iGEM 2023 — Team stuttgart

# iGEM Lab Notebook - June to November 2023

##### To see what was completed on each day, click the date in the following calender.

# June

| Monday | Tuesday | Wednesday | Thursday | Friday | Saturday | Sunday |
| --- | --- | --- | --- | --- | --- | --- |
|  |  |  | 1 | 2 | 3 | 4 |
| 5 | 6 | 7 | 8 | 9 | 10 | 11 |
| 12 | 13 | 14 | 15 | 16 | 17 | 18 |
| 19 | 20 | 21 | 22 | 23 | 24 | 25 |
| 26 | 27 | 28 | 29 | 30 |

## Date: Monday, 05.06.2023

**Participants:** Anna, Jana, Sumohit

## Summary:

Preparation of mediums and vectors

## Preparation of medium

* 3x 500 ml LB Medium prepared
* 2x 500 ml LB-Agar Medium prepared

## Resuspension of AB\_Pp-pAOX1,BBF10K\_000181:

From the iGEM Distrubion Kit plate, K4 (AB\_Pp-pAOX1,BBF10K\_000181) was resuspended with 10 µl of water. It was then incubated for 5 minutes and transferred into a PCR tube labeled "Pichia Vector".

## PCR for the Backbone of AB\_Pp-pAOX1,BBF10K\_000181

2 µl of the resuspended AB\_Pp-pAOX1,BBF10K\_000181 was transferred into a PCR tube. 6 µl of ddH2O was then added and mixed in. Four PCR reactions were set up as follows:

1. On the left thermocycler with the 1st Q5 MasterMix
2. On the left thermocycler with the 2nd Q5 MasterMix
3. On the right thermocycler with the 1st Q5 MasterMix
4. On the right thermocycler with the 2nd Q5 MasterMix

The PCR was carried out following the protocol and as described in the table.

| Step | Temperature | Time |
| --- | --- | --- |
| Initial Denaturation | 98°C | 30 s |
| Cycle Denaturation | 98°C | 10 s |
| Cycle Annealing | 64°C | 30 s |
| Cycle Elongation | 72°C | 105 s |
| GoTo Step 2 | 30 times |  |
| Final Extension | 70°C | 150 s |
| Hold | 4°C | ever |

## Notes:

A big thank you to the Freiburg team for allowing us to borrow their plate, as our iGEM package had been delayed.

## Date: Tuesday, 07.06.2023

**Participants:** Jana, Sarah V., Sumohit

## Summary:

Due to issues with the autoclave and unavailability of TAE buffer, the planned work for the day had to be canceled and delayed.

## Date: Wednesday, 08.06.2023

**Participants:** Jana, Jessica, Sumohit

## Summary:Medium autoklav. Ampicillin solution preparation. Agar plates poured.

## Medium autoklav

1x LB-Medium 500 ml
1x LB-Medium 250 ml
1x LB-Agar 500 ml
Autoclaved at IBVT

## Ampicillin solution preparation

5 ml Ampicillin prepared (100 mg/ml)

## Agar plates poured

Agar plates poured:
5x without Amp.
~13 / 14 plates with Amp. (not sure about the exact count)
Stored in refrigerator 1.366

## Date: Tuesday, 13.06.2023

**Participants:** Jana, Sarah V., Sumohit

## Summary:Agarose gel electrophoresis.

## Agarose gel electrophoresis

0.8% gel for PCR check of Pichia vector from Distribution Kit
Use of Loading dye with SDS
10 μl of sample from the PCR tubes
See protocol

Gel loading:
1kb plus on right vector block
Vektor Block links
M
MM1
MM2
MM1
MM2

Must repeat gel (without pBad vector)

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230613-pcrpichiavektor-1.png)

## Transformation of iGEM Biobricks in *E. coli* Sure cells.

Heatshock Transformation in *E. coli* SURE cells
(Procedure as per protocol)
2 setups of Pichia vector (from iGEM Kit)
1 setup of Pichia vector dilution
2 setups of pBad vector (iGEM 2021)
plated on LB-Amp plates & incubated at 30°C

## Date: Wednesday, 14.06.2023

**Participants:** Jana, Sarah S.

## Summary:Investigation of the transformation.

## Investigation of the transformation

Examined plates
Some colonies grew on pBAD 44.5
Only 2 colonies on pBAD 27.5
No colonies on all Pichia vectors
Plates further incubated

## Date: Thursday, 15.06.2023

**Participants:** Jana, Anna

## Summary:

Reviewed plates
No changes/status since Wednesday, June 14th
Medium
PCR of Pichia vector
Same progress as on June 5th
PCR product from June 5th used as a template
Agarose gel
0.8% gel for PCR check of Pichia vector from Distribution Kit
Used Loading dye with SDS
10 μl of sample from the PCR tubes
See protocol
Gel image & loading scheme:
1kb plus
Pichia Vector (MM1 2021)
Pichia Vector (MM1 2021)
Pichia Vector (MM2 2021)
Pichia Vector (MM2 2021)
M

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230615-pcrpichiavektor-1.png)

PCR clean-up (NEB Kit)
Setups 3 and 4 (total 45 μl)
Followed the instructions in the kit (-> DNA under 2 kb, centrifugation at 13000 rpm)
Elution in 12 μl
Measure concentration on NanoDrop!
Inoculate MiniPrep (from the transformation on June 13)
5 ml LB-Amp medium
Pick 3 colonies (pBAD-vector 44.5 ng/ml)
Overnight incubation at 37°C in shaker
New heat-shock transformation
3 setups each in SURE cells and NEB cells
Heat shock for 45 sec (standard protocol)
Heat shock for 30 sec
with centrifugation after recovery (13000 rpm, 1 min, 4°C -> discard supernatant and resuspend in 100 μl outgrowth medium)
Plate on Amp plates (200 μl or all for centrifuged sample)
Incubation at 30°C
Plate NEB and SURE cells without transformation on Agar plates (without Amp)
Incubation at 30°C

## Notes:

A big thank you to the Freiburg team for allowing us to borrow their plate, as our iGEM package had been delayed.

## Date: Friday, 16.06.2023

**Participants:**: Anna, Sumohit

## Summary:Plasmid isolation of the pBAD vector. Transformation with a new protocol. Sequencing.

Received Falcons and tip boxes + tips from Stutz, as well as large tip boxes from Biochemistry
Poured new agar plates with 100 μg/ml ampicillin

## Plasmid isolation

Plasmid isolation from ÜN cultures of the pBAD vector (elution with 12 ul) and measurement of DNA concentration of the Pichia vector (2 setups) from PCR clean-up

| Setup | Method | Concentration [ng/ul] | 260/280 | 230/260 |
| --- | --- | --- | --- | --- |
| pBAD 1 | PI | 76.2 | 1.85 | 2.13 |
| pBAD 2 | PI | 94.6 | 1.87 | 2.23 |
| pBAD 3 | PI | 65.9 | 1.89 | 2.19 |
| Pichia Vector PCR | PCR | 108.0 | 1.91 | 2.39 |
| Pichia Vector PCR | PCR | 121.6 | 1.88 | 2.37 |

## Transformation of 3 iGEM Pichia vectors

Transformation with a new protocol (see protocol) of 3 iGEM Pichia vectors
Plates left at room temperature over the weekend

## Sequencing

Sequencing of Pichia vectors from PCR or PCR clean-up and pBAD vectors

## Date: Monday, 19.06.2023

**Participants:** Jana, Sarah H.

## Summary: Investigation of transformation. Plasmid isolation

## Investigation of transformation

Colonies grown on all plates (3 Pichia vectors in NEB stable cells)
Plates in the refrigerator

Inoculation in 5 ml LB-medium + 100 μg/ml Amp (2 setups per vector)
Overnight incubation at 30°C in the shaker

## Task1

[Description of what happened in task 1]

## Task 2

[Description of what happened in task 2]

## Date: Tuesday, 20.06.2023

**Participants:** Jana, Sarah V., Sumohit

## Summary: Plasmid isolation. Sequencing of the isolated plasmids.

## Plasmid isolation

Plasmid isolation (MiniPrep)
Conducted as per Kit Manual (see protocols)
Eluted in 50 μl
DNA concentration measured using NanoDrop

| Sample | Concentration [ng/ul] | A260/280 | A230/260 |
| --- | --- | --- | --- |
| Pichia Vector 1.1 | 19.5 | 2.50 | 2.03 |
| Pichia Vector 1.2 | 37.1 | 2.15 | 2.27 |
| Pichia Vector 2.1 | 32.4 | 2.28 | 2.07 |
| Pichia Vector 2.2 | 31.9 | 2.27 | 2.41 |
| Pichia Vector 3.1 | 35.6 | 2.13 | 2.28 |
| Pichia Vector 3.2 | 41.7 | 2.33 | 2.34 |

Pichia Vector 1: well K4
Pichia Vector 2: well E14
Pichia Vector 3: well G14

## Sequencing

Sequencing
50-100 ng/μl DNA & 5 μM Primer
Since the concentration is so low:
7.5 μl Plasmid
2.5 μl Primer (10 μM)
Sample 1.2 forward (1): Seq. ID EMX401
Sample 1.2 reverse (2): Seq. ID EMX399
Sample 3.2 forward (1): Seq. ID EMX400
Sample 3.2 reverse (2): Seq. ID EMX398

## Date: Wednesday, 21.06.2023

**Participants:** Jana, Sarah S.

## Summary:

Overnight culture as on Monday, June 19th

## Date: Thursday, 22.06.2023

**Participants:** Sarah S., Sumohit

## Summary:Overnight culture

Overnight culture as on Monday, June 19th

## Date: Friday, 23.06.2023

**Participants:** Sarah H., Sumohit

## Summary: Restriction digestion of Minipreps with BSAI. Agarose gel electrophoresis. Plasmid isolation.

## Restriction digestion of Minipreps with BSAI

Approach:

| Component | Volume |
| --- | --- |
| ddH2O | ad 10 ul |
| BSAI | 0.5 ul |
| CutSmart | 1 ul |
| DNA (100 ng) | 1.1: 5.13 ul  1.2: 2.7 ul  2.1: 3.09 ul  2.2: 3.13 ul  3.1: 2.8 ul  3.2: 2.4 ul |

Incubation for 20 min at 37°C in PCR machine

## Agarose gel electrophoresis

0.7% Agarose Gel according to the protocol
Scheme:
M M1.1 M1.2 M2.1 M2.2 M3.1 M3.2 M

## Plasmid isolation (MiniPrep)

Conducted as per Kit Manual (see protocols)
Eluted in 50 μl
DNA concentration measured using NanoDrop

| Sample | Concentration [ng/ul] | A260/280 | A230/260 |
| --- | --- | --- | --- |
| Pichia Vector 1.1 | 53.8 | 1.87 | 1.90 |
| Pichia Vector 1.2 | 45.1 | 1.70 | 1.48 |
| Pichia Vector 2.1 | 92.1 | 1.93 | 2.24 |
| Pichia Vector 2.2 | 63.8 | 1.97 | 2.36 |
| Pichia Vector 3.1 | 69.4 | 1.82 | 1.90 |
| Pichia Vector 3.2 | 76.5 | 1.99 | 2.10 |

Pichia Vector 1: well K4
Pichia Vector 2: well E14
Pichia Vector 3: well G14

## Sequencing

50-100 ng/μl DNA & 5 μM Primer
Since the concentration is so low:
5 μl Plasmid
5 μl Primer (5 μM)
Sample 1.1 forward (1): Seq. ID EMX368
Sample 1.1 reverse (2): Seq. ID EMX367
Sample 2.1 forward (1): Seq. ID EMX366
Sample 2.1 reverse (2): Seq. ID EMX371
Sample 3.1 forward (1): Seq. ID EMX370
Sample 3.1 reverse (2): Seq. ID EMX369

## Date: Monday, 26.06.2023

**Participants:** Jana, Jessica, Sarah H.

## Summary: Preparation for competent cells. Preparation for PCR.

## Preparation for competent cells

Prepared RF1 and RF2 buffer for competent cells as per the protocol
Operated and calibrated the pH meter from the laboratory; calibration solutions available at IBVT
Coordination with IBVT regarding Falcon centrifuge, cold room, and liquid nitrogen
Items rearranged as needed
Protocol for competent cells was specified; for inquiries, contact Sarah H.

## Preparation for competent cells

Primer and fragment for AMP gel were dissolved and frozen

## Notes:

[Additional notes]

## Date: Tuesday, 27.06.2023

**Participants:** Jana, Sarah V.

## Summary: Gibson Assembly of AMP-0 (PCR and Agarose gel electrophoresis). Preparing for making component cells

## PCR

PCR: Gibson-Overhang Fragment
Fragment: Pre-experiment BB-41
Primers: Frag-AMP-forw & Alternative Fragment (diluted 1:10 -> 10μM)
Reaction Setup:
1.25 μl forward Primer
1.25 μl reverse Primer
1 μl Template DNA
12.5 μl Q5 High-Fidelity 2X Master Mix
9 μl water
PCR Program: AMPOVERH
(PCR Machine 1 -> right side)

| Step | Temperature [°C] | Duration [sec] | Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 98 | 30 |  |
| Denaturation | 98 | 10 | 30 |
| Annealing | 69 | 30 |  |
| Elongation | 72 | 18 |  |
| Final Extension | 72 | 120 |  |
| Hold | 4 | for ever |  |

## Agarose gel electrophoresis

Agarose Gel (1%) at 100V for 1 hour
1: 5 μl Marker
2: 10 μl PCR sample

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230627-pcrfragmentgibbsonoverhang-1.png)

## Preparing for making component cells

NEB stable -> Preculture
Pick 1 culture
in 4 ml LB-Medium + 1% Glucose (without Amp!)
overnight at 37°C in shaker

## Date: Wednesday, 28.06.2023

**Participants:** Jana, Jessica, Sarah S.

## Summary: Making of competent cells

## Making of competent cells

Preparation of competent cells as per the protocol.
OD values for cultivation:

| Time | OD 600 Culture 1 | OD 600 Culture 2 |
| --- | --- | --- |
| 0h 0 min | 0.04 | 0.04 |
| 45 min | 0.08 | 0.08 |
| 90 min | 0.23 | 0.24 |
| 105 min | 0.33 | 0.34 |
| 120 min | 0.43 | 0.54 |

50 μl aliquots of the competent NEB Stable cells are stored in the -70 Freezer, Box 5, in the bottom left of the tower in unlabeled boxes. The freezer is located in the laboratory.

## Date: Thursday, 29.06.2023

**Participants:** Jessica, Sarah H.

## Summary: Verification of the competence with different concetration and methods.

## Verification of the competence.

Verification of the competence of self-made NEB Stable cells
Transformation of pESP plasmid according to the protocol with various plating volumes or centrifugation

Plating volumes:

* 100 μl plating volume
* 200 μl plating volume
* Centrifuge in 50-100 μl and plate

Different plasmid amounts:

* 5 ng → 5 μl of 1ng/ul dilution
* 1 ng → 1 μl of 1ng/ul dilution
* 500 pg → 0.5 μl of 1ng/ul dilution

## Date: Friday, 30.06.2023

**Participants:** Jessica, Sarah H.

## Summary: Maintance.

## Maintance

Cleaning and autoclaving various items → Clearing items from the dishwasher and drying cabinet on Monday

Transformation efficiency results:

| Plate | Colony Count | Cfu/μg (Transformation Efficiency) |
| --- | --- | --- |
| 500pg\_100 ul | 1 | 2000 |
| 1ng\_100ul | 0 | 0 |
| 5ng\_100ul | 4 | 800 |
| 500pg\_200ul | 0 | 0 |
| 1ng\_200ul | 0 | 0 |
| 5ng\_200ul | 7 | 1400 |
| 500pg\_centrifuged | 1 | 2000 |
| 1ng\_centrifuged | 8 | 8000 |
| 5ng\_centrifuged | 16 | 3200 |

Average Transformation Efficiency: 1933.33 cfu/μg

# July

| Monday | Tuesday | Wednesday | Thursday | Friday | Saturday | Sunday |
| --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  | 1 | 2 |
| 3 | 4 | 5 | 6 | 7 | 8 | 9 |
| 10 | 11 | 12 | 13 | 14 | 15 | 16 |
| 17 | 18 | 19 | 20 | 21 | 22 | 23 |
| 24 | 25 | 26 | 27 | 28 | 29 | 30 |
| 31 |  |  |  |  |  |  |

## Date: Monday, 03.07.2023

**Participants:** Jana, Jessica, Sarah H.

## Summary: Dissolving enzyme fragments.

## Dissolving enzyme fragments

Dissolving enzyme fragments
1000 ng -> + 100 µl -> 10 ng/µl in ddH2O
Vortex
1-hour incubation (shaker under the bench)
Primers not found for this purpose / not yet arrived, hence no PCR

## Notes:

[Additional notes]

## Date: Tuesday, 04.07.2023

**Participants:** Anna, Sarah S., Sarah V.

## Summary: Competent cells. PCR for Gibson Assembly. Overnight cultures.

## Competent cells

Competent cells (buffer + o/n cultures)
Preparation of buffers for competent cells (new protocol by Antonin)
CC Buffer: 200 ml
CaCl2 (50 mM, 5.5 g/l) -> 1.1 g (dissolve first, then add Glycerol)
10% Glycerol (20 ml -> (Density = 1.25 g/ml) 25 g
CaCl2 (50 mM, 5.5 g/l) -> 1.1g in 200 ml
Sterile filtration (using vacuum)

## PCR for Gibson Assembly

Primers for enzymes have arrived (dissolved + frozen)
PCR: Overhang for Gibbson from His4, Dextranase Split1, and Split 2
Primer dissolution for enzymes (100 µM)
Vektor Gibson Primer for + rev
His4 for + rev
Mutanase for + rev
Dispersin B for + rev
Dextranase Split1 for + rev
Dextranase Split 2 for + rev
PCR: His4, Dextranase Split1, Dextranase Split2

His4 [2576 bp] -> His4over program (Device: PCR 1)

| Action | Temperature | Time |
| --- | --- | --- |
| Initial Denaturation | 98 °C | 30 s |
| Denaturation | 98 °C | 8 s (30 cycles) |
| Annealing | 69 °C | 20 s (30 cycles) |
| Elongation | 72 °C | 80 s (30 cycles) |
| Final Extension | 72 °C | 2 min |
| Hold | 4 °C |  |

Dextranase Split 1 [2166 bp] -> DEX1OH program (Device: PCR 2)

| Action | Temperature | Time |
| --- | --- | --- |
| Initial Denaturation | 98 °C | 30 s |
| Denaturation | 98 °C | 8 s (30 cycles) |
| Annealing | 70 °C | 30 s (30 cycles) |
| Elongation | 72 °C | 70 s (30 cycles) |
| Final Extension | 72 °C | 2 min |
| Hold | 4 °C |  |

Dextranase Split 2 [1808 bp] -> DEX2OH program (Device: PCR 2)

| Action | Temperature | Time |
| --- | --- | --- |
| Initial Denaturation | 98 °C | 30 s |
| Denaturation | 98 °C | 8 s (30 cycles) |
| Annealing | 72 °C | 30 s (30 cycles) |
| Elongation | 72 °C | 60 s (30 cycles) |
| Final Extension | 72 °C | 2 min |
| Hold | 4 °C |  |

## Overnight cultures

Overnight cultures for competent cells (NEB Stable)
Preculture from the cold room
Prepare 2x 10 ml LB-Medium (16:00)
Pick one colony each

## Notes:

[Additional notes]

## Date: Wednesday, 05.07.2023

**Participants:** Michael, Sarah S.

## Summary: Making of competent cells. PCR

## Making of competent cells

Competent cells were prepared according to Antonin's protocol. 200µl aliquots are stored at -80°C. A note is included in the bottom box of the rack.

## PCR

| Action | Temperature | Time |
| --- | --- | --- |
| Initial Denaturation | 98 °C | 30 s |
| Denaturation | 98 °C | 8 s (30 cycles) |
| Annealing | 70 °C | 30 s (30 cycles) |
| Elongation | 72 °C | 80 s (30 cycles) |
| Final Extension | 72 °C | 2 min |
| Hold | 4 °C |  |

## Date: Thursday, 06.07.2023

**Participants:**

## Summary: PCR. Verification of competence. Gel electrophoresis of PCR products.

## PCR:

* 25 µl setup
* DNA template had no concentration specified - used 1 µl
* Possibly used the wrong DNA template
* Primer at 10 µM each - used 1.25 µl
* 2x Q5 High Fidelity Mix 12.5 µl

| Action | Temperature | Time |
| --- | --- | --- |
| Initial Denaturation | 98 °C | 30 s |
| Denaturation | 98 °C | 8 s (30 cycles) |
| Annealing | 67 °C | 30 s (30 cycles) |
| Elongation | 72 °C | 95 s (30 cycles) |
| Final Extension | 72 °C | 2 min |
| Hold | 4 °C |  |

## Verification of competence of self-made NEB Stable Cells

Transformation of PESP plasmid following protocol (used 6 Eppis) with various plating volumes or centrifugation
- 3 Eppies centrifuged, discarded supernatant leaving 50 µl, resuspended in 300 µl, and plated with various volumes on different plates:
- Plating volume 200 µl
- Plating volume 100 µl
- 3 Eppies centrifuged, discarded supernatant leaving 50 µl, no new medium added, resuspended, and plated
Different plasmid amounts:
- 5 ng --> 5 µl of 1 ng/µl dilution
- 1 ng --> 1 µl of 1 ng/µl dilution
- 500 pg --> 0.5 µl of 1 ng/µl dilution

## Gel electrophoresis of PCR products:

- Gel loaded with 10 µl sample and 2 µl loading dye
- Gel loading:
Ladder - His 4 - Mut - Dis B - Dex 1 - Dex 2 - Vec

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230706-pcrfragmente-1.jpg)

## Date: Friday, 07.07.2023

**Participants:** Michael, Sumohit

## Summary: PCR.

**PCR for Dispersin B, His4, and Mutanase with the recommended temperatures from NEB and SnapGene.**

* PCR Setup:

+ 50 µl reaction volume
+ 1 µl DNA template
+ Primer MasterMix with Rev and For Primer 5 µl
+ 2x Q5 High Fidelity mix 25 µl
+ ddH2O 19 µl

**Dispersin B and His4 (NEB)**

| Action | Temperature | Time |
| --- | --- | --- |
| Initial Denaturation | 98 °C | 30 s |
| Denaturation | 98 °C | 10 s (30 cycles) |
| Annealing | 69 °C | 30 s (30 cycles) |
| Elongation | 72 °C | 85 s (30 cycles) |
| Final Extension | 72 °C | 3 min |
| Hold | 4 °C |  |

**Dispersin B and His4 (SnapGene)**

| Action | Temperature | Time |
| --- | --- | --- |
| Initial Denaturation | 98 °C | 30 s |
| Denaturation | 98 °C | 10 s (30 cycles) |
| Annealing | 72 °C | 30 s (30 cycles) |
| Elongation | 72 °C | 85 s (30 cycles) |
| Final Extension | 72 °C | 3 min |
| Hold | 4 °C |  |

**Mutanase (NEB)**

| Action | Temperature | Time |
| --- | --- | --- |
| Initial Denaturation | 98 °C | 30 s |
| Denaturation | 98 °C | 10 s (30 cycles) |
| Annealing | 70 °C | 30 s (30 cycles) |
| Elongation | 72 °C | 90 s (30 cycles) |
| Final Extension | 72 °C | 3 min |
| Hold | 4 °C |  |

**Mutanase (SnapGene)**

| Action | Temperature | Time |
| --- | --- | --- |
| Initial Denaturation | 98 °C | 30 s |
| Denaturation | 98 °C | 10 s (30 cycles) |
| Annealing | 70 °C | 30 s (30 cycles) |
| Elongation | 72 °C | 90 s (30 cycles) |
| Final Extension | 72 °C | 3 min |
| Hold | 4 °C |  |

**PCR clean-up of Pichia Vector:**

* Starting volume: 10 µl
* Nanodrop measurement: -1.5 µl

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230707-pcrfragmente-neb-snapgene-1.png)

![Nanodrop](https://static.igem.wiki/teams/4575/wiki/20230707-pcrcleanup-pichivector-1.png)

## Date: Monday, 10.07.2023

### Participant:

## Summary: PCR for Gibson Assembly

 **PCR**

Dextranase Split 1:

* 2 setups:

+ 2 different PCR programs
+ Concentrations in setups identical (see PCR protocol) - used fragment concentration: 10 ng/µl

Dex 1 [2166 bp] -> Program Dex1a (Device: PCR 1)

| Step | Action | Temperature | Time |
| --- | --- | --- | --- |
| 1 | Initial Denaturation | 98 °C | 30 s |
| 2 | Denaturation | 98 °C | 8 s (30 cycles) |
| 3 | Annealing | 70 °C | 25 s |
| 4 | Elongation | 72 °C | 80 s |
| 6 | Final Extension | 72 °C | 2 min |
| 7 | Hold | 4 °C |  |

Dex 1 [2166bp] -> DEX1BIGEM (Device: PCR in IBVT)

| Step | Action | Temperature | Time |
| --- | --- | --- | --- |
| 1 | Initial Denaturation | 98 °C | 30 s |
| 2 | Denaturation | 98 °C | 8 s (30 cycles) |
| 3 | Annealing | 68 °C | 25 s |
| 4 | Elongation | 72 °C | 80 s |
| 6 | Final Extension | 72 °C | 2 min |
| 7 | Hold | 4 °C |  |

Dextranase Split 2:

+ 2 setups:

Dex 2 [1808bp] -> Program His4over (Device: PCR 1)

| Step | Action | Temperature | Time |
| --- | --- | --- | --- |
| 1 | Initial Denaturation | 98 °C | 30 s |
| 2 | Denaturation | 98 °C | 8 s (30 cycles) |
| 3 | Annealing | 72 °C | 30 s |
| 4 | Elongation | 72 °C | 80 s |
| 6 | Final Extension | 72 °C | 2 min |
| 7 | Hold | 4 °C |  |

Dex 2 [1808bp] -> Program His4over (Device: PCR 1)

| Step | Action | Temperature | Time |
| --- | --- | --- | --- |
| 1 | Initial Denaturation | 98 °C | 30 s |
| 2 | Denaturation | 98 °C | 8 s (30 cycles) |
| 3 | Annealing | 72 °C | 25 s |
| 4 | Elongation | 72 °C | 80 s |
| 6 | Final Extension | 72 °C | 2 min |
| 7 | Hold | 4 °C |  |

## Notes:

[Additional notes]

## Date: Tuesday, 11.07.2023

**Participants:** Sumohit, Wintana

## Summary: Maintance.

Laboratory cleaned up

## Notes:

[Additional notes]

## Date: Wednesday, 12.07.2023

## Date: Thursday, 13.07.2023

**Participants:** Sarah S., Sarah V., Sumohit

## Summary: PCR.

 **PCR for pichia vector and His4.**

PCR: Pichia Vector (1 ng / 3 ng / 5 ng)

[Description of the PCR with Pichia Vector at different concentrations]

PCR: HIS4

* Fragment concentration: 5 ng/µl
* Annealing at 67°C for 30 seconds, otherwise similar conditions

## Notes:

[Additional notes]

## Date: Friday, 14.07.2023

**Participants:** Sarah S., Sarah V., Sumohit

## Summary: pH Verification of TAE Buffers. PCR. Agarose gel electrophoresis. PCR Gel Clean Up.

**pH Verification of TAE Buffers**

* All three bottles are at ~8.25 pH.
* It should ideally be between 8.3 and 8.5 (Antonin suggests we can still use them).

**PCR: Dispersin Overhang**

* Fragment concentration: 5 ng/µl
* Annealing at 67°C for 30 seconds, otherwise similar conditions

**Agarose Gel 0.8%:**

Samples include 3x Pichia Vector (from Thu, July 13), + His4 (from Thu, July 13) + Dispersin (from today, July 14).

* Large gel chamber: 80 V for 1 h 45 min --> we made a mistake and then ran it again at 120 V for 30 min (next time, run at 120 V for ~1 h).
* GelRed: Add 2 µl to 50 ml Gel, shortly before pouring.
* Here: large gel (250 ml) -> 10 µl GelRed.
* Add 5 µl Loading Dye -> load entire sample onto the gel.
* Expected sizes with overhangs [without OH]:
  + Pichia Vector: 1990 bp [3062]
  + His4: 2616 bp [2576]
  + Dispersin: 2756 bp [2715]
* Gel order: Marker - Dispersin B - His4 - PichiaVektor 1 ng - PichiaVektor 3 ng - PichiaVektor 5 ng - Marker

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230714-pcroverhang-pichia-his-disp-1.jpeg)

**PCR Gel Clean Up:**

In Eppi, there are 10 µl.

* PichiaV (1 ng): 36.7 ng/µl (260/280 = 1.99; 260/230 = 0.14)
* PichiaV (3 ng): 46.5 ng/µl (260/280 = 2.05; 260/230 = 1.27)
* PichiaV (5 ng): 33.3 ng/µl (260/280 = 1.98; 260/230 = 1.73)
* His4: 4.2 ng/µl (260/280 = 2.91; 260/230 = 0.52)

## Date: Monday, 17.07.2023

**Participants:** Michael, Sarah H.

## Summary: PCR for Gibson Assembly

 **PCR**

Mutanase
PCR Programm Muto

| Action | Temperature | Time |
| --- | --- | --- |
| Initial Denaturation | 98 °C | 30 s |
| Denaturation | 98 °C | 8 s (30 cycles) |
| Annealing | 68 °C | 30 s (30 cycles) |
| Elongation | 72 °C | 90 s (30 cycles) |
| Final Extension | 72 °C | 2 min |
| Hold | 4 °C |  |

PCR Programm for His4 [2576 bp] - Programm His4over (Gerät: PCR 1)

| Action | Temperature | Time |
| --- | --- | --- |
| Initial Denaturation | 98 °C | 30 s |
| Denaturation | 98 °C | 8 s (30 cycles) |
| Annealing | 67 °C | 30 s (30 cycles) |
| Elongation | 72 °C | 80 s (30 cycles) |
| Final Extension | 72 °C | 2 min |
| Hold | 4 °C |  |

| Substance | Volume [µl] |
| --- | --- |
| Q5 2xMM | 25 |
| fwd Primer | 2.5 |
| rev Primer | 2.5 |
| Template | 0.6 |
| ddH2O | 19.4 |

PCR Programm for His4 [2576 bp] - Programm His4over (Gerät: PCR 1)

| Action | Temperature | Time |
| --- | --- | --- |
| Initial Denaturation | 98 °C | 30 s |
| Denaturation | 98 °C | 8 s (30 cycles) |
| Annealing | 67 °C | 30 s (30 cycles) |
| Elongation | 72 °C | 80 s (30 cycles) |
| Final Extension | 72 °C | 2 min |
| Hold | 4 °C |  |

## Notes:

[Additional notes]

## Date: Tuesday, 18.07.2023

**Participants:** Sarah V., Wintana

## Summary:

**Dispersin B Optimization Strategy:**

Several temperature approaches for Dispersin B PCR (2 above, 2 below) - Gradient in PCR machine at IBVT.

