each time 10 min at RT
Adding primary antibody which was diluted 1:2000 in 10% milkpowdersolution
1h RT incubation
3x washing with PBST for wach time 10 min at RT
Adding for each blot directly for taking the picture the solution for HRP (2mL)

[ ]

19
**(2)**

Test Miniprep of used strains for Measurement Part

|  |  |
| --- | --- |
| Aim / Task | Test Miniprep of used strains for Measurement Part |
| Protocol | Miniprep |
| Participants | Leon Altmann |

## Protocol

**Notes**

3x washing with PBST for each time 10 min at RT
Adding primary antibody which was diluted 1:2000 in 10% milkpowdersolution
1h RT incubation
3x washing with PBST for wach time 10 min at RT
Adding for each blot directly for taking the picture the solution for HRP (2mL)

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The following protocols cover our methods used for cloning, cell culture, protein analysis, and measurement.

[ ]
Puring gels

**Puring gels:**

*Why did we choose this method?*

For Coomassi staining and western blots gels are needed. The choice of
gel and % of the gel is dependent on the protein.

**Procedure:**

**SDS Gel for 2 Gels:**

Resolving Gel:

| Percentage Gel | 8% | 10% | 15% |
| --- | --- | --- | --- |
| H2O | 4.6 | 4.0 | 2.3 |
| 30% acrylamidmix | 2.7 | 3.3 | 5.0 |
| 1,5 M Tris (pH 8.8) | 2.5 | 2.5 | 2.5 |
| 10% SDS | 0.1 | 0.1 | 0.1 |
| 10 % APS | 0.1 | 0.1 | 0.1 |
| TEMED | 0.004 | 0.004 | 0.004 |

Stacking gel:

| Percentage Gel | 5% |
| --- | --- |
| H2O | 2.1 |
| 30% acrylamidmix | 0.33 |
| 1 M Tris (pH 6.8) | 0.25 |
| 10% SDS | 0.02 |
| 10 % APS | 0.02 |
| TEMED | 0.002 |

**BisTris Gel for 2 Gels:**

Resolving Gel:

| Percentage Gel | 8% | 10% | 12% |
| --- | --- | --- | --- |
| H2O | 8.74 | 7.41 | 6.07 |
| 30% acrylamidmix | 5.33 | 6.67 | 8.0 |
| 1,5 M BisTris | 5.72 | 5.72 | 5.72 |
| 10 % APS | 0.2 | 0.2 | 0.2 |
| TEMED | 0.008 | 0.008 | 0.008 |

Stacking gel:

| Percentage Gel | 5% |
| --- | --- |
| H2O | 7.13 |
| 30% acrylamidmix | 1.43 |
| 1,5 M BisTris | 1.33 |
| 10 % APS | 0.1 |
| TEMED | 0.01 |

[ ]
Handling of IDT sequences

## **Handling of IDT sequences:**

**Procedure:**

1. spin down aliquots
2. add required volume of TE buffer (20 µl)
3. vortex thoroughly
4. incubate on the heating block at 50°C for 20 min
5. Store at -20°C

[ ]
Preparation of all buffers

## **Preparation of all buffers:**

**LB Medium:**

| 10 g/L | Tryptone |
| --- | --- |
| 5 g/L | Yeast extract |
| 10 g/L | NaCl |

* Sterilize by autoclaving and store at room temperature.

**CC buffer:**

| 10mM | Hepes | 2.38 g/L |
| --- | --- | --- |
| 15 mM | CaCl2 | 2.21 g/L |
| 55 mM | MnCl2xH2O | 10.89 g/L |
| 250 mM | KCl | 18.64 g/L |

* Dissolve all components except MnCl2 and adjust the pH to 6.7 with
  KOH. Then add the MnCl2 an filter sterilize the solution over a 0.22
  µm filter

**SOC/SOB medium:**

1. To 950 mL of deionized H2O, add:

| Tryptone | 20g |
| --- | --- |
| Yeast extract | 5g |
| NaCl | 0.5g |

1. SOC medium is identical to SOB medium, except that it contains 20 mM
   glucose. To prepare SOB medium, combine the above ingredients and
   shake until the solutes have dissolved. Add 10 mL of a 250 mM
   solution of KCl. (This solution is made by dissolving 1.86 g of KCl
   in 100 mL of deionized H2O.)
2. Adjust the pH of the medium to 7.0 with 5 N NaOH (∼0.2 mL). Adjust
   the volume of the solution to 1 L with deionized H2O. Sterilize by
   autoclaving for 20 min at 15 psi (1.05 kg/cm2) on liquid cycle.
3. Just before use, add 5 mL of a sterile solution of 2 M MgCl2 and 5ml
   of MGSO4. (This solution is made by dissolving 40,66 g of MgCl2
   (Hydrate) and 49,3g MgSO4 (Hydrate) respectively in 90 mL of
   deionized H2O. Adjust the volume of the solution to 100 mL with
   deionized H2O and sterilize by autoclaving for 20 min at 15 psi
   [1.05 kg/cm2] on liquid cycle.)

   After the SOB medium has been autoclaved, allow it to cool to 60°C or
   less.
4. Add 20 mL of a sterile 1 M solution of glucose. (This solution is
   made by dissolving 18 g of glucose in 90 mL of deionized H2O. After
   the sugar has dissolved, adjust the volume of the solution to 100 mL
   with deionized H2O and sterilize by passing it through a 0.22-µm
   filter.)

**10% Glycerol**

| Glycerol | Glycerol |
| --- | --- |
| ddH2O | ddH2O |
| Total: | 1L of 10% Glycerol |

**Medium for 293T HEK cells (10%) (HEK medium):**

| DMEM - Dulbecco’s Modified Eagle Medium | 500 ml |
| --- | --- |
| FCS | 50 ml |
| L-Glutamin | 5ml |

[ ]
Cultivation of E.coli strains

## **Cultivation of *E.coli* strains:**

**Freezing of *E.coli* strains:**

1. Spinned for 10 minutes at 2,500 rpm
2. Discarded supernatant without disturbing the pellet, while leaving
   ~1ml for every sample
3. Resuspend the pellet in the remaining supernatant.
4. Added 900μl cell culture and 300μl 99% glycerol, to a final
   glycerol concentration of ~25%. 6.
5. Store in -80 °C freezer

[ ]
Preparation of chemically competent bacteria

## **Preparation of chemically competent bacteria:**

*Why did we choose this method?*

Competent cells have an increased cell permeability. This is an important
characteristic for the following transformation in which the aim is to
transfer DNA into the bacteria.