* 1 setup with GC Enhancer
* 1 reaction with DMSO at 70°C

**Dispersin B PCR Reactions (25 μl setups; approximately 3 ng Template):**

* 66°C Annealing
* 68°C Annealing
* 70°C Annealing with 4% DMSO (1µl) (MOBINDING DMSO should also be in the freezer, but it's almost empty, otherwise ask Antonin)
* 70°C with 5 µl GC-Enhancer (Q5 High GC Enhancer should be somewhere in the freezer)
* 71°C Annealing
* 72°C Annealing

**DMSO:** 8 µl ddH2O
**GC-Enhancer:** 4 µl ddH2O

[Note: 9 ng of Dispersin Fragment used (oops)]

## Date: Wednesday, 19.07.2023

**Participants:** Sarah H., Sarah S.

## Summary: PCR Optimazation. Gel Extraction.

**Repetition of Dispersin Optimization PCR from Tuesday due to loading dye in the PCR tubes on Tuesday.**

PCR Setup:

* Reaction A:

  + Volumes [µl]
  + Q5 2x MM: 12.5
  + Forward Primer: 1.25
  + Reverse Primer: 1.25
  + Template: 1 of 3, 10x dilution from 10 ng/µl Eppi
  + ddH2O: 9
* Reaction B:

  + Q5 2x MM: 12.5
  + Forward Primer: 1.25
  + Reverse Primer: 1.25
  + Template: 1 of 3, 10x dilution from 10 ng/µl Eppi
  + ddH2O: 8
  + DMSO: 1
* Reaction C:

  + Q5 2x MM: 12.5
  + Forward Primer: 1.25
  + Reverse Primer: 1.25
  + Template: 1 of 3, 10x dilution from 10 ng/µl Eppi
  + ddH2O: 4
  + GC-Enhancer: 5

PCR Conditions:

| Action | Temperature | Time |
| --- | --- | --- |
| Initial Denaturation | 98 °C | 30 s |
| Denaturation | 98 °C | 8 s (30 cycles) |
| Annealing | 72/71/70/68/66 °C | 30 s (30 cycles) |
| Elongation | 72 °C | 80 s (30 cycles) |
| Final Extension | 72 °C | 2 min |
| Hold | 4 °C |  |

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230719-pcrfragmente-1.png)

**Gel Extraction of Dex1a and Dex2b, Mut, and His4:**

| Fragment | Weight [mg] | Diss Buffer [µl] |
| --- | --- | --- |
| Dex1a | 49.5 | 196.8 |
| Dex2b | 55.4 | 221.6 |
| Mut | 72.3 | 289.2 |
| His4 | 35.7 | 142.8 |

Elution in 10 µl

Sample Concentrations:

| Sample | Concentration [ng/µl] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| Dex 1a | 3.3 | 5.57 | -0.19 |
| Dex 2b | 4.8 | 2.4 | -0.25 |
| Mutanase | 13.4 | 2.16 | 0.19 |
| His4 | 2.8 | 4.62 | -0.21 |

## Date: Thursday, 20.07.2023

**Participants:** Sarah H., Sarah S., Sumohit

## Summary:

**PCR of Gel Extraction Sample for Mutanase and IDT Fragments of His4, Dex1, and Dex2**
**PCR Setup:**

* **His4, Dex1, and Dex2 and Mut:**
  + **Reaction A:**
    - Volumes [µl]
    - Q5 2x MM: 12.5
    - fwd Primer: 1.25
    - rev Primer: 1.25
    - Template: 1 of 3, 10x dilution from 10 ng/µl Eppi
    - ddH2O: 9
  + **Reaction B:**
    - Q5 2x MM: 12.5
    - fwd Primer: 1.25
    - rev Primer: 1.25
    - Template: 1 of 3, 10x dilution from 10 ng/µl Eppi
    - ddH2O: 8
    - DMSO: 1
* **Mut:**
  + **PCR Program Muto:**
    - Initial Denaturation: 98 °C for 30 s
    - Denaturation: 98 °C for 8 s, 30 cycles
    - Annealing: 68°C for 30s, 30 cycles
    - Elongation: 72 °C for 90s, 30 cycles
    - Final Extension: 72 °C for 2 min
    - Hold: 4 °C
* **Dex1:**
  + **Dex 1 [2166bp] - DEX1BIGEM (Machine: PCR in IBVT):**
    - Initial Denaturation: 98 °C for 30 s
    - Denaturation: 98 °C for 8 s, 30 cycles
    - Annealing: 68 °C for 30 s, 30 cycles
    - Elongation: 72 °C for 90 s, 30 cycles
    - Final Extension: 72 °C for 2 min
    - Hold: 4 °C
* **Dex2:**
  + **Dex 2 [1808bp] - Programm His4over (Machine: PCR 1):**
    - Initial Denaturation: 98 °C for 30 s
    - Denaturation: 98 °C for 8 s, 30 cycles
    - Annealing: 72 °C for 30 s, 30 cycles
    - Elongation: 72 °C for 90 s, 30 cycles
    - Final Extension: 72 °C for 2 min
    - Hold: 4 °C

**Agarose-Gel 0.8%:**

* All samples from July 20th
* Small gel chamber: 80 V for 1 h
* MidoriGreen: 2.5 µl in 50 ml Gel
* 1 µl Loading Dye added to 5 µl sample and then applied to the gel
* Gel Order: Marker - Dextranase 1 - Dextranase 1 DMSO - His4 - Mutanase (Gel extraction template) - Mutanase (Gel extraction template) DMSO - Dextranase 2 - Marker

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230720-pcrfragmente-1.png)

**PCR Clean-up for Dextranase Split 2 Fragment:**

* Elution with Elution Buffer in 10 µl

**Sample Concentrations:**

| Sample | Concentration [ng/µl] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| Dextranase Split 2 | 35.3 | 1.85 | 2.5 |

## Date: Friday, 21.07.2023

**Participants:** Anna, Sumohit

## Summary:Primer Test

**Primer Test for Mutanase, Dispersin B, and Dextranase 1.**

Primer Test for Mutanase, Dispersin B, and Dextranase 1 using BB41 Vorexp AMP Fragment. AMP Rev was used as the reverse primer for Dextranase. BB41 was used as a positive control.

Alternative PCR for Dispersin B using Mutanase's Primer and PCR Program:

* Reaction Setup:

+ 1.25 μl forward Primer
+ 1.25 μl reverse Primer
+ 1 μl Template DNA (AMP 10 ng, rest 3 ng)
+ 12.5 μl Q5 High-Fidelity 2X Master Mix
+ 9 μl Water

* PCR Program: AMPOVERH (PCR Machine: iGEM)

| Step | Temperature [°C] | Duration [sec] | Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 98 | 30 |  |
| Denaturation | 98 | 10 | 30 |
| Annealing | 69 | 30 | 30 |
| Elongation | 72 | 18 | 30 |
| Final Extension | 72 | 120 |  |
| Hold | 4 | forever |  |

**PCR Program for Mutanase:**

* PCR Program: Mutanase68 (PCR Machine: IBVT)

| Step | Temperature [°C] | Duration [sec] | Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 98 | 30 |  |
| Denaturation | 98 | 8 | 30 |
| Annealing | 68 | 30 | 30 |
| Elongation | 72 | 90 | 30 |
| Final Extension | 72 | 120 |  |
| Hold | 4 | forever |  |

**Sample Designations and Purposes:**

* 2107-1: Mutanase Primer Test
* 2107-2: Dispersin B Primer Test
* 2107-3: Dextranase 1 Primer Test
* 2107-4: Mutanase Normal Repeat (Positive Control)
* 2107-5: Dispersin B Fragment Test
* 2107-6: BB41 AMP Normal Repeat (Positive Control)

**Agarose-Gel 0.8%:**

* All samples from July 21
* Small gel chamber: 80 V for 1 h
* MidoriGreen: 2.5 µl in 50 ml Gel
* 1 µl Loading Dye added to 5 µl sample and then applied to the gel
* Gel Order: Marker - Mutanase Primer Test - Dispersin B Primer Test - Dextranase 1 Primer Test - Mutanase - Dispersin B Fragment Test - BB41 AMP - Marker

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230721-pcrfragmente-1.png)

## Date: Monday, 24.07.2023

**Participants:**

## Date: Tuesday, 25.07.2023

**Participants:** Anna, Sarah S.

## Summary: Perform PCR and gel electrophoresis.

**Perform PCR and gel electrophoresis**

Preparation:

### Mutanase H2O Template PCR

**Reaction Mix (25µl):**

* 1µl Template -> ddH2O
* 1.25µl of both Primers
* 12.5µl Mastermix
* 9µl ddH2O

### His4 PCR

**Reaction Mix (50µl):**

* 2µl DNA (3ng/µl) -> total 6ng
* 2.5µl of both Primers
* 25µl Mastermix
* 18µl ddH2O

**PCR Program:**

* Initial Denaturation: 98°C for 30s
* Denaturation: 98°C for 8s
* Annealing: 68°C, 67°C for 30s
* Elongation: 72°C for 80s
* Final Elongation: 72°C for 2min
* Hold: 4°C

### Dex2 PCR

**Reaction Mix (50µl):**

* 2µl DNA (3ng/µl) -> total 6ng
* 2.5µl of both Primers
* 25µl Mastermix
* 18µl ddH2O

## Preparation:

Dye was added to the DNA tubes. 100 μL for Mutanase and 200 μL for His-4 and Dex-2. These were shaken to mix the dye better on the walls.

## Order in Gel Electrophoresis:

* Ladder
* Mutanase
* His-4
* Dex-2

## Notes:

U.V. table was used from the top, then cleaned and returned. DNA placed back in the top refrigerator.

## Gel Result:

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230725-pcrfragmente-1.jpg)

## Date: Wednesday, 26.07.2023

**Participants:** Jana, Michael, Ronja

## Goals: PCR. Agarose Gel Electrophoresis

**Mut PCR - Gel Extraction - Determine Concentration**

**DispB PCR - Gel Extraction - Determine Concentration**

**Dex2 PCR Clean-up - Determine Concentration**

## Preparation:

### Mut PCR

**Reaction Mix (2x 50µl):**

* 0.5µl DNA (10ng/µl) -> total 5ng
* 2.5µl of both Primers
* 25µl Mastermix
* 19.5µl ddH2O

**PCR Program (Muto):**

* Initial Denaturation: 98°C for 30s
* Denaturation: 98°C for 8s, 30 cycles
* Annealing: 68°C for 30s, 30 cycles
* Elongation: 72°C for 90s, 30 cycles
* Final Extension: 72°C for 2min
* Hold: 4°C

### DispB PCR

**Reaction Mix (2x 50µl):**

* 0.5µl DNA (10ng/µl) -> total 5ng
* 2.5µl of both Primers
* 25µl Mastermix
* 19.5µl ddH2O

**PCR Program:**

* Initial Denaturation: 98°C for 30s
* Denaturation: 98°C for 8s, 30 cycles
* Annealing: 68°C for 30s, 30 cycles
* Elongation: 72°C for 80s, 30 cycles
* Final Extension: 72°C for 2min
* Hold: 4°C

### Agarose Gel Electrophoresis

**Gel Image:**

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230726-pcrfragmente-1.jpg)

### Gel Extraction of Mutanase (new Zymo Kit)

**Eluted in 10μl Elution Buffer**

### NanoDrop Concentration

**Concentration [ng/ul] A260/280 A260/230**

* 16.7 2.20 1.05

### PCR Clean-up Dex2 (PCR from 25.07)

**Eluted in 10μl**

### NanoDrop Concentration

**Concentration [ng/ul] A260/280 A260/230**

* 77.7 1.86 3.04

## Date: Thursday, 27.07.2023

**Participants:** Jana, Jessica

## PCR His4

**Reaction Mix (2x 50µl):**

* 2 µl DNA (3ng/µl) -> total 6 ng
* 2.5 µl rev Primer
* 2.5 µl for Primer
* 25 µl Q5 Mastermix
* 18 µl ddH2O

**PCR Program:**

* Initial Denaturation: 98°C for 30s
* Denaturation: 98°C for 8s, 30 cycles
* Annealing: 67°C for 30s, 30 cycles
* Elongation: 72°C for 80s, 30 cycles
* Final Extension: 72°C for 2min
* Hold: 4°C

## PCR Dispersin B

**Reaction Mix (2x 50µl):**

* 1 µl DNA (3ng/µl) -> total 6 ng
* 2.5 µl rev Primer (Mutanase)
* 2.5 µl for Primer (Mutanase)
* 25 µl Q5 Mastermix
* 19 µl ddH2O

**PCR Program (Mutanase68):**

* Initial Denaturation: 98°C for 30s
* Denaturation: 98°C for 8s, 30 cycles
* Annealing: 68°C for 30s, 30 cycles
* Elongation: 72°C for 90s, 30 cycles
* Final Extension: 72°C for 2min
* Hold: 4°C forever

## Agarose Gel Electrophoresis

**Gel Image:**

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230727-pcrfragmente-1.png)

## Gel Extraction Dispersin B

**Method:** Zymo Kit, eluted in 10 µl Elution Buffer (preheated to 50°C)

### NanoDrop Concentration

**Concentration [ng/ul] A260/280 A260/230**

* 9.3 1.83 0.24

## Amplification Pichia Vector

**Reaction Mix (25 µl each for Pichia-Vector E14 and G14):**

* 1 µl DNA (Pichia-Vektor E14 or G14)
* 1.25 µl rev Primer (Vektor\_Gibson\_Primer\_reverse)
* 1.25 µl for Primer (Vektor\_Gibson\_Primer\_forward)
* 12.5 µl Q5 Mastermix
* 9 µl ddH2O

**PCR Program:**

* Initial Denaturation: 98°C for 30s
* Denaturation: 98°C for 8s, 30 cycles
* Annealing: 67°C for 30s, 30 cycles
* Elongation: 72°C for 95s, 30 cycles
* Final Extension: 72°C for 2min
* Hold: 4°C forever

## Date: Friday, 28.07.2023

**Participants:**: Sumohit 09:00-18:00; Jessica 09-12:30; Annie 12:30-18:00

## Experiment Title: PCR for His4 and Dextranase 1; Freezer Cleanup

## Summary:

Various approaches were attempted for His4 PCR which did not yield successful results. The same holds true for Dextranase 1. Many items were removed and disposed of from the freezer during the cleanup.

## Materials and Methods:

**PCR Protocol**
**Agarose Gel Electrophoresis Protocol**
**PCR Purification Protocol**
**Components:**

* Q5 2x Master Mix: 12.5 µl
* 10 µM Primer fw: 1.25 µl (0.5 µM)
* 10 µM Primer rv: 1.25 µl (0.5 µM)
* ddH2O: 9 µl
* DNA Template: 1 µl (10 ng DNA)

**Primers for Mutanase, His4, Dextranase Split 1**
**Agarose Gel:**

* Agarose
* 1X TAE Buffer
* Gel Chamber
* Midori Green

**PCR Variants:**

* His4 with His4 Primer:

| Sample | Program Description | Step | Temperature [°C] | Duration [sec] | Cycles |
| --- | --- | --- | --- | --- | --- |
| His4 [2576 bp] -> His4 Program | Initial Denaturation | 98 | 30 |  |  |
| His4 [2576 bp] -> His4 Program | Denaturation | 98 | 8 | 30 |  |
| His4 [2576 bp] -> His4 Program | Annealing | 69 | 20 | 30 |  |
| His4 [2576 bp] -> His4 Program | Elongation | 72 | 80 | 30 |  |
| His4 [2576 bp] -> His4 Program | Final Extension | 72 | 120 |  |  |
| His4 [2576 bp] -> His4 Program | Hold | 4 |  |  | for ever |

| Program Description | Step | Temperature [°C] | Duration [sec] | Cycles |
| --- | --- | --- | --- | --- |
| Initial Denaturation | 98 | 30 |  |  |
| Denaturation | 98 | 8 | 30 |  |
| Annealing | 67 | 30 | 30 |  |
| Elongation | 72 | 80 | 30 |  |
| Final Extension | 72 | 120 |  |  |
| Hold | 4 |  |  | for ever |

| Program Description | Step | Temperature [°C] | Duration [sec] | Cycles |
| --- | --- | --- | --- | --- |
| Initial Denaturation | 98 | 30 |  |  |
|> Denaturation | 98 | 8 | 30 |  |
| Annealing | 68 | 30 | 30 |  |
| Elongation | 72 | 80 | 30 |  |
| Final Extension | 72 | 120 |  |  |
| Hold | 4 |  |  | for ever |

| Program Description | Step | Temperature [°C] | Duration [sec] | Cycles |
| --- | --- | --- | --- | --- |
| Initial Denaturation | 98 | 30 |  |  |
| Denaturation | 98 | 8 | 30 |  |
| Annealing | 69 | 30 | 30 |  |
| Elongation | 72 | 80 | 30 |  |
| Final Extension | 72 | 120 |  |  |
| Hold | 4 |  |  | for ever |

| Program Description | Step | Temperature [°C] | Duration [sec] | Cycles |
| --- | --- | --- | --- | --- |
| Initial Denaturation | 98 | 30 |  |  |
| Denaturation | 98 | 8 | 30 |  |
| Annealing | 70 | 30 | 30 |  |
| Elongation | 72 | 80 | 30 |  |
| Final Extension | 72 | 120 |  |  |
| Hold | 4 |  |  | for ever |

* Dextranase Split 1 with Mutanase For Primer and Dextranase 1 Rev Primer / Dextranase 1 Primer:

| Program Description | Step | Temperature [°C] | Duration [sec] | Cycles |
| --- | --- | --- | --- | --- |
| Initial Denaturation | 98 | 30 |  |  |
| Denaturation | 98 | 8 | 30 |  |
| Annealing | 69 | 20 | 30 |  |
| Elongation | 72 | 80 | 30 |  |
| Final Extension | 72 | 120 |  |  |
| Hold | 4 |  |  | for ever |

| Program Description | Step | Temperature [°C] | Duration [sec] | Cycles |
| --- | --- | --- | --- | --- |
| Initial Denaturation | 98 | 30 |  |  |
| Denaturation | 98 | 8 | 30 |  |
| Annealing | 67 | 30 | 30 |  |
| Elongation | 72 | 80 | 30 |  |
| Final Extension | 72 | 120 |  |  |
| Hold | 4 |  |  | for ever |

| Program Description | Step | Temperature [°C] | Duration [sec] | Cycles |
| --- | --- | --- | --- | --- |
| Initial Denaturation | 98 | 30 |  |  |
| Denaturation | 98 | 8 | 30 |  |
| Annealing | 68 | 30 | 30 |  |
| Elongation | 72 | 80 | 30 |  |
| Final Extension | 72 | 120 |  |  |
| Hold | 4 |  |  | for ever |

| Program Description | Step | Temperature [°C] | Duration [sec] | Cycles |
| --- | --- | --- | --- | --- |
| Initial Denaturation | 98 | 30 |  |  |
| Denaturation | 98 | 8 | 30 |  |
| Annealing | 69 | 30 | 30 |  |
| Elongation | 72 | 80 | 30 |  |
| Final Extension | 72 | 120 |  |  |
| Hold | 4 |  |  | for ever |

| Program Description | Step | Temperature [°C] | Duration [sec] | Cycles |
| --- | --- | --- | --- | --- |
| Initial Denaturation | 98 | 30 |  |  |
| Denaturation | 98 | 8 | 30 |  |
| Annealing | 70 | 30 | 30 |  |
| Elongation | 72 | 80 | 30 |  |
| Final Extension | 72 | 120 |  |  |
| Hold | 4 |  |  | for ever |

## Agarose Gel Electrophoresis with Pichia PCR Samples from 27th July

**Gel Image:**
![Gel Image](Gel.jpg)

## Results:

**Preparation:**
A 0.8% agarose gel was used for agarose gel electrophoresis and run at 80 V for 60 minutes.
**Gel Electrophoresis Order:**
Ladder - Pichia Vector 1 - Pichia Vector 2 - His-4 - Dex-1 - Marker

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230728-pcrfragmente-1.png)

![Nanodrop](https://static.igem.wiki/teams/4575/wiki/20230728-nanodrop-pichiavector1-1.jpeg)

![Nanodrop](https://static.igem.wiki/teams/4575/wiki/20230728-nanodrop-pichiavector2-1.jpeg)

![Nanodrop](https://static.igem.wiki/teams/4575/wiki/20230728-nanodrop-his4-1.jpeg)

![Nanodrop](https://static.igem.wiki/teams/4575/wiki/20230728-nanodrop-dex1-1.jpeg)

...

**Discussion:**
The PCR for the Pichia vector was successful, showing clear bands in the gel and appropriate concentrations. In the His4 69°C 20 sec approach, a faint band is visible at the expected height, though uncertain. Hence, various annealing temperatures (67-70°C) were tested for 30 sec but were not applied to the gel due to time constraints. The wrong program was used (69°C 20 sec) as the previous entries were a bit cryptic, and the successful PCR was not clearly identified. For Dextranase 1 with Mutanase For Primer and Dextranase 1 Split Rec Primer, multiple bands are visible. The wrong size was read from a document (2367 instead of 2167 bp), and thus everything was merged with His4. Also, a gradient from 67°C to 70°C was created with two different primer combinations (Mut For-Dex1 Rev and Dex1 For-Dex1 Rev). The error was later identified.

**For the Next Days:**
- Repeat Dextranase 1 approaches with the correct elongation time of 60 sec.
- Apply all samples to the gel and analyze.

## Date: Monday, 31.07.2023

**Participants:**: Sharkey and Sarah H.

## Summary: Medium preparation. Dextranase 1 Gradient PCR. Gel Electrophoresis.

## Medium preparation

Preparation of 500 ml YPD+His Medium and 200 ml YPD+His Agar using the following recipe:

![Rezept](https://static.igem.wiki/teams/4575/wiki/20230731-agarrezept-1.png)

Plates poured with YPD+His Agar and stored in the refrigerator at -U2.

YPD+His Medium stored in the laboratory.

Also, plates with yeast can be stored with bacteria in the refrigerator.

## Dextranase 1 Gradient PCR

Gradient PCR from 62°C to 72°C using Mutanase forward primer and Dextranase1 reverse primer:

| Step | Action | Temperature (°C) | Duration | Cycles |
| --- | --- | --- | --- | --- |
| 1 | Initial Denaturation | 98 | 30 s | 1 |
| 2 | Denaturation | 98 | 8 s | 30 |
| 3 | Annealing | 62-72°C in 1°C increments | 30 s | 30 |
| 4 | Elongation | 72 | 65 s | 30 |
| 5 | Final Extension | 72 | 2 min | 1 |
| 6 | Hold | 4 |  |  |

Total reaction volume: 25 µl

## Components

| Component | Volume | Concentration/Mass |
| --- | --- | --- |
| Q5 2x Master Mix | 12.5 µl | - |
| 10 µM Mutanase Primer fw | 1.25 µl | 0.5 µM |
| 10 µM Dextranase1 Primer rv | 1.25 µl | 0.5 µM |
| ddH2O | 9 µl | - |
| DNA Template | 1 µl | 5 ng DNA |

## Gel Electrophoresis

Small gel with 15 wells at 100V for 1 hour.

5 µl of each reaction loaded onto the gel.

Gel loading:

* His 4 + His4 Primer: 67 68 69 70
* Dex1 + MutPrimer(fr)?: 67 68
* Marker Dex1 + MutPrimer(mon): 65 66 67 68 69 70 71 72

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230731-pcr-his-mut-1.png)

## Notes:

# August

| Monday | Tuesday | Wednesday | Thursday | Friday | Saturday | Sunday |
| --- | --- | --- | --- | --- | --- | --- |
|  | 1 | 2 | 3 | 4 | 5 | 6 |
| 7 | 8 | 9 | 10 | 11 | 12 | 13 |
| 14 | 15 | 16 | 17 | 18 | 19 | 20 |
| 21 | 22 | 23 | 24 | 25 | 26 | 27 |
| 28 | 29 | 30 | 31 |  |  |  |

## Date: Tuesday, 01.08.2023

**Participants:** Jessica, Sarah H., Sumohit

## Summary: PCR. Pichia Pastoris Strain GS115. Gel Electrophoresis.

Performed His4 PCR with annealing at 67°C and 35 cycles.

Prepared fresh template and primer dilution for His4 PCR.

## His4 PCR

| Component | Volume (ul) |
| --- | --- |
| Q5 2x Master Mix | 25 |
| Forward Primer | 2.5 |
| Reverse Primer | 2.5 |
| Template | 1 |
| ddH2O | 19 |

Program for His4 PCR:

| Step | Action | Temperature (°C) | Duration | Cycles |
| --- | --- | --- | --- | --- |
| 1 | Initial Denaturation | 98 | 30 s | 1 |
| 2 | Denaturation | 98 | 8 s | 35 |
| 3 | Annealing | 67 | 30 s | 35 |
| 4 | Elongation | 72 | 80 s | 35 |
| 5 | Final Extension | 72 | 2 min | 1 |
| 6 | Hold | 4 |  |  |

## Dex1 PCR

Performed Dex1 PCR with annealing at 68-72°C, 30 cycles, and Mutanase forward primer.

Used fresh Mut Primer, fresh Dex1 reverse primer, and fresh Template Sample.

| Component | Volume (ul) |
| --- | --- |
| Q5 2x Master Mix | 25 |
| Forward Primer | 2.5 |
| Reverse Primer | 2.5 |
| Template | 1 |
| ddH2O | 19 |

Program for Dex1 PCR:

| Step | Action | Temperature (°C) | Duration | Cycles |
| --- | --- | --- | --- | --- |
| 1 | Initial Denaturation | 98 | 30 s | 1 |
| 2 | Denaturation | 98 | 8 s | 30 |
| 3 | Annealing | 68-72°C in 1°C increments | 30 s | 30 |
| 4 | Elongation | 72 | 65 s | 30 |
| 5 | Final Extension | 72 | 2 min | 1 |
| 6 | Hold | 4 |  |  |

## Pichia Pastoris Strain GS115

Spread Pichia Pastoris Strain GS115 on YPD+His plates with 3-streak and "Christmas tree" streaking, incubated at 30°C in the MiBi laboratory.

* Christmas tree streaking

![Abbildung](https://static.igem.wiki/teams/4575/wiki/20230801-ausplattieren-1.png)

* 3-streak

![Abbildung](https://static.igem.wiki/teams/4575/wiki/20230801-ausplattieren-2.png)

## Gel Electrophoresis

Performed gel electrophoresis on remaining Dex1 PCR samples from Monday and Friday, as well as the Dex1 fragment sample, to check for purity.

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230801-pcrfragmente-1.png)

## Notes:

## Date: Wednesday, 02.08.2023

**Participants:** Jana, Jessie

## Summary:

Performed agarose gel electrophoresis for His4 and Dex1 samples from August 1st.

Loaded 10 μl of His4 and 50 μl of Dex1 (68-72°C) into the gel, divided into 2 wells.

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230802-pcrfragmente-1.png)

## Task1: Gel Extraction for Dex1

Eluted Dex1 in 10 μl of elution buffer.

## Task2: Concentration Analysis

| Concentration [ng/ul] | A260/280 | A260/230 |
| --- | --- | --- |
| 114.0 | 1.90 | 2.08 |

## Task3: Pichia Pastoris Overnight Culture

Prepared 4x 10 ml cultures in YPD + His Medium for Pichia Pastoris.

Inoculated at 4:30 PM.

Incubated overnight at 30°C on a shaker.

Note: Plates are stored in the IBVT refrigerator.

## Notes:

## Date: Thursday, 03.08.2023

**Participants:** Sumohit 09:30-18:00; Jana 09:30-18:00;

## Summary:

Experiment: Gel for Münster; His4 PCR Variants; Competent Pichia Cells; Dex 2 PCR and Gel Electrophoresis to test the gel extraction kit.

## Gel Preparation for Münster

Loaded samples of His4 and Dex1 (68-72°C) onto the agarose gel.

![Gel Image](image.png)

## Gel Extraction for Dex1

Eluted Dex1 using 10 μl of elution buffer.

## Concentration Analysis

Concentration [ng/ul] A260/280 A260/230

114.0 1.90 2.08

## Pichia Pastoris Overnight Culture

Prepared 4x 10 ml cultures in YPD + His Medium for Pichia Pastoris.

Inoculated at 4:30 PM.

Incubated overnight at 30°C on a shaker.

Note: Plates are stored in the IBVT refrigerator.

## His4 Variants

His4 samples with GC Enhancer 2 µl; His4 samples with 1µl 100% DMSO; His4 with both AMP BB41 Primers; His4 with BB41 Rev and His4 For; AMP BB41 with His4 Primer;

## PCR Program for His4:

| Step | Action | Temperature (°C) | Duration | Cycles |
| --- | --- | --- | --- | --- |
| 1 | Initial Denaturation | 98 | 30 s | 1 |

## Loading Scheme

Marker – pBAD18 – BB41 – BB41 with His4 Primer – Marker – Pichia Vector – Mutanase – Dispersin B – Dex1 excess from previous days – Dex1 Jana Gel Extraction – His4 100 ng from stock – Marker – His4 GC – His4 DMSO End Concentration 4% – His4 with BB41 Primer – His4 with BB41 Rev and His4 For – Marker – Dex2 – Dex2 – Dex2 – Dex2 – Marker

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230803-bb41.png)

## Competent Pichia Pastoris (Jana)

Allowed overnight cultures to grow at OD600 of 0.9 (at 30°C on shaker).

Prepared competent cells following the protocol in the Frozen Yeast Transformation Kit by Zymo.

Aliquoted competent cells in 200 μl aliquots. Cells can be refrozen after thawing (according to the kit) if the complete volume is not needed.

Stored in the -70°C freezer.

![Bild](https://static.igem.wiki/teams/4575/wiki/20230803-freezer-1.png)

## Notes:

## Date: Friday, 04.08.2023

## Date: Monday, 07.08.2023

**Participants:** Sarah 09:30-18:00; Sumohit 09:30-18:00;

Summary:Experiment: His4 PCR with different DMSO concentrations and adjusted annealing temperature and AMP; PCR for AMP Gibson Assembly; Testing old gel extraction kits and comparing with the new Zymo kit; Attempt to lower the A230 value using DNA Clean-Up and Concentrator Kit.

## pBAD18 PCR

pBAD18

| Step | Temperature [°C] | Dauer [sec] | Cycles |
| Initial Denaturation | 98 | 30 |  |
| Denaturation | 98 | 10 | 40 Zyklen |
| Annealing | 70 | 30 | 40 Zyklen |
| Elongation | 72 | 180 | 40 Zyklen |
| Final Extension | 72 | 3:30 min |  |
| Hold | 4 |  |  |

## AMP BB41 Pre-Experiment PCR

Alle BB41 (AMPOVERH)

| Step | Temperature [°C] | Dauer [sec] | Cycles |
| Initial Denaturation | 98 | 30 |  |
| Denaturation | 98 | 10 | 30 |
| Annealing | 69 | 30 | 30 |
| Elongation | 72 | 18 | 30 |
| Final Extension | 72 | 120 |  |
| Hold | 4 | for ever |  |

## Task3: His4 PCR

Alle His4

| Step | Temperature [°C] | Dauer [sec] | Cycles |
| Initial Denaturation | 98 | 30 |  |
| Denaturation | 98 | 8 | 30 Zyklen |
| Annealing | 67 | 30 | 30 Zyklen |
| Elongation | 72 | 80 | 30 Zyklen |
| Final Extension | 72 | 120 |  |
| Hold | 4 |  |  |

### DMSO Concentration - Annealing Temperature [°C]

| DMSO Concentration | Annealing Temperature [°C] |
| --- | --- |
| 10% | 61-63 |

## Agarose Gel Preparation

Prepared a 0.6% agarose gel for His4 PCRs with peptide primers and loaded 25 ul in each well.

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230807-pcr-his4-1.png)

Prepared a 1.2% agarose gel for His4 DMSO PCRs, pBad, and AMP PCRs. Loaded 10 ul of His4 DMSO PCRs and 5 ul of pBad and AMP PCRs.

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230807-pcr-his-pbad-amp-1.png)

## Testing Old and New Gel Extraction Kits

Used the old NEB Kit for extraction from 3 samples and the Zymo kit for 2 samples from the samples of Thursday, 3rd August.

### Results

DNA Concentration and Ratios

| Sample | DNA Concentration [ng/µl] | A260/A280 | A260/A230 | Kit |
| His4-Sample3 | 33,0 | 2,02 | 1,86 | Alt NEB |
| Dex2-Sample1 | 33,2 | 1,88 | 0,57 | Alt NEB |
| Dex2-Sample2 | 23,7 | 2,00 | 1,70 | Zymo |
| Dex2-Sample3 | 15,0 | 2,01 | 1,03 | Alt NEB |
| Dex2-Sample4 | 14,2 | 1,89 | 0,21 | Zymo |

![Results Image](WhatsApp Image 2023-08-07 at 16.19.08 (1).jpeg)

## Task6: DNA Clean-Up

Used DNA Clean-Up and Concentrator Kit to improve A260/A230 ratios for selected samples.

### Results

DNA Concentration and Ratios

| Sample | DNA Concentration [ng/µl] | A260/A280 | A260/A230 |
| Dex2-Sample1 | 7,9 | 2,03 | 3,11 |
| Dex2-Sample2 | 3,9 | 1,80 | 11,05 |
| Dex2-Sample3 | -7,1 | 1,44 | 0,70 |

![DNA Clean-Up Results Image](WhatsApp Image 2023-08-07 at 16.19.10.jpeg)

## Discussion:

Both the old NEB Kit and the Zymo Kit worked well for one sample but showed poor A260/A230 ratios for another. Concentrations were above 15 ng/µl for all samples, and the A260/A280 ratios looked good. Considering possible organic contamination in some samples, DNA clean-up was performed. However, only Dex2-Sample1 showed good values after clean-up; other samples were unusable and were discarded. In the future, either the old NEB Kit or the Zymo Kit could be used for DNA extraction from gels.

## Upcoming Tasks

Continue testing His4

Gibson Assembly for AMP BB41 in pBAD18

## Date: Tuesday, 08.08.2023

**Participants:** Sumo, Jana, Sarah H.

Summary:Maintenance activities were conducted, including washing and autoclaving equipment. Additionally, PCR clean-up was performed for pBAD and AMP fragments with elution in 10 ul. Concentrations and ratios for the samples were measured.

DNA Concentration and Ratios

| Sample | Concentration [ng/ul] | 260/280 | 260/230 |
| AMP | 57,3 | 1,84 | 2,01 |
| pBAD1 | 96,6 | 1,84 | 1,68 |
| pBAD2 | 94,1 | 1,90 | 2,24 |

## Gibson Assembly with NEBuilder for AMP BB41 in pBAD18

| Components | Volume [ul] | Concentration (ng) |
| --- | --- | --- |
| pBAD18 | x (75 ng) |
| BB41 | 18.77 ng (OKS) |
| Water | Up to 10 ul |
| NEBbuilder | 10 ul |

## Transformation

Transformation was carried out as per the protocol using 3µl and 5µl Gibson product from Lila Eppi, and 3µl Gibson product with NEBuilder, along with 1 µl pBAD18 as a control.

## PCR with His4 Gel Extraction Product

| Components | Volume [ul] |
| --- | --- |
| Q5 2xMM | 25 |
| fwd Primer | 2.5 |
| rev Primer | 2.5 |
| His4 Gel Extraction Template (100pg Verd.) | 1/0.5/1 (1 ng/500 pg/100 pg) |
| ddH2O | 19/19.5/19 |

His4 Gel Product (33.33 ng/ul) was diluted 1:33 in 33.0 ul to achieve a concentration of approximately 1 ng/ul. An additional 1:10 dilution was made from this dilution to obtain a concentration of 100 pg/ul.

### PCR Program for His4

PCR Program for His4 Amplification

| Step | Temperature [°C] | Duration [sec] | Cycles |
| Initial Denaturation | 98 | 30 |  |
| Denaturation | 98 | 8 | 35 |
| Annealing | 67 | 30 | 35 |
| Elongation | 72 | 80 | 35 |
| Final Extension | 72 | 120 |  |
| Hold | 4 |  |  |

## Date: Wednesday, 09.08.2023

**Participants:** Jana 09:30-17:00; Sumohit 09:30-17:00;

Summary: The Gibson Assembly plates were examined. Due to a high number of colonies on all three plates, 8 colonies from each plate were used for Colony-PCR and plated on a separate plate. All enzymes for BioBrick Assembly were checked for functionality. PCR was conducted with AMP primers for all fragments.

## Plating after Gibson Assembly of BB41 into pBAD18

![Bild](https://static.igem.wiki/teams/4575/wiki/20230809-platten-gibbsonassembly-1.jpeg)

During Colony-PCR, 8 colonies from each plate were taken and checked using the AMP PCR program to confirm the presence of the desired insert in the cells.

### Colony-PCR Program

PCR Program for iGEM PCR Machine

| Step | Temperature [°C] | Dauer [sec] | Cycles |
| Initial Denaturation | 98 | 30 |  |
| Denaturation | 98 | 10 | 30 |
| Annealing | 69 | 30 | 30 |
| Elongation | 72 | 18 | 30 |
| Final Extension | 72 | 120 |  |
| Hold | 4 |  | forever |

## Mutanase, Dispersin B, Dextranase 1 and 2 PCR with AMP BB41 Primers

This was done to enable cloning into pBAD18 for BioBrick Assembly. For Dextranase 1, Dex1 Rev primer was used instead of AMP BB41 Rev, and for Dex2, Dex2 For primer was used instead of AMP BB41 For.

PCR Program for Mutanase and Dispersin B (PCR-Maschine IBVT)

| Step | Temperature [°C] | Dauer [sec] | Cycles |
| Initial Denaturation | 98 | 30 |  |
| Denaturation | 98 | 8 | 30 |
| Annealing | 68 | 30 | 30 |
| Elongation | 72 | 90 | 30 |
| Final Extension | 72 | 120 |  |
| Hold | 4 |  | forever |

PCR Program for Dex2

| Step | Temperature [°C] | Dauer [sec] | Cycles |
| Initial Denaturation | 98 °C | 30 s |  |
| Denaturation | 98 °C | 8 s | 30 Zyklen |
| Annealing | 72 °C | 30 s | 30 Zyklen |
| Elongation | 72 °C | 90 s | 30 Zyklen |
| Final Extension | 72 °C | 2 min |  |
| Hold | 4 °C |  |  |

## Preparation of 1.0% Agarose Gel for Colony-PCR and Mutanase, Dispersin B, and Dex2 with AMP Primers

Complete 10 µl was applied.

### Application Scheme

Marker – 8 Colonies Jana 3 µl – Marker – 8 Colonies Jana 5 µl - Marker
Marker – 8 Colonies Sumo 3 µl – Marker – Mut – Dsp B – Dex2 – Marker

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230809-colonypcr-1.png)

As the Colony PCR did not show any bands, overnight cultures were set with additional ampicillin to ensure its presence in case it was depleted from the plates.

## Functionality Test of All Required Restriction Enzymes for BioBrick Assembly (except SpeI)

pBAD18 has restriction sites for EcoRI, XbaI as unique cutters, and PstI as dual cutters. To observe bands on the gel with EcoRI or XbaI digestion, an additional approach was made using BsaI, resulting in two bands on the gel. 500 ng of the sample was used for digestion.

Theoretical Expectation:

Order scheme: Marker- undigested (supercoii) - digest with EcoRi - digest with XbaI - digest with PstI - digest with EcoRI and BsaI - digest with XbaI and BsaI - marker

![Bild](https://static.igem.wiki/teams/4575/wiki/20230809-restriktionsverdau-erwartungen-1.png)

## Preparation of 1% Agarose Gel for Functionality Test of All Required Restriction Enzymes for BioBrick Assembly (except SpeI and Dex1)

10 µl was applied.

Application Scheme:

Application scheme: Marker- undigested (supercoii) - Digest with EcoRi - Digest with XbaI - Digest with PstI - Digest with EcoRI and BsaI - Digest with XbaI and BsaI - Dex1 with AMP BB41 For and Dex1 Rev Primer

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230809-restriktionsverdau-1.png)

## Discussion:

There were a high number of cells on the plates after Gibson Assembly, far exceeding the expected count. As a result, 8 colonies were selected from each plate for Colony-PCR to check if the insert was successfully cloned into the plasmid. The Colony-PCR and the corresponding gel confirmed that the Gibson Assembly was not successful. To validate the Colony-PCR results, overnight cultures were initiated and incubated. The functionality test of the restriction enzymes was successful and produced the expected results. The PCR with AMP BB41-pBAD18 overhangs worked for Dispersin B, Dex1 and 2, but not for Mutanase, and the reason is unknown. Considering potential issues with Gibson Assembly, preparations were made for BioBrick Assembly.

## Next Steps:

* Plasmid Isolation from the 24 overnight cultures and Restriction Analysis
* Mutanase PCR
* Preparation for BioBrick Assembly

## Date: Thursday, 10.08.2023

## Experiment: LB Agar Medium and LB Medium with Ampicillin and Chloramphenicol

Performed plasmid isolation, restriction analysis, and Gibson Assembly without transformation for subsequent BioBrick Assembly. PCR for BioBrick Assembly.

**Participants:** Jessica 09:30-19:00; Sumohit 09:30-19:00;

Summary: Suspecting that Ampicillin was not depleted, new plates and fresh Ampicillin medium were prepared. Plasmid isolation was performed on the 30 overnight cultures from the previous day, followed by restriction analysis. Gibson Assembly without transformation was conducted with all enzyme fragments into pBAD18. PCR was performed with AMP primers for Mutanase.

## Plasmid Isolation Concentrations

Sample Concentrations and Ratios

| Sample | Concentration | A260/A280 | A260/A230 |
| S1 | 36.31 | 1.93 | 2.22 |
| S2 | 45.17 | 1.99 | 2.10 |
| S3 | 36.5 | 1.88 | 2.05 |
| S4 | 26.78 | 1.51 | 0.80 |
| S5 | 24.97 | 2.00 | 1.96 |
| S6 | 38.56 | 1.81 | 0.83 |
| S7 | 37.37 | 2.01 | 2.36 |
| S8 | 38.06 | 1.89 | 2.24 |
| S9 | 36.98 | 1.95 | 1.96 |
| S10 | 22.15 | 1.89 | 1.51 |
| J1 | 28.88 | 1.93 | 2.03 |
| J2 | 45.25 | 2.05 | 2.04 |
| J3 | 40.32 | 2.01 | 1.93 |
| J4 | 30.54 | 2.08 | 2.01 |
| J5 | 30.48 | 2.02 | 2.04 |
| J6 | 15.95 | 2.05 | 2.23 |
| J7 | 25.94 | 2.05 | 2.11 |
| J8 | 38.19 | 1.97 | 2.25 |
| J9 | 35.54 | 2.10 | 2.08 |
| J10 | 37.34 | 2.04 | 1.92 |
| J11 | 31.71 | 2.00 | 1.75 |
| J12 | 33.17 | 1.98 | 2.20 |
| J13 | 45.25 | 2.11 | 2.11 |
| J14 | 34.99 | 2.06 | 2.15 |
| J15 | 32.37 | 2.03 | 2.16 |
| J16 | 32.67 | 1.89 | 2.23 |
| J17 | 55.52 | 2.09 | 2.11 |
| J18 | 39.4 | 2.00 | 2.06 |
| J19 | 26.96 | 1.93 | 2.16 |
| J20 | 34.16 | 2.05 | 2.17 |

## Restriction Digest for Plasmid Isolates

500 ng of each plasmid was used for restriction digestion with XbaI and BsaI. Two bands were observed in both correct and incorrect assemblies, allowing distinction between the two cases.

## Gibson Assembly without Transformation

Gibson Assembly was performed in pBAD18 with all enzymes. The required volumes are listed in the table below.

Amounts for Gibson Assembly

| Sample | Amount for Gibson [ng] | Volume Sample [µl] | Water | NEBuilder |
| Mut | 110,5 | 3,44 | 5,76 | 10 |
| Dsp B | 103,5 | 2,29 | 6,91 | 10 |
| Dex 1 | 82,09 | 0,83 | 7,68 | 10 |
| Dex 2 | 68,66 | 0,69 |  |  |
| HIs4 | 109,5 | 3,32 | 5,88 | 10 |
| pBAD18 | 75 | 0,8 |  |  |

## Restriction Digest Analysis for Gibson BB41 Samples

Theoretical expectation:

![Bild](https://static.igem.wiki/teams/4575/wiki/20230810-restriktionsverdau-erwartung-1.png)

## Preparation of 1% Agarose Gel for AMP BB41 Gibson Restriction Analysis

10 µl for PCR samples and 15 µl for restriction analysis were loaded onto the gel.

### Application Scheme

Application scheme
Marker - 8 colonies Jana 3 µl - Marker - 8 colonies Jana 5 µl - Marker
Marker - 8 colonies Sumo 3 µl - Marker - Dex1 - Marker

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230810-restriktionsverdau-1.png)

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230810-restriktionsverdau-2.jpg)

## Preparation of 1% Agarose Gel for Mutanase and Dex1 with BB41 Overhangs and His4 Variants

10 µl was loaded for PCR samples.

### Application Scheme

Auftragungsschema
Marker – Mut - Dex1 - Marker - His4 - His4-V1 - His4-V2 - His4-V3 - Marker

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230810-pcrfragmente-1.png)

## Discussion:

Suspecting Ampicillin depletion, fresh Ampicillin was added. All 30 overnight cultures showed growth, and plasmid isolation was successful. Restriction digestion with XbaI and BsaI was performed to identify the plasmids within the cells. Several samples indicated the presence of the desired Gibson plasmid. PCRs and PCR purification were successful, although the values were slightly lower than expected. These samples were later used for Gibson Assembly.

For the agarose gel simulation, incorrect plasmids were initially used. Upon reanalysis, it was determined that J1, J2, J8, J12, J13, J15, J17, J18, S1, S2, and S8 could be correct, as the bands were slightly above 3000 bp.

## Date: Friday, 11.08.2023

## Experiment: Transformation of Gibson and pSB1C3, pSB1A2, and pBAD18. Restriction Analysis of Gibson BB41. Sending for Sequencing.

**Participants:** Jessica 09:30-14:00; Sarah 09:30 - 16:30; Sumohit 09:30-17:00;

## Summary:

Transformation of Gibson constructs from the previous day and empty vectors PSB1C3, and pSB1A2 to ensure sufficient material for BioBrick Assembly. Relevant samples were sent for sequencing.

An additional restriction analysis was performed on samples J3, J4, J5, J6, J7, J11, J14, J15, J16, S3, S4, S5, S6, and S7 to further validate the data. For this, 700 ng DNA was used with the restriction enzyme SpeI. If the plasmid is the correct construct, two bands should be visible in the gel, otherwise only one band.

## Mixing Details for Restriction Analysis:

Sample Volume and Components

| Sample | Volume Sample | Restriktion enzyme je | 2.1 Buffer | Water |
| S3 | 19.18 | 0.50 | 5.00 | 24.82 |
| S4 | 26.14 | 0.50 | 5.00 | 17.86 |
| S5 | 28.03 | 0.50 | 5.00 | 15.97 |
| S6 | 18.15 | 0.50 | 5.00 | 25.85 |
| S7 | 18.73 | 0.50 | 5.00 | 25.27 |
| J3 | 17.36 | 0.50 | 5.00 | 26.64 |
| J4 | 22.92 | 0.50 | 5.00 | 21.08 |
| J5 | 22.97 | 0.50 | 5.00 | 21.03 |
| J6 | 43.89 | 0.50 | 5.00 | 0.11 |
| J7 | 26.99 | 0.50 | 5.00 | 17.01 |
| J11 | 22.08 | 0.50 | 5.00 | 21.92 |
| J14 | 20.01 | 0.50 | 5.00 | 23.99 |
| J15 | 21.62 | 0.50 | 5.00 | 22.38 |
| J16 | 21.43 | 0.50 | 5.00 | 22.57 |

## Analysis of Restriction Digest for Gibson BB41 Samples

Theoretical expectation:

Ordering scheme: Marker - Digestion with SpeI pBAD18-BB41 - Digestion with SpeI pBAD18 LV

![Bild](https://static.igem.wiki/teams/4575/wiki/20230811-restriktionsverdau-erwartung-1.png)

## Preparation of 1% Agarose Gel for Mutanase and Dex1 with AMP BB41 Primer and His4

10 µl loaded for PCR reactions.

Application Scheme:

Application scheme: Marker samples Digested with SpeI - Marker

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230811-restriktionsverdau-1.png)

## Transformation of Various Empty Vectors and Gibson Constructs

Transformation was performed with the empty vectors pSB1C3, pSB1A2 at positions A1 and A5, and pBAD18 into the respective plates. Additionally, transformation was conducted with four Gibson constructs from the previous day. Negative control samples without plasmids were prepared for each plate.

The transformation was carried out as per the protocol.

## Sequencing

As the results from the previous restriction analysis were inconclusive, samples S3, S4, J4, J7, and J11 were sent for sequencing. For sequencing, 2.5 µl of the sample and 2.5 µl of 10 µM primer were used and topped up to 10 µl with ddH2O.

Sample Concentration, Quantity for Sequencing, and Primer Information

| Sample | Concentration | Amount for Seq | Primer | Seq ID |
| S3 | 36.5 | 91.25 | BB41 For | EMX383 |
| BB41 Rev | EMX378 |
| S4 | 26.78 | 66.95 | BB41 For | EMX379 |
| BB41 Rev | EMX380 |
| J4 | 30.54 | 76.35 | BB41 For | EMX375 |
| BB41 Rev | EMX376 |
| J7 | 25.94 | 64.85 | BB41 For | EMX377 |
| BB41 Rev | EMX372 |
| J11 | 31.71 | 79.28 | BB41 For | EMX373 |
| BB41 Rev | EMX374 |

## Preparation of Pichia Pastoris Minimal Agar

See protocol for details.

Autoclaving of Glycerol, ddH20, and Agar solutions for Pichia Pastoris Minimal Medium. Autoclave had technical issues and had to be turned off.

Preparation of new and old His4 primers and His4 enzyme sequencing primers. Adding the appropriate amount of autoclaved ddH2O and placing the primer solution on the shaker for 1 hour at 20-25°C.

## Discussion:

The second restriction analysis did not proceed as expected, and the data could not be interpreted. Therefore, five samples were selected and sent for sequencing.

## For the Next Few Days:

* Plating of the empty vectors overnight cultures
* Evaluation of Gibson Assembly Transformation results
* Analysis of the sequencing results

## Date: Monday, 14.08.2023

**Participants:** Sarah H., Jana

## His4 PCR with New and Old Primers

### Reaction Setup:

| Components | Volume [ul] |
| --- | --- |
| Q5 2xMM | 25 |
| fwd Primer | 2.5 |
| rev Primer | 2.5 |
| Template | 1 |
| ddH2O | 19 |

### PCR Program for His4:

PCR Programm

| Step | Temperature [°C] | Dauer [sec] | Cycles |
| 1 | Initial Denaturation | 98 °C | 30 s |
| 2 | Denaturation | 98 °C | 8 s |
| 3 | Annealing | 67 °C | 30 s |
| 4 | Elongation | 72 °C | 80 s |
| 6 | Final Extension | 72 °C | 2 min |
| 7 | Hold | 4 °C |  |

## PCRs with Enzyme Fragments to Insert AMP Overhangs

### Reaction Setup:

Reaction Setup:

| A | Volume [µl] | 5x MM |
| Q5 2xMM | 12.5 | 62.5 |
| BB41-fwd Primer | 1.25 | 6.25 |
| BB41-rev Primer | 1.25 | 6.25 |
| Template | 1 | - |
| ddH2O | 9 | 45 |

### PCR Program for Enzyme Fragment PCRs:

PCR Program

| Step | Action | Temperature | Duration | Cycles |
| 1 | Initial Denaturation | 98 °C | 30 s |
| 2 | Denaturation | 98 °C | 8 s | 30 cycles |
| 3 | Annealing | 67°C | 30 s | 30 cycles |
| 4 | Elongation | 72 °C | His4: 80s; Mutanase: 90s; Dispersin B: 85s; | 30 cycles |
| 6 | Final Extension | 72 °C | 2 min |
| 7 | Hold | 4 °C |

## Agarose Gel

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230814-pcrfragmente-ampoverhangs-1.png)

## Gel Extraction and PCR Clean-up

Gel extraction was performed for Mutanase and Dispersin B bands, along with PCR clean-up for His4 (with AMP Overhangs). The concentrations and ratios for the extracted DNA were measured.

### Gel Extraction Elution:

Sample Concentration and Ratios

| Sample | Concentration (ng/µl) | 260/280 | 230/260 |
| Mutanase | 8.4 | 2.67 | 0.02 |
| Dispersin B | 60.9 | 10.72 | 4.46 |

### PCR Clean-up Elution:

Sample Concentration and Ratios

| Sample | Concentration (ng/µl) | 260/280 | 230/260 |
| His4 | 12.9 | 1.79 | 4.55 |

## Colony PCR for pBAD Enzyme Fragments Gibson Assembly

Colony PCR was conducted for pBAD enzyme fragment Gibson Assembly using colonies from various plates.

### Reaction Setup:

Reaction Setup

| A | Volume [µl] | 15x MM |
| Q5 2xMM | 12.5 | 187.5 |
| BB41-fwd Primer | 1.25 | 18.75 |
| BB41-rev Primer | 1.25 | 18.75 |
| Template | 1 Colony | - |
| ddH2O | 10 | 150 |

### PCR Program for Colony PCR:

PCR Program

| Step | Temperature | Duration | Cycles |
| 1 | Initial Denaturation | 98 °C | 30 s |
| 2 | Denaturation | 98 °C | 8 s | 30 cycles |
| 3 | Annealing | 67°C | 30 s | 30 cycles |
| 4 | Elongation | 72 °C | 90 s | 30 cycles |
| 6 | Final Extension | 72 °C | 2 min |
| 7 | Hold | 4 °C |

### Agarose Gel:

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230814-colonypcr-pbad-1.png)

## Overnight Cultures of pSB1C3 Vector

Three overnight cultures were prepared using 5ml LB medium and 5 ul Com(?) antibiotic for each.

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230814-uebernachtkultur-psb1c3-1.jpg)

## Date: Tuesday, 15.08.2023

**Participants:** Jessica, Sarah H.

### Experiment: Repeating AMP Overhang PCRs and His4 PCRs with New Primers from 14.08. with 5ng Template

## His4 PCR

### Reaction Setup:

| Components | Volume [ul] |
| --- | --- |
| Q5 2xMM | 25 |
| fwd Primer | 2.5 |
| rev Primer | 2.5 |
| Template | 1 |
| ddH2O | 19 |

### PCR Program for His4:

| Step | Temperature [°C] | Duration [s] | Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 98 | 30 | - |
| Denaturation | 98 | 8 | 35 cycles |
| Annealing | 67 | 30 | 35 cycles |
| Elongation | 72 | 80 | 35 cycles |
| Final Extension | 72 | 2 min | - |
| Hold | 4 | - | - |

## AMP Overhang PCRs

### Reaction Setup:

| A | Volume [µl] | 4x MM |
| --- | --- | --- |
| Q5 2xMM | 25 | 100 |
| BB41-fwd Primer | 2.5 | 10 |
| BB41-rev Primer | 2.5 | 10 |
| Template | 1 | - |
| ddH2O | 19 | 76 |

### PCR Program for AMP Overhang PCRs:

| Step | Temperature | Duration | Cycles |
| --- | --- | --- | --- |
| 1 | Initial Denaturation | 98 °C | 30 s |
| 2 | Denaturation | 98 °C | 8 s |
| 3 | Annealing | 69°C | 30 s |
| 4 | Elongation | 72 °C | 90 s |
| 6 | Final Extension | 72 °C | 2 min |
| 7 | Hold | 4 °C | - |

## Task3: 1% Agarose Gel for PCRs

10 ul of each PCR sample was loaded onto the agarose gel.

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230815-pcrfragmente-ampoverhangs-1.png)

## Task4: PCR Clean-up of His4 and Enzyme Fragments with AMP Overhangs

Gel extraction was performed for His4 and enzyme fragments with AMP Overhangs, and eluted in 10 ul.

### Gel Extraction Elution:

| Sample | Concentration [ng/µl] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| His4 new | 56.19 | 1.84 | 1.99 |

## Miscellaneous

Pipette tips and Eppendorf tubes at IBVT were autoclaved, and ddH2O and glycerol were prepared for autoclaving. Started organizing the refrigerator compartment categorically.

## Date: Wednesday, 16.08.2023

## Date: Thursday, 17.08.2023

## Experiment: Colony PCR, ÜN Cultures, Restriction Digest for BioBrick Assembly, PCR for Gibson Assembly of Enzyme Fragments in Pichia Vector, His4 PCR for Gibson Assembly, Linkage PCR of Dextranase

**Participants:** Sharkey 10:00-15:00; Sumohit 10:00-19:30;

## Summary:

For Colony-PCR, the samples were heated at 95°C for 5 minutes before conducting the Colony PCR. ÜN cultures were prepared from picked colonies of pBAD18-enzyme fragments. PCR for Gibson Assembly of enzyme fragments in the Pichia vector was performed for all fragments except His4, followed by purification of the samples post-PCR. Restriction digestion was performed on the purified PCR samples for BioBrick Assembly. His4 PCR for Gibson Assembly of enzyme fragments in the Pichia vector was conducted. Linkage PCR for Dextranase was also carried out.

## Colony-PCR

Since the previous day's colony PCR did not work, it was observed how Colony PCR is conducted, and online suggestions were found to heat the samples at 95°C for 5 minutes to denature the cells before the PCR. Mutanase primers were used for Mutanase and Dispersin B, following the Mutanase PCR program. For Dextranase, Mutanase primers were used along with Dex1 Primer and Dex2 Primer for Linkage PCR.

### Mutanase and Dispersin B PCR Program (Mutanase68):

| Step | Temperature [°C] | Duration [sec] | Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 98 | 30 |  |
| Denaturation | 98 | 8 | 30 |
| Annealing | 68 | 30 | 30 |
| Elongation | 72 | 90 | 30 |
| Final Extension | 72 | 120 |  |
| Hold | 4 | for ever |  |

### Linkage PCR for Dextranase (Dex):

| Dex | Dex | Dex | Dex |
| --- | --- | --- | --- |
| 1 | Initial Denaturation | 98 °C | 30 s |
| 2 | Denaturation | 98 °C | 8 s |
| 3 | Annealing | 68 °C | 30 s |
| 4 | Elongation | 72 °C | 120 s |
| 6 | Final Extension | 72 °C | 2 min |
| 7 | Hold | 4 °C |  |

## PCR for Gibson Assembly of Enzyme Fragments in Pichia Vector

Since the old PCRs with Mutanase primers or Mutanase primers – Dex1 Primer/Dex2 Primer were not found, 50 µl setups were prepared twice. Mutanase primers were used for Mut and Dsp B, Mut For Primer and Dex 1 Rev Primer were used for Dex 1, Dex2 For Primer and Mut Rev Primer for Dex 2, and Pichia primers for the Pichia vector. After PCR, 10 µl of the samples were loaded, and the remaining 40 µl were used for PCR purification.

## Preparation of 1.0% Agarose Gel for Colony-PCR and Mutanase, Dispersin B, Dex1, Dex2, and Pichia Vector

Complete 10 µl was loaded from the PCR for Gibson Assembly and 25 µl from the Colony-PCR samples.

### Load Order

Marker – Mut - Mut - DspB - DspB - Dex1 - Dex1 - Dex2 - Dex2 - Pichia Vector - Pichia Vector - Marker - Pichia Vector - Pichia Vector

Marker – Colonies Mut – Colonies DspB – Colonies Dex – Marker

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230817-colonypcr-pbad18-1.png)

Overnight cultures were made from pBAD18-enzyme fragment colonies and incubated at 37°C overnight.

## PCR Purification and Restriction Digest for BioBrick Assembly

After PCR purification, the purified samples were used for restriction digestion. The digestion was carried out as per the NEB BioBrick Protocol. For the digestion, 500 ng of Mutanase, Dispersin B, and Pichia vector were taken, and 400 ng of His4.

## Concentration Analysis (ng/µl) and Ratios

| Sample | Concentration [ng/ul] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| Mutanase 1 | 109.83 | 1.9 | 2.26 |
| Mutanase 2 | 88.5 | 1.9 | 2.34 |
| DspB1 | 72.87 | 1.89 | 2.17 |
| DspB2 | 33.66 | 1.7 | 1.01 |
| Dex1-1 | 204.1 | 1.93 | 2.37 |
| Dex1-2 | 205.1 | 1.92 | 2.26 |
| Dex2-1 | 127.5 | 1.92 | 2.33 |
| Dex2-2 | 139.3 | 1.9 | 2.18 |
| pichia Vektor-1 | 63.1 | 1.74 | 1.07 |
| pichia Vektor-2 | 113.5 | 1.79 | 1.97 |

## Volumes Used for BioBrick Assembly Restriction Digest

| Sample | Volume of Sample [µl] | Restriction Enzyme 1 [µl] | Restriction Enzyme 2 [µl] | Buffer 2.1 [µl] | Water [µl] |
| --- | --- | --- | --- | --- | --- |
| Mutanase | 4.55 | EcoRI-HF | SpeI | 5 | 38.45 |
| Dispersin B | 6.86 | EcoRI-HF | SpeI | 5 | 36.14 |
| His4 Over | 10 | XbaI | PstI | 5 | 33 |
| pSB1C3 | 3.86 | EcoRI-HF | PstI | 5 | 39.14 |

## His4 PCR for Gibson Assembly

Due to insufficient quantity of His4 for Gibson Assembly, His4 PCR was performed 5 times with the newly designed His4 primers, and 10 µl of each was loaded onto the gel.

## Preparation of 1.0% Agarose Gel for His4 and pBAD18-His4 Gibson Colony-PCR

Complete 10 µl was loaded from the PCR for Gibson Assembly.

### Load Order

His 4 Colonies - Marker – His4 – His4 – His4 – His4 – His4

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230817-colonypcr-his4-1.png)

## Linkage PCR for Dextranase

Since Dextranase is divided into two fragments, they need to be combined as one fragment. There are two methods for this, 1. Gibson Assembly and 2. Linkage PCR. Linkage PCR was chosen as it is more cost-effective than Gibson Assembly. For the Linkage PCR, the PCR-purified Dex1 and Dex2 fragments were taken, and PCR was performed with 5 ng DNA using Mutanase For and Rev Primer.

### Linkage PCR Dex

| Step | Temperature [°C] | Duration [sec] | Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 98 | 30 |  |
| Denaturation | 98 | 8 | 30 cycles |
| Annealing | 68 | 30 | 30 cycles |
| Elongation | 72 | 120 | 30 cycles |
| Final Extension | 72 | 2 min |  |
| Hold | 4 |  |  |

## Discussion:

Unfortunately, the Colony PCR did not work, even though the simultaneously run PCR for Gibson Assembly did. Lysing the cells before PCR might be necessary for successful PCR. The His4 PCRs did not work, although they had worked in previous days; the reason remains unknown.

## Next Steps:

* Plasmid isolation from the overnight cultures and restriction analysis
* His4 PCR
* Gibson Assembly and BioBrick Assembly

## Date: Friday, 18.08.2023

**Participants:** Sarah H (09:30:00-14:00); Sumohit (10:00-20:00);

## Summary:

PCR amplification of His4 with old and new His4 for Gibson Assembly. Since colony PCR yielded no results, plasmids were isolated from overnight cultures and analyzed through restriction digestion. Gibson Assembly of Mutanase into the Pichia vector and BioBrick Assembly of Mutanase and Dispersin B into pSB1C3 with transformation.

## His4 PCR

Since the PCR for His4 from the previous day did not work, the PCR for His4 was repeated using old and new His4 fragments and His4 primers. 10 µl of the PCR product was loaded onto the gel. The gel also included the Linkage PCR Sample from the previous day.

## Creation of a 1.0% Agarose Gel for His4 and Linkage PCR

Complete 10 µl was loaded from the PCR for Gibson Assembly.

Gel Loading Scheme

Marker - His4 - His4 Overhang - Marker - Dex Linkage PCR - Marker

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230818-pcr-his4-overhang-1.png)

Since the Colony PCR from the previous day did not work, the plasmids were isolated from the ÜN cultures and analyzed with PstI restriction digest. Cells were grown in all samples and plasmid was isolated from the cells as in the protocol.

| Sample | Concentration [ng/µl] | A260/A280 | A260/A230 |
| --- | --- | --- | --- |
| Mut1 | 23.48 | 2.02 | 2.34 |
| Mut2 | 27.5 | 1.87 | 2.19 |
| Mut3 | 27.61 | 1.91 | 2.12 |
| Mut4 | 20.05 | 2.07 | 2.29 |
| Mut5 | 20.25 | 1.92 | 2.14 |
| DspB1 | 21.61 | 2.18 | 2.12 |
| DspB2 | 36.03 | 2.17 | 2.26 |
| DspB3 | 21.61 | 2.09 | 2.42 |
| HIs4-1 | 27.34 | 2.13 | 2.29 |
| HIs4-2 | 22.42 | 1.99 | 2.18 |
| HIs4-3 | 27.43 | 2.03 | 2.10 |
| Dex1 | 26.94 | 1.98 | 2.29 |
| Dex2 | 39.56 | 2.02 | 2.21 |
| Dex3 | 29.42 | 1.98 | 1.67 |

After plasmid isolation, 500 ng of plasmid was used for restriction digestion with PstI. If the plasmids are cloned correctly or incorrectly, two bands can be seen, which are different in both approaches and thus it can be decided which situation prevails. The table below shows how they were all mixed together. After analysis, the samples Mutanase Colony 3, Dextranase Colony 3 and His4 Colony 3 were sent for sequencing.

| Sample | Volume of Sample [µl] | Restriction Enzyme PstI [µl] | Buffer 2.1 [µl] | Water [µl] |
| --- | --- | --- | --- | --- |
| Mut1 | 17.04 | 1.00 | 5.00 | 25.96 |
| Mut2 | 14.55 | 1.00 | 5.00 | 28.45 |
| Mut3 | 14.49 | 1.00 | 5.00 | 28.51 |
| Mut4 | 19.95 | 1.00 | 5.00 | 23.05 |
| Mut5 | 19.75 | 1.00 | 5.00 | 23.25 |
| DspB1 | 18.51 | 1.00 | 5.00 | 24.49 |
| DspB2 | 11.10 | 1.00 | 5.00 | 31.90 |
| DspB3 | 18.51 | 1.00 | 5.00 | 24.49 |
| HIs4-1 | 14.63 | 1.00 | 5.00 | 28.37 |
| HIs4-2 | 17.84 | 1.00 | 5.00 | 25.16 |
| HIs4-3 | 14.58 | 1.00 | 5.00 | 28.42 |
| Dex1 | 14.85 | 1.00 | 5.00 | 28.15 |
| Dex2 | 10.11 | 1.00 | 5.00 | 32.89 |
| Dex3 | 13.60 | 1.00 | 5.00 | 29.40 |

## Colony PCR Analysis from Gibson Assembly and BioBrick Assembly

Analysis of restriction digestion of the Gibson Enzyme samples
theoretical expectation
Assignment scheme: Marker- PstI with pBAD18 mutanase - PstI with pBAD18 dispersin B - PstI with pBAD18-His4 - PstI with pBAD18 dextranase - PstI with pBAD18 LV - Marker

![Bild](https://static.igem.wiki/teams/4575/wiki/20230818-restriktionsverdau-erwartung-1.png)

Preparation of a 1% agarose gel for restriction analysis in Gibson. 25 µl applied during the restriction analysis.

Agarose gel preparation

Prepared a 1% agarose gel for restriction analysis. Applied 25 µl during the restriction analysis.

Application scheme
Application scheme: Marker- PstI with pBAD18-mutanase (5x) - PstI with pBAD18-dispersin B (3x) - PstI with pBAD18-His4 (3x) - PstI with pBAD18-dextranase (3x) - Marker

![Bild](https://static.igem.wiki/teams/4575/wiki/20230818-restriktionsverdau-1.png)

Gibson Assembly of Mutanase in Pichia vector

Performed Gibson Assembly of Mutanase in the Pichia vector. The required volumes are provided in the table below and followed the protocol accordingly.

| Sample | Amount for Gibson [ng] | Volume of Sample [µl] | Water | NEBuilder |
| --- | --- | --- | --- | --- |
| Mut | 220 | 2 | 2.07 | 10 |
| HIs4 | 295.9 | 5.27 |  | 10 |
| Pichia Vector | 75 | 0.66 |  |  |

**BioBrick Assembly of Mutanase and Dispersin B in pBAD18 Vector**

BioBrick assembly was made with mutanase and dispersin B in pBAD18 as stated in the NEB protocol.