**Procedure:**

1. Inoculate 5 ml LB medium with the appropriate antibiotics with the
   E. coli strain of which you want to make competent cells and incubate
   overnight at 37°C. When preparing DH5a competent cells it is better to
   use SOB medium instead of LB.
2. Use the overnight culture to inoculate 500 ml LB medium and incubate
   at 30°C until the absorbance at 600 nm is between 0.4-0.6. Optional: add
   2.5 ml 2M MgCl2 to the medium (to a final concentration of 10 mM) at the
   start of the cultivation.
3. Chill the culture for at least 10 min on ice. In the following steps
   the cell suspension should be kept on ice as much as possible.
4. Spin the cell suspension for 10 min at 6000 rpm (Sorvall GSA rotor)
   or 4000 rpm when harvested in 50-ml Falcon tubes.
5. Gently resuspend the pellet in 100 ml ice-cold CC buffer in 50-ml
   Falcon tubes. Resuspend with a 10-ml serological pipette and avoid
   introducing bubbles.
6. Incubate the cell suspension on ice for at least 10 min.
7. Spin for 10 min at 4000 rpm at 4°C.
8. Gently resuspend the pellet in 18.6 ml ice-cold CC buffer and add 1.4 ml DMSO.
9. Incubate the cell suspension on ice for at least 10 min.
10. Distribute the cell suspension in 100-200 µl aliquots in 0.5 or
    1.5-ml microfuge tubes.
11. Flash freeze the cell suspension in liquid nitrogen and store the
    tubes at -80°C. At -80°C the cell will be competent for at least 6
    months.

[ ]
Preparation of electro-competent cells

## **Preparation of electro-competent cells**

*Why did we choose this method?*

Competent cells have a increased cell permeability. This is an important
characteristic for the following transformation in which the aim is to
transfer DNA into the bacteria.

**Prepare first:**

* 2 liter of LB without NaCl (10 g tryptone, 5 g yeast extract)
* 250 ml of 8.7 % v/v glycerol autoclaved/sterile
* 2 liter of milliQ water autoclaved/sterile
* 250 ml cylinder closed with aluminum foil containing
  autoclaved/sterile
* 4x 2 L Erlenmeyer flasks autoclaved/sterile
* 4x 250 ml centrifuge tubes for Beckam rotor J14 autoclaved/sterile
* 5 ml pipet tips autoclaved/sterile
* 1 ml pipet tips autoclaved/sterile
* 200 µl pipet tips autoclaved/sterile
* At least 500x 0.5 ml eppendorf tubes autoclaved/sterile
* Required: Centrifuge Beckam with rotor J14 or similar
* Work very sterile because we do not use any antibiotics!

**Procedures:**

1. Reserve 15 ml LB in a small bottle at 4 °C
2. Inoculate 2 L LB Medium with a starter culture and mix well
3. Spread to 4x 2 L Erlenmeyer flasks
4. Incubate at 37 °C shaking 200 rpm
5. Measure OD 600 of all four flasks every 45 min
6. If the first flask reaches OD 0.40 put all flasks for 15 min on an
   ice-water bath
7. Pellet (1.) at 3500 G for 20 min at 4 °C
8. Discard supernatant and resuspend in 6x 150 ml ice cold water
9. Pellet (2.) at 5000 G for 20 min at 4 °C
10. Discard supernatant and resuspend in 6x 150 ml ice cold water
11. Pellet (3.) at 5000 G for 20 min at 4 °C
12. Discard supernatant and resuspend in 2x 150 ml ice cold water
    Attention: 3 buckets are now pooled in one!
13. Pellet (4.) at 5000 G for 20 min at 4 °C
14. Discard supernatant and resuspend in 2x 100 ml ice cold 8.7 %
    glycerol
    Attention: use now 8.7 % glycerol, not water!
15. Pellet (5.) at 5000 G for 20 min at 4 °C
16. Discard supernatant and resuspend in 2x 2.5 ml ice cold 8.7 %
    glycerol
17. Pool both buckets in one 50 ml tube
18. Make 50 µl aliquots in 0.5 ml eppendorf tubes
19. Freeze and store at -80 °C

[ ]
Polymerase chain reaction PCR

## **Polymerase chain reaction (PCR):**

*Why did we use this method?*

Within the method of PCR it is possible to amplify small fragments of
DNA which is very useful for further investigation. Furthermore, one
task of the project was to perform Gibson Assembly and thereby it was
necessary to fuse overhangs to the DNA fragments. Another method for
cloning was the restriction enzyme digest. Therefore, suitable
restriction sites had to be added to the ends of our DNA fragments. All
these tasks were performed by different programs of PCR using the
Phusion High-Fidelity PCR master mix.

**PCR Mix:**

| Template DNA | 5µl (250ng) |
| --- | --- |
| Forward primer | 1.25µl (0.5µM) |
| Reverse primer | 1.25µl (0.5µM) |
| 2xPhusion Master Mix | 12.5µl |
| Autoclaved H2O | 25µl |

**Distinct programs for PCR:**

*Amplification of DNA constructs:*

| Cycle Step | Temperature [°C] | Time [min.] | Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 98 | 00:30 | X1 |
| Denaturation | 98 | 00:10 | X40 |
| Annealing | 56.0 | 00:30 |  |
| Extension | 72.0 | 00:30 |  |
| Final Extension | 72.0 | 2:00 | X1 |
|  | 4.0 | hold |  |

*Adding of restriction sites with PCR:*

| Cycle Step | Temperature [°C] | Time [min.] | Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 98 | 5:00 | X1 |
| Denaturation | 98 | 00:10 | X40 |
| Annealing | 53.7 | 00:30 |  |
| Extension | 72.0 | 1:00 |  |
| Final Extension | 72.0 | 10:00 | X1 |
|  | 4.0 | hold |  |

*Adding overhangs for Gibson Assembly:*

| Cycle Step | Temperature [°C] | Time [min.] | Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 98 | 3:00 | X1 |
| Denaturation | 98 | 00:10 | X40 |
| Annealing | 59.8 | 00:30 |  |
| Extension | 72.0 | 1:00 |  |
| Final Extension | 72.0 | 10:00 | X1 |
|  | 4.0 | hold |  |

[ ]
Restriction digest

## **Restriction digest**

*Why did we choose this method?*

We used a restriction digest for our cloning strategy.

**Materials:**

- Template DNA

- Restriction Enzyme of choice

- 10x Cutsmart Buffer (NEB)

- ddH2O Procedure

**Procedure:**

1. Mix the following reagents.

| Concentration | Reagent |
| --- | --- |
| 4-5µg | Template DNA |
| 1µl | Restriction enzyme(s) |
| 4µl | Cutsmart Buffer (NEB) |
| Fill to 40µl | ddH2O |

2. Incubate 60 minutes 37°C. (respectively to enzyme)

3. Inactivate 20 minutes at 65°C

[ ]
Dephosphoryllation of the digested DNA fragments

## **Dephosphoryllation of the digested DNA fragments**

*Why did we choose this method?*

After vector digestion, the newly formed fragments could religate, which would prevent the insertion of our sequence.
Removing the phosphate groups of the fragment ends by dephosphorylation probits that issue.