**Transformation of empty vectors and Gibson/BioBrick constructs**

One transformation each was made from the empty vector pichia vector in the corresponding plates, as well as from the Gibson construct and two BioBrick Assembly constructs. For this purpose, a negative sample was made with cells without plasmids, in the Amp plate.
The transformation was performed as in the protocol

**Sequencing**

Since the results from the previous restriction are inconclusive the samples mutanase colony 3, dextranase colony 3 and His4 colony 3 were sent for sequencing. For sequencing, 2.5 µl of sample and 2.5 µl of 10 µM primer were used and made up to 10 µl with ddH2O.

**Preparation of Pichia minimal agar selection plates.**

See protocol

The agar solution was heated in a microwave and cooled to below 60°C. Then YNB, biotin and glucose solutions were added according to protocol. Plates are in cold storage of IBVT; only one agar solution a 400ml was used up the 2nd still exists in case you need to pour again.

**Glycerol stock Pichia Pastoris GS115**

See protocol.

Culture with YPD+His medium

## Discussion:

Unfortunately, the His4 PCR did not succeed again. The Linkage PCR did show the desired PCR band, but it was very weak, and with Dex1 and Dex2 bands, which were also very weak. Plasmid isolation worked well for all samples. The restriction analysis shows mixed results.

## Tasks for the Next Days:

* Analyze the colonies from Gibson Assembly and BioBrick Assembly.
* Analyze the sequencing results.

## Date: Monday, 21.08.2023

## Experiment: Repeat Transformation of Cloning from Friday. Colony PCR of His4, Dex and dispersin B-pBAD18 colonies. His4 PCR. Gibson assembly of enzyme fragments and pichia vector. BioBrick assembly of mutanase and dispersin B with the new purified PCR. Transformer of new Gibson and BioBrick assembly. Linkage PCR of Dex and gel extraction.

**Participants:** Sumohit 08:00-20:00

### Summary:

Check plates from Friday. Perform colony-PCR of His4, Dex, and Dispersin B-pBAD18 colonies. His4 PCR. Gibson Assembly of enzyme fragments and pichia vector. BioBrick Assembly of Mutanase and Dispersin B with the newly purified PCR products. Transformation of new Gibson and BioBrick Assemblies. Linkage PCR of Dex and gel extraction.

### Summary of Friday Plates Observation

The plates from Friday did not show any colonies, not even the positive control. Hence, everything needs to be repeated.

![Bild](https://static.igem.wiki/teams/4575/wiki/20230821-platten-1.jpeg)

## Colony PCR

Colony PCR was attempted again using iGEM 2021 Stuttgart sequencing primers for pBAD18. For the colony PCR, all three His4 samples, all three Dex samples, Dispersin B, and pBAD18 were taken as the positive control. PCR programs that work for normal PCR were used. An annealing temperature of 66 or 67 was used.

## Linkage PCR of Dextranase

Since Dextranase is divided into two fragments, Dex1 and Dex2, an attempt was made to combine these two fragments through a Linkage PCR, enabling BioBrick Assembly. A PCR was performed and loaded onto the gel. Subsequently, the Dex band at approximately 4000 bp was extracted from the gel.

## His4 with Overhangs PCR

Since there was insufficient His4 available, an attempt was made to perform a PCR again for the His4 fragment with the overhangs.

## Preparation of a 1.0% Agarose Gel for His4 PCR, Linkage PCR, and Colony PCR

A total of 10 µl was loaded from the PCR for Gibson Assembly.

## Loading Scheme

Marker - Colony PCR - Dex Linkage PCR - Marker – His4 Overhang - His4 Overhang - Marker

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230821-pcr-his4-overhang-1.png)

## PCR Cleanup

PCR cleanup was performed on these PCR samples. The values are listed in the table below.

| Sample | Concentration [ng/µl] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| His4 1 | 24.4 | 1.92 | 2.3 |
| His4 2 | 47.1 | 1.78 | 2.29 |
| Dex | 2.53 | 0.09 |  |

Since the concentrations were satisfactory, a Gibson Assembly was performed with Mut, Dex, and Dispersin B from one PCR with a 1:1 Fragment:Vector ratio.

## Gibson Assembly

Gibson Assembly was performed as outlined in the Mutanase protocol. Gibson Assembly was carried out in a pichia vector with Mutanase, Dispersin B, and Dextranase. The table below shows the required volumes and the procedure was followed as per the protocol. Since the required DNA quantities were excessive, a 1:1 Fragment:Vector ratio was used for Gibson Assembly.

| Sample | Amount for Gibson [ng] | Sample Volume [µl] | Water | NEBuilder |
| --- | --- | --- | --- | --- |
| Mut | 110 | 1 | 6.17 | 10 |
| Dsp B | 103.9 | 1.43 | 5.74 | 10 |
| Dex1 | 82.4 | 0.41 | 6.22 | 10 |
| Dex2 | 69.0 | 0.54 |  |  |
| His4 | 98.6 | 2.17 |  |  |
| pichia Vector | 75 | 0.66 |  |  |

## BioBrick Assembly of Mutanase and Dispersin B in pBAD18 Vector

For the BioBrick, 450 ng of His4 DNA digested with XbaI and PstI were used for BioBrick Assembly. BioBrick Assembly was performed in a pichia vector with Mutanase and Dispersin B, following the NEB protocol.

## Transformation of Empty Vectors and Gibson/BioBrick Constructs

The DNA constructs from BioBrick Assembly and Gibson Assembly were transformed into E. coli cells and plated on respective antibiotic-containing plates. pSB1C3 was used as a positive control for the transformation and plated on CMR-containing plates. Cells without plasmids were treated as a blank in the transformation process, plated on CMR-containing plates, and served as a negative control for the transformation. The transformation was carried out as per the protocol.

## Transformation of Clones from Friday

The transformations from Friday were repeated following the procedure from Friday, August 18.

## Date: Monday, 21.08.2023

## Experiment: Repeat Transformation of Cloning from Friday

**Participants:** Sumohit 08:00-20:00

### Summary:

Check plates from Friday. Perform colony-PCR of His4, Dex, and Dispersin B-pBAD18 colonies. His4 PCR. Gibson Assembly of enzyme fragments and pichia vector. BioBrick Assembly of Mutanase and Dispersin B with the newly purified PCR products. Transformation of new Gibson and BioBrick Assemblies. Linkage PCR of Dex and gel extraction.

### Summary of Friday Plates Observation

The plates from Friday did not show any colonies, not even the positive control. Hence, everything needs to be repeated.

![Bild](https://static.igem.wiki/teams/4575/wiki/20230821-platten-1.jpeg)

## Colony PCR

Colony PCR was attempted again using iGEM 2021 Stuttgart sequencing primers for pBAD18. For the colony PCR, all three His4 samples, all three Dex samples, Dispersin B, and pBAD18 were taken as the positive control. PCR programs that work for normal PCR were used. An annealing temperature of 66 or 67 was used.

## Linkage PCR of Dextranase

Since Dextranase is divided into two fragments, Dex1 and Dex2, an attempt was made to combine these two fragments through a Linkage PCR, enabling BioBrick Assembly. A PCR was performed and loaded onto the gel. Subsequently, the Dex band at approximately 4000 bp was extracted from the gel.

## His4 with Overhangs PCR

Since there was insufficient His4 available, an attempt was made to perform a PCR again for the His4 fragment with the overhangs.

## Preparation of a 1.0% Agarose Gel for His4 PCR, Linkage PCR, and Colony PCR

A total of 10 µl was loaded from the PCR for Gibson Assembly.

## Loading Scheme

Marker - Colony PCR - Dex Linkage PCR - Marker – His4 Overhang - His4 Overhang - Marker

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230821-pcr-his4-overhang-1.png)

## PCR Cleanup

PCR cleanup was performed on these PCR samples. The values are listed in the table below.

| Sample | Concentration [ng/µl] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| His4 1 | 24.4 | 1.92 | 2.3 |
| His4 2 | 47.1 | 1.78 | 2.29 |
| Dex | 2.53 | 0.09 |  |

Since the concentrations were satisfactory, a Gibson Assembly was performed with Mut, Dex, and Dispersin B from one PCR with a 1:1 Fragment:Vector ratio.

## Gibson Assembly

Gibson Assembly was performed as outlined in the Mutanase protocol. Gibson Assembly was carried out in a pichia vector with Mutanase, Dispersin B, and Dextranase. The table below shows the required volumes and the procedure was followed as per the protocol. Since the required DNA quantities were excessive, a 1:1 Fragment:Vector ratio was used for Gibson Assembly.

| Sample | Amount for Gibson [ng] | Sample Volume [µl] | Water | NEBuilder |
| --- | --- | --- | --- | --- |
| Mut | 110 | 1 | 6.17 | 10 |
| Dsp B | 103.9 | 1.43 | 5.74 | 10 |
| Dex1 | 82.4 | 0.41 | 6.22 | 10 |
| Dex2 | 69.0 | 0.54 |  |  |
| His4 | 98.6 | 2.17 |  |  |
| pichia Vector | 75 | 0.66 |  |  |

## BioBrick Assembly of Mutanase and Dispersin B in pBAD18 Vector

For the BioBrick, 450 ng of His4 DNA digested with XbaI and PstI were used for BioBrick Assembly. BioBrick Assembly was performed in a pichia vector with Mutanase and Dispersin B, following the NEB protocol.

## Transformation of Empty Vectors and Gibson/BioBrick Constructs

The DNA constructs from BioBrick Assembly and Gibson Assembly were transformed into E. coli cells and plated on respective antibiotic-containing plates. pSB1C3 was used as a positive control for the transformation and plated on CMR-containing plates. Cells without plasmids were treated as a blank in the transformation process, plated on CMR-containing plates, and served as a negative control for the transformation. The transformation was carried out as per the protocol.

## Transformation of Clones from Friday

The transformations from Friday were repeated following the procedure from Friday, August 18.

## Date: Tuesday, 22.08.2023

## Experiment: Media Preparation, Overnight Cultures for Plasmid Isolation, Dex Linkage PCR, Sequencing, Colony PCR

**Participants:** Jana 09:00-18:00; Sumohit 09:00-18:00;

### Summary:

LB medium with Amp, TB medium, and LB Agar with Amp and Cmr were prepared. Overnight (ÜN) cultures of the Gibson enzyme fragments in the pichia vector were created. Dex Linkage PCR was repeated. Colony PCR was performed with 10 minutes of boiling. Sequencing of Dex C2 was sent.

### Observation of Plates from Monday

The plates from Monday showed all colonies except the BioBrick samples from Friday and the Mutanase-pichia vector 1:2. ÜN cultures were also prepared with 4 or 3 colonies per plate, so that ÜN cultures can be made the next day.

![Bild](https://static.igem.wiki/teams/4575/wiki/20230822-platten-1.jpeg)

![Bild](https://static.igem.wiki/teams/4575/wiki/20230822-platten-2.jpeg)

![Bild](https://static.igem.wiki/teams/4575/wiki/20230822-platten-3.jpeg)

## Media

Media were prepared and autoclaved as per the protocols.

## His4 PCR

Performed as on Monday, August 21, 2023.

## Preparation of 1.0% Agarose Gel for His4

10 µl was loaded from the PCR samples.

### Loading Scheme

Marker - His 4 - His4 - Marker

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230822-pcr-his4-1.png)

## PCR Purification from His4

PCR purification was performed on these PCR samples. Since the first and second His4 PCR did not show intense bands, the samples were pooled and purified. The values are listed in the table below.

| Sample | Concentration [ng/µl] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| His4 12 | 95.4 | 1.85 | 2.22 |
| His4 3 | 60.0 | 1.86 | 2.48 |
| His4 4 | 82.2 | 1.80 | 2.08 |

## Linkage PCR Dex

A PCR was performed from the gel-extracted sample using Mutanase primers.

## Colony PCR with Heating

Since the colony PCR from the previous day did not work, colony PCR was again performed with J1, J11, and S1. This time the samples were heated for 10 minutes at 98°C before PCR, and then PCR was performed. pBAD18 was used as a positive control, and AMP primers were used as primers.

### Loading Scheme

Marker - J1 - J11 - S1 - Marker

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230822-colonypcr-1.png)

## Sequencing

For sequencing, pBAD18-Mutanase colony 4 was sent with Mutanase Forward and Reverse primers, as well as pBAD18-Dextranase with pBAD18 seq Primers Forward and Reverse.

## Discussion:

The plates from Monday showed all colonies except the negative plate, Mut BioBrick old, and DspB BioBrick old. It was expected on the negative plate since they had small selection markers and were plated on selection plates. However, with Mut BioBrick old and DspB BioBrick old plates, colonies were expected, but none were visible. His4 worked well, and PCR purification was also good. Due to time constraints, no gel was made for Linkage Dex. The colony PCR with 10 minutes of heating at 98°C did not work.

## Date: Wednesday, 23.08.2023

## Experiment: Quick Analysis of Gibson Plasmids. Plasmid Isolation of Gibson Plasmids. Colony PCR with Lysozyme

**Participants:** Jana 09:00-18:00; Sumohit 09:00-18:00

### Summary:

Quick analysis of Gibson plasmids. Plasmid isolation of Gibson plasmids. Colony PCR with 10 minutes of lysozyme treatment.

## Quick Analysis of Gibson Plasmids

A small amount of cells was taken from the backup plates and dissolved in 15 µl of sterile ddH2O. Then proceeded as per the protocol.

A master mix of 2x NSEB solution was prepared, totaling 500 µl. After 1 hour of incubation, the cells were mixed with 25% Ficoll and applied to the gel.

## Preparation of 1.0% Agarose Gel for Plasmid Quick Analysis

Completely loaded 25 µl of the samples.

### Loading Scheme

pBAD18 (negative control) - Mutanase BioBrick new - DspB BioBrick new - Mutanase 1:1 Gibson - Dispersin B 1:1 Gibson - Dextranase 1:1 Gibson - empty - pESP - pREP2

The gel ran overnight at 15 V without Midori Green.

## Plasmid Isolation from Gibson Colonies

Plasmids were isolated from the ÜN cultures according to the protocol. The DNA concentrations in the plasmid isolation samples are listed in the table below.

| Sample | Concentration [ng/µl] | A260/A280 | A260/A230 |
| --- | --- | --- | --- |
| Mut 1 | 61.4 | 1.82 | 1.13 |
| Mut 2 | 51.6 | 1.92 | 2.55 |
| Mut 3 | 61.9 | 1.91 | 2.16 |
| Disp 1 | 47.8 | 1.96 | 2.45 |
| Disp 2 | 48.4 | 2.01 | 2.16 |
| Disp 3 | 61.1 | 1.90 | 1.48 |
| Mut 1:1 1 | 32.1 | 2.07 | 2.29 |
| Mut 1:1 2 | 18.1 | 2.01 | 2.03 |
| Mut 1:1 3 | 27.0 | 2.09 | 2.41 |
| Mut 1:1 4 | 26.5 | 2.26 | 2.76 |
| Dex 1:1 1 | 48.0 | 1.78 | 0.94 |
| Dex 1:1 2 | 53.1 | 1.93 | 1.85 |
| Dex 1:1 3 | 40.8 | 2.00 | 1.48 |
| Dex 1:1 4 | 33.7 | 2.11 | 1.93 |
| Disp 1:1 1 | 56.7 | 1.91 | 1.41 |
| Disp 1:1 2 | 35.4 | 2.00 | 1.96 |
| Disp 1:1 3 | 40.5 | 1.99 | 1.52 |
| Disp 1:1 4 | 40.9 | 1.74 | 0.99 |
| Mut C4 | 60.3 | 1.96 | 1.53 |

## Colony PCR with Lysozyme

Since the colony PCR did not work, a colony PCR was performed, where initially 15 µl of lysozyme solution was added to the PCR tubes. Then cells were added, and the sample was incubated at 37°C for 10 minutes. After incubation, a regular PCR was performed with AMP primers. J1, J2, and J12 were used as samples, and AMP Fragment BB41 was taken as a positive control for PCR.

## AMP BB41 Pre-experiment

PCR Program: AMPOVERH (PCR machine iGEM)

| Step | Temperature [°C] | Duration [sec] | Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 98 | 30 |  |
| Denaturation | 98 | 10 | 30 |
| Annealing | 69 | 30 | 30 |
| Elongation | 72 | 18 | 30 |
| Final Extension | 72 | 120 |  |
| Hold | 4 |  | forever |

## Discussion:

The plasmid isolation worked well, and the concentrations are above the target value.

## Date: Thursday, 24.08.2023

## Experiment: Restriction Analysis of Gibson Plasmids. Post-staining of Quick Analysis Gel of Gibson Plasmids.

**Participants:** Sharkey 09:30-16:00; Sumohit 09:30-16:00

### Summary:

Restriction analysis of Gibson plasmids using PstI. Post-staining with Midori Green of the quick analysis gel of Gibson plasmids.

## Post-staining of Quick Analysis Gel of Gibson Plasmids

Since the gel ran overnight without Midori Green, in the morning the gel was placed in a Midori Green bath with 200 ml of Midori Green solution, which contains 40µl of Midori, and shaken for 1 hour. Then Midori Green was collected, and the gel was placed in water twice for 30 minutes each, with shaking.

### Loading Scheme

pBAD18 (negative control) - Mutanase BioBrick new - DspB BioBrick new - Mutanase 1:1 Gibson - Dispersin B 1:1 Gibson - Dextranase 1:1 Gibson - empty - pESP - pREP2

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230824-gibsonplasmide-1.png)

## Restriction Analysis of Gibson Plasmids with PstI

Restriction digestion with PstI was performed on the plasmid-isolated samples. 500 ng of plasmid DNA was taken and digested with 1 µl of PstI. If the plasmids are cloned correctly, two bands are expected to be seen in all Gibson constructs, and if cloning is unsuccessful, only one band will be visible. The samples from the colony PCR from the previous day and the linkage PCR from Tuesday were also loaded on the gel.

| Sample | Concentration | A260/A280 | A260/A230 |
| --- | --- | --- | --- |
| Mut 1 | 23.48 | 2.02 | 2.34 |
| Mut 2 | 27.5 | 1.87 | 2.19 |
| Mut 3 | 27.61 | 1.91 | 2.12 |
| Disp 1 | 47.8 | 1.96 | 2.45 |
| Disp 2 | 48.4 | 2.01 | 2.16 |
| Disp 3 | 8.18 | 1.00 | 5.00 | 34.82 |
| Mut 1:1 1 | 15.58 | 1.00 | 5.00 | 27.42 |
| Mut 1:1 2 | 27.62 | 1.00 | 5.00 | 15.38 |
| Mut 1:1 3 | 18.52 | 1.00 | 5.00 | 24.48 |
| Mut 1:1 4 | 18.87 | 1.00 | 5.00 | 24.13 |
| Dex 1:1 1 | 10.42 | 1.00 | 5.00 | 32.58 |
| Dex 1:1 2 | 9.42 | 1.00 | 5.00 | 33.58 |
| Dex 1:1 3 | 12.25 | 1.00 | 5.00 | 30.75 |
| Dex 1:1 4 | 14.84 | 1.00 | 5.00 | 28.16 |
| Disp 1:1 1 | 12.35 | 1.00 | 5.00 | 30.65 |
| Disp 1:1 2 | 14.12 | 1.00 | 5.00 | 28.88 |
| Disp 1:1 3 | 12.35 | 1.00 | 5.00 | 30.65 |
| Disp 1:1 4 | 12.22 | 1.00 | 5.00 | 30.78 |
| pBAD18 | 7.59 | 1.00 | 5.00 | 35.41 |

## Analysis of Restriction Digestion of Gibson Enzyme Samples

Theoretical Expectation

### Loading Scheme

Marker - PstI with pichia - PstI with pichia-Mutanase - PstI with pichia-Dex - PstI with pSB1C3-BBa\_I20270 - PstI with pSB1C3-Mut - PstI with pSB1C3-Dispersin B - PstI with pSB1C3-Dextranase

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230824-restriktionsverdau-erwartung-1.png)

## Preparation of 1% Agarose Gel for Restriction Analysis of Gibson

25 µl was loaded for restriction analysis and other samples as well.

### Loading Scheme

Above: pBAD18 (negative control) - Mutanase BioBrick new - DspB BioBrick new - Mutanase 1:1 Gibson - Dispersin B 1:1 Gibson - Dextranase 1:1 Gibson - empty - pESP - pREP2

Below: Marker - Colony-PCR - Positive Control - Marker - Dextranase Linkage PCR with Dex 1 and Dex2 - Dextranase Linkage PCR with Dex Linkage from Gel Extraction - Marker

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230824-restriktionsverdau-1.png)

## Discussion:

The gel from the quick analysis of Gibson plasmids is not evaluable and must be repeated on Friday. The gel from the restriction analysis also looks bad, and the gel electrophoresis with the restriction enzymes, colony PCR with lysozyme, and Dex linkage PCR need to be repeated.

## Date: Friday, 25.08.2023

## Experiment: Restriction Analysis of Gibson Plasmids. Quick Analysis Gel of Gibson Plasmids. Colony PCR with Lysozyme and Heating. Linkage PCR. Transformation in Pichia Cells. Sequencing of Samples.

**Participants:** Ronja 08:30-18:30; Sumohit 08:30-19:30

### Summary:

Restriction analysis of Gibson plasmids using PstI. Post-staining with Midori Green of the quick analysis gel of Gibson plasmids.

## Gel for Restriction Samples from the Previous Day

Since the gel did not run well, the gel for restriction analysis was repeated.

## Analysis of Restriction Digestion of Gibson Enzyme Samples

Theoretical Expectation

### Loading Scheme

Marker- PstI with Pichia - PstI with Pichia-Mutanase - PstI with Pichia-Dex - PstI with pSB1C3-BBa\_I20270 - PstI with pSB1C3-Mut - PstI with pSB1C3-Dispersin B - PstI with pSB1C3-Dextranase - PstI with pBAD18 LV

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230825-restriktionsverdau-erwartung-1.png)

## Preparation of 1% Agarose Gel for Restriction Analysis of Gibson

25 µl was loaded for the restriction analysis.

### Loading Scheme

Above: Mutanase BioBrick new - DspB BioBrick new - Mutanase 1:1 Gibson - Dispersin B 1:1 Gibson - Dextranase 1:1 Gibson - Marker - Marker - pBAD18 (negative control)

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230825-restriktionsverdau-1.png)

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230825-restriktionsverdau-2.png)

## Repeating Gel for Quick Analysis of Gibson and BioBrick Plasmids

Since the quick analysis from previous days was not successful, it was repeated for all samples.

| Sample | Concentration | A260/A280 | A260/A230 |
| --- | --- | --- | --- |
| Mut 1 | 23.48 | 2.02 | 2.34 |
| Mut 2 | 27.5 | 1.87 | 2.19 |
| Mut 3 | 27.61 | 1.91 | 2.12 |
| Disp 1 | 47.8 | 1.96 | 2.45 |
| Disp 2 | 48.4 | 2.01 | 2.16 |
| Disp 3 | 8.18 | 1.00 | 5.00 | 34.82 |
| Mut 1:1 1 | 15.58 | 1.00 | 5.00 | 27.42 |
| Mut 1:1 2 | 27.62 | 1.00 | 5.00 | 15.38 |
| Mut 1:1 3 | 18.52 | 1.00 | 5.00 | 24.48 |
| Mut 1:1 4 | 18.87 | 1.00 | 5.00 | 24.13 |
| Dex 1:1 1 | 10.42 | 1.00 | 5.00 | 32.58 |
| Dex 1:1 2 | 9.42 | 1.00 | 5.00 | 33.58 |
| Dex 1:1 3 | 12.25 | 1.00 | 5.00 | 30.75 |
| Dex 1:1 4 | 14.84 | 1.00 | 5.00 | 28.16 |
| Disp 1:1 1 | 12.35 | 1.00 | 5.00 | 30.65 |
| Disp 1:1 2 | 14.12 | 1.00 | 5.00 | 28.88 |
| Disp 1:1 3 | 12.35 | 1.00 | 5.00 | 30.65 |
| Disp 1:1 4 | 12.22 | 1.00 | 5.00 | 30.78 |
| pBAD18 | 7.59 | 1.00 | 5.00 | 35.41 |

Z cells were taken from the backup plates and dissolved in 15 µl of sterile ddH2O. Then proceeded as per the protocol.

## Repeating Gel for Restriction Analysis of Gibson and BioBrick Assembly

25 µl was loaded for the restriction analysis and other samples.

### Loading Scheme (Large Gel)

Above: Quick analysis Samplen pBAD18 (negative control)- Mutanase BioBrick new - DspB BioBrick new - Mutanase 1:1 Gibson - Dispersin B 1:1 Gibson - Dextranase 1:1 Gibson - empty - pESP - pREP2

Below: Marker - Mutanase 1:1 Gibson - Dispersin B 1:1 Gibson - Dextranase 1:1 Gibson - pBAD18 (negative control) - Marker

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230825-restriktionsverdau-unten-1.png)

### Loading Scheme (Small Gel)

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230825-colonypcr-kleinesgel-1.png)

## Sequencing and Transformation in Pichia Cells

Since the Dispersin B 1:1 Gibson constructs Sample 1 looked correct, it was sent for sequencing. Additionally, transformation in Pichia cells was performed with the samples Dispersin B 1:1 Gibson constructs Samples 1 and 2 following the EZ yeast transformation kit protocol and incubated at RT and 30°C over the weekend.

## Date: Monday, 28.08.2023

## Experiment: Colony PCR with Lysozyme and Different Cycles. Restriction Analysis. Observing Pichia Plates. Fluorescence Microscopy. BioBrick Assembly with Transformation. Quick Analysis.

**Participants:** Lukas 08:30 - 16:00, Sumohit 08:30-20:30

### Summary:

Observing plates from Friday. Colony PCR with Lysozyme at 40 and 45 cycles. Examining the restriction samples from Friday. Fluorescence microscopy with BioBrick samples. New BioBrick Assembly of Mutanase and Dispersin B.

## Observing Plates from Friday

On the pichia selection plates at 30°C, a few small colonies of Dsp B can be seen, but not on the others.

![Bild](https://static.igem.wiki/teams/4575/wiki/20230828-platten-1.jpeg)

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230828-colonypcr-1.png)

## Colony PCR

Since slight bands were visible in the colony PCR from last week with Lysozyme, two colony PCRs were repeated for S1, J1, and J2 with 40 cycles and 45 cycles.

| Step | Temperature [°C] | Duration [sec] | Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 98 | 30 |  |
| Denaturation | 98 | 10 | 40/45 |
| Annealing | 69 | 30 | 40/45 |
| Elongation | 72 | 18 | 40/45 |
| Final Extension | 72 | 120 |  |
| Hold | 4 | for ever |  |

## Restriction Analysis of BioBrick Samples

The restriction samples from Friday for BioBrick Assembly, which were not loaded onto the gel, were loaded onto the gel. 500 ng DNA was taken for the restriction digestion.

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230828-colonypcr-2.png)

## Fluorescence Microscopy

First, we were instructed by Martina on fluorescence microscopy. Then, the BioBrick samples were examined on the fluorescence microscope for GFP.

Positiv controll GFP

![Fluoreszenzbild](https://static.igem.wiki/teams/4575/wiki/20230828-fluoreszenz-1.jpeg)

Negativ controll GFP

![ Fluoreszenzbild ](https://static.igem.wiki/teams/4575/wiki/20230828-fluoreszenz-2.jpeg)

Mut1

![Fluoreszenzbild ](https://static.igem.wiki/teams/4575/wiki/20230828-fluoreszenz-dispb-1.jpeg)

Mut2

![Fluoreszenzbild ](https://static.igem.wiki/teams/4575/wiki/20230828-fluoreszenz-dispb-2.jpeg)

Mut3

![Fluoreszenzbild ](https://static.igem.wiki/teams/4575/wiki/20230828-fluoreszenz-dispb-3.jpeg)

Dispersin B1

![Fluoreszenzbild ](https://static.igem.wiki/teams/4575/wiki/20230828-fluoreszenz-mut-1.jpeg)

Dispersin B2

![Fluoreszenzbild ](https://static.igem.wiki/teams/4575/wiki/20230828-fluoreszenz-mut-2.jpeg)

Dispersin B3

![Fluoreszenzbild ](https://static.igem.wiki/teams/4575/wiki/20230828-fluoreszenz-mut-3.jpeg)

## BioBrick Assembly

GFP fragment bands or signals were observed on the gel and fluorescence microscope. After reviewing old posts, it was determined that the pSB1C3 restriction digestion with EcoRI and PstI needs to be purified. Therefore, the BioBrick Assembly was repeated. For this, pSB1C3 was digested again with EcoRI and PstI.

### Restriction Digestion of pSB1C3

The correct band was cut and extracted from the gel.

| Sample | Concentration [ng/ul] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| pSB1C3 | 16.8 | 1.71 | 1.65 |
| pSB1C3 | 20.2 | 1.68 | 0.05 |

## Transformation of Empty Vectors and BioBrick Constructs

The DNA constructs from BioBrick Assembly were transformed into E. coli cells and plated on respective antibiotic plates. pSB1C3 was used as a positive control for the transformation and plated on CMR-containing plates. Cells without plasmids were treated as a blank in the transformation, plated on CMR-containing plates, and served as a negative control for the transformation.

The transformation was carried out as per the protocol.

## Date: Tuesday, 29.08.2023

## Experiment: Restriction Digestion of pBAD18 Enzyme Fragments. Restriction Analysis of pichia Vector Enzyme Fragments. UV Irradiation. Fluorescence Microscopy. Setting up Overnight Cultures. AMP Cloning.

**Participants:** Lukas 09:30 - 16:00, Sumohit 09:30-21:00

### Summary:

Restriction digestion of pBAD18 enzyme fragments. Observing plates from Friday. Restriction digestion of pichia vector enzyme fragments to detect the fragments. UV irradiation. Fluorescence microscopy. Setting up overnight cultures. Gibson Assembly of AMP1 and AMP2 into pBAD18.

## Observing Plates from Friday

On the pichia selection plates at 30°C and RT, a few small colonies of Dsp B can be seen, but not on the others.

![Bild](https://static.igem.wiki/teams/4575/wiki/20230829-platten-1.jpeg)

![Bild](https://static.igem.wiki/teams/4575/wiki/20230829-platten-2.jpeg)

## Restriction Digestion of pBAD18 Enzyme Fragments

Since hardly any colonies were visible on the plates by tomorrow 10 AM and there was growth in the positive control, it was assumed that the restriction digestion of the Gibson overhangs is not sufficient for BioBrick Assembly. Therefore, pBAD18-Mut and -Dsp B were digested with EcoRI and SpeI, and pBAd18-His4 was digested with XbaI and PstI so that BioBrick Assembly can be done with these samples. After digestion, the samples were loaded onto the gel.

## Restriction Digestion of pichia Enzyme Fragments

Since so far only the His4 fragment could be detected on the gels, a new restriction analysis with EcoRI and PstI was performed so that the upper band in the previous gels can separate into the enzyme fragment band and backbone band. 500 ng DNA was digested with EcoRI and PstI.

## PCR for AMP1 and AMP2 Gibson

The AMP1 and AMP2 fragments were diluted in water to achieve an end mass concentration of 10ng/µl and then a PCR was performed with them.

PCR programm for AMP1

| Step | Temperature [°C] | Duration [sec] | Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 98 | 30 |  |
| Denaturation | 98 | 10 | 40/45 |
| Annealing | 69 | 30 | 40/45 |
| Elongation | 72 | 18 | 40/45 |
| Final Extension | 72 | 120 |  |
| Hold | 4 | for ever |  |

PCR programm for AMP2

| Step | Temperature [°C] | Duration [sec] | Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 98 | 30 |  |
| Denaturation | 98 | 10 | 40/45 |
| Annealing | 69 | 30 | 40/45 |
| Elongation | 72 | 40 | 40/45 |
| Final Extension | 72 | 120 |  |
| Hold | 4 | for ever |  |

## Creating a 1.0% Agarose Gel for Restriction Digestion and AMP PCR

25 µl was loaded from the restriction digestion and PCR samples onto the gel.

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230829-restriktionsverdau-amppcr-1.png)

## UV Irradiation of BioBrick Cells

Cells have GFP if the colonization was not successful. Therefore, the cells were examined under a UV lamp in Mibi, and colonies 4, 15, and 17 from Mutanase BioBrick plate and colonies 14 and 21 from Dextranase plate did not show any or very weak signals. Therefore, these samples were taken and examined under a fluorescence microscope.

## Fluorescence Microscopy of BioBrick Cells

The cells that showed no or very weak signal were examined under the fluorescence microscope and GFP was observed in the cells.

## AMP1 and AMP2 Gibson Assembly

Due to communication issues during sample loading in the previous gels, the entire PCR approach was loaded onto the gel. Therefore, the PCR for AMP1 and AMP2 was repeated, and for AMP2, two PCR approaches with 50 µl each were made.

PCR programm for AMP1

| Step | Temperature [°C] | Duration [sec] | Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 98 | 30 |  |
| Denaturation | 98 | 10 | 40/45 |
| Annealing | 69 | 30 | 40/45 |
| Elongation | 72 | 18 | 40/45 |
| Final Extension | 72 | 120 |  |
| Hold | 4 | for ever |  |

PCR programm for AMP2

| Step | Temperature [°C] | Duration [sec] | Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 98 | 30 |  |
| Denaturation | 98 | 10 | 40/45 |
| Annealing | 69 | 30 | 40/45 |
| Elongation | 72 | 40 | 40/45 |
| Final Extension | 72 | 120 |  |
| Hold | 4 | for ever |  |

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230829-pcramp-1.png)

PCR purification was made from these PCRs. The values are listed in the table below.

| Sample | Concentration [ng/ul] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| AMP1 | 116.9 | 1.85 | 2.27 |
| AMP2-1 | 93.0 | 1.90 | 2.46 |
| AMP2-2 | 92.3 | 1.84 | 2.4 |

## Quick Analysis of BioBrick Colonies

Performed as documented in the protocol for the BioBrick samples.

## Preparation of a 1.0% Agarose Gel

The complete samples were loaded onto the gel.

## Application Scheme

* Marker
* Mutanase BB
* Dispersin B BB
* Dextranase BB
* His4 BB
* pBAD18
* pSB1C3
* Marker

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230829-biobrickcolony-1.png)

## Gibson Assembly

Performed as described in the protocol. The table below shows the DNA quantities and sample volumes used for Gibson Assembly.

| Sample | Amount for Gibson [ng] | Volume Sample [µl] | Water | NEBuilder |
| --- | --- | --- | --- | --- |
| AMP1 | 19.63 | 0.1679213003 | 9.0320786997 | 10 |
| AMP2 | 45.47 | 0.4889247312 | 8.7110752688 | 10 |
| pBAD18 Vektor | 75 | 0.80 |  |  |

## Transformation of Empty Vectors and Gibson Constructs

The DNA constructs from the Gibson Assembly were transformed into E. coli cells and plated on the corresponding antibiotic plates. pBAD18 was used as a positive control for transformation and plated on Amp-containing plates. Cells without plasmids were treated as a blank in the transformation, plated on Amp-containing plates, and served as a negative control for transformation.

The transformation was carried out as per the protocol.

## Setting Up Overnight Cultures

Overnight cultures were made from all BioBrick colonies from the previous cloning. Overnight cultures were also made from the samples that showed no signal during UV irradiation and from the colonies from the Gibson Assembly of enzyme fragments into the pichia vector, which are most likely correct.

## Date: Wednesday, 30.08.2023

## Experiment Summary

Experiment: Plasmid Isolation and Restriction Analysis of Pichia Enzyme Fragments and BioBrick Assembly Samples. Setting up Overnight Cultures. Maintenance.

**Participants:** Sharkey 10:00 - 15:30, Sumohit 10:00-15:30

### Summary

View plates from Tuesday. Perform plasmid isolation and restriction analysis of Pichia enzyme fragments and BioBrick assembly samples (old and new). Set up overnight cultures. Maintenance.

#### View Plates from Tuesday

Samples were visible on AMP plates as well as on the positive control.

#### Plasmid Isolation

Plasmids were isolated from the overnight cultures according to the protocol. The table below shows the DNA concentrations in the plasmid isolation samples.

| Sample | Concentration [ng/ul] | A260/280 | A260/A230 |
| --- | --- | --- | --- |
| 4;1 | 39.1 | 2.07 | 2.29 |
| 4;2 | 54.5 | 2.09 | 2.75 |
| 14;1 | 58.1 | 2.17 | 2.7 |
| 14;2 | 57.4 | 2.17 | 2.71 |
| 15;1 | 49.4 | 2.1 | 2.51 |
| 15;2 | 41.9 | 2.06 | 2.26 |
| 17;1 | 29.6 | 2.12 | 2.54 |
| 17;2 | 36.6 | 2.12 | 2.44 |
| 21;1 | 26.4 | 2.14 | 2.79 |
| 21;2 | 16.9 | 2.17 | 2.42 |
| Dex2 | 24.3 | 2.49 | 2.69 |
| Dex4 | 18.1 | 2.04 | 2.73 |
| BB Mut1 | 44.5 | 2.13 | 2.45 |
| BB Mut2 | 42.7 | 2.25 | 2.89 |
| BB Mut3 | 43.5 | 1.99 | 2.13 |
| BB Mut4 | 58 | 2.18 | 2.42 |
| BB Mut5 | 24 | 2.22 | 1.92 |
| BB Mut6 | 37.1 | 2.22 | 2.03 |
| BB Dsp B 1 | 51 | 2 | 1.55 |
| BB Dsp B 2 | 29.5 | 2.17 | 1.95 |
| BB Dsp B 3 | 33.6 | 2.21 | 2.23 |
| BB Dsp B 4 | 24.8 | 2.13 | 2.11 |
| Dsp B1 | 42.7 | 2.08 | 1.92 |
| Dsp B2 | 41.9 | 2.11 | 2.24 |

#### Restriction Analysis of Plasmids

Since the restriction analysis from the previous day did not show the desired bands, the restriction analysis was repeated with different enzymes. BsaI was added to Dispersin B samples from the previous day, and HindIII was added to Dextranase samples from the previous day. Additionally, a restriction analysis was performed with the old BioBrick samples and the BioBrick samples from the latest cloning using EcoRI and PstI.

| Sample | Amount for Gibson [ng] | Volume Sample [µl] | Water | NEBuilder |
| --- | --- | --- | --- | --- |
| 4;1 | 12.79 | 2.00 | 5.00 | 28.21 |
| 4;2 | 9.17 | 2.00 | 5.00 | 31.83 |
| 14;1 | 8.61 | 2.00 | 5.00 | 32.39 |
| 14;2 | 8.71 | 2.00 | 5.00 | 32.29 |
| 15;1 | 10.12 | 2.00 | 5.00 | 30.88 |
| 15;2 | 11.93 | 2.00 | 5.00 | 29.07 |
| 17;1 | 16.89 | 2.00 | 5.00 | 24.11 |
| 17;2 | 13.66 | 2.00 | 5.00 | 27.34 |
| 21;1 | 18.94 | 2.00 | 5.00 | 22.06 |
| 21;2 | 29.59 | 2.00 | 5.00 | 11.41 |
| Dex2 | 20.58 | 2.00 | 5.00 | 20.42 |
| Dex4 | 27.62 | 2.00 | 5.00 | 13.38 |
| BB Mut1 | 11.24 | 2.00 | 5.00 | 29.76 |
| BB Mut2 | 11.71 | 2.00 | 5.00 | 29.29 |
| BB Mut3 | 11.49 | 2.00 | 5.00 | 29.51 |
| BB Mut4 | 8.62 | 2.00 | 5.00 | 32.38 |
| BB Mut5 | 20.83 | 2.00 | 5.00 | 20.17 |
| BB Mut6 | 13.48 | 2.00 | 5.00 | 27.52 |
| BB Dsp B 1 | 9.80 | 2.00 | 5.00 | 31.20 |

| Sample | Conc. [ng/ul] | Restriktion enzyme | Puffer 2.1 | Water |
| --- | --- | --- | --- | --- |
| BB Dsp B 2 | 16.95 | 2.00 | 5.00 | 24.05 |
| BB Dsp B 3 | 14.88 | 2.00 | 5.00 | 26.12 |
| BB Dsp B 4 | 20.16 | 2.00 | 5.00 | 20.84 |
| Dsp B1 | 11.71 | 2.00 | 5.00 | 29.29 |
| Dsp B2 | 11.93 | 2.00 | 5.00 | 29.07 |

#### Analysis of Restriction Digests

Preparation of a 1% Agarose Gel for restriction analysis of Gibson. 25 µl loaded for the restriction analysis and other samples as well.

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230830-restriktionsverdau-1.png)

#### Setting up Overnight Cultures

Set up overnight cultures from 5 colonies from the AMP1 plate, 5 colonies from the AMP2 plate, and 18 different colonies from the pichia-DspB plate, and streaked onto additional plates.

## Date: Thursday, 31.08.2023

## Experiment Summary

Experiment: Plasmid Isolation and Restriction Digest. Samples sent for sequencing. Quick analysis. pSB1C3 Restriction Digest and Gel Extraction.

**Participants:** Sharkey 09:30 - 15:30, Sumohit 09:30-22:00

### Summary

Plasmid Isolation and Restriction Analysis of AMP1 and AMP2 colonies. Quick analysis of AMP1 and AMP2 colonies. pSB1C3 Restriction Digest and Gel Extraction. Sequencing.

#### Plasmid Isolation

Plasmids were isolated from the overnight cultures according to the protocol. The table below shows the DNA concentrations in the plasmid isolation samples.

| Sample | Conc. [ng/ul] | A260/280 | A260\\230 |
| --- | --- | --- | --- |
| AMP1-1 | 52.6 | 1.94 | 2.3 |
| AMP1-2 | 46.6 | 2.0 | 2.39 |
| AMP1-3 | 8.9 | 1.76 | 2.16 |
| AMP1-4 | 79.7 | 1.98 | 1.99 |
| AMP1-5 | 62.9 | 2.02 | 2.28 |
| Mut-4 | 16.1 | 1.9 | 2.08 |
| Kontrolle | 19.2 | 1.96 | 1.72 |
| AMP2-1 | 45.2 | 2.0 | 2.4 |
| AMP2-2 | 36.5 | 1.94 | 2.14 |
| AMP2-3 | 25.1 | 1.98 | 1.89 |
| AMP2-4 | 58.4 | 2.0 | 2.16 |
| AMP2-5 | 58.9 | 2.02 | 2.34 |

#### Restriction Analysis of Gibson Plasmids with PstI

The AMP samples were digested with PstI for the restriction analysis. 1000 ng of plasmid DNA was taken and digested with 1 µl PstI. If the plasmids are properly cloned, two bands can be seen in all Gibson constructs, and if the cloning was unsuccessful, only one band will be visible. Quick analysis samples for AMP Gibson were also loaded onto the gel. Since the gel ran overnight, 5 µl of all AMP samples were loaded onto a small gel so that some samples can be sent for sequencing.

| Sample | Conc. [ng/ul] | A260/280 | Puffer 2.1 |
| --- | --- | --- | --- |
| AMP1-1 | 52.6 | 1.94 | 2.3 |
| AMP1-2 | 46.6 | 2.0 | 2.39 |
| AMP1-3 | 8.9 | 1.76 | 2.16 |
| AMP1-4 | 79.7 | 1.98 | 1.99 |
| AMP1-5 | 62.9 | 2.02 | 2.28 |
| Mut-4 | 16.1 | 1.9 | 2.08 |
| Kontrolle | 19.2 | 1.96 | 1.72 |
| AMP2-1 | 45.2 | 2.0 | 2.4 |
| AMP2-2 | 36.5 | 1.94 | 2.14 |
| AMP2-3 | 25.1 | 1.98 | 1.89 |
| AMP2-4 | 58.4 | 2.0 | 2.16 |
| AMP2-5 | 58.9 | 2.02 | 2.34 |

#### Analysis of Restriction Digests of Gibson Enzyme Samples

Theoretical Expectations

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230831-restriktionsverdau-erwartung-1.png)

##### Gel Preparation for Restriction Analysis of Gibson

25 µl were loaded for the restriction analysis and other samples in the large gel (Ready on Friday after post-staining). 5 µl were loaded for the restriction analysis in small gel.

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230831-restriktionsverdau-1.png)

#### Quick Analysis

With the latest BioBrick cloning, a quick analysis of the plasmids was performed and loaded onto the gel. Unfortunately, the negative control ran out of the well, and it was decided to repeat the entire quick analysis as the evaluation can be difficult without control. A quick analysis of a colony from pSB1C3 was also performed, serving as a negative control for the plasmid quick analysis.

#### Sequencing

After some samples appeared successful for cloning, a few samples were sent for sequencing.

#### pSB1C3 Restriction and Gel Extraction

Since the amount of pSB1C3 seemed insufficient and more was needed, 500 ng of pSB1C3-BBaI20270 was digested with EcoRI and PstI and loaded onto the gel.

![Gelbild](https://static.igem.wiki/teams/4575/wiki/20230831-restriktionsverdau-psb1c3-1.png)

# September

| Monday | Tuesday | Wednesday | Thursday | Friday | Saturday | Sunday |
| --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  | 1 | 2 | 3 |
| 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| 11 | 12 | 13 | 14 | 15 | 16 | 17 |
| 18 | 19 | 20 | 21 | 22 | 23 | 24 |
| 25 | 26 | 27 | 28 | 29 | 30 |  |

## Date: Friday, 01.09.2023

## Experiment Summary

**Experiment:** Post-staining of Quick Analysis and Restriction Analysis Gel of Gibson Plasmids. Pelleting of Pichia with Dispersin B. Pichia Transformation. Colony PCR of Pichia samples.

**Participants:**Sumohit 09:30-17:30

### Summary

Post-staining with Midori Green of the Quick Analysis and Restriction Analysis Gel of Gibson Plasmids. The Pichia cell cultures were centrifuged and stored as pellets. Transformations were done in Pichia with Mutanase 1:1 Colony 4 and Dextranase 1:1 Colony 2 and 4. Colony PCR of Pichia with Dispersin B.

#### Post-staining of Quick Analysis and Restriction Analysis Gel of Gibson Plasmids

Since the gel ran overnight without Midori Green, the gel was placed in a Midori Green bath in the morning with 200 ml Midori Green solution, containing 40µl of Midori, and shaken for 1 hour. Then the Midori Green was collected and the gel was placed in water twice for 30 minutes each and shaken.

#### Ordering Scheme

**Top**

* pBAD18 (negative control) - mutanase BioBrick new - DspB BioBrick new - mutanase 1:1 Gibson - dispersin B 1:1 Gibson - dextranase 1:1 Gibson - empty - pESP -pREP2

**Bottom**

* Marker - pBAD18 (negative control) - Mutanase BioBrick old - DspB BioBrick old - pESP -pREP2 - Marker

**Top**

#### Pelleting of Pichia-Dispersin B Cultures

Attempts were made to pellet from the Pichia cultures. Unfortunately, it seemed like nothing had grown in the Pichia cultures, and after centrifuging at 14,000 rpm for 5 minutes, no pellet was visible.

#### Pichia Transformation

In the Pichia cells, according to the protocol, Mutanase 1:1 Colony 4 and Dextranase 1:1 Colonies 2 and 4 were transformed. Additionally, a few Pichia cells without transformation were streaked onto a plate, serving as a negative control for the transformation.

#### Colony PCR Pichia

Since the cultures were not growing, colony PCR was performed to confirm whether Dispersin B has integrated into the genome of the cells or is present in the cells. For this, an online protocol was followed where the cells were lysed with 20µl of 20 nM NaOH at 98°C for 45 minutes. The supernatant was collected after centrifugation for PCR. PCR was performed using 1 µl of the supernatant with Mutanase For and Rev Primers for confirmation. Additionally, a PCR with Dispersin B from IDT Eppi was used as a template, serving as a positive control for the PCR itself.

##### Mutanase and Dispersin B PCR Program: Mutanase68

| Step | Temperature [°C] | Dauer [sec] | Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 98 | 30 |  |
| Denaturation | 98 | 8 | 30 |
| Annealing | 68 | 30 | 30 |
| Elongation | 72 | 90 | 30 |
| Final Extension | 72 | 120 |  |
| Hold | 4 | for ever |  |

## Date: Monday, 11.09.2023

## Experiment: Colony-PCR of Pichia Samples. Restriction Analysis of pBAD18-AMP1 and -AMP2. Preparing Overnight Cultures.

**Participants:** Sumohit 09:00-19:30

**Summary:** Colony-PCR of Pichia samples with 45 minutes of heating beforehand. Restriction analysis of pBAD18-AMP1 and -AMP2 with PstI. Setting up ÜN cultures for AMP1, AMP2, Dex, and Mut samples.

### Colony-PCR Samples from Friday

Colony-PCR was performed on the samples from Friday and loaded onto a gel.

**Preparation of 1% Agarose Gel for Colony-PCR of Pichia Dispersin B Cells**

15 µl of the samples were loaded.

**Application Scheme**

*Gel1*

* Marker - White Pichia Colonies - Dsp B Control PCR - Small White Pichia Colonies - Marker

*Gel2*

* Marker - Beige Pichia Colonies - Dsp B Control PCR - Marker - Mutanase digested with SalI - DspB digested with SalI - Dextranase digested with SalI - Marker

### Restriction Analysis of pBAD18-AMP1 and -AMP2 Gibson

Since the restriction analysis from last week with AMP1 and AMP2 did not show many bands and the ones shown were weak, the restriction analysis was repeated.

| Sample | Conc. [ng/ul] | A260/280 | A260/A230 |
| --- | --- | --- | --- |
| AMP1-1 | 52.6 | 1.94 | 2.3 |
| AMP1-2 | 46.6 | 2 | 2.39 |
| AMP1-3 | 8.9 | 1.76 | 2.16 |
| AMP1-4 | 79.7 | 1.98 | 1.99 |
| AMP1-5 | 62.9 | 2.02 | 2.28 |
| Mut-4 | 16.1 | 1.9 | 2.08 |
| Kontrolle | 19.2 | 1.96 | 1.72 |
| AMP2-1 | 45.2 | 2 | 2.4 |
| AMP2-2 | 36.5 | 1.94 | 2.14 |
| AMP2-3 | 25.1 | 1.98 | 1.89 |
| AMP2-4 | 58.4 | 2 | 2.16 |
| AMP2-5 | 58.9 | 2.02 | 2.34 |

**Analysis of Restriction Digestion of Gibson Enzyme Samples**

*Theoretical Expectation*

**Application Scheme:** Marker - PstI with pBAD18-AMP2 - PstI with pBAD18-AMP1 - PstI with pBAD18 LV

**Preparation of 1% Agarose Gel for Restriction Analysis of Gibson**

25 µl applied for restriction analysis

**Application Scheme**

Marker - AMP1-1 - AMP1-2 - AMP1-4 - AMP1-6 - Marker - AMP2-1 - AMP2-2 - AMP2-3 - AMP2-4 - AMP2-5 - Marker

### Overnight Cultures

Overnight cultures were set up for all old AMP1 and AMP2 backup colonies and 3 new colonies each. ÜN was also set up for Dex Colony 2 and Dex Colony 4 1:1 Pichia Gibson twice, and Mut Colony 4 1:1 Gibson.

## Date: Tuesday, 05.09.2023

## Experiment: Colony-PCR of S1 samples. Colony-PCR Disp B Pichia. Plasmid Isolation and Restriction Analysis

**Participants:** Sumohit 08:00-18:30

**Summary:** Progress of Colony-PCR of S1 samples with heating and variations. Colony-PCR of Pichia samples with Dsp B after NaOH lysis. Plasmid isolation. Restriction analysis of Mutanase and Dextranase samples.

### Colony-PCR Trial

Since some bands were observed in the Pichia samples, it was decided to treat the E. coli cells in a similar manner. For the trial, S1 sample colonies were taken, known for correct cloning. For the trial, a colony was picked, dissolved in 20µl 20mM NaOH, and heated at 98°C for 45 minutes. Another colony was taken, dissolved in 20µl ddH2O, and heated at 98°C for 45 minutes. Another colony was dissolved in 20µl 20 Lysozyme and heated at 37°C for 45 minutes. As a control, 1 µl BB41 was dissolved in 20 µl 20mM NaOH to observe if it can damage the DNA. Additionally, a PCR was performed with 1 µl BB41 in water, serving as a positive control for the PCR.

#### AMP PCR Program: AMPOVERH

| Step | Temperature [°C] | Duration [sec] | Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 98 | 30 |  |
| Denaturation | 98 | 10 | 40/45 |
| Annealing | 69 | 30 | 40/45 |
| Elongation | 72 | 18 | 40/45 |
| Final Extension | 72 | 120 |  |
| Hold | 4 | for ever |  |

**Preparation of 1% Agarose Gel for Colony-PCR Trial**

15 µl of samples were loaded.

**Application Scheme**

Marker - Colony in NaOH - Colony in Water - Colony in Lysozyme - DNA in NaOH - AMP Positive Control - Marker

### Colony-PCR Pichia-Dsp B

The colony-PCR from the previous day was repeated since not even the positive control showed the correct band size, and the Colony-PCR trial showed very good results. The cells were lysed again in 20 µl 20 NaOH and used for the PCR. A positive control for the PCR was also prepared.

#### Mutanase and Dispersin B PCR Program

| Step | Temperature [°C] | Duration [sec] | Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 98 | 30 |  |
| Denaturation | 98 | 8 | 30 |
| Annealing | 68 | 30 | 30 |
| Elongation | 72 | 90 | 30 |
| Final Extension | 72 | 120 |  |
| Hold | 4 | for ever |  |

**Preparation of 1% Agarose Gel for Colony-PCR Pichia Dispersin B Cells**

15 µl of samples were loaded.

**Application Scheme**

Marker - Colonies from white and small white Pichia samples - Marker - Dsp B Positive Control - Marker

### Plasmid Isolation

Plasmids were isolated from the ÜN culture following the protocol. The DNA concentrations in the plasmid isolation samples are provided in the table below.

| Sample | Conc. [ng/ul] | A260/280 | A260/A230 |
| --- | --- | --- | --- |
| AMP1-1 | 52.6 | 1.94 | 2.3 |
| AMP1-2 | 46.6 | 2 | 2.39 |
| AMP1-3 | 8.9 | 1.76 | 2.16 |
| AMP1-4 | 79.7 | 1.98 | 1.99 |
| AMP1-5 | 62.9 | 2.02 | 2.28 |
| Mut-4 | 16.1 | 1.9 | 2.08 |
| Kontrolle | 19.2 | 1.96 | 1.72 |
| AMP2-1 | 45.2 | 2 | 2.4 |
| AMP2-2 | 36.5 | 1.94 | 2.14 |
| AMP2-3 | 25.1 | 1.98 | 1.89 |
| AMP2-4 | 58.4 | 2 | 2.16 |
| AMP2-5 | 58.9 | 2.02 | 2.34 |

### Restriction Analysis of Gibson Plasmids (Mutanase; Dispersin B; Dextranase) with EcoRI and PstI

It was suspected that the restriction analysis last week did not work for unknown reasons, so it was repeated with EcoRI and PstI for Mutanase Colony 4, Dextranase Colony 2, and Colony 4. For the restriction analysis, 500 ng of DNA was used with 1µl of restriction enzyme each.

**Preparation of 1% Agarose Gel for Restriction Analysis of Gibson**

25 µl were loaded for the restriction analysis.

**Application Scheme**

Marker - PstI with Pichia vector-Mutanase - PstI with Pichia vector-Dispersin B - PstI with Pichia vector-Dextranase - PstI with Pichia vector

**Preparation of 1% Agarose Gel for Restriction Analysis of Gibson**

25 µl were loaded for the restriction analysis.

**Application Scheme**

Marker - PstI with Pichia vector-Mutanase I - PstI with Pichia vector-Mutanase II - PstI with Pichia vector-Dispersin B I - PstI with Pichia vector-Dispersin B II - PstI with Pichia vector-Dextranase I - PstI with Pichia vector-Dextranase II - Marker

Unfortunately, there is no image. Only the His4 band was visible again along with the enzyme fragment band and the backbone band. Ideally, this band should be seen as two bands, one for the backbone and one for the enzyme fragment in the gel.

## Date: Wednesday, 06.09.2023

## Experiment: Colony-PCR of AMP1 and AMP2 samples; Colony-PCR of Mutanase and Dispersin B; Gibson Assembly; Media

**Participants:** Sarah 13:00-15:00; Sumohit 09:00-19:30

**Summary:** Colony-PCR of AMP1 and AMP2 samples. Colony-PCR of Mutanase and Dispersin B samples. Gibson Assembly of enzyme fragments in Pichia vector. Preparation of YPD medium.

### Colony-PCR of Mutanase and Dispersin B samples for Pichia

Colony-PCR was performed for Pichia vector-Mutanase colony 4 and Dispersin B colonies 1 and 2. The Colony-PCR was conducted as per the protocol. Additionally, a positive control was included for each, with 1 µl from the respective fragment stock.

#### Mutanase and Dispersin B PCR Program

| Step | Temperature [°C] | Dauer [sec] | Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 98 | 30 |  |
| Denaturation | 98 | 8 | 30 |
| Annealing | 68 | 30 | 30 |
| Elongation | 72 | 90 | 30 |
| Final Extension | 72 | 120 |  |
| Hold | 4 | for ever |  |

Colony-PCR of AMP1 and AMP2 samples

Colony-PCR was performed for pBAD18-AMP1 and -AMP2. The Colony-PCR was conducted as per the protocol. Additionally, a positive control was included for each, with 1 µl from the respective fragment stock.

#### PCR programm for AMP1

| Step | Temperature [°C] | Duration [sec] | Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 98 | 30 |  |
| Denaturation | 98 | 10 | 40/45 |
| Annealing | 69 | 30 | 40/45 |
| Elongation | 72 | 18 | 40/45 |
| Final Extension | 72 | 120 |  |
| Hold | 4 | for ever |  |

#### PCR programm for AMP2

| Step | Temperature [°C] | Duration [sec] | Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 98 | 30 |  |
| Denaturation | 98 | 10 | 40/45 |
| Annealing | 69 | 30 | 40/45 |
| Elongation | 72 | 40 | 40/45 |
| Final Extension | 72 | 120 |  |
| Hold | 4 | for ever |  |

### Preparation of 1% Agarose Gel for Colony-PCR AMP1 Colonies

15 µl were loaded for the samples.

**Application Scheme - Gel 1**

AMP1-1 - AMP1-2 - AMP1-3 - AMP1-4 - AMP1-5 - AMP1-7 - Positive Control

**Application Scheme - Gel 2**

Marker - AMP1-1 - AMP1-2 - AMP1-3 - AMP1-4 - AMP1-5 - AMP1-7 - Positive Control - Marker

**Application Scheme - Gel 3**

Marker - AMP2-1 - AMP2-2 - AMP2-3 - AMP2-4 - AMP2-5 - AMP2-6 - AMP2-7 - AMP2-8 - Marker - Mutanase Colony 4 - Mutanase + Control - Dispersin B Colony 1 - Dispersin B Colony 2 - Dispersin B + Control - Marker - AMP2 + Control (200 µl PCR Tube) - AMP2 + Control (500 µl PCR Tube) - AMP2 DNA in 20 µl 20 NaOH - Marker

### Gibson Assembly

Since there was suspicion of errors in the cloning of enzyme fragments into the Pichia vector, the 1:1 vector:fragment Gibson Assembly was repeated. The required volumes are shown in the table below and were followed as per the protocol.

| Sample | Amount for Gibson [ng] | Sample Volume [µl] | Water [µl] | NEBuilder |
| --- | --- | --- | --- | --- |
| Mut | 110 | 1 | 6.17 | 10 |
| Dsp B | 103.9 | 1.43 | 5.74 | 10 |
| Dex1 | 82.4 | 0.41 | 6.22 | 10 |
| Dex2 | 69.0 | 0.54 |  |  |
| HIs4 | 98.6 | 2.17 |  |  |
| pichia Vektor | 75 | 0.66 |  |  |

### Transformation of Empty Vectors and Gibson Constructs

The DNA constructs from the Gibson Assembly were transformed into E. coli cells and plated on respective antibiotic-containing plates. pBAD18 was used as a positive control for transformation and plated on Amp-containing plates. Cells without plasmids were treated as a blank during transformation, plated on Amp-containing plates, and served as a negative control for transformation. The transformation was conducted as per the protocol.

### Media

500 ml of YPD medium was prepared twice.

## Date: Thursday, 07.09.2023

## Experiment: Colony-PCR AMP1 and AMP2 Samples; Sequencing; Colonies Plating; Colony-PCR for Enzyme Fragments and Peptide Fragments; Genomic Isolation; Restriction Digestion of AMP Samples; GFP and AMP2 Expression

**Participants:** Sumohit 09:00-22:30

**Summary:** Gel for Colony-PCR for AMP1 and AMP2. Sending samples for sequencing. Plating colonies on backup plates. Colony-PCR for pichia vector-Mutanase, -Dextranase, -Dispersin B. Preparation for genomic isolation and setting up ÜN cultures. Restriction digestion of AMP samples. GFP and AMP2 expression.

### Colony-PCR for Pichia Vector-Dextranase old Gibson Assembly

Colony PCR was performed for Dextranase Colony 2 and Colony 4 from the old Gibson Assembly for the enzyme fragments in the Pichia vector. The samples were loaded on the AMP gel.

#### Linkage PCR for Dextranase (Dex)

| Step | Temperature [°C] | Duration [sec] | Cycles |
| --- | --- | --- | --- |
| 1 | Initial Denaturation | 98 °C | 30 s |
| 2 | Denaturation | 98 °C | 8 s (30 cycles) |
| 3 | Annealing | 68 °C | 30 s (30 cycles) |
| 4 | Elongation | 72 °C | 150 s (30 cycles) |
| 6 | Final Extension | 72 °C | 3 min |
| 7 | Hold | 4 °C | Continuous |

### Gel for Colony PCR AMP1 and AMP2

As the bands in the gels for AMP1 and AMP2 were too smeary, we discussed this issue with MiBi and they mentioned that this happens when GelRed is used in the gel. In MiBi, they do post-staining with GelRed, and the same was done here.

**Preparation of 1% Agarose Gel for Colony-PCR AMP Colonies and Dextranase Colony from old Gibson Assembly**

15 µl were loaded for the samples.

**Application Scheme - Gel**

* Marker - AMP1-1- AMP1-6 - AMP1-7 - AMP1-8 - AMP1 + Control - Marker - AMP2-3 - AMP2-6 - AMP2-7 - AMP2-8 - Positive Control - Marker - Dextranase Colony 2 - Dextranase Colony 4

The gel ran overnight at 15 V.

### Sequencing

Samples AMP1-1; AMP1-6; AMP1-7; AMP2-6; AMP2-7; and AMP2-8 were sent for sequencing using AMP forward and reverse primers. All samples look correct except AMP2-8, which, according to both primers, has a mismatch in GFP resulting in a sequence change.

### Examining Plates with Gibson Assembly from Wednesday

Images were forgotten to be taken. The plates are there and will be done later. Colonies have grown everywhere as expected except in the negative control.

### Colonies Plating

To avoid waiting for long, 12 colonies from each pichia enzyme fragment were plated on a backup plate and incubated at 37°C for 6 hours until enough cell material is available to perform a colony-PCR.

### Colony PCR with Pichia Vector-Enzyme Fragment new Gibson Assembly

The cells from the previously plated colonies were used to perform colony PCR. The cells were dissolved in 20µl 20 mM NaOH and heated at 98°C for 45 minutes. After that...

### Preparation for Genomic Isolation and overnight Cultures

5.99 g of lithium acetate was procured by Rebekka and a 200 mM lithium acetate solution in water (final volume 50 ml) was prepared. ÜN cultures from Biege 1 to 4 of the pichia cells with Dsp B were made in the self-made YPD medium and incubated overnight at 37°C.

### Restriction Digestion of pBAD18-AMP1 and -AMP2 Gibson

Since the restriction analysis last week with AMP1 and AMP2 did not show many bands and the ones shown were weak, the restriction analysis was repeated. Here, 1000 ng of DNA was used and after the restriction digestion, the samples will be purified so that...

| Sample | Amount [ng] | Restriktion enzyme | Puffer 2.1 | Water |
| --- | --- | --- | --- | --- |
| AMP1-1 | 52.6 | 1.94 | 2.3 |  |
| AMP1-2 | 46.6 | 2 | 2.39 |  |
| AMP1-3 | 8.9 | 1.76 | 2.16 |  |
| AMP1-4 | 79.7 | 1.98 | 1.99 |  |
| AMP1-5 | 62.9 | 2.02 | 2.28 |  |
| Mut-4 | 16.1 | 1.9 | 2.08 |  |
| Kontrolle | 19.2 | 1.96 | 1.72 |  |
| AMP2-1 | 45.2 | 2 | 2.4 |  |
| AMP2-2 | 36.5 | 1.94 | 2.14 |  |
| AMP2-3 | 25.1 | 1.98 | 1.89 |  |
| AMP2-4 | 58.4 | 2 | 2.16 |  |
| AMP2-5 | 58.9 | 2.02 | 2.34 |  |

### GFP and AMP2 Expression

After the colony PCR and sequencing provided the desired results, 10 ml TB medium with AMP2 colonies (AMP2-3, AMP2-6, AMP2-7, AMP2-8) was inoculated. Due to time constraints, the cells were induced with 100 µl 2% arabinose at OD600 0.257; 0.452; 0.289; and 0.313 and incubated further at 37°C overnight.