**Master Mix:**

| Name | Amount |
| --- | --- |
| DNA | 3.3 µl |
| CutSmart x10 NEB | 5 µl |
| Arctic Buffer 10x NEB | 5 µl |
| Arctic Phosphatase NEB | 1 µl |
| SmaI NEB | 0.5 µl |
| H2O to 50 µl | 35.2 µl |

**Procedure:**

1. Incubate the samples at 37°C for 1 h;
2. Inactivate at 80  °C for 20 min (80 °C necessary to inactivate the
   phosphatase, SmaI itself 65 °C for 20 min would be sufficient)

[ ]
Electroporation

## **Electroporation**

*Why did we choose this method?*

This method makes cell membranes permeable, which allows to transform DNA into the
cells. The permeabilization is done by short pulses

**Procedure:**

1. Prepare 17 mm x 100 mm round-bottom culture tubes (e.g. VWR
   #60818-667) at room temperature. Place SOC recovery medium in a
   37°C water bath. Pre-warm selective plates at 37°C for 1 hour.
2. Place electroporation cuvettes (1 mm) and microcentrifuge tubes on
   ice.
3. As a positive control for transformation, dilute the control pUC19
   by 1:5 to a final concentration of 10 pg/μl using sterile water.
   Heat-denatured ligation reactions can be used for electroporation
   directly; however, column purification is recommended.
4. Thaw NEB Turbo Electrocompetent cells on ice (about 10 min) and mix
   cells by flicking gently. Transfer 25 μl of the cells (or the amount
   specified for the cuvettes) to a chilled microcentrifuge tube. Add 1
   μl of the DNA solution.
5. Carefully transfer the cell/DNA mix into a chilled cuvette without
   introducing bubbles and make sure that the cells deposit across the
   bottom of the cuvette. Electroporate using the following conditions
   for BTX ECM 630 and Bio-Rad GenePulser electroporators: 2.1 kV,
   100 Ω, and 25 μF. The typical time constant is ~2.6 milliseconds.
6. Immediately add 975 µl of 37°C SOC to the cuvette, gently mix up and
   down twice, then transfer to the 17 mm x 100 mm round-bottom culture
   tube.
7. Shake vigorously (250 rpm) or rotate at 37°C for 1 hour.
8. Dilute the cells as appropriate then spread 100-200 μl cells onto a
   pre-warmed selective plate.
9. Incubate plates 8 hours to overnight at 37°C.

[ ]
Heat Shock

## **Heat Shock**

*Why did we choose this method?*

Heat shock is performed to insert our produced plasmids into our prepared chemically competent cells.

**Procedure:**

1. Thaw competent cells on ice.
2. Chill approximately 5 ng (2 μl) of the ligation mixture in a 1.5 ml
   microcentrifuge tube.
3. Add 50 µl of competent cells to the DNA. Mix gently by pipetting up
   and down or flicking the tube 4–5 times to mix the cells and DNA. Do
   not vortex.
4. Place the mixture on ice for 30 minutes. Do not mix.
5. Heat shock at 42°C for 30 seconds. Do not mix.
6. Add 950 µl of room temperature media to the tube.
7. Place tube at 37°C for 60 minutes. Shake vigorously (250 rpm) or
   rotate.
8. Warm selection plates to 37°C.
9. Spread 50–100 µl of the cells and ligation mixture onto the plates.
10. Incubate overnight at 37°C.

[ ]
Isolation of plasmid DNA from transformed bacteria (Mini – Midi Prep)

## **Isolation of plasmid DNA from transformed bacteria (Mini – Midi Prep)**

*Why did we choose this method?*

Isolating the plasmid from the transformed bacteria allows us to verify the cloning and transformation success.

**Procedure:**

**Mini Prep:**

1. Pick a single colony from a freshly streaked selective plate and
   inoculate a starter culture of 2–5ml LB medium containing the
   appropriate selective antibiotic. Incubate for approximately 8 h at 37°C
   with vigorous shaking (approx. 300 rpm). Use a tube or flask with a
   volume of at least 4 times the volume of the culture.
2. Dilute the starter culture 1⁄500 to 1⁄1000 into 3 ml selective LB
   medium. Grow at 37°C for 12–16 h with vigorous shaking (approx. 300
   rpm). Use a flask or vessel with a volume of at least 4 times the volume
   of the culture. The culture should reach a cell density of approximately
   3–4 x 109 cells per ml, which typically corresponds to a pellet wet
   weight of approximately 3 g/liter.
3. Harvest the bacterial cells by centrifugation at 6000 x g for 15 min
   at 4°C.
   If you wish to stop the protocol and continue later, freeze the cell pellets at –20°C.
4. Resuspend the bacterial pellet in 0.3 ml of Buffer P1. Ensure that
   RNase A has been added to Buffer P1.
5. Add 0.3 ml of Buffer P2, mix thoroughly by vigorously inverting the
   sealed tube 4–6 times, and incubate at
6. Add 0.3 ml of chilled Buffer P3, mix immediately and thoroughly by
   vigorously inverting 4–6 times, and incubate on ice for 5 min.
   Precipitation is enhanced by using chilled Buffer P3 and incubating on
   ice. After addition of Buffer P3, a fluffy white material forms and the
   lysate becomes less viscous.
7. Centrifuge at maximum speed in a microcentrifuge for 10 min. Remove
   supernatant containing plasmid DNA promptly. Before loading the
   centrifuge, the sample should be mixed again. Centrifugation should be
   performed at maximum speed in 1.5 ml or 2 ml microcentrifuge tubes
   (e.g., 10,000–13,000 rpm in a microcentrifuge).
8. Equilibrate a QIAGEN-tip 20 by applying 1 ml Buffer QBT, and allow
   the column to empty by gravity flow.
9. Apply the supernatant from step 7 to the QIAGEN-tip 20 and allow it
   to enter the resin by gravity flow.
10. Wash the QIAGEN-tip 20 with 2 x 2 ml Buffer QC. Allow Buffer QC to
    move through the QIAGEN-tip by gravity flow.
11. Elute DNA with 0.8 ml Buffer QF. Collect the eluate in a 1.5 ml or
    2 ml microcentrifuge tubes (not supplied).
12. Precipitate DNA by adding 0.7 volumes (0.56 ml per 0.8 ml of
    elution volume) of room-temperature isopropanol to the eluted DNA. Mix
    and centrifuge immediately at≥15,000xg rpm for 30 min in a
    microcentrifuge. Carefully decant the supernatant.
13. Wash DNA pellet with 1ml of 70 % ethanol and centrifuge at 15,000xg
    for 10min. Carefully decant the supernatant without disturbing the
    pellet.
14. Air-dry the pellet for 5–10min, and redissolve the DNA in a
    suitable volume of buffer (e.g., TE buffer, pH 8.0, or 10mM Tris·Cl, pH
    8.5)