## Date: Friday, 08.09.2023

## Experiment: Fluorescence Microscopy; Genomic Isolation; Purification of Restriction Digestion; Post-staining; Preparation for AMP Expression; Preparation for Genomic Integration; Colony-PCR; PCR

**Participants:** Sarah 09:30-17:00; Sumohit 10:00-19:00

**Summary:** Fluorescence microscopy of expression samples; Genomic isolation of pichia cells with DspB; Purification of AMP1 and AMP2 restriction digestion samples; Post-staining; Preparation for AMP expression; Preparation for genomic integration; Colony-PCR of Mutanase, Dispersin B, and Dextranase; PCR of AMP2 samples for sequencing

### Post-staining

A 3% GelRed solution was prepared, and the gel from the previous day was immersed in 200 µl GelRed solution and incubated on the shaker for 20 minutes. Afterward, the gel was washed twice with water for 20 minutes and analyzed.

**Application Scheme - Gel**

* Marker - AMP1-1- AMP1-6 - AMP1-7 - AMP1-8 - AMP1 + Control - Marker - AMP2-3 - AMP2-6 - AMP2-7 - AMP2-8 - Positive Control - Marker - Dextranase Colony 2 - Dextranase Colony 4

### Fluorescence Microscopy

All AMP samples showed a GFP signal, including the AMP2-3 sample, although no signal was expected here. The saline control showed no GFP signal.

Images were taken, but it's difficult to see much in the pictures.

### Genomic DNA Isolation from Pichia Cells

The genomic DNA from the ÜN culture samples from the previous day was isolated and stored as per the protocol by iGEM Team tjusls China 2022.

Lithium acetate was dissolved in water instead of acetic acid as found in all relevant papers.

### Purification of AMP Restriction Samples

The AMP restriction samples from the previous day were purified using the Zymo Kit, as recommended by the manufacturer. The concentrations are listed in the table below.

### Preparation for AMP Expression

Flasks were autoclaved, and the shaker in the MiBi laboratory was checked for functionality.

### Colony PCR of Mutanase and Dispersin B

PCR was performed for Mutanase and Dispersin B using the lysed cells from Thursday. A 25 µl reaction was prepared with 1µL from the lysate. After PCR, the Mutanase samples were loaded on the gel. Additionally, a positive control for the PCR was made and loaded.

| Step | Temperature [°C] | Duration [sec] | Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 98 | 30 |  |
| Denaturation | 98 | 8 | 30 |
| Annealing | 68 | 30 | 30 |
| Elongation | 72 | 90 | 30 |
| Final Extension | 72 | 120 |  |
| Hold | 4 | for ever |  |

After gel electrophoresis, the gel was restained with GelRed and analyzed.

### PCR of AMP2 Samples for Sequencing

Since the data from the AMP2-6 and AMP2-7 sequencing with the AMP Forward Primer did not arrive and there was not much plasmid available, a PCR was performed with AMP2-6 and AMP2-7. The samples were purified with the PCR purification kit and sent for sequencing with the AMP Forward Primer.

| Step | Temperature [°C] | Dauer [sec] | Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 98 | 30 |  |
| Denaturation | 98 | 10 | 30 |
| Annealing | 69 | 30 | 30 |
| Elongation | 72 | 40 | 30 |
| Final Extension | 72 | 120 |  |
| Hold | 4 | for ever |  |

| Sample | Concentration [ng/ul] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| AMP2-6 | 45.9 | 1.90 | 2.46 |
| AMP2-7 | 51.2 | 1.84 | 2.4 |

## Date: Monday, 11.09.2023

**Participants:**Participants: Sarah H.

## Summary: Making of TB Medium. Restriktion digestion. Overbnight culture.

**Preparation of 3L TB Medium:**

Following the protocol: [TB Medium Protocol](https://static.igem.org/mediawiki/2019/9/9d/T--TU_Kaiserslautern--TB-Medium.pdf)

**Autoclaving of TB Media**

**Preparation of KPO4 Buffer (not autoclaved)**

**Drying Oven**

Dry items were removed from the drying oven, wet items were left, and the oven was heated again.

**SalI-HF Restriction Digestion for Dex Colony 2 and 4, as well as Mut Colony 4:**

* 500 ng per plasmid
* 10x Cut Smart
* SalIHF (20 U/ul) --> 0.5 ul
* ddH2O

| Component | Volume [ul] | Comments |
| --- | --- | --- |
| Plasmid | Dex2-1: 6.7 ; Dex2-2: 11.6 ; Dex4-1: 11.7 ; Dex4-2: 6.8; Mut4-1: 11.23 ; Mut4-2: 13.7 |  |
| Cut Smart | 2.5 |  |
| SalIHF | 0.5 |  |
| ddH2O | Dex2-1: 15.3 ; Dex2-2: 10.4 ; Dex4-1: 10.3 ; Dex4-2: 15.2; Mut4-1: 10.8 ; Mut4-2: 8.3 |  |

10 min digestion at 37°C, followed by 20 min heat inactivation at 65°C.

**ÜN Cultures of AMP Colonies:** AMP2-6, AMP2-8, AMP1-6, and AMP1-7

5 ml TB Medium with salt + 5 ul Amp (100mg/ml)

## Date: Tuesday, 12.09.2023

**Participants:** Sarah (09:30-17:00), Sumohit (08:00-20:30)

### AMP Expression

After detecting GFP in the AMP2 samples, it was decided to start the expression of samples AMP1-6, -7, and AMP2-6, -7. For expression, 1 ml of the overnight culture was taken and added to 100 ml of TB medium. The cells were incubated at 30°C on a shaker, continuously shaking. During expression, the optical density (OD600) of the samples was continuously measured. At an OD600 of 0.4-0.6, the expression was induced by adding 1 ml of 2% arabinose solution. Expression was started at 08:30 AM, induced at 12:45 PM, and ended at 08:00 PM.

### Gibson Assembly of Enzyme Fragments in pichia Vector

Was performed as per the protocol. Gibson Assembly was performed in the pichia vector with Mutanase, Dispersin B, and Dextranase. Additionally, Gibson Assembly was performed in pBAD18 with Mutanase and Dispersin B. The required volumes are listed in the table below. Since the 1:1 Gibson cloning did not yield the desired results, a 1:2 vector-to-fragment Gibson Assembly was performed for both pichia vector and pBAD18.

| Sample | Amount for Gibson [ng] | Volume Sample [µl] | Water | NEBuilder |
| --- | --- | --- | --- | --- |
| Mut | 220 | 2 | 5.27 | 10 |
| Dsp B | 207.8 | 2.86 | 4.41 | 10 |
| Dex1 | 164.8 | 0.84 | 5.35 | 10 |
| Dex2 | 138.0 | 1.08 |  |  |
| HIs4 | 197.2 | 2.07 |  |  |
| pichia Vektor | 75 | 0.66 |  |  |

| Sample | Amount for Gibson [ng] | Volume Sample [µl] | Water | NEBuilder |
| --- | --- | --- | --- | --- |
| Mut | 110.5 | 1.44 | 6.67 | 10 |
| Dsp B | 101.9 | 1.31 | 6.8 | 10 |
| HIs4 | 98.2 | 1.09 |  |  |
| pBAD18 Vektor | 75 | 0.80 |  |  |

### Transformation of Empty Vectors and Gibson Constructs for pichia Gibson

Only the DNA constructs from the Gibson Assembly for pichia Gibson were transformed into E. coli cells and plated on the corresponding antibiotic-containing plates. Due to a shortage of LB-agar plates with Amp, not enough were available for all samples. pBAD18 was used as a positive control for transformation and plated on Amp-containing plates. Cells without plasmids were treated as blanks in the transformation, plated on Amp-containing plates, and used as a negative control for the transformation. The transformation was performed as per the protocol.

### Preparation for Improvement of Existing Part

Over the past years, many teams have successfully worked with Dispersin B. Therefore, it was decided to search for plasmids in the iGEM distribution kit containing Dispersin B. In the iGEM distribution kit 2021, on Plate 5, three fragments with Dispersin B were identified, found at positions 15D, 15F, and 17D. These fragments were resuspended from the corresponding wells and transformed into E. coli. For the transformation, 1 µl of the solution was taken and transformed into the cells. The transformed solution was then plated on CMR-containing agar plates. The transformation was performed as per the protocol.

| Component | Volume [ul] | 14 x MM |
| --- | --- | --- |
| Q5 2xMM | 12.5 | 175 |
| Mut fwd Primer | 1.25 | 17.5 |
| His4 rev neu Primer | 1.25 | 17.5 |
| Template | 1 | - |
| ddH2O | 9 | 126 |

| Step | Temperature [°C] | Dauer [sec] | Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 98 | 30 |  |
| Denaturation | 98 | 8 | 30 |
| Annealing | 68 | 30 | 30 |
| Elongation | 72 | Dex: 165; Mut: 140; DispB: 140 | 30 |
| Final Extension | 72 | Mut und Disp: 120; Dex: 300 |  |
| Hold | 4 | forever |  |

### Gel Preparation for Colony PCRs and SalI-HF Restriction Digest (Mo)

1% agarose gel was prepared for Colony PCRs and SalI-HF restriction digest.

**Gel Details:**

* **Concentration:** 1%
* **Voltage:** 10V
* **Duration:** 16 hours
* **Stain Used:** Gel-Red

**Sample Order on the Gel:**

Mut2 - Mut4 - Mut7 - Mut9 - DispB2 - DispB5 - DispB8 - DispB10 - M - Dex2 - Dex3 - Dex5 - Dex10 - RVDex2-1

## Date: Wednesday, 13.09.2023

**Participants:** Jessica, Sarah H.

#### Post-staining of Colony PCR Gel from Tuesday

Sample Order: M-Mut2-Mut4-Mut7-Mut9-DispB2-DispB5-DispB8-DispB10-M-Dex2-Dex3-Dex5-Dex10-RVDex2-1

![Gel Image](image.png)

#### Centrifugation and Resuspension in Saline of E. coli Cells with Amp1 and Amp2

E. coli cultures were centrifuged at 5000g for 20 minutes and resuspended in 3ml of 0.9% saline. The samples included Amp1-6, Amp1-7, Amp2-6, Amp2-7, and 0.9% saline.

![E.coli in Saline under UV light](20230913_E.coli mit AMP1 un AMP2 in Saline unter UV Licht2.jpg)

The E. coli solutions were stored in the -20°C freezer.

#### Sequencing of Some Minipreps from August 23rd with the Correct Sequencing Primer

1:2 dilution of Enzyme-His4 Sequencing Primer was prepared. 5 ul sample was mixed with 5 ul of the primer (1:2 dilution).

| Name | Seq ID |
| --- | --- |
| Disp 1:1 1 | DEW028 |
| Disp1 | DEW018 |
| Disp3 | DEW017 |
| Mut3 | DEW022 |
| Dex 1:1 3 | DEW021 |
| Mut 1:1 2 | DEW020 |
| Disp 1:1 3 | DEW025 |
| Dex 1:1 2 | DEW024 |

#### Restriction Digestion with SalIHF of Mut and Dex

5 µL input, post-staining with GelRed. Order: Marker, Mut 4-1, Mut 4-2, Dex 2-2, Dex 4-1, Dex 4-2, Marker, Mut 4-2, Dex 4-2, Dex 2-1

![Digestion Image](image.png)

#### Colony PCR with M13 reverse (21nt) and Enzym-His4 fwd Primer

| A | Volume [ul] | 14 x MM |
| --- | --- | --- |
| Q5 2xMM | 12.5 | 175 |
| Enzym-His4 fwd Sequencing-Primer | 1.25 | 17.5 |
| M13 rev (21nt) Primer | 1.25 | 17.5 |
| Template | 1 | - |
| ddH2O | 9 | 126 |

| Step | Temperature [°C] | Dauer [sec] | Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 98 | 30 |  |
| Denaturation | 98 | 8 | 30 |
| Annealing | 62 | 30 | 30 |
| Elongation | 72 | 165 | 30 |
| Final Extension | 72 | 300 |  |
| Hold | 4 | for ever |  |

Sample Order: M-Mut2-Mut3-Mut4-Mut8-Disp2-Disp3-Disp8-Disp10-M-Dex2-Dex3-Dex14

![PCR Image](IMG-20230913-WA0011.jpg)

Expected Length: 2702 bp

#### Checking Biobrick and Gibson Assembly Plates

##### Gibson Assembly

##### Biobrick Assembly

Biobrick plates are still in the incubator, all others are in the refrigerator.

#### ÜN Culture of E. Coli BW...

5ml TB medium with salts. E. coli picked from a plate without antibiotics. Incubation at 37°C.

## Date: Thursday, 14.09.2023

### Experiment Summary: Colony-PCR, GFP Analysis in AMP2, PCR Tests, Media Preparation, Gibson Assembly

**Participants:** Sumohit 09:30-21:30

Summary: Colony-PCR with Gibson Assembly of enzyme fragments in pichia vector. GFP investigation in AMP2 samples. PCR with irradiation primers. Media preparation. Gibson Assembly for pBAD18 vector.

#### Colony-PCR with Gibson Assembly of enzyme fragments in pichia vector

Colony Picking: To expedite the process, 6 colonies of each pichia enzyme fragment were streaked onto a backup plate and incubated at 37°C for 8 hours to obtain sufficient cell material for Colony-PCR. After the 8-hour incubation, cell lysis was performed.

#### Colony PCR with pichia vector enzyme fragment for new Gibson Assembly

Cells from the streaked colonies were used to perform Colony PCR. The cells were dissolved in 20µl of mM NaOH and heated at 98°C for 45 minutes. Due to insufficient PCR block availability, PCR was not performed and everything was stored at -20°C.

#### GFP Analysis in AMP2 Samples

After expression, the samples (AMP2-6 and -7) were examined under a fluorescence microscope to check for the presence of GFP. For this, 2 µl of cell suspension from AMP2-6 and -7 samples, as well as 2 µl of saline, were applied to a slide and examined under the microscope.

A GFP-like signal was observed in the AMP2 samples, and there was no signal in the saline Sample. Unfortunately, good images could not be captured, but Caro also observed a GFP-like signal in the AMP2 samples.

#### Colony PCR with Irradiation Primers

Due to uncertainty about the functionality of Colony PCR using highly redundant primers, it was decided to use some older primers that attach to the Ori. For the test PCR, AMP2-6 was chosen as a positive control for successful cloning, pBAD18 as a positive control, and the samples Mutanase Colony 3, 4, 7, Dispersin B Colony 10, and Dextranase Colony 5. Different PCR runs were performed due to the different sizes of the plasmids. Additionally, a PCR was conducted with Mutanase Colony 3 using AMP primers, suspecting that this colony might represent a successful cloning.

#### PCR Programs for Various Samples

Program for AMP2-6 and pBAD18:

| Step | Temperature [°C] | Dauer [sec] | Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 98 | 30 |  |
| Denaturation | 98 | 8 | 30 |
| Annealing | 66 | 30 | 30 |
| Elongation | 72 | 150 | 30 |
| Final Extension | 72 | 180 |  |
| Hold | 4 | for ever |  |

Program for Mut Colony 3:

| Step | Temperature [°C] | Dauer [sec] | Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 98 | 30 |  |
| Denaturation | 98 | 8 | 30 |
| Annealing | 66 | 30 | 30 |
| Elongation | 72 | 90 | 30 |
| Final Extension | 72 | 120 |  |
| Hold | 4 | for ever |  |

Program for Mutanase and Dispersin B:

| Step | Temperature [°C] | Dauer [sec] | Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 98 | 30 |  |
| Denaturation | 98 | 8 | 30 |
| Annealing | 66 | 30 | 30 |
| Elongation | 72 | 240 | 30 |
| Final Extension | 72 | 420 |  |
| Hold | 4 | for ever |  |

Program for Dextranase Colony 5:

| Step | Temperature [°C] | Dauer [sec] | Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 98 | 30 |  |
| Denaturation | 98 | 8 | 30 |
| Annealing | 66 | 30 | 30 |
| Elongation | 72 | 270 | 30 |
| Final Extension | 72 | 480 |  |
| Hold | 4 | for ever |  |

#### Gel Electrophoresis

Following PCR, the samples were loaded onto a 1% agarose gel and left to run overnight.

#### Sample Loading Schema

Marker - AMP2-6 - pBAD18 - Dextranase PCR-Colony - Mutanase PCR-Colony - Dispersin B PCR-Colony - Dextranase PCR-Colony - Mutanase Colony 3 (AMP) - Marker

#### Media Preparation

LB-Agar medium was prepared for Amp-resistant plates following the protocol.

#### Transformation of pBAD18-Mut, pBAD18-Disp B, and Empty Vector

Since previously only the DNA constructs from the Gibson Assembly for pichia Gibson were transformed into E. coli cells and plated on the appropriate antibiotic plates, and there were too few LB-Agar plates with Amp, not sufficient for all samples, the Gibson Assembly of enzyme fragments into pBAD18 vector was transformed into E. coli. pBAD18 was used as a positive control for the transformation and plated on Amp-containing plates. Cells without plasmids were treated as a blank during transformation, plated on Amp-containing plates, and used as a negative control for the transformation. The transformation was performed according to the protocol.

## Date: Friday, 15.09.2023

**Participants:** Jessica, Sarah H., Sumohit

### Colony PCRs of New Gibson Assemblies with Mut Fwd and His4 New Rev, and Enzym-His4 Sequencing Fwd and M3 (21nt) Rev

#### PCR Setup for Enzym-His4 Sequencing Fwd and M13 Rev (21nt) Primer

| A | Volume [ul] | 8 x MM |
| --- | --- | --- |
| Q5 2xMM | 12.5 | 100 |
| Enzym-His4 fwd Sequencing-Primer | 1.25 | 10 |
| M13 rev (21nt) Primer | 1.25 | 10 |
| Template | 1 | - |
| ddH2O | 9 | 72 |

PCR Program:

| Step | Temperature [°C] | Dauer [sec] | Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 98 | 30 |  |
| Denaturation | 98 | 8 | 30 |
| Annealing | 62 | 30 | 30 |
| Elongation | 72 | 115 | 30 |
| Final Extension | 72 | 300 |  |
| Hold | 4 | for ever |  |

#### PCR Setup for Mut Fwd and His4 New Rev Primer

| A | Volume [ul] | 8 x MM |
| --- | --- | --- |
| Q5 2xMM | 12.5 | 100 |
| Mut fwd Primer | 1.25 | 10 |
| His4 rev neu Primer | 1.25 | 10 |
| Template | 1 | - |
| ddH2O | 9 | 72 |

PCR Program:

| Step | Temperature [°C] | Dauer [sec] | Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 98 | 30 |  |
| Denaturation | 98 | 8 | 30 |
| Annealing | 68 | 30 | 30 |
| Elongation | 72 | 165 | 30 |
| Final Extension | 72 | 300 |  |
| Hold | 4 | for ever |  |

#### Linkage PCR: His4 with Dispersin B and His4 with Mutanase

| A | Volume [ul] | MM |
| --- | --- | --- |
| Q5 2xMM | 25 |  |
| Mut fwd Primer | 2.5 |  |
| His4 rev neu Primer | 2.5 |  |
| Templates (His4 und Mut oder DispB) | Je 3ng |  |
| ddH2O | x |  |

PCR Program:

| Step | Temperature [°C] | Dauer [sec] | Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 98 | 30 |  |
| Denaturation | 98 | 8 | 30 |
| Annealing | 67 | 30 | 30 |
| Elongation | 72 | 140 | 30 |
| Final Extension | 72 | 300 |  |
| Hold | 4 | for ever |  |

#### Maintenance

* Liquid waste autoclaved
* Equipment cleaned

#### Cell Lysis

The cells in AMP2-6 and AMP1-7 samples were lysed using ultrasound. After lysis, the cells were centrifuged at 14000 rpm for 5 minutes at 4°C, and the supernatant was collected.

#### Post-Staining Gel for Gibson Plasmids

Since the gel ran overnight without GelRed, in the morning the gel was placed in a GelRed bath with 200 ml GelRed solution containing 60µl Midori, and shaken for 20 minutes. Then GelRed was collected, and the gel was placed in water twice for 30 minutes each, shaking in between.

#### Sample Loading Schema

Marker - AMP2-6 - pBAD18 - Dextranase PCR-Colony - Mutanase PCR-Colony - Dispersin B PCR-Colony - Dextranase PCR-Colony - Mutanase Colony 3 (AMP) - Marker

#### GFP Analysis in AMP2 Samples

After cell lysis, the supernatant from AMP2-6 and AMP1-7 samples was examined under a fluorescence microscope to check for the presence of GFP. For this, 2 µl of cell suspension from AMP2-6 and AMP1-7 samples, as well as 2 µl of saline, were applied to a slide and examined under the microscope.

A GFP-like signal was observed in the AMP2 samples, and there was no signal in the saline Sample. Unfortunately, good images could not be captured.

## Date: Monday, 18.09.2023

### Experiment: Improvement of Existing Part; Colony PCR pBAD18-Mut/pBAD18-DspB; Competent Cells Preparation

**Participants:**Jessica, Sumohit (16:30-20:00)

#### Summary

Transformation for Improvement of Existing Part. Cell lysis of pBAD18-Mut/-DspB samples. Preparation of competent cells culture.

#### Media

LB-Agar medium was prepared for CMR-containing plates.

Prepared as per the protocol.

#### Transformation of Improvement Plasmids and Empty Vector

As the transformation didn't work well last time and very few cells were visible in the positive control, the procedure was repeated for sample 15D as per the protocol. pBAD18 plasmid was used as the positive control.

#### Colony PCR of pBAD18-Mut/-DspB Samples

A small amount of cells was taken from the plates streaked on Friday and dissolved in 20 µl 20 mM NaOH. The mixture was incubated for 45 minutes at 98°C. After incubation, the samples were centrifuged, the supernatant was collected, and stored.

#### Competent Cells Preparation

LB Medium (ÜN) was prepared from BW25113 to create competent cells.

## Date: Tuesday, 19.09.2023

### Experiment: Competent Cells; Transformation; Colony PCR Progress

**Participants:** (10:00 - 16:00), Sumohit (09:30 - 18:00)

#### Summary

Competent BW25113 cells; Transformation of AMP constructs; Progress of Colony-PCR testing.

#### Competent Cells

The BW25113 cells were made competent as per the provided protocol and stored at -70°C.

#### Transformation of AMP Constructs and Empty Vector into Competent BW25113 Cells

AMP1 Colony 7 and AMP2 Colony 6 were transformed into competent BW25113 cells following the provided protocol. pBAD18 plasmid was used as a positive control.

#### Images

#### Colony-PCR Progress

Since the previous PCRs didn't work well, new primers that attach to the AOX promoter and AOX terminator were obtained and tested. For the test, Mutanase and Dispersin from the stock were used as DNA templates.

#### Mutanase and Dispersin B

PCR Program: AOX

PCR Machine: iGEM

| Step | Temperature [°C] | Duration [sec] | Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 98 | 30 | - |
| Denaturation | 98 | 8 | 30 |
| Annealing | 66 | 30 | 30 |
| Elongation | 72 | 120 | 30 |
| Final Extension | 72 | 150 | - |

#### Preparation of a 1.0% Agarose Gel for Colony-PCR and Mutanase, Dispersin B, Dex1, Dex2, and pichia Vector

Complete 10 µl were loaded from the PCR for Gibson Assembly and 25 µl from the Colony-PCR samples.

#### Sample Layout

Marker – Mut AOX Test - DspB AOX Test - Marker

![Screenshot](Screenshot.png)

## Date: Wednesday, 20.09.2023

### Experiment: Expression; Colony PCR pBAD18-Mut; Colony-PCR pBAD18-Mut/pBAD18-DspB; PCR for Genomic DNA Integration; PCR Purification and Sequencing; SalI Restriction Digest of His4 Fragments; Overnight Culture; 50x TAE Buffer

**Participants:**Sarah (09:30-18:00), Sumohit (06:00-22:00)

#### Summary

Expression of AMP2 and AMP1; Colony-PCR of pBAD18-Mut; PCR for genomic DNA integration; SalI restriction digest of His4 fragments. Overnight culture of Mutanase-Pichia vector colony 1 and pBAD-Dispersin B colony 5 for minipreps. Preparation of 50x TAE buffer.

#### Expression of AMPs

An expression was performed using AMP2 Colony 6, AMP1 Colony 7, wild-type BW25113 strain, and pSB1C3-GFP-BW25113. One colony was taken and added to 500 ml TB-medium for expression. The cells were incubated at 30°C on a shaker, shaking continuously. During expression, the optical density (OD600) of the samples was continuously measured. Expression was induced by adding 5 ml of 2% arabinose solution at an OD600 of 0.4-0.6. Expression was initiated at 07:00, induced at 13:30, and ended at 21:30.

![Image](image.png)

## Date: Thursday, 21.09.2023

### Experiment: Expression; Harvest Cells; Colony PCR; Overnight Culture; Media; Fluorescence

**Participants:** (09:30-17:00), Sumohit (09:30-19:00)

#### Summary:

#### GFP Examination

After expression, the samples were examined for GFP in TECAN. Unfortunately, there was no or hardly any difference observed in the fluorescence of AMP2 samples compared to wildtype GFP after normalization.

![Screenshot 1](Screenshot1.png)

Above are non-frozen samples and below are frozen samples.

#### Cell Harvesting

The cells were harvested after expression.

#### Colony PCR pBAD18-Mut

Since the gel from the previous day did not work well, gel electrophoresis was repeated.

#### Preparation of a 1.0% Agarose Gel for Colony-PCR

10 µl of the Colony-PCR samples

#### Sample Layout

Marker – Colony PCR Mut - Mut AOX Control - Marker - gDNA - Marker

![Screenshot 2](Screenshot2.png)

#### Maintenance

Preparation of 1L TB Medium

Performed as per the protocol

Autoclaving the TB Media

![210923SalI](210923SalI.png)

Correct bands but much weaker than expected.

#### His4 Gibson Overhang PCR to Generate New Gibson Material

| A | Volume [ul] |
| --- | --- |
| Q5 2xMM | 25 |
| fwd Primer new | 2.5 |
| rev Primer new | 2.5 |
| Template | 1 |
| ddH2O | 19 |

#### PCR Program: His4over

| Step | Temperature [°C] | Duration [sec] | Cycles |
| --- | --- | --- | --- |
| 1 | Initial Denaturation | 98 | 30 s |
| 2 | Denaturation | 98 | 8 s | 35 cycles |
| 3 | Annealing | 66 | 30 s | 35 cycles |
| 4 | Elongation | 72 | 80 s | 35 cycles |
| 5 | Final Extension | 72 | 2 min |
| 6 | Hold | 4 °C |

![210923His](210923His.png)

Correct bands.

#### Miniprep of Mut-Vector Colony 1 and DispersinB-pBAD Colony 5

Miniprep was performed using the plasmid isolation kit from Analytik Jena.

Elution in 55 ul.

| Sample | Conc. [ng/ul] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| Disp B-pBAD Col 5 | 57.6 | 1.99 | 2.31 |
| Mut-Pichia Vektor Col 1 | 31.1 | 2.16 | 2.25 |

#### Sequencing of the Minipreps

| Seq ID | Sample |
| --- | --- |
| DEW029 | Mut1 + AOX5' Primer |
| DEW030 | Disp B5 + Seq. Primer |
| DEW031 | Mut 1 + Seq Primer |
| DEW026 | Sumo 3' AOX Primer |
| DEW027 | Sumo 5' AOX Primer |

## Date: Friday, 22.09.2023

### Experiment: Expression; GFP Enzyme Kinetics; Pichia Transformation

**Participants:** Sarah (09:30-18:00), Sumohit (09:30-19:00)

#### Summary:

Restriction digestion of His4 fragments with SalI. Overnight culture of Mutanase-Pichia vector Colony 1 and pBAD-Dispersin B Colony 5 for minipreps. Preparation of 50x TAE buffer.

#### Expression and GFP Enzyme Kinetics

Since the wrong L-arabinose end concentration was used, and the literature suggests a range of L-arabinose end concentration from 0.01% to 1%, multiple expression attempts were made, and GFP enzyme kinetics were investigated. L-arabinose end concentrations ranging from 0.02% to 2% were used for expression and GFP analysis.

#### Remaining Sequencing Reactions at Microsynth

![Image](image.png)

#### Clean-up of His4 Fragments

Performed with a kit from NEB. Elution in 8 ul.

| Sample | Conc. [ng/ul] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| His4 1 | 51.4 | 1.93 | 1.73 |
| His4 2 | 60.7 | 1.87 | 1.61 |

#### Disposal of n-Hexane and possibly tert-Butanol

Fill into a container, store, and fill out the form together with Martina.

#### Restriction Digestion of Minipreps with SalI-HF for Pichia Transformation

| Component | Volume [ul] | Comments |
| --- | --- | --- |
| Minipreps (approx. 300ng) | Disp B Col5: 6 ul; Mut Col1: 12 ul |
| Cut Smart | 1.5 |
| SalI-HF | 0.5 |
| ddH2O | Disp B Col5: 7 ul; Mut Col1: 1 |

Digest for 15 minutes at 37°C, then heat inactivate for 20 minutes at 65°C. No gel!

#### Transformation of Digested Plasmids into Pichia pastoris

Transformation was carried out as described in the Micro scale protocol of the Frozen-EZ yeast Kit, with the deviation that 20 ul of competent cells were used instead of 10 ul.

## Date: Monday, 25.09.2023

### Experiment Update: Pichia Transformation Results and Overnight Culture

Checked Pichia transformation plates - no colonies but filamentous fungal growth observed.

#### Overnight Culture of pBAD Mut3

5 ml LB+Amp with 0.5% (250 mg) arabinose inoculated with 50 ul overnight culture. Sent to Herzberg for TECAN measurement.

## Date: Tuesday, 26.09.2023

### Experiment Update: Gibson Assembly and Expression Troubleshooting

**Participants:** Sarah; Sumohit (09:30-19:00)

#### Gibson Assembly of Dispersin B and Dextranase in pBAD18 Vector

Since there was a suspicion that the Dispersin B cloning was not successful (there was a suspicion that His4 was not cloned properly), the Dispersin B cloning was repeated from the PCR step. An attempt was made to clone dextranase into pBAD18. On Tuesday, only the PCR and gel electrophoresis were performed.

| Sample | Amount for Gibson [ng] | Volume of Sample [µl] | Water [µl] | NEBuilder [µl] |
| --- | --- | --- | --- | --- |
| Mut | 110.5 | 1.44 | 6.67 | 10 |
| Dsp B | 101.9 | 1.31 | 6.8 | 10 |
| Dex 1 |  |  |  |  |
| Dex 2 |  |  |  |  |
| His4 | 98.2 | 1.09 |  |  |
| pBAD18 Vector | 75 | 0.80 |  |  |

**Gel Electrophoresis:**

Marker - Disp B - Dex 1 - Dex 2 - Marker

#### Expression Troubleshooting

Since the expression did not work, a setup was prepared with AMP2-6 in BW25113 and NEB stable cells. The same cells were seeded in wildtype and with pSB1C3-GFP in TB medium at OD 0.4 with various L-arabinose concentrations ranging from 0.02% to 2%. Enzyme kinetics were measured in a reader after induction. 5 ml setups were also prepared so that if successful, the cells can be lysed and analyzed on SDS-PAGE. Unfortunately, after 4 hours of induction, no increase in fluorescence was observed in any of the samples.

**3x 5ml Overnight Cultures for pBAD-Dispersin B Kol5**

## Date: Wednesday, 27.09.2023

### Experiment Update: Cell Lysis and Minipreps

**Cell Lysis of Various Samples by Sumo**

Cells were pelleted at 5000g and RT for 5 minutes, resuspended in 4 ml 0.9% saline, and subjected to ultrasound pulses on ice for 30 seconds, repeated 5 times. A 1-minute break was taken between cycles to prevent overheating. The cell extracts were transferred to Eppendorf tubes and centrifuged at 14000 rpm and 4°C for 60 minutes. The pellet and supernatant were separated.

**Gel Electrophoresis:**

Marker - Disp B - Dex 1 - Dex 2 - Marker

![Gel Image](20230927_144942.jpg)

#### Minipreps of pBAD-Dispersin B Kol5 and pBAD-Mutanase Kol3

Performed using the Analytik Jena kit, where 3 overnight cultures of Dispersin B were loaded onto a column. Eluted in 60 µl.

| Sample | Concentration [ng/ul] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| pBAD-Dispersin B Kol5 | 117.7 | 1.96 | 2.05 |
| pBAD-Mutanase Kol3 | 76.3 | 1.94 | 2.06 |

#### Preparation of LB Medium

Prepared 500ml of LB medium following the standard recipe.

## Date: Thursday, 28.09.2023

### Experiment Update: Gibson Assembly, Expression Troubleshooting, Bradford, Sequencing, Restriction Digest

**Participants: Sarah, Sumohit (09:30 - 18:30)**

**Summary:** Gibson Assembly of Dispersin B and Dextranase in pBAD18 vector. Expression troubleshooting with various concentrations of L-arabinose. Bradford assay for the previous expression. Sequencing of Gibson samples. Restriction digestion of Gibson samples. DNA cleanup of the restriction-digested Gibson samples.