**Midi-Prep:**

1. Harvest overnight bacterial culture by centrifuging at 6000 x g for
   15 min at 4°C.
2. Resuspend the bacterial pellet in 4 ml Buffer P1.
3. Add 4 ml Buffer P2, mix thoroughly by vigorously inverting 4–6 times
   and incubate at room temperature (15–25°C) for 5 min. If using LyseBlue
   reagent, the solution will turn blue.
4. Add 4 ml Buffer P3, mix thoroughly by vigorously inverting 4–6
   times. Incubate on ice for 15 min. If using LyseBlue reagent, mix the
   solution until it is colorless.
5. Centrifuge at ≥20,000 x g for 30 min at 4°C. Re-centrifuge the
   supernatant at ≥20,000 x g for 15 min at 4°C. 6. Equilibrate a
   QIAGEN-tip 100 by applying 4 ml Buffer QBT, and allow column to empty by
   gravity flow.
6. Apply the supernatant from step 5 to the QIAGEN-tip and allow it to
   enter the resin by gravity flow.
7. Wash the QIAGEN-tip with 2 x 10 ml Buffer QC. Allow Buffer QC to
   move through the QIAGEN-tip by gravity flow.
8. Elute DNA with 5 ml Buffer QF into a clean 15 ml vessel. For
   constructs larger than 45 kb, prewarming the elution buffer to 65°C may
   help to increase the yield.
9. Precipitate DNA by adding 3.5 ml roomtemperature isopropanol to the
   eluted DNA and mix. Centrifuge at ≥15,000 x g for 30 min at 4°C.
   Carefully decant the supernatant.
10. Wash the DNA pellet with 2 ml room-temperature 70% ethanol and
    centrifuge at ≥15,000 x g for 10 min. Carefully decant supernatant.
11. Air-dry pellet for 5–10 min and redissolve DNA in a suitable volume
    of appropriate buffer (e.g., TE buffer, pH 8.0, or 10 mM Tris·Cl, pH
    8.5).

[ ]
Colony PCR

## **Colony PCR:**

*Why did we choose this method?*

Polymerase chain reaction is an in vitro technique to exponentially
amplify DNA of interest. Here, we exploit this technique to confirm the
transformation and insertion of our sequence into our plasmids.

**Materials:**

* Template DNA
* Forward Primer
* Reverse Primer
* OneTaq Polymerase
* LB-Agar plate

**Procedure:**

1. Pick a clone with the pipet.

2. Putting the pipette in the PCR tube and then into the LB with
antibiotics.

1. Mix the following reagents.
2. Run a PCR (see protocol PCR)

| Concentration | Reagents |
| --- | --- |
| 5µl | OneTaq buffer |
| 0.25µM | Forward Primer |
| 0.25µM | Reverse Primer |
| 0.125µl | OneTaq Polymerase |
| Bacteria from 1 colony | Template |
| Fill to 25µl | ddH2O |

[ ]
Agarose-Gel electrophoresis

## **Agarose-Gel electrophoresis**

*Why did we choose this method?*

This method is used as a quality control of enzymatic reactions on DNA
or RNA. It is also useful for the separation of DNA or RNA fragments of
different lengths.

**Materials**

* DNA of Interest
* Gel Ladder
* 6x purple loading dye
* Agarose
* 1x TAE buffer
* Gel chamber
* Sybr Safe DNA stain
* UV illuminator + camera Procedure

1. Prepare an agarose gel with an appropriate concentration for the
   fragment (0.5%-3% (w/v)) in TAE buffer.
2. Heat solution until it is fully dissolved.
3. Add DNA stain.
4. Cast the gel in a gel chamber, add an appropriate comb and wait at
   least 20 minutes until the gel is fully polymerized.
5. Mix at least 100ng DNA with loading dye.
6. Load the gel with your sample.
7. Let it run at 120V, 400mA for 20 minutes.
8. Image the gel under a Camera with UV illuminator.

[ ]
Gibson Assembly

## **Gibson Assembly**

*Why did we choose this method?*

Gibson assembly is a very useful tool for cloning, because it is
possible to fuse several fragments. Therefore, Gibson assembly was chosen as one of our cloning strategies.

**Procedure:**

1. Set up the following reaction on ice:

|  | Rcommended Amount of Fragments Used for Assembly |  |  |
| --- | --- | --- | --- |
|  | 2-3 fragments | 4-6 fragments | Positive control |
| Total Amount of Fragments | 0.02-0.05 pmol \* x µl | 0.2-1 pmol \* x µl | 10 µl |
| Gibson Assembly Master Mix (2x) | 10 µl | 10 µl | 10 µl |
| Deionized H2O | 10-X µl | 10-X µl | 0 |
| Total volume: | 20 µl | 20 µl | 20 µl |

1. assembled or 60 minutes when 4-6 fragments are being assembled.
   Following incubation, store samples on ice or at –20°C for
   subsequent transformation.

[ ]
Ligation

## **Ligation:**

*Why did we choose this method?*

With the help of the T4 Ligase, it is possible to connect DNA fragments
which were previous digested by restriction digestion. Thus, blunt-end cloning was performed with this method.

**Procedure:**

1. Set up the following reaction in a microcentrifuge tube on ice.

| Component | 20µl reaction |
| --- | --- |
| T4 DNA Ligase Buffer (10x) | 2 µl |
| Vector DNA (4kb) | 50 ng (0,020 pmol) |
| Insert DNA (1kb) | 37.5 ng (0.060 pmol) |
| Nuclease-free water | To 20µl |
| T4 DNA Ligase | 1 µl |

1. The T4 DNA Ligase Buffer should be thawed and resuspended at room
   temperature.
2. Gently mix the reaction by pipetting up and down and microfuge
   briefly.
3. For cohesive (sticky) ends, incubate at 16°C overnight or room
   temperature for 10 minutes.
4. For blunt ends or single base overhangs, incubate at 16°C
   overnight or room temperature for 2 hours (alternatively, high
   concentration T4 DNA Ligase can be used in a 10 minute ligation).
5. Heat inactivate at 65°C for 10 minutes.
6. Chill on ice and transform 1-5 μl of the reaction into 50 μl
   competent cells.

[ ]
Gelextraction

## **Gelextraction**

*Why did we choose this method?*

Gelextraction was performed to isolate specific DNA fragments from agarose gels.
Thereby, with the help of gelextraction it was possible for us to carry out further experiments with the desired DNA fragments.

**Materials:**

QIAquick® Gel Extraction Kit

**Procedure:**

1. Excise the DNA fragment from the agarose gel with a clean, sharp
   scalpel.
2. Weigh the gel slice in a colorless tube. Add 3 volumes Buffer QG to
   1 volume gel (100 mg ~ 100 μl). For >2% agarose gels, add 6 volumes
   Buffer QG.
3. Incubate at 50°C for 10 min (or until the gel slice has completely
   dissolved). Vortex the tube every 2–3 min to help dissolve gel.
4. After the gel slice has dissolved completely, check that the color
   of the mixture is yellow (similar to Buffer QG without dissolved
   agarose)
5. Add 1 gel volume of isopropanol to the sample and mix.
6. Place a QIAquick spin column in a provided 2 ml collection tube
7. To bind DNA, apply the sample to the QIAquick column and centrifuge
   for 1 min until all the samples have passed through the column. Discard
   flow-through and place the QIAquick column back into the same tube.
8. If the DNA will subsequently be used for sequencing, in vitro
   transcription, or microinjection, add 0.5 ml Buffer QG to the QIAquick
   column and S centrifuge for 1 min. Discard flow-through and place the
   QIAquick column back into the same tube.
9. To wash, add 0.75 ml Buffer PE to QIAquick column and centrifuge for
   1 min. Discard flow-through and place the QIAquick column back into the
   same tube.
10. Centrifuge the QIAquick column once more in the provided 2 ml
    collection tube for 1 min at 17,900 x g (13,000 rpm) to remove residual
    wash buffer.
11. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
12. To elute DNA, add 50 μl water to the center of the QIAquick
    membrane and centrifuge the column for 1 min.
13. If the purified DNA is to be analyzed on a gel, add 1 volume of
    Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting
    up and down before loading the gel.

[ ]
Cultivation of HEK 293T cells

## **Cultivation of HEK 293T cells:**

\*Why did we choose this method?

HEK 293T cells expressed our constructs optimized for eukaryotic expression. Cultivation is performed to obtain a sufficient number of cells for transfection and protein expression.

**Procedure:**

**Apply frozen 293T cells to cell culture**

1. Fill 10ml medium into a 75 cm² culture flask
2. Frozen cells in cryotubes should be defrosted quickly under warm water.
3. Transfer cells into centrifuge tubes and add 5ml HEK medium (see preparation of all buffers)
4. Centrifuge the tubes at 900 U for 5 min and discard the supernatant
5. Resuspend the pellet in 5ml HEK medium and put the suspension the the
   prepared culture flask from step 1

**Change medium of the adherent 293T cells**

1. Discard HEK medium
2. Add new HEK medium into the culture flask

**Trypsinization of the 293T cells to detach them from the culture flask
wall**

1. Discard HEK medium
2. Washing with 3 ml trypsin
3. Incubation with 3 ml trypsin
4. When cells are detached mixing with 7 ml HEK medium
5. Counting in neubauer chamber
6. Add respective amount of HEK medium

[ ]
FACS (fluorescence activated cell sorting)

## **FACS (fluorescence activated cell sorting):**

*Why did we choose this method?*

With the fluorescence-activated cell sorting (FACS) method it is possible to send cells trough a laser sensor
and check the condition of the cells. We used FACS for the part characterization.

**Procedure:**

1. Overnight culture of the bacteria
2. For prohibiting clumbing of the cells, they were treated 1:1 with
   ELISA buffer (with 0,05% Tween) direct before the measurement
3. Cells (*E.coli* bacteria) were not stained before FACS analysis
4. Cells were checked for fluorescence to measure CFP expression

   1. Excitation at 405nm
   2. Detection of CFP at 485nm
   3. Gain: FSC 165, SSC 400

[ ]
Protein purification by His SpinTrap

## **Protein purification by His SpinTrap**

*Why did we choose this method?*

We integrated Histidine (His)-tags in all our composite parts for detection and purification. This allows us to isolate our desired proteins via His-tag-purification. His-tag purification uses the purification technique of immobilized metal affinity chromatography. In this technique, transition metal ions are immobilized on a resin matrix using a chelating agent such as iminodiacetic acid. The most common ion for the his-purification is Ni2+. The His-tag has high affinity for these metal ions and binds strongly to the column. Imidazole competes with the His-tag for binding to the metal-charged resin and thus is used for elution of the protein.

Binding buffer: 50mM NaH2PO4, 500 mM NaCl, 20 mM Imidazol, pH 8,0

Elution buffer: 50 mM NaH2PO4, pH 8,0, 500 mM NaCl, 500 mM Imidazol

**Procedure:**

1. Place the column in a 2 ml microcentrifuge tube to collect the
   liquid during centrifugation.
2. Remove storage solution

* Invert and shake the column repeatedly to resuspend the medium.
* Loosen the top cap one-quarter of a turn and twist off the bottom
  closure.
* Place the column in a 2 ml microcentrifuge tube and centrifuge for
  30 s at 70 to 100 × g.
* Remove and discard the top cap.

1. Column equilibration

* Add 600 µl binding buffer.
* Centrifuge for 30 s at 70 to 100 × g

1. Sample application

* Add up to 600 µl sample in one application. • Centrifuge for 30 s at
  70 to 100 × g.

1. Wash

* Add 600 µl binding buffer.
* Centrifuge for 30 s at 70 to 100 × g.

1. Elution

* Add 200 µl elution buffer.
* Centrifuge for 30 s at 70 to 100 × g and collect the purified
  sample.
* Add 200 µl elution buffer.
* Centrifuge for 30 s at 70 to 100 × g and collect the purified sample

[ ]
Western Blotting

## **Western Blotting**

*Why did we choose this method*

Different purification tags are commonly used to isolate fusion proteins or to detect certain proteins within lysates or other protein mixtures. Western Blotting is a technique used to identify small amounts of tagged proteins.
A primary antibody detects and binds a specific target protein present in the sample and is then recognized by a secondary antibody, that is coupled to a fluorescent protein, radioactive substance, or an enzyme, which catalyzes a light emitting reaction. Then, detection systems are able to visualize the target proteins.