**Gibson Assembly of Dispersin B and Dextranase in pBAD18 Vector**

After observing bands on the gel, 10 µl of the PCR samples were purified and further processed with Gibson Assembly.

| Sample | Concentration [ng/µl] | A260/A280 | A260/A230 |
| --- | --- | --- | --- |
| Dispersin B | 33 | 1.79 | 2.41 |
| Dex 1 | 28.5 | 1.72 | 1.99 |
| Dex 2 | 28.8 | 1.84 | 2.53 |
| His4 | 34.6 | 1.82 | 1.93 |

| Sample | Gibson Amount [ng] | Sample Volume [µl] | Water | NEBuilder |
| --- | --- | --- | --- | --- |
| Dispersin B | 110.5 | 3.35 | 3.01 | 10 |
| Dex 1 | #VALUE! | #VALUE! | 10 |  |
| Dex 2 | #VALUE! |  |  |  |
| His4 | 98.2 | 2.838150289 |  |  |
| pBAD18 Vector | 75 | 0.80 |  |  |

**Transformation of Gibson Constructs for pBAD18 Gibson**

Gibson Assembly for pBAD18 Gibson constructs was transformed into E. coli cells and plated on respective antibiotic-containing plates. pBAD18 was used as a positive control for transformation and plated on amp-containing plates. Cells without plasmids were treated as blank during transformation, plated on amp-containing plates, and serve as the negative control for transformation. The transformation was carried out as per the protocol.

**Expression Test from Tuesday - Bradford Assay**

To be able to do SDS-PAGE later, protein concentration was measured from the expression samples. The protein concentration was measured as in the protocol.

|  | BW25113 | BW-1 | BW-2 | BW-3 | BW-4 | BW-pSB1c3 |
| --- | --- | --- | --- | --- | --- | --- |
| Dilution | 1 | 1 | 1 | 1 | 1 | 1 |
| Protein [µg/ml] | 28.7578863 | 19.6601932 | 21.6451444 | 15.6489375 | 15.4835249 | 18.5023049 |
| On SDS Page | 7.5 | 7.5 | 7.5 | 7.5 | 7.5 | 7.5 |
| Amount on SDS | 215.684147 | 147.451449 | 162.338583 | 117.367032 | 116.126437 | 138.767287 |

|  | BW25113 | N-1 | N-2 | N-3 | N-4 | N-pSB1C3 |
| --- | --- | --- | --- | --- | --- | --- |
| Dilution | 1 | 1 | 1 | 1 | 1 | 1 |
| Protein [µg/ml] | 24.2090397 | 5.84824082 | 13.8707521 | 10.3557342 | 10.3143811 | 17.427123 |
| On SDS Page | 7.5 | 7.5 | 7.5 | 7.5 | 7.5 | 7.5 |
| Amount on SDS | 181.567798 | 43.8618061 | 104.03064 | 77.6680068 | 77.3578582 | 130.703423 |

**B Stands for BW25113; N for NEB Stable; 1 for 0.02% L-Arabinose; 2 for 0.5% L-Arabinose; 3 for 1% L-Arabinose; 4 for 2% L-Arabinose.**

**No SDS-PAGE was conducted because no differences in fluorescence were observed among all samples.**

### Expression Test 2

Since the previous expressions did not work, a test was conducted. BW25113 cells with AMP2-6 and NEB stable cells with AMP2-6 were cultured in LB medium. The cells were induced with varying L-Arabinose concentrations from 0.02% to 0.2% L-Arabinose. After induction, the cells were incubated overnight at 37°C on a shaker.

### Sequencing pBAD-Mutanase Kol3 and pBAD-Dispersin B Kol5

**Target Concentration for Sample:** 80 ng/µl

**pBAD Mut:** 12 µl Sample + 3 µl Primer (20 uM)

**pBAD Dispersin B:** 8.2 µl Sample + 3.8 µl ddH2O + 3 µl Primer (20uM)

| Seq. ID | Sample | Primer |
| --- | --- | --- |
| 94796806 | pBAD-Dispesin B Kol5 | Enzym-His4 Sequencing Primer |
| 94796807 | pBAD-Mutanase Kol3 | Enzym-His4 Sequencing Primer |
| 94796809 | pBAD-Mutanase Kol3 | 5' AOX |
| 94796808 | pBAD-Mutanase Kol3 | 3' AOX |

### SalI HF Digestion of pBAD-Dispersin Kol5 and pBAD-Mutanase Kol3 for Transformation

**1.5 µg Plasmid/ Digestion**

| Component | Volume [µl] | Comments |
| --- | --- | --- |
| Minipreps (Disp: 1.5 µg and Mut: approx. 1.3 µg) | Disp: 12.7 ; Mut: 17 |  |
| Cut Smart | 5 |  |
| SaliHF | 1 |  |
| ddH2O | Disp: 31.3 ; Mut: 27 |  |

**15 min digestion at 37°C and then 20 min heat inactivation at 65°C.**

### PCR Cleanup of Restriction Digests

PCR cleanup of the restriction digests was unsuccessful because Zymo was used instead of NEB, and Zymo requires a larger elution volume. Therefore, two overnight cultures (ÜN Kulturen) of pBAD-Dispersin B Kol5 and pBAD-Mutanase Kol3 were prepared.

## Date: Friday, 29.09.2023

### Experiment: Expression Troubleshooting, Cell Harvesting, Bradford, Transformation, Restriction Digest

**Participants:** Sarah (09:30-16:45), Sharkey (09:30-15:00), Sumohit (10:00-17:30)

#### Summary:

Expression Troubleshooting, Cell Harvesting, Bradford, Transformation, Restriction Digest.

### Fluorescence Measurement of Expression

After expression, OD600 and fluorescence of the expression samples were measured.

| Sample | BW | 1b | 2b | 3b | 4b | 5b | 1n | 2n | 3n | 4n | 5n | b-p | n-p |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Fluo | 413.00 | 525 | 477.000 | 514.000 | 643.000 | 547.000 | 559.000 | 502.000 | 504.000 | 641.000 | 590.000 | 10071 | 6937.000 |
| Fluo | 384.00 | 485 | 498.000 | 493.000 | 661.000 | 490.000 | 512.000 | 503.000 | 502.000 | 617.000 | 561.000 | 10034 | 7148.000 |
| Fluo | 410.00 | 503 | 486.000 | 528.000 | 698.000 | 505.000 | 512.000 | 499.000 | 499.000 | 695.000 | 536.000 | 9940.000 | 7234.000 |
| MW Fluo | 402.33 | 504.33 | 487.00 | 511.67 | 667.33 | 514.00 | 527.67 | 501.33 | 501.67 | 651.00 | 562.33 | 10015.00 | 7106.33 |
| OD600 | 5.89 | 2.90 | 2.68 | 2.69 | 3.82 | 2.84 | 1.97 | 2.06 | 1.86 | 2.93 | 4.93 | 5.42 | 4.13 |
| Fluo/OD | 68.31 | 173.91 | 181.72 | 190.21 | 174.69 | 180.99 | 267.85 | 243.37 | 269.71 | 222.18 | 114.06 | 1847.79 | 1720.66 |
| Fold Change | 1.00 | 2.55 | 2.66 | 2.78 | 2.56 | 2.65 | 3.92 | 3.56 | 3.95 | 3.25 | 1.67 | 27.05 | 25.19 |

For pSB1C3-GFP, a strong GFP signal was seen in both BW25113 and NEB stable approach compared to wild type samples. In the other samples, only a slightly increased GFP signal was seen compared to the BW25113 wild type.

### Cell Harvesting

After the cells, especially the pSB1C3-GFP samples, showed GFP, the cells were lysed. The cell lysis was performed as per the protocol.

### Bradford

To perform SDS-PAGE later, the protein concentration of the expression samples was measured.

| Sample | BW | 1b | 2b | 3b | 4b | 5b | 1n | 2n | 3n | 4n | 5n | b-p | n-p |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Fluo | 413.00 | 525 | 477.000 | 514.000 | 643.000 | 547.000 | 559.000 | 502.000 | 504.000 | 641.000 | 590.000 | 10071 | 6937.000 |
| Fluo | 384.00 | 485 | 498.000 | 493.000 | 661.000 | 490.000 | 512.000 | 503.000 | 502.000 | 617.000 | 561.000 | 10034 | 7148.000 |
| Fluo | 410.00 | 503 | 486.000 | 528.000 | 698.000 | 505.000 | 512.000 | 499.000 | 499.000 | 695.000 | 536.000 | 9940.000 | 7234.000 |
| MW Fluo | 402.33 | 504.33 | 487.00 | 511.67 | 667.33 | 514.00 | 527.67 | 501.33 | 501.67 | 651.00 | 562.33 | 10015.00 | 7106.33 |
| OD600 | 5.89 | 2.90 | 2.68 | 2.69 | 3.82 | 2.84 | 1.97 | 2.06 | 1.86 | 2.93 | 4.93 | 5.42 | 4.13 |
| Fluo/OD | 68.31 | 173.91 | 181.72 | 190.21 | 174.69 | 180.99 | 267.85 | 243.37 | 269.71 | 222.18 | 114.06 | 1847.79 | 1720.66 |
| Fold Change | 1.00 | 2.55 | 2.66 | 2.78 | 2.56 | 2.65 | 3.92 | 3.56 | 3.95 | 3.25 | 1.67 | 27.05 | 25.19 |

BW stands for BW25113;NEB stands fpr NEB stable cells, 4 stands for 0.15% L-arabinose induction: 5 stands for 0.2% L-arabinose induction:

### Expression Troubleshooting 3

Since this expression provided better results, a new setup was arranged without aluminum foils as it showed better results in Expression Troubleshooting 2 with these measures. Overnight cultures were set up, inoculated, and allowed to grow at room temperature over the weekend for expression.

### Minipreps of pBAD-Dispersin B Kol5 and pBAD-Mutanase Kol3

Sharkey

### Restriction Digest for Transformation in Pichia pastoris

| Component | Volume [ul] | Comments |
| --- | --- | --- |
| Minipreps (Disp: 1.5 µg and Mut: approx. 1.5 µg) | Disp: 12.7 |  |
|  | Mut: 14.4 |  |
| Cut Smart | 5 |  |
| SaliHF | 1 |  |
| ddH2O | Disp: 31.3 |  |
|  | Mut: 29.6 |  |

**15 minutes at 37°C followed by 20 minutes heat inactivation at 65°C.**

### PCR Cleanup of a pBAD-Dispersin B Restriction Digest

Performed with NEB kit. Eluted in 6 µl.

| Sample | Conc. [ng/ul] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| pBAD-Dispersin B Kol5 | Approx. 80 ng/ul? (as written on Eppi) | ? | ? |

### Gel after Restriction Digest

0.7% Gel

Expected bands: Disp: approx. 9362 bp and Mut: approx. 9551 bp

150 ng per lane

Not cleaned-up Restriction Digest: 5 µl each

## Transformation in Pichia pastoris

For the transformation in Pichia pastoris, the EZ-Kit protocol was followed with the following adjustments:

**Transformation Setup Details**

* For pBAD-Dispersin B Kol5 (without clean-up):

+ 30 ul competent cells + 25 ul digested DNA

* For pBAD-Dispersin B Kol5 (with clean-up) and pBAD-Mutanase Kol3:

+ 50 ul competent cells + 5 ul DNA

* For ddH2O Control (instead of DNA):

+ 50 ul competent cells + 5 ul ddH2O

# October

| Monday | Tuesday | Wednesday | Thursday | Friday | Saturday | Sunday |
| --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  | 1 |
| 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| 9 | 10 | 11 | 12 | 13 | 15 | 16 |
| 17 | 18 | 19 | 20 | 21 | 22 | 23 |
| 24 | 25 | 26 | 27 | 28 | 29 | 30 |
| 31 |  |  |  |  |  |  |

## Date: Monday, 02.10.2023

## Experiment Details: Medium; Expression; Harvesting; Fluorescence Measurement; Sequencing; Maintenance

**Participants:**Sarah: 09:30-17:00, Sumohit: 06:00-23:30

**Summary:**

This experiment involved activities related to medium preparation, expression, harvesting, fluorescence measurement, sequencing, and maintenance.

### Medium Preparation

Since the available media appeared turbid, as if something had grown in them, 1 L of LB medium was prepared and autoclaved.

### Expression

After autoclaving and cooling the medium, the expression process began. 200 ml of LB medium with the appropriate antibiotics were added to a flask, followed by the addition of 2.22 ml of the preculture to the main culture. Expressions were performed using AMP2 colony 6, AMP1 colony 7, wild-type BW25113 strain, BW25113 with pBAD18 empty vector, and pSB1C3-GFP-BW25113. The cells were incubated at 37°C on a shaker with continuous shaking. The optical density (OD600) of the samples was continuously measured during the expression. When the OD600 reached 0.7-0.75, the expression was induced by adding 4 ml of 10% arabinose solution. The expression was started at 10:00 AM, induced at 12:30 PM, and concluded at 9:30 PM.

![Expression data](image.png)

### Harvesting

After the expression, the cells were harvested following the protocol.

### E. coli NightSeq Sequencing of Gibson Colonies

For sequencing, 10 colonies from both Dex and Disp plates were picked using Enzyme-His4 Sequencing Primer and 5' AOX Primer in separate tubes. Microsynth tubes were labeled and spun down, and colonies were picked and resuspended in the Microsynth tube. Subsequently, they were streaked on an LB-Amp plate using toothpicks. Additionally, 100 ul of 10uM primer solutions were prepared in separate tubes.

![Sequencing data](image.png)

*Note: Sequencing quality was not satisfactory, likely due to the colonies being too small.*

## Date: Tuesday, 03.10.2023

## Date: Wednesday, 04.10.2023

**Participants:**Sarah H.

## Cell Lysis of Various E. coli Cell Samples

Samples 26, 26R, 17, and 17R were resuspended in 6 ml of 0.9% saline. Samples B, P, and TB were transferred to Falcon tubes. The cells were lysed using ultrasound with 5 pulses of 30 seconds each and 1-minute intervals between the pulses. The solutions were centrifuged at 13,000 rpm for 60 minutes at 4°C. The supernatant was collected and frozen along with the pellets.

## OD Measurement of Shake Flask Cultures and Pelleting of Samples

| Kulture | OD(600) 1:50 Dilution | OD(600) Undiluted | Dilution for OD=6 | 30 ml Approach (Culture + ddH2O) |
| --- | --- | --- | --- | --- |
| AMP 1-7 | 0.142 | 7.1 | 1:1.18 | 25.4 + 4.6 |
| AMP 2-6 | 0.117 | 5.85 | - | - |
| AMP 1-7 R | 0.151 | 7.55 | 1:1.26 | 23.8 + 6.2 |
| AMP 2-6 R | 0.158 | 7.9 | 1:1.31 | 22.9 + 7.1 |
| Gr.1 ? | 0.085 | 4.25 | - | - |
| ASB ? | 0.178 | 8.9 | 1:1.48 | 20.3 + 9.7 |
| Gr.6? | 0.176 | 8.8 | 1:1.46 | 20.5 + 9.5 |

**Pelleting of Cell Samples:** Centrifuged at 5000g for 15 minutes.

## Date: Thursday, 05.10.2023

**Participants: Jana, Sarah H., Sumohit**

## Cell Lysis of Various E. coli Cell Samples

Cell samples were resuspended in 6ml of 0.9% saline. The samples were transferred to Falcon tubes. The cells were lysed using ultrasound with 5 pulses of 30 seconds each and 1-minute intervals between the pulses. The solutions were centrifuged at 13,000 rpm for 60 minutes at 4°C. The supernatant was collected and frozen along with the pellets.

## Bradford Assay for Protein Concentration Determination

### Preparation of the Calibration Curve:

| BSA Amount [ug] | BSA Stock [ul] | H2O [ul] |
| --- | --- | --- |
| 0 | 0 | 100 |
| 2 | 2 | 98 |
| 5 | 5 | 95 |
| 7 | 7 | 93 |
| 10 | 10 | 90 |
| 15 | 15 | 85 |
| 20 | 20 | 80 |

900 ul of Bradford reagent added, mixed, and incubated for 5 minutes. Measurement at 595 nm in the spectrophotometer.

### Measurement of Samples:

Approach in cuvettes:

* 100ul of a 1:10 dilution of the samples
* 900ul Bradford reagent

Mix and incubate for 5 minutes. Measurement at 595 nm in the spectrophotometer.

| Sample | B | C | D | E |
| --- | --- | --- | --- | --- |
| Dilution | 1 | 1 | 1 | 1 |
| Protein [ug/ml] |  |  |  |  |
| On SDS PAGE (7.5%) |  |  |  |  |
| Quantity on SDS |  |  |  |  |

## Date: Friday, 06.10.2023

**Participants:** Jana, Sarah H., Sumohit

## Sample Preparation for SDS PAGE

A total of 15ul of samples were prepared as follows:

* 7.5 ul of sample
* 7.5 ul of 2x loading buffer

Heated at 98°C for 10 minutes. Samples were frozen for future use.

## Date: Monday, 02.10.2023

**Participants:** Jana, Sarah H.

## Sample Preparation of Pellets for SDS PAGE

For each sample:

* - 150 / OD = Volume of the respective cell sample
* - Centrifugation: 14000g, 4°C, 20 min
* - Discard the supernatant
* - Resuspend the pellet in 7.5 ul of water
* - Add 7.5 ul of 2x SDS loading buffer
* - Heat at 98°C for 10 minutes
* - Samples were frozen until further use (-20°C)

## Date: Tuesday, 03.10.2023

**Participants:**Jana, Sumohit

## Procedure:

Arrangement:

(Top: Gel1, Bottom: Gel2)

![Gel1](image1.png)
![Gel2](image2.png)

Apply 120V for 1 hour.

Stain the gel in Coomassie for 30 minutes and destain for 1 hour.

# Engineering Success in Combatting Biofilms using Antimicrobial Peptides

In our iGEM project, our goal was to disrupt existing biofilms, prevent their reformation, and suppress their growth using antimicrobial peptides (AMPs), enzymes, and lipids. To achieve this objective, we needed to investigate the efficacy of peptides, enzymes, and lipids in attacking and eradicating the biofilm. We initiated a literature search for the most prevalent strains according to existing literature and identified *S. mutans* as a promising target. *S. mutans* produces three distinct types of exopolysaccharides, leading us to choose three different enzymes to break down each type of exopolysaccharide. For the peptides, we sought those that specifically target *S. mutans*, and we found various papers highlighting different *S. mutans*-specific peptides. From these peptides, we selected α-5 and BB-41 due to their high specificity for attacking *S. mutans* and their low minimum inhibitory concentration (MIC).

Once we determined the enzymes and peptides for our project, we began devising a plan on how to work with them. Given the lack of prior work demonstrating peptide synthesis within cells by an iGEM team or other research groups, we aimed to experiment with this aspect in our project. In this Engineering Success section, we describe our thought process and intentions regarding the AMPs, focusing on their expression within cells and subsequent purification for utilization in our project.

To achieve this, we integrated AMP BB-41 into a DNA fragment containing a pBAD promoter, an efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989), a spacer, a glycine-serine-rich linker, a His tag, and a T7 terminator, as illustrated in Figure 1.

![BB-41](https://static.igem.wiki/teams/4575/wiki/bb-41-map.png)

Upon successful cloning into the pBAD18 plasmid, we realized that the purification process and efficiency of the AMP will be challenging due to its deviation from the structure described in the literature. Our constructed fragment contains, in addition to the AMP BB-41, a glycine-serine-rich linker and a His tag, which change the length and amino acid sequence of our purified peptide. Consequently, we designed a new DNA construct containing the α-5 AMP. We selected α-5 due to its high specificity for *S. mutans*, similar to BB41, and its low MIC concentration. Furthermore, α-5 possesses an α-helical structure. We anticipated utilizing this property in the future to demonstrate successful peptide expression and proper folding by circular dichroism spectroscopy. In comparison to the DNA fragment for AMP BB-41, which we labelled as AMP-0, the DNA fragment for AMP α-5, denoted as AMP-1, included enterokinase, cyanogen bromide, and carboxypeptidase cleavage sites. After expression and purification using His-Tag affinity chromatography, the purified proteins were to be treated with enterokinase, removing all amino acids before the initial amino acid. Cyanogen bromide will remove all amino acids after methionine, leaving only methionine at the end of the desired AMP, as depicted in Figure 2. Carboxypeptidase A will remove methionine at the end of the wanted AMP, leaving the AMP in the desired size and sequence.

![AMP-1](https://static.igem.wiki/teams/4575/wiki/amp-1-map.png)

***Figure 2: a-5 AMP Fragment (Labelled as AMP-1). This figure comprehensively depicts all essential elements within the DNA fragment.***  The DNA fragment encompasses vital features, including a pBAD promoter, a highly efficient ribosome binding site sourced from bacteriophage T7 gene 10 (Olins and Rangwala, 1989), a spacer, an enterokinase cleavage site, a glycine-serine-rich linker, a cyanogen bromide cleavage site, a carboxypeptidase cleavage site, a His tag, and a T7 terminator.

Concerned that the peptide might not be translated by the ribosomes, due to its small size, we designed a DNA fragment called AMP-2. It contains a GFP coding frame in addition to AMP-1. A glycine-serine linker was attached to the C-terminus of GFP, linked to enterokinase and AMP α-5, as shown in Figure 3. The concept behind this was that cells would initiate transcription and translation with GFP, subsequently transcribing and translating AMP α-5. Following transcription and translation, AMP-2 would be purified and treated similarly to AMP-1 to obtain the AMP.

![AMP-2](https://static.igem.wiki/teams/4575/wiki/amp-2-map.png)

***Figure 3: GFP with a-5 AMP Fragment (Labelled as AMP-2). This figure provides a comprehensive representation of all critical elements found within the DNA fragment.***  The DNA fragment encodes both the green fluorescent protein (GFP) gene and AMP a-5. It encompasses essential components, including a pBAD promoter, a highly efficient ribosome binding site sourced from bacteriophage T7 gene 10 (Olins and Rangwala, 1989), a spacer, the open reading frame of GFP, an enterokinase cleavage site, a glycine-serine-rich linker, a cyanogen bromide cleavage site, a carboxypeptidase cleavage site, a His tag, and a T7 terminator.

After completing the cloning of AMP-1 and AMP-2, we contemplated ways to enhance our system. We noticed that cells with AMP-2 would transcribe and translate excessive amino acids that were unnecessary and decreases the AMP-yield. To address this, we explored whether we could improve the payload-to-load ratio by either increasing the length of the payload sequence (AMP sequence) or reducing the length of the load sequence (non-AMP sequence).

To enhance the payload-to-load ratio, we designed two new DNA fragments, AMP-3 and AMP-4. In both new DNA fragments, the open frame of GFP was replaced with AMPs. In the AMP-3 fragment, the open frame of GFP was replaced by an 8-fold linker-enterokinase site-α-5-cyanogen bromide site, aiming to improve the payload-to-load ratio from 5% to X%. In an ideal state, this alteration could potentially generate nine times more AMP α-5 compared to AMP-2, as illustrated in Figure 4. Considering the potential limitations of AMP-3 due to the repetitive nature of α-5 in the fragment we designed another DNA fragment. This fragment called AMP-4 contains nine different AMPs, as depicted in Figure 5. Our goal with AMP-4 was to produce not only one *S. mutans*-specific AMP but also numerous AMPs capable of killing *S. mutans* cells and potentially other organisms present in the biofilm, contributing to biofilm formation and caries development. We are aiming to improve the payload-to-load ratio from 5% to Y% with AMP-4.

![AMP-3](https://static.igem.wiki/teams/4575/wiki/amp-nr3-map.png)

***Figure 4: AMP DNA-Fragment with ninefold α-5 (Labelled as AMP-3). This figure provides a detailed depiction of the DNA fragment featuring ninefold AMP a-5, each with a distinct nucleotide sequence.***  TThe DNA fragment encodes nine instances of AMP a-5, each characterized by a unique nucleotide sequence. Notable components of this fragment include a pBAD promoter, a highly efficient ribosome binding site sourced from bacteriophage T7 gene 10 (Olins and Rangwala, 1989), a spacer, nine enterokinase cleavage sites, nine different linkers, nine cyanogen bromide cleavage sites, nine carboxypeptidase cleavage sites, a His tag, and a T7 terminator.

![AMP-4](https://static.igem.wiki/teams/4575/wiki/amp-nr4-map.png)

***Figure 5: AMP DNA-Fragment with nine different AMPs (Labelled as AMP-4). This figure provides a comprehensive representation of the DNA fragment, highlighting nine distinct antimicrobial peptides.***  The DNA fragment encodes nine unique AMPs, each characterized by a distinct amino acid sequence and varying efficiency against *S. mutans*. Noteworthy components of this fragment include a pBAD promoter, a highly efficient ribosome binding site sourced from bacteriophage T7 gene 10 (Olins and Rangwala, 1989), a spacer, nine enterokinase cleavage sites, nine different linkers, nine cyanogen bromide cleavage sites, nine carboxypeptidase cleavage sites, a His tag, and a T7 terminator.

Unfortunately, we were unable to clone AMP-3 and AMP-4. Nonetheless, we believe that our ideas lay the foundation for future iGEM teams as well as other research groups to demonstrate the ribosomal production of diverse AMPs, which could ultimately aid individuals facing various challenges.

![experiments banner](https://static.igem.wiki/teams/4575/wiki/experiments.png)

# **Agar and Media**

##

## LB-Agar-Medium (+ Amp)

* 10 g/L tryptone
* 10 g/L NaCL
* 5 g/L yeast extract
* 20 g/L agar
* Fill up with water to 1 L
* Autoclave the bottle with medium
* Add 1 ml of 100 mg/mL ampicillin solution into 1 liter medium

## LB-Medium (+ Amp)

* 10 g/L tryptone
* 10 g/L NaCl.
* 5 g/L yeast extract
* Fill up with water to 1 L
* Autoclave the bottle with medium.
* Add 1ml of 150 mg/mL of ampicillin solution into 1 liter medium.

## TB-Medium

* 12 g tryptone in 900 ml
* 5 g (4 ml) glycerol in 900 ml
* 24 g yeast extract in 900 ml
* Fill up with water to 900 ml
* Autoclave the bottle with medium
* Add 100 ml 10x TB-salt to 900 ml TB media

## TB-Salt

* 23.1 g/L KH2PO4
* 125.4 g/L K2HPO4
* Autoclave the bottle.

## SOC-Medium

* 20 g/L tryptone
* 0.584 g/L NaCl
* 0.186 g/L KCl
* 2.4 g/L MgSO4
* 5 g/L yeast extract
* Fill up with water to 1 L
* Autoclave the bottle with medium
* Per 1L of SOB, add 20mL of filter-sterilized 20% glucose solution

## Pichia Pastoris YPD

* 20 g/L tryptone/peptone
* 20 g/L glucose
* 10 g/L yeast extract
* Fill up with water to 1 L
* Autoclave the bottle with medium

## Pichia Pastoris YPD Agar Plates

* 20 g/L tryptone/peptone
* 20 g/L glucose
* 10 g/L yeast extract
* 20 g/L agar
* Fill up with water to 1 L
* Autoclave the bottle with medium

## Pichia Pastoris YPD + Histidin Agar Plates

* 20 g/L tryptone/peptone
* 20 g/L glucose
* 10 g/L yeast extract
* 1.56 g/L histidin
* 20 g/L agar
* Fill up with water to 1 L
* Autoclave the bottle with medium

## Pichia Pastoris minimal Agar Plates

* 15 g/L agar
* Fill up with water to 800 ml
* Autoclave the bottle with medium
* Cool solution to below 60°C and add YNB, Dextrose and Biotin solutions.
* 100 ml YNB solution (13.4%)
* 100 ml dextrose (20%)
* 2 ml Biotion (0.02%)

---

# **Buffers**

## 50x TAE-Buffer

* 242 g Tris base
* 57.1 ml Glacial acetic acid
* 37.2 g EDTA
* Fill up with distilled water to approximately 950 ml
* Measure the pH of the solution using a pH meter. Adjust the pH to approximately 8.2 with concentrated acetic acid or concentrated NaOH as needed.
* Stirring plate or magnetic stir bar
* Fill up with distilled water to 1000 ml

## 10x SDS running-Buffer

* 30.3 g Tris base
* 144 g glycin
* 100g SDS (Sodium Dodecyl Sulfate)
* Fill up with distilled water to approximately 950 ml
* Fill up with distilled water to 1000 ml

---

# **Cloning**

## Processing of primers and fragments

* Centrifuge the fragments and primers with Short Spin for approx. 10 sec.
* Dilute the fragments and primers with ddH2O (final concentrations 50 nM for fragments and 100 µM for primers).
* Vortex thoroughly
* Short spin for approx. 10 sec.

## PCR Protocol for 50 µl Approach

## Materials:

* Q5 2x Master Mix
* 10 µM Primer fw
* 10 µM Primer rv
* Distilled water (ddH2O)
* DNA template
* PCR tubes/strips
* PCR machine

## Procedure:

1. **Preparation of PCR Reaction Mix (50 µl):**
   * Mix the following components in a PCR tube to achieve a total volume of 50 µl per reaction:

   | Components | Volume | Concentration |
   | --- | --- | --- |
   | Q5 2x Master Mix | 25 µl | - |
   | 10 µM Primer fw | 2.5 µl | 0.5 µM |
   | 10 µM Primer rv | 2.5 µl | 0.5 µM |
   | ddH2O | 19 µl | - |
   | DNA Template | 1 µl | - |
2. **PCR Amplification (Cycling Conditions):**

   Set up the PCR machine with the following cycling conditions listed in Table 2.
3. **PCR Cycling Conditions (Table 2):**

   | Step | Temp for Fragment | Time for Fragment | Temp for Vector | Time for Vector |
   | --- | --- | --- | --- | --- |
   | Initial Denaturation | 98°C | 30 s | 98°C | 30 s |
   | Cycle: Denaturation | 98°C | 10 s | 98°C | 10 s |
   | Cycle: Annealing | 70°C | 30 s | 70°C | 30 s |
   | Cycle: Elongation | 72°C | 90 s | 72°C | 180 s |
   | Final Extension | 70°C | 150 s | 70°C | 210 s |
   | Hold | 8°C | - | 8°C | - |
4. **PCR Amplification:**

   Run the PCR program for the specified number of cycles, 30 times for the fragments and 40 times for the vector.
5. **PCR Product Analysis:**

   Analyze the PCR products by running a portion of the reaction on an agarose gel for size comparison.

## Linkage PCR

## Materials:

* DNA samples containing the target DNA sequences
* Q5 MasterMix
* Primers specific to the target DNA sequences
* PCR tubes/strips
* PCR machine
* Agarose gel
* Gel electrophoresis equipment
* DNA ladder

## Procedure:

1. **DNA Extraction and Quantification:**
   * Extract DNA from the respective sources and quantitate the DNA using a suitable method.
2. **Primer Design:**
   * Design primers specific to the target DNA sequences that will be used to amplify the linkage between the two DNA regions.
3. **PCR Reaction Mix Preparation:**
   * Calculate the amount of each component for a single PCR reaction based on the number of reactions you plan to perform and prepare the PCR reaction mix accordingly.
4. **PCR Reaction Setup:**
   * Place a PCR tube or strip into a PCR tube rack.
   * Dispense an appropriate volume of the PCR reaction mix into each PCR tube according to the calculations.
5. **DNA Template Addition:**
   * Add the DNA templates to the respective PCR tubes, ensuring the appropriate amount of DNA is added for each sample.
6. **PCR Amplification:**
   * Load the PCR tubes into the PCR machine.
   * Set up the PCR program, including denaturation, annealing, and extension temperatures and times based on the primer specifications and target DNA.
7. **PCR Product Analysis:**
   * After PCR amplification, analyze the PCR products by running a portion of the reaction on an agarose gel with a DNA ladder for size comparison.
   * Visualize the DNA bands under UV light using a gel documentation system.

## 0,8 % Gel

* 0,4 g Agarose
* 50 ml 1x TAE-Puffer

1. Heat in microwave until the agarose is completely dissolved.

-Use 3,0 μl Midori-Green (Advance DNA stain) per 50 ml.

2. Allow to harden for 30 min.

For 1% Gel use 0,5g Agarose, etc.

For a larger gel-chamber and 0,8% gel: use 250 ml TAE-Puffer on 2 g Agarose

## Agarose Gel

## Materials:

* Agarose powder
* 1x TAE buffer
* Loading dye (6x)
* PCR product

## Procedure:

1. **Preparation of 0.8% w/v Agarose Gel:**
   * Measure the required amount of agarose powder (w/v) for a 0.8% gel according to the desired gel size/volume.
   * Add the measured agarose powder to an appropriate volume of 1x TAE buffer in a microwave-safe flask or container.
   * Mix well to ensure the agarose is completely dissolved.
   * Microwave the mixture until the agarose is fully dissolved, but be cautious to avoid boiling over.
   * Allow the mixture to cool to around 50-60°C.
2. **Addition of Loading Dye to PCR Product:**
   * Add 5 µl or 10 µl of loading dye (6x) to 25 µl or 50 µl of the PCR product, respectively.
   * Mix the mixture well using a pipette to ensure thorough mixing of the PCR product and loading dye.
3. **Loading PCR Product on Agarose Gel:**
   * Prepare the agarose gel mold and comb to create gel pockets suitable for loading the PCR product.
   * Once the agarose mixture has cooled to around 50-60°C, pour it into the gel mold to create the gel.
   * Allow the gel to solidify by cooling at room temperature.
4. **Gel Electrophoresis:**
   * Carefully remove the comb from the solidified agarose gel to create wells for loading the DNA samples.
   * Place the gel in the gel electrophoresis chamber and add enough 1x TAE buffer till the fill band.
   * Load the PCR product mixed with loading dye into the wells created in the gel.
5. **Gel Electrophoresis Running:**
   * Run the gel at 100 V for 60 minutes.
6. **DNA Fragment Visualization:**
   * After electrophoresis, carefully remove the gel from the gel chamber.
   * Visualize the DNA fragments by placing the gel on a UV table and turning on the UV light.
   * Observe and document the DNA fragments based on their migration pattern and size.