**Materials**

* Anti-6xHis Antibody (mouse)
* HRP anti-mouse (donkey)
* Washing buffer (1xPBS, 0.1% Tween-20)
* Transfer Buffer
* PVDF blotting membrane
* Blocking Buffer (10% milkpowder solution)
* Filter paper
* Methanol
* Proteinmarker: Page ruler: Protein Ladder Thermo Fisher

**Procedure**

1. Preperation of a 10% Bis-tris Gel.
2. Mixing of 10 µl harvested cells with 2x loading buffer
3. 95°C for 5 min
4. Loading of the gel with 5µL marker
5. Running gel for 1 h at 130V in 1x MOPS buffer
6. Blotting to the membrane at 65mA for 1h30
7. Blocking membrane in 10% milkpowder for 1h30
8. Washing with PBST
9. Adding primary antibody (mouse anti-His in 1:2000 dilution) 10mL in
   10% milkpowder
10. Shaking overnight at 4°C
11. 3 times washing for 10 min with PBST
12. Adding secondary antibody (HRP anti mouse in 1:2000 dilution) 10 ml
    in 10% milkpowder
13. Shaking at RT for 1h
14. 3 times washing for 10 min with PBST
15. Adding 2mL of the reaction mix directly before taking the picture
    of the blot

---

For the picture select channel “Chemiluminescence” to detect the bands.

For the marker select “Copy Marker” and don’t move the membrane between
the two pictures!

[ ]
Transfection of 293T cells

## **Transfection of 293T cells**

*Why did we choose this method?*

To insert our DNA of interest within our plasmids into eukaryotic cells, transfection was
performed with calcium-phosphate (calcium-phosphate transfection) and lipofectamine (lipofection).

**Conduction:**

**Calcium-phosphate transfection**

Day 0: Seed 4x106 HEK 293T cells in 10ml medium on a 10cm
dish

Day 1: One hour prior to transfection, change the medium (+8ml medium)

Plasmid should be diluted to a concentration od 1µg/µl in TE buffer

**Perpare two  mixes:**

A) 1 ml of 2x HBS in a 15 ml Falcon;

B)  DNA Mix:

| Water | 860 µl |
| --- | --- |
| Plasmid | 20 µl (1 µg/µl) |
| 2,5 M CaCl2 | 100 µl |
| 25 mM Chloroquine | 20 µl |

* Carefully apply the DNA mix to the 2x HBS over the time of 1 min,
  while constantly blowing air into the HBS with a Pipette boy
* Vortex the resulting mix thorougly and apply it dropwise to the
  cells
* Change medium 6-10 hours after transfection (+10ml medium)

Day 2: Change medium and collect the old medium

Day 3: Collect supernatant

Day 4: Collect supernatant

* For day 2-4 the harvest should be centrifuged and the remaining
  supernatant should be filtered through 0.22 µm filter.

**Lipofection:**

Day 0: Seed 4x10^6 HEK 293T cells in 10ml medium on a 10cm dish.

Day 1: Medium change 1 hour before transfection

* Preparation of following solutions

  + A: 500µl pure medium with 2µg DNA
  + B: 500µl pure medium with 4µl genjet (twice the amount of DNA)
* Mixing and incubation for 10 minutes at room temperature
* Add the solution dropwise to the cells

Day 3: Harvesting of the cells

* Centrifugation at 250rpm for 5 min.
* Filtering through

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<p>For the measurement, we chose the Part BBa\_E0020, which is an engineered cyan fluorescent protein (CFP) derived from *A. victoria* GFP designed by Caitlin Conboy and Jennifer Braff. We performed the cloning with the restriction enzymes XbaI and EcoRI into the vector pUC19. CFP was under control of the lac promotor and the lac operator. For the amplification, the cloned plasmid was transformed by heat shock into the E.coli cloning strain DH5α. After plasmid preparation, the construct was transformed into the E.coli expression strains BL21 Star (DE3) and BL21 (DE3). The clones for overnight cultures were picked under a fluorescence microscope to ensure that they contain the CFP insert.
To assure the same amount of cells, the OD of the distinct strains of the overnight cultures was measured.

<thead>| Culture | OD |
| --- | --- |
</thead>
<tbody>| Star CFP transformed | 2.59 |
| BL21 CFP transformed | 1.76. |
| Tuner CFP untransformed | 1.97 |
| Star CFP untransformed | 1.77 |
| BL21 CFP untransformed | 1.67 |
</tbody>

*Table 1: Measurement of the OD of the overnight cultures (Star, BL21, Tuner) of transformed and untransformed bacteria with the protein CFP by photometer*

Afterwards, they were set to an OD of 0.5 in a total volume of 5 ml. For induction of the expression, IPTG was used. The bacteria were induced for 2 h 20 min with 0 mM, 0.3 mM and 0.5 mM IPTG.

The fluorescence of the transformed bacteria was measured by fluorescence activated cell sorting (FACS). Therefore, the bacterial cells were separated from each other by treating them with 1:1 ELISA buffer (including 0,05% Tween). Afterwards, they were measured unstained within the FACS machine. Gain settings of the FACS were set to FSC 165 and SSC 400. The measuring part CFP was excited by the 405 nm laser line and detected by 485 nm.

# Results

The gating strategy for the identification of the *E.coli* cells by FACS machine can be seen in Figure 1. For the identification of the *E.coli* cells, the backgating method was used. The cell’s physical properties were investigated by using the forward and the sideward scatter, analysing the size and the granularity of the cells.

```
   <img class="w-2/3 mx-auto" src="![T--FAU_Erlangen--Identification_of_E.coli.png](https://static.igem.org/mediawiki/2019/5/5d/T--FAU_Erlangen--Identification_of_E.coli.png)
```

Figure 1

: Gating strategy for the identification of E.coli cells for further analysis of expressed CFP

```
   <img class="w-2/3 mx-auto" src="![T--FAU_Erlangen--IPTG_induction_of_BL21_and_Star.png](https://static.igem.org/mediawiki/2019/2/29/T--FAU_Erlangen--IPTG_induction_of_BL21_and_Star.png)" />
```

Figure 2

: Comparison of IPTG inductions with different concentrations (0mM, 0.3mM, 0.5mM) in BL21 (Fig. A) and Star (Fig. B)

The CFP expression was analysed afterwards by measuring the fluorescence signal. For the analysis, the cell counts were normalized to mode. Different concentrations of IPTG were tested. In Figure 2 it can be seen that IPTG induction had no effect on the CFP expression. Nevertheless, CFP was expressed indicating a leaky expression independent of IPTG. According to the literature, leaky expression is a characteristic of lac promotors, which is a component of our chosen vector (Rosano und Ceccarelli 2014).

```
   <img class="w-2/3 mx-auto" src="![T--FAU_Erlangen--Comparison_of_transformed_and_untransformed_cells.png](https://static.igem.org/mediawiki/2019/1/11/T--FAU_Erlangen--Comparison_of_transformed_and_untransformed_cells.png)" />
```

Figure 3

: Comparison of IPTG inductions with different concentrations (0mM, 0.3mM, 0.5mM) in BL21 (Fig. A) and Star (Fig. B)

In Figure 3, a clear difference between transformed and non-transformed bacteria can be detected. Transformed bacteria were able to express CFP, which results in a higher fluorescence signal, whereas non-transformed bacteria showed no CFP signal. Therefore, it can be suggested that CFP is a non-toxic protein for the cells upon they survived the protein expression.

The fluorescence signal of BL21, Star and the negative control (Tuner) is compared in Figure 4.

```
   <img class="w-2/3 mx-auto" src="![T--FAU_Erlangen--Comparison_of_BL21%2C_Tuner_and_Star.png](https://static.igem.org/mediawiki/2019/f/f5/T--FAU_Erlangen--Comparison_of_BL21%2C_Tuner_and_Star.png)" />
```

Figure 4

: Comparison of BL21, Tuner and Star

BL21 shows the highest fluorescence signal meaning that CFP expression is stronger in this strain compared to Star. This result is contrary to our expectations, because Star has mutations in the RNAseE (rne131), which results in reduced mRNA degradation and therefore a higher protein expression was suggested (Lopez et al. 1999).

The FACS method can also be used to analyse a whole cell culture.

```
   <img class="w-2/3 mx-auto" src="![T--FAU_Erlangen--Cell_culture_analysis_by_FACS.png](https://static.igem.org/mediawiki/2019/3/3b/T--FAU_Erlangen--Cell_culture_analysis_by_FACS.png)" />
```

Figure 5

: Cell culture analysis by FACS

In Figure 5 BL21(DE3) is depicted, which was analysed by FACS as a preliminary experiment to investigate the properties of the strain. It was expected that the culture is monoclonal, derived from one single clone. The result of the FACS experiment demonstrates that the bacteria of this culture did not all origin from one clone, because two peaks at different fluorescent levels can be noticed. This suggests a special application of the FACS method to define the assets of a bacterial culture.

In summary, it can be said that BL21 Star (DE3) and BL21 (DE3) are able to express functional CFP. Furthermore, analysis of the bacterial cells can be conducted via the FACS method and cell cultures can be investigated upon their properties, like their uniformity.

## References

Lopez, P. J.; Marchand, I.; Joyce, S. A.; Dreyfus, M. (1999): The C-terminal half of RNase E, which organizes the Escherichia coli degradosome, participates in mRNA degradation but not rRNA processing in vivo. In: *Molecular microbiology* 33 (1), S. 188–199. DOI: 10.1046/j.1365-2958.1999.01465.x.

Rosano, Germán L.; Ceccarelli, Eduardo A. (2014): Recombinant protein expression in Escherichia coli: advances and challenges. In: *Frontiers in microbiology* 5, S. 172. DOI: 10.3389/fmicb.2014.00172.

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# Results

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# Molecular Dynamics Simulations

The MD-simulations of the two linker types showed the average end-to-end distance of each considered linker.
The results provide two interest insights. First, for every motif repeat *n*, the *(EA3K)n* linker always possessed a longer average end-to-end distance than the according *(G4S)n* (Fig 1).

![](https://static.igem.org/mediawiki/2019/2/21/T--FAU_Erlangen--g4s_hist.png)

![](https://static.igem.org/mediawiki/2019/6/6a/T--FAU_Erlangen--eak_hist.png)

Figure 1b

: Length distribution of the (EA3K)n linker.

Second, the end-to-end distance of both linkers showed a linear dependence from the number of amino acids. Here, the *(EA3K)n* revealed a steeper slope than the *(G4S)n* linker, which agrees with the aforementioned relation between the average lengths of those two linker types.

![](https://static.igem.org/mediawiki/2019/0/0b/T--FAU_Erlangen--I_vs_n.png)

Figure 2

: Average length against number of amino acids.

In combination with existing literature our simulations allowed us to choose a linker sequence, leading to the successful expression of our bispecific antibody.

# Wet-lab

## Sequencing Data

To confirm the accuracy of the HEK293T transfection, sequencing of the constructs was performed.
To cover the complete construct, the sequencing was done in both directions.
The result are depicted in figures 3-5 and matched to the designed sequences.

![](https://static.igem.org/mediawiki/parts/thumb/a/a4/T--FAU_Erlangen--Bild_Sequencing_K1_Results_Composite_part.png/800px-T--FAU_Erlangen--Bild_Sequencing_K1_Results_Composite_part.png)

Figure 3

: Sequencing data of K1 (complete bispecific antibody)

![](https://static.igem.org/mediawiki/parts/thumb/6/6a/T--FAU_Erlangen--Bild_Sequencing_K2a_Results_Composite_part.png/850px-T--FAU_Erlangen--Bild_Sequencing_K2a_Results_Composite_part.png)

Figure 4

: Sequencing data of K2a (GPA33+SpyCatcher)

![](https://static.igem.org/mediawiki/parts/thumb/a/a8/T--FAU_Erlangen--Bild_Sequencing_K2b_Results_Composite_part.png/800px-T--FAU_Erlangen--Bild_Sequencing_K2b_Results_Composite_part.png )

Figure 5

: Sequencing data of K2b (CD3+SpyTag)

## Transport of the protein into the medium with the Igκ leader

For the detection of the protein a western blot was conducted.
The primary antibody was chosen against His-Tag, since all constructs include one or two HisTags.
All three constructs - K1 (the complete bispecific antibody), K2a (GPA33+SpyCatcher), K2b (CD3+SpyTag) - were successfully expressed in HEK 293T cells.
The western blot of the harvest is shown in figure 6, which confirmed the proteins at expected height (K1: 54 kDa, K2a: 42 kDa, K2b: 41 kDa).
The presence of the protein was able to show the ability of the Igk leader to transport the protein into the medium.
In lane K2a two bands can be seen. In the literature a second line below the expected line could be explained by the use of an additional Start codon downstream of the sequence (Kochetov, 2008).
This phenomenon is unlikely for K2a, since the Igκ leader sequence wouldn’t be transcripted and thereby the protein not transported out of the cell.
This second band and the blurry band on the top in the line of K2a might be an effect of protease digestion.

![](https://static.igem.org/mediawiki/parts/thumb/d/d5/T--FAU_Erlangen--Bild_Sequencing_Ernte_Results_Composite_part.png/200px-T--FAU_Erlangen--Bild_Sequencing_Ernte_Results_Composite_part.png)

Figure 6

: Western blot of the harvest after transfection of HEK 293T cells with an anti His-Tag antibody of the constructs K1, K2a and K2b.

## Purification via HisTag

For the further use of the protein purification is necessary.
The presence of the His-Tag in the constructs provides the possibility to detect the protein in a western blot and in addition to purify the protein.
In figure 7 the western blot after the purification of the proteins over a HisTrap column can be seen.
All constructs were still present after the purification. This shows that all constructs can be purified via their His-Tag.
Blurry bands in the western blot might have been the result of protease degradation.

![](http://parts.igem.org//wiki/images/thumb/4/49/T--FAU_Erlangen--Bild_Sequencing_Aufreinigung_Results_Composite_part.png/150px-T--FAU_Erlangen--Bild_Sequencing_Aufreinigung_Results_Composite_part.png)

Figure 7

: Western blot of the purified constructs K1, K2a and K2b with an anti His-Tag antibody

## SpyTag/SpyCatcher reaction

By combining K2a and K2b in a 1:1 ratio, the basis for the SpyTag/SpyCatcher reaction was provided.
In the later on performed western blot, which is depicted in Fig. 8. A new specific band could be detected at ~120 kDa.
A band of 83 kDa was expected. Self-reactivity of the constructs K2a and K2b, which is described in the literature [Keeble et al., 2017] can be excluded,
due to the lack of this specific band in the lines of the constructs themselves.
Regarding the size of the protein, which can be detected after combining K2a and K2b our suggestion is that a trimeric protein was produced. Polymeric proteins in the usage of the SpyTag/SpyCatcher system is described in the literature as a result of low level intramolecular interaction of the construct (Schoene, Fierer, Bennett, & Howarth, 2014). It is also described that scFvs may form multimers depending on the linker length connecting VH and VL [Atwell et al., 1999]

![](https://static.igem.org/mediawiki/parts/thumb/f/f9/T--FAU_Erlangen--Bild_Sequencing_Aufreinigung_TagCatcher_Composite_part.png/150px-T--FAU_Erlangen--Bild_Sequencing_Aufreinigung_TagCatcher_Composite_part.png)

Figure 8

: Western blot of the constructs K2a, K2b and K2a+b (1:1 ratio K2a with K2b) with an anti His-Tag antibody

## CD3 binding

For analysing the binding of the anti CD3 scFv to CD3 flow cytometry on T cells was performed.
The used gating strategy is exemplary depicted in figure 9 for the negative control.

![](https://static.igem.org/mediawiki/2019/4/45/T--FAU_Erlangen--FACS_Gating.png)

Figure 9

: Gating strategy of the FACS analysis to determining CD3 binding. After gating for IgG negative cells T cells were selected by CD4/CD8 expression.

K1, K2b and the fused K2ab should possess the ability to bind CD3.
In fact this is supported by the FACS analysis depicted in figure 10, visible as a nearly complete shift of the T cell population,
showing a positive signal for anti His-Tag antibody binding.
Just K2a, designed to bind GPA33 instead of CD3, was not able to bind the T cells.

![](https://static.igem.org/mediawiki/2019/a/a6/T--FAU_Erlangen--FACS_Konstrukte.png)

Figure 10

: FACS analysis of the constructs K1, K2a, K2b and K2ab for binding to CD3 on T cells.

## GPA33 binding

Binding of the anti GPA33 scFv to GPA33, should result in an increased anti His-Tag detection.
Mirroring the previously described CD3 binding, GPA33 should be bound by K1, K2a and K2ab.
Contrary to this presumption no shift of the signal in the corresponding channel is visible in figure 11.

This might be explained, if the CaCo-2 cells did not express GPA33 or if the GPA33 binding domain was defect.
CaCo-2 cells are known to vary their GPA33 expression in a cell cycle dependent manner (Frey, 2011).
The sequence of the GPA33 binding region was derived from the patent EP1801208A1, in which a successful binding was reported.
To confirm the transfected DNA, previously described sequencing was performed.
In addition to the consideration of the protein sequence for the lack of binding,
the protein might not folded properly or was damaged prior to the measurement as a result of unfavorable transport or buffer conditions.

![](https://static.igem.org/mediawiki/2019/c/c7/T--FAU_Erlangen--Histogram_CaCo.png)

Figure 11

: Histogram of all constructs (K1, K2a, K2b, K2ab) added to CaCo cells to determine binding

## Bispecific antibody functionality

To test our bispecific antibodies’ functionality a chromium-51 release assay was performed.
As the tested constructs do not possess a crystallizable fragment (Fc fragment) only a composite of the GPA33 binding and the CD3 binding scFv may facilitate killing.
Tested were K1, K2a, K2b and the fused K2ab. Additional Rituximab, an anti CD20 binding antibody (Plosker & Figgitt, 2003) was used as negative control and Cetuximab,
an epidermal growth factor receptor (EGFR) binding antibody was used as a positive control.
As described in Jonker et al., 2007 CaCo2 cells should express EGFR. However no sample showed an increasing amount of Cr-51,
depicted in figure 12, indicating no killing in any sample.
This is corroborated by the result that no GPA33 binding could be detected, as described previously.

![](https://static.igem.org/mediawiki/2019/e/e9/T--FAU_Erlangen--Cr-51.png)

Figure 12

: Testing of the four constructs and two monoclonal antibodies in a Cr-51 release assay on CaCo cells

# Artificial Neural Network “allerGEM”

Our allgerGEM achieved an accuracy of 99%. However, due to a bias in the relation of sequences with ten times more non-allergenic sequences than allergenic sequences, there are more reliant metrics for the performance of a binary classifier, like precision and recall.

![](https://static.igem.org/mediawiki/2019/9/9a/T--FAU_Erlangen--statistics-overview.svg)

Figure 13

: Precision and recall.

Our metric of choice was the area under curve (AUC) of the correctly allergenic labeled sequences plotted against the incorrectly allergenic labeled sequences. While an AUC value of 0.5 presents a random labeling and 1 perfectly labeled test data, our model scored a 0.985, and can thus be regarded as a quite successful classifier.

![](https://static.igem.org/mediawiki/2019/1/15/T--FAU_Erlangen--roc-final.png)

Figure 14

: Receiver operator curve

## References

Atwell, J. L., et al., “scFv multimers of the anti-neuraminidase antibody NC10: length of the linker between VH and VL domains dictates precisely the transition between diabodies and triabodies.“, *Protein engineering*, 1999:12(7), 597-604.

Frey, D. “Charakterisierung von gpA33 und Eignung als Antikörper-Target”, 2011

Jonker, D. J., et al., “Cetuximab for the treatment of colorectal cancer.”, *New England Journal of Medicine*, 2007:357(20), 2040-2048.

Keeble, A. H., et al., “Evolving accelerated amidation by SpyTag/SpyCatcher to analyze membrane dynamics.”, *Angewandte Chemie International Edition*, 2017:56(52), 16521-16525.

Kochetov, A. V. “Alternative translation start sites and hidden coding potential of eukaryotic mRNAs.”, *Bioessays*, 2008:30(7), 683-691.

Plosker, G. L., & Figgitt, D. P. “Rituximab. Drugs”, 2003:63(8), 803-843.

Schoene, C., et al., “SpyTag/SpyCatcher cyclization confers resilience to boiling on a mesophilic enzyme.”, *Angewandte Chemie International Edition*, 2014:53(24), 6101-6104.

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