## Gel Extraction

## Materials:

* Agarose gel containing DNA fragments
* NEB Monarch® DNA Gel Extraction Kit (or similar)
* Midori Green solution
* Sterile water
* Microcentrifuge tubes
* Microcentrifuge
* Vortex mixer
* Refrigerated centrifuge
* UV table

## Procedure:

1. **Preparation of the Gel:**
   * Prepare an agarose gel containing DNA fragments using standard agarose gel electrophoresis procedures.
   * Stain the gel with Midori Green according to the manufacturer's instructions.
   * Visualize the DNA bands using a UV table.
2. **Gel Excision:**
   * Using a clean scalpel or razor blade, carefully cut out the DNA band of interest from the gel.
3. **Gel Extraction Preparation:**
   * Weigh the gel slice to accurately determine the amount of gel for subsequent steps.
   * Transfer the gel slice into a microcentrifuge tube.
4. **Gel Dissolution:**
   * Add 3-4 volumes (in μl) of Monarch Gel Dissolving Buffer to the gel slice, based on the weight of the gel slice.
   * Incubate the tube at 50°C for 5-10 minutes or until the gel slice is completely dissolved.
   * Mix the contents by inverting the tube periodically.
5. **DNA Binding:**
   * Place a Monarch DNA Gel Extraction Column in a collection tube.
   * Transfer the dissolved gel mixture onto the column.
   * Centrifuge at 16,000 x g for 1 minute to bind the DNA to the column.
6. **Wash Steps:**
   * Discard the flow-through and place the column back into the collection tube.
   * Wash the column by adding 200 μl of Monarch DNA Wash Buffer to the column.
   * Centrifuge at 16,000 x g for 1 minute. Discard the flow-through and repeat this wash step once more.
7. **DNA Elution:**
   * Transfer the column to a clean 1.5 ml microcentrifuge tube.
   * Add 10 μl of Monarch DNA Elution Buffer directly onto the center of the column membrane.
   * Incubate at room temperature for 1 minute.
   * Centrifuge at 16,000 x g for 1 minute to elute the purified DNA.
8. **DNA Quantification:**
   * Measure the concentration and purity of the purified DNA using a spectrophotometer or Nanodrop.

## PCR Purification

## Materials:

* PCR reaction mixture
* NEB Monarch® PCR & DNA Cleanup Kit (or similar)
* Ethanol (95-100%)
* Sterile water
* Microcentrifuge tubes
* Microcentrifuge
* Vortex mixer

## Procedure:

1. **PCR Amplification:**
   * Perform a standard PCR amplification according to the PCR protocol for your target DNA.
2. **Preparation of PCR Products:**
   * After PCR amplification, proceed with the purification process immediately or store the PCR reaction mixture at -20°C for later purification.
3. **Sample Preparation for Cleanup:**
   * Add 5 volumes of Monarch Binding Buffer to the PCR reaction mixture for dsDNA below 2 kb.
   * Add 2 volumes of Monarch Binding Buffer to the PCR reaction mixture for dsDNA above 2 kb.
4. **DNA Binding:**
   * Mix the sample thoroughly by pipetting or vortexing.
   * Transfer the mixture to a Monarch column placed in a collection tube.
   * Centrifuge at 16,000 x g for 1 minute to bind the DNA to the column.
5. **Wash Steps:**
   * Discard the flow-through and place the column back into the collection tube.
   * Add 200 μl of Monarch Wash Buffer to the column.
   * Centrifuge at 16,000 x g for 1 minute. Discard the flow-through and repeat this wash step once more.
6. **DNA Elution:**
   * Transfer the column to a clean 1.5 ml microcentrifuge tube.
   * Eluate with 10 μl of Monarch Elution Buffer by putting it directly onto the center of the column membrane.
   * Incubate at room temperature for 1 minute.
   * Centrifuge at 16,000 x g for 1 minute to elute the purified DNA.
7. **DNA Quantification:**
   * Measure the concentration and purity of the purified DNA using a spectrophotometer or Nanodrop.

## Gibson Assembly

## Materials:

* DNA fragments to be assembled
* NEBuilder® HiFi DNA Assembly Master Mix
* Distilled water
* PCR machine or thermal cycler

## Procedure:

1. **Calculate Amounts of DNA Fragments:**
   * Calculate the amount of each DNA fragment to be used in the Gibson Assembly based on their respective lengths and concentrations. Typically, equimolar amounts of fragments are used.
   * In the table below are the molar concentrations used.

cool

2. **Mix DNA Fragments and Master Mix:**
   * In a sterile microcentrifuge tube, combine the DNA fragments in equimolar amounts. The total DNA amount should be within the recommended range for Gibson Assembly.
   * Add an appropriate volume of NEBuilder® HiFi DNA Assembly Master Mix based on the total DNA amount. Refer to the manufacturer's instructions for the correct ratio.
3. **Incubation:**
   * Mix the DNA fragments and Master Mix thoroughly by gentle pipetting.
   * Incubate the reaction at the 50°C for the 15 minutes.
4. **Transformation:**
   * Use 3 µl of the Gibson Assembly reaction to transform into 25 µl competent *E. coli* cells as per standard transformation protocols.
5. **Plating and Selection:**
   * Plate the transformed cells on selective agar plates containing the appropriate antibiotic(.
   * Incubate the plates overnight at the 37°C.

## BioBrick Assembly

## Materials:

* BioBrick DNA parts (recipient and insert)
* Restriction enzymes (EcoRI, XbaI, SpeI, PstI)
* DNA ligase
* 10X restriction enzyme buffer
* Gel extraction kit
* Sterile water
* PCR machine
* Incubator set to 37°C

## Procedure:

1. **Preparation of DNA Parts:**
   * Retrieve the BioBrick DNA parts (recipient and insert) from the freezer and thaw on ice.
   * Quantify the DNA concentration using a spectrophotometer or Nanodrop.
2. **Digestion of DNA Parts:**
   * Set up separate digestion reactions for the recipient and insert DNA parts.
   * Mix 1000 ng of each DNA part, 5 μl of 10X restriction enzyme buffer, and appropriate restriction enzymes according to RFC10 (EcoRI and XbaI for recipient, SpeI and PstI for insert).
   * Incubate the reactions at 37°C for 15 minutes and after that at 80°C for 20 minutes.
3. **Gel Electrophoresis:**
   * Prepare a 1% agarose gel and load the digested DNA samples alongside a DNA ladder.
   * Run the gel at 100 V for about 60 minutes.
   * Visualize the bands using a UV table.
4. **Gel Extraction:**
   * Cut out the gel bands corresponding to the digested DNA fragments.
   * Purify the DNA fragments using a gel extraction kit following the manufacturer's protocol.
5. **Ligation:**
   * Mix the purified insert DNA (SpeI-PstI digested) and the purified recipient DNA (EcoRI-XbaI digested) in a 1:1 molar ratio.
   * Add DNA ligase and ligase buffer according to the manufacturer's instructions.
   * Incubate the ligation reaction at 16°C overnight.
6. **Transformation:**
   * Heat-shock E. coli competent cells with the ligated DNA according to standard protocols.
   * Plate the transformed cells on appropriate selective agar plates.
7. **Colony PCR and Sequencing:**
   * Pick colonies from the selective agar plates and perform colony PCR using BioBrick-specific primers to confirm successful assembly.
   * Sequence the PCR products to validate the correct assembly.

>

## DNA cleanup

## Materials:

* DNA sample to be purified
* Monarch® PCR & DNA Cleanup Kit
* Microcentrifuge tubes

## Procedure:

1. **Binding DNA to Monarch® Columns:**
   * Place a Monarch® Column in a provided collection tube.
   * Add the DNA sample (not exceeding 5 μg) to the Monarch® Column.
2. **DNA Binding and Washing:**
   * Add 2 volumes of Binding Buffer to the DNA sample in the Monarch® Column.
   * Mix the contents by pipetting gently.
   * Centrifuge at maximum speed for 30 seconds.
   * Discard the flow-through.
3. **Washing Steps:**
   * Add 700 µl of Wash Buffer to the Monarch® Column.
   * Centrifuge at maximum speed for 30 seconds.
   * Discard the flow-through.
   * Repeat the wash step once.
4. **Elution of DNA:**
   * Transfer the Monarch® Column to a clean microcentrifuge tube.
   * Add the provided Elution Buffer or distilled water directly to the center of the column membrane (min. 10 µl and up to 100 µl) for DNA elution.
   * Incubate at room temperature for 1 minute.
   * Centrifuge at maximum speed for 30 seconds to elute the DNA.
5. **Checking DNA Concentration:**
   * Measure the DNA concentration using a Nanodrop.

## Overnight culture

## Materials:

* LB (Luria-Bertani) or TB (Terrific Broth) medium
* Ampicillin solution (5 µL per culture tube)
* Sterile pipette tips
* Bacterial culture plate

## Procedure:

1. **Preparation of Culture Medium:**
   * Depending on the purpose, select either LB or TB medium for the bacterial culture.
   * Measure 5 mL of the chosen medium and add it to a sterile culture tube.
2. **Addition of Ampicillin:**
   * Measure 5 µL of ampicillin using a micropipette and add it to the culture tube containing the medium. Ampicillin is added to promote the growth of bacteria harboring a plasmid with ampicillin resistance.
3. **Colony Selection and Inoculation:**
   * Using a sterile pipette tip, carefully pick a single bacterial colony from the culture plate that you want to inoculate.
   * Transfer the picked colony into the culture tube containing the medium and ampicillin.
4. **Incubation:**
   * Immerse the pipette tip with the picked colony into the medium-antibiotic mixture in the culture tube.
   * Incubate the culture tube at 37°C to promote bacterial growth and propagation. The culture can be incubated in an appropriate incubator or shaker.

## Competent cell production *E. coli*

### Materials:

* Bacterial culture in LB medium (10 mL)
* LB medium
* CaCl2 (50 mM)
* CC Buffer
* Liquid Nitrogen

### Procedure:

1. #### Overculture in LB Medium:

   Prepare an overculture by incubating the bacterial culture in 10 mL of LB medium at the appropriate temperature and conditions until reaching the desired growth phase.
2. #### Inoculation and Growth:

   * Inoculate 50 mL of fresh LB medium with the overculture to achieve an initial OD600 of 0.1.
   * Incubate the culture at 37°C until the OD600 reaches approximately 0.6.
3. #### Centrifugation and Supernatant Discard:

   * Centrifuge the bacterial culture at 5000 rpm and 4°C for 5 minutes.
   * Discard the supernatant.
4. #### Resuspension in CaCl2 (50 mM):

   * Resuspend the bacterial pellet in 25 mL of CaCl2 (50 mM).
   * Centrifuge at 5000 rpm and 4°C for 5 minutes.
   * Discard the supernatant.
5. #### Resuspension in CC Buffer:

   * Resuspend the bacterial pellet in 10 mL of CC Buffer.
   * Centrifuge at 5000 rpm and 4°C for 5 minutes.
   * Discard the supernatant.
6. #### Division into Aliquots:

   * Resuspend the bacterial pellet in 6 mL of CC Buffer.
   * Divide the suspension into 200 µl aliquots.
   * Freeze each aliquot immediately in liquid nitrogen.

## Competent cell production *P. pastoris*

### Materials:

* Pichia pastoris cells (grown to mid-log phase)
* 1 M Sorbitol solution
* Lithium chloride (LiCl) solution (0.1 M)
* Ice
* Centrifuge
* Sterile microcentrifuge tubes
* Luria-Bertani (LB) agar plates

### Procedure:

#### 1. Prepare Pichia pastoris Culture:

Grow Pichia pastoris cells in YPD (Yeast extract Peptone Dextrose) medium to mid-log phase (OD600 ≈ 0.6-1.0) by incubating at 30°C with shaking at 220 rpm.

#### 2. Harvest the Cells:

* Transfer the culture to a sterile centrifuge tube and centrifuge at 3000 rpm for 5 minutes at 4°C.
* Discard the supernatant and resuspend the pellet gently in an equal volume of 1 M sorbitol.

#### 3. Wash and Resuspend the Cells:

* Centrifuge the cells again at 3000 rpm for 5 minutes at 4°C.
* Discard the supernatant and resuspend the pellet gently in an equal volume of ice-cold 1 M sorbitol.

#### 4. Centrifuge and Resuspend in LiCl:

* Centrifuge the cells at 3000 rpm for 5 minutes at 4°C.
* Discard the supernatant and resuspend the pellet in an equal volume of ice-cold 0.1 M LiCl.
* Incubate the cells on ice for 1 hour.

#### 5. Centrifuge and Wash with LiCl:

* Centrifuge the cells again at 3000 rpm for 5 minutes at 4°C.
* Discard the supernatant and resuspend the pellet in 1 mL of ice-cold 0.1 M LiCl.
* Centrifuge the cells at 3000 rpm for 5 minutes at 4°C.
* Discard the supernatant and resuspend the pellet in a small volume (e.g., 100-200 μL) of ice-cold 0.1 M LiCl.

## Transformation

## Materials:

* Competent cells
* Plasmid DNA (positive control)
* Gibson construct (3 µl)
* BioBrick construct (2 µl)
* LB medium
* LB agar culture plates with appropriate antibiotic
* Ice
* Thermocycler
* Centrifuge

## Procedure:

1. **Thawing Competent Cells:**
   * Thaw 25 µL of competent cells on ice.
2. **Pre-incubation of Competent Cells:**
   * Leave the cells on ice for 25 to 30 minutes.
3. **DNA Addition:**
   * Add the following to the competent cells while keeping the tube on ice:
     + 1 µL of plasmid DNA (positive control)
     + 3 µL of Gibson construct
     + 2 µL of BioBrick construct
4. **Incubation on Ice:**
   * Incubate the cells with DNA for 30 minutes on ice.
5. **Heat Shock Treatment:**
   * Place the tube with the cells and DNA in a thermocycler at 42°C for 45 seconds.
6. **Cooling on Ice:**
   * Place the cells on ice for 1 minute to cool down.
7. **Addition of LB Medium:**
   * Add 450 µL of LB medium to the cells.
8. **Incubation:**
   * Incubate the cells for at least 60 minutes at 37°C.
9. **Centrifugation (for Gibson Assembly):**
   * Centrifuge the cells with Gibson Assembly constructs at 13,000 rpm for 1 minute.
   * Discard 350 µL of the supernatant, resuspend the pellet, and retain the remainder.
10. **Plating of Cells:**
    * Plate out 100 μL of the cell suspension directly onto LB agar culture plates with the appropriate antibiotic.
11. **Overnight Incubation:**
    * Incubate the culture plates overnight at 37°C.

## Quick plasmid analysis

## Objective:

The objective of this protocol is to quickly analyze Gibson-assembled plasmids.

## Materials:

* Cell samples from the backup plates
* Sterile ddH2O (deionized water)
* 2x NEB solution (NSEB)
* 25% Ficoll
* Agarose gel

## Procedure:

1. **Cell Preparation:**
   * Extract a small amount of cells from the backup plates and dissolve them in 15 µl of sterile ddH2O.
2. **Master Mix Preparation:**
   * Prepare a Master Mix using 2x NSEB solution.
3. **Incubation:**
   * Incubate the cell samples with the Master Mix for 1 hour at 98°C.
4. **Ficoll Mixing:**
   * Mix the incubated cells with 25% Ficoll.
5. **Gel Application:**
   * Apply the mixture onto an agarose gel for analysis.

## Colony-PCR

## Materials:

* Bacterial colonies
* NEB Q5 MasterMix
* Primers specific to the target DNA
* Sterile water
* PCR tubes/strips
* PCR machine
* Agarose gel
* Gel electrophoresis equipment
* DNA ladder

## Procedure:

1. **Colony Selection:**
   * Select bacterial colonies from a freshly streaked agar plate that contains the desired DNA fragment.
2. **Preparation of PCR Reaction Mix:**
   * Prepare a master mix for the PCR reaction by combining the following components in a sterile microcentrifuge tube:
     + NEB Q5 MasterMix
     + Primers (forward and reverse) specific to the target DNA
     + Sterile water
3. **PCR Reaction Setup:**
   * Place a PCR tube or strip into a PCR tube rack.
   * Dispense an appropriate volume of the PCR reaction mix (calculated for each reaction) into each PCR tube.
4. **Collection of Bacterial Cells:**
   * Using a sterile pipette tip, pick a small amount of a bacterial colony from the streaked agar plate.
5. **Inoculation into PCR Reaction Mix:**
   * Transfer the picked colony directly into the PCR tube containing the reaction mix. Mix gently by pipetting up and down.
6. **PCR Amplification:**
   * Load the PCR tubes into the PCR machine.
   * Set up the PCR program, including denaturation, annealing, and extension temperatures and times based on the primer specifications and target DNA.
7. **PCR Program:**
   * Perform PCR amplification using the following program as a general guideline:
     + Initial denaturation: 98°C for 30 seconds
     + Denaturation: 98°C for 10 seconds
     + Annealing: Optimal temperature for primer pair (e.g., 55-65°C) for 20 seconds
     + Extension: 72°C for appropriate time per kb of expected PCR product (e.g., 30 seconds per kb)
     + Final extension: 72°C for 2-5 minutes
8. **PCR Product Analysis:**
   * After PCR amplification, analyze the PCR products by running a portion of the reaction on an agarose gel with a DNA ladder for size comparison.
   * Visualize the DNA bands under UV light using a gel documentation system or UV table.

## Colony-PCR

## Materials:

* Bacterial colonies
* Sterile water
* 20 µl of 20 mM NaOH
* PCR master mix
* PCR tubes/strips
* PCR machine
* Agarose gel
* Gel electrophoresis equipment
* DNA ladder

## Procedure:

1. **Colony Selection and Lysis:**
   * Select bacterial colonies from an agar plate.
   * In a sterile microcentrifuge tube, add a single colony from each selected bacterial colony to 20 µl of 20 mM NaOH.
   * Mix gently to ensure thorough mixing of cells with NaOH.
   * Incubate the mixture at 98°C for 45 minutes for cell lysis.
2. **Neutralization:**
   * After the incubation, allow the lysed cells to cool down to room temperature.
3. **PCR Setup:**
   * Add 1-2 µl of the lysed cell suspension to the PCR tubes containing the PCR master mix.
4. **PCR Amplification:**
   * Load the PCR tubes into the PCR machine.
   * Set up the PCR program, including denaturation, annealing, and extension temperatures and times based on the primer specifications and target DNA.
5. **PCR Product Analysis:**
   * After PCR amplification, analyze the PCR products by running a portion of the reaction on an agarose gel with a DNA ladder for size comparison.
   * Visualize the DNA bands under UV light using a gel documentation system or UV table.

## Plasmid isolation

## Materials:

* E. coli culture containing the plasmid of interest
* innuPREP Plasmid Mini Kit 2.0 (Jena Analytik)
* Lysis buffer
* Neutralization buffer
* Wash buffer (I and II)
* Elution buffer
* Microcentrifuge tubes
* Microcentrifuge
* Centrifuge

## Procedure:

1. **Harvesting Bacterial Culture:**
   * Inoculate a suitable volume of E. coli culture containing the plasmid of interest and grow it to the desired cell density.
2. **Pellet Cells:**
   * Transfer 1.5–2 mL of the bacterial culture into a microcentrifuge tube.
   * Centrifuge at 8,000 × g for 2 minutes to pellet the bacterial cells.
3. **Resuspension of Cell Pellet:**
   * Discard the supernatant carefully.
   * Resuspend the cell pellet in 250 µL of lysis buffer by gentle vortexing or pipetting.
   * Ensure thorough mixing to achieve a homogenous cell lysate.
4. **Lysis of Cells:**
   * Incubate the resuspended cells in lysis buffer at room temperature for 10 minutes.
5. **Neutralization:**
   * Add 300 µL of neutralization buffer and mix gently by inverting the tube several times.
   * Incubate at room temperature for 5 minutes.
6. **Binding Plasmid DNA:**
   * Load the lysate onto the innuPREP spin filter placed in a collection tube.
   * Centrifuge at 13,000 × g for 1 minute.
   * Discard the flow-through.
7. **Washing:**
   * Add 500 µL of wash buffer I to the spin filter.
   * Centrifuge at 13,000 × g for 1 minute.
   * Discard the flow-through.
   * Add 700 µL of wash buffer II to the spin filter.
   * Centrifuge at 13,000 × g for 1 minute.
   * Discard the flow-through.
8. **Elution of Plasmid DNA:**
   * Place the spin filter in a clean microcentrifuge tube.
   * Add 50 µL of elution buffer to the center of the spin filter.
   * Incubate at room temperature for 2 minutes.
   * Centrifuge at 13,000 × g for 1 minute to elute the plasmid DNA.
9. **Quantification and Storage:**
   * Quantify the isolated plasmid DNA using a Nanodrop.

## Sequencing

Materials:

* DNA samples in a concentration of at least 50 ng/µl
* Barcode labels

## Procedure:

1. **Prepare DNA Samples:**
   * Ensure you have DNA samples available, each with a concentration of at least 50 ng/µl.
2. **Labeling with Barcode:**
   * Affix a barcode label to each tube containing the DNA sample.
3. **Volume Measurement:**
   * Measure and pipette 5 µL of the DNA sample into each labeled tube.
   * Pipette 5 µL of the 5 µM primer into each labeled tube.
   * Send the labeled tube.

## Post-staining Midori-Green

## Materials:

* Agarose gel with separated DNA fragments
* Midori Green DNA staining solution
* Gel electrophoresis chamber
* UV table

## Procedure:

1. **Gel Preparation:**
   * Perform agarose gel electrophoresis to separate DNA fragments based on size and charge.
2. **Preparing Midori Green Staining Solution:**
   * Dilute Midori Green DNA staining solution as per the manufacturer's instructions to the desired concentration for staining.
3. **Poststaining:**
   * After completing the gel electrophoresis, remove the gel from the gel electrophoresis chamber.
4. **Midori Green Staining:**
   * Submerge the gel in the prepared Midori Green staining solution, ensuring the gel is completely covered.
5. **Staining Duration:**
   * Allow the gel to stain for 20-30 minutes. The staining time can be adjusted based on the intensity of staining required.
   * Wash the gel twice for 30 minutes with water.
6. **Visualization:**
   * After staining, visualize the DNA bands under a UV table.

## Post-staining GelRed

## Materials:

* Agarose gel with separated DNA fragments
* GelRed nucleic acid stain
* Gel electrophoresis chamber
* UV table

## Procedure:

1. **Gel Preparation:**
   * Perform agarose gel electrophoresis to separate DNA fragments based on size and charge.
2. **Preparing GelRed Staining Solution:**
   * Dilute the GelRed nucleic acid stain according to the manufacturer's instructions to the desired concentration for staining.
3. **Poststaining:**
   * After completing the gel electrophoresis, remove the gel from the gel electrophoresis chamber.
4. **GelRed Staining:**
   * Submerge the gel in the prepared GelRed staining solution, ensuring the gel is completely covered.
5. **Staining Duration:**
   * Allow the gel to stain for 15 minutes. The staining time can be adjusted based on the intensity of staining required.
   * Wash the gel twice for 30 minutes with water.
6. **Visualization:**
   * After staining, visualize the DNA bands under a UV transilluminator. GelRed fluoresces under UV light, allowing for easy visualization of DNA bands.

---

# **Expression**

## Protein expression

## Materials:

* Plasmid containing the protein of interest
* E. coli strain BW25113
* Antibiotic selection plates (ampicillin)
* TB medium (Terrific Broth)
* TB salt
* 10% arabinose
* Spectrophotometer (measuring OD at 600 nm)
* Shaking incubator

## Procedure:

1. **Plasmid Transformation and Overnight Culture:**
   * Transform the plasmid containing the protein of interest into BW25113 E. coli cells.
   * Plate the transformed cells on antibiotic selection plates (ampicillin) and incubate overnight at 37°C.
2. **Preparing Overnight Culture:**
   * Pick a single colony from the antibiotic selection plate and inoculate it into 5 ml of TB medium containing TB salt and ampicillin.
   * Incubate the culture overnight at 37°C with shaking.
3. **Scaling Up the Culture:**
   * Transfer the entire overnight culture into 500 ml of TB medium in a larger flask.
4. **Incubation and Growth Monitoring:**
   * Incubate the culture at 37°C with shaking.
   * Continuously measure the optical density (OD) at 600 nm at regular intervals and record the values.
5. **Induction:**
   * When the OD value is between 0.7 and 0.9, induce protein expression.
   * Add 10 ml of 10% arabinose to reach the desired concentration for induction. Adjust the total volume accordingly.
6. **Expressing Protein:**
   * Continue incubation after induction for the desired period at the designated temperature:
   * 37°C or 30°C.
7. **Protein Harvesting:**
   * Harvest the cells by centrifugation at an 14000 rpm and 15 minutes to obtain the cell pellet.

## Bradford

## Materials:

* Bovine Serum Albumin (BSA)
* Distilled water (ddH2O)
* Bradford reagent
* Cuvettes
* Spectrophotometer (set to 595 nm)
* Parafilm

## Procedure:

### Preparation of BSA Standard Curve:

1. **Prepare BSA Standard Solutions:**
   * Dissolve 1 mg of BSA in 1 mL of ddH2O to prepare a 1 mg/mL BSA standard solution.
2. **Prepare Triplicate Standard Samples:**

   | BSA [µg] | BSA Standard [µL] | H2O [µL] |
   | --- | --- | --- |
   | 0 | 0 | 100 |
   | 2 | 2 | 98 |
   | 5 | 5 | 95 |
   | 7 | 7 | 93 |
   | 10 | 10 | 90 |
   | 15 | 15 | 85 |
   | 20 | 20 | 80 |
   | 25 | 25 | 75 |
3. **Prepare Cuvettes:**
   * Pipette the triplicate standard samples directly into cuvettes as per the table.
4. **Spectrophotometer Setup:**
   * Set the spectrophotometer to measure at 595 nm.
5. **Add Bradford Reagent:**
   * Add the Bradford reagent to each cuvette and gently invert the samples using Parafilm.
   * Measure the absorbance at exactly 5 minutes.
6. **Record Absorbance:**
   * Note the absorbance values at 595 nm for each standard sample.

### Preparing Unknown Samples:

1. **Sample Preparation:**
   * Use 100 µL of the diluted sample and add 900 µL of Bradford reagent.
2. **Measurement:**
   * Measure the absorbance at 595 nm following steps 3-5 of the BSA standard curve preparation.
3. **Adjust Sample Volume:**
   * Adjust the used sample volume as needed to stay within the linear range of the standard samples (2-25 µg/mL).

## SDS PAGE

## Materials:

* Protein samples
* 2x SDS loading buffer
* Marker
* SDS-PAGE gel
* SDS running buffer
* Coomassie staining solution
* 20% acetic acid
* Distilled water (ddH2O)
* Electrophoresis apparatus
* Power supply

## Procedure:

1. **Sample Preparation:**
   * Apply approximately 10 µg of protein per sample. If needed, dilute the sample to achieve the desired protein concentration.
2. **Mixing with SDS Loading Buffer:**
   * Mix 7.5 µL of each sample with 7.5 µL of 2x SDS loading buffer.
3. **Boiling:**
   * Boil the sample-buffer mixture at 95°C for 10 minutes.
4. **Applying Marker and Samples:**
   * Apply 5 µL of the marker and 15 µL of each sample mixed with the loading buffer onto the SDS-PAGE gel.
5. **Running SDS-PAGE:**
   * Run the SDS-PAGE at 100 V and 200 mA for 1.5 hours.
6. **Staining with Coomassie:**
   * Stain the gel with Coomassie staining solution for 40 minutes. Ensure the gel is completely covered.
7. **Destaining:**
   * Destain the gel with 20% acetic acid for 60 minutes.
   * Subsequently, destain the gel with distilled water (ddH2O) overnight.
8. **Visualization:**
   * Visualize the protein bands on the gel.
   * Document the results using an appropriate imaging system.

## Genomic DNA Isolation

## Protocol for Isolating Genomic DNA from Pichia pastoris

### Materials:

* Pichia pastoris cells (grown culture)
* TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0)
* Lysis buffer (50 mM Tris-HCl, 50 mM EDTA, 1% SDS, pH 8.0)
* Phenol:chloroform:isoamyl alcohol (25:24:1)
* Chloroform:isoamyl alcohol (24:1)
* Ethanol (ice-cold)
* Isopropanol (ice-cold)
* 70% ethanol (ice-cold)
* Nuclease-free water

### Procedure:

#### 1. Cell Lysis:

* Harvest Pichia pastoris cells and wash with TE buffer.
* Resuspend the cells in lysis buffer and incubate at 65°C for 30-60 minutes.

#### 2. DNA Extraction:

* Extract genomic DNA using phenol:chloroform:isoamyl alcohol (25:24:1) and centrifuge to separate phases.
* Transfer the aqueous phase to a new tube.
* Precipitate DNA with ice-cold ethanol or isopropanol and centrifuge at maximum speed for 10-15 minutes.

#### 3. DNA Wash:

* Discard the supernatant and wash the DNA pellet with 70% ethanol.
* Centrifuge at maximum speed for 5 minutes and carefully remove the ethanol.

#### 4. DNA Resuspension:

* Allow the DNA pellet to air dry briefly and then resuspend in TE buffer or nuclease-free water.
* Incubate at 65°C for 10-15 minutes to aid in resuspension.

#### 5. DNA Quantification:

* Quantify the isolated genomic DNA using a spectrophotometer.

---

# **Monolaurin**

## Monolaurin acid

## Materials:

* n-Hexane
* tert-Butanol
* Lauric Acid
* Candida Antarctica Lipase B (CALB) immobilized on beads
* Glycerol
* Orbital shaker
* Hot water (80°C)
* Volumetric flasks
* Ice bath
* Whatman filter paper
* Glycerol enzyme assay kit (Genaro)
* Spectrophotometer (measuring at 340 nm)

## Procedure:

1. **Preparation of Reaction Mixture:**
   * In a shaking flask (200 ml end volume) with a cap, prepare a mixture containing:
     + 5.21 g (0.13 mol) lauric acid dissolved in hexane
     + 2.3944 g (0.13 mol) glycerol
     + Immobilized lipase (160 mg, do not dissolve in water)
     + Add hexane (or 1:1 hexane/tert-butanol) to make a total volume of 200 ml.
   * Prepare a control without enzyme.
2. **Incubation:**
   * Incubate the mixture using an orbital shaker at 300 rpm/min at two different temperatures: 40°C and 65°C.
3. **Sample Collection:**
   * Take samples (1.5 ml) every 30 minutes and keep them on ice until further measurement or fat extraction.
4. **Hot Water Extraction for Fat Separation:**
   * For samples with high fat content:
     + Weigh the samples into a volumetric flask.
     + Extract with hot water (80°C).
     + Allow the sample solution to cool for fat separation (e.g., 15 min in an ice bath).
     + Fill the volumetric flask up to the mark with water.
     + Filter the aqueous solution before testing using Whatman filter paper.
5. **Sample Dilution:**
   * Dilute the samples 1:100 at the beginning of the measurements to achieve a linear range of 0.04 to 0.4 g/l.
6. **Spectrophotometric Measurement:**
   * Measure the samples at 340 nm using a spectrophotometer.
7. **Glycerol Enzyme Assay:**
   * Perform the glycerol enzyme assay using the Glycerol enzyme assay kit, following the provided manual.

---

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

![description banner](https://static.igem.wiki/teams/4575/wiki/results.png)

## Goals of our project

#### Enzymes

* Cloning of a plasmid for each enzyme that allows for genome integration and expression as well as secretion of the produced enzymes in Pichia pastoris
* Expression in Pichia pastoris
* Purification of secreted enzymes
* Validation of enzyme activity and function as well as investigation of potency
* Investigation of cytotoxicity of enzymes

#### Peptides

* Cloning of the AMP DNA fragments into the pBAD18 plasmid
* Investigation of the cloning
* Transformation in an L-arabinose deficient *E. coli* strain
* Expression in BW25113 E. coli strain (L-arabinose deficient *E. coli* strain)
* Verification of successful expression by SDS-PAGE and fluorescence measurement
* Purification of the expressed AMP by affinity chromatography
* Verification of successful purification by SDS-PAGE
* Digestion of the purified AMPs by enterokinase, bromocyanine and carboxypeptidase A
* Validation of the activity of AMP against *S. mutans*
* Cytotoxicity assay of AMP against eukaryotic cells

#### Monolaurin

* Synthesis of monolaurin from lauric acid and glycerol through an enzymatic reaction
* Validation of antimicrobial activity and function as well as investigation of potency

## Enzymes results summary

We generated the necessary fragments with overhangs for Gibson assembly. These fragments were employed to perform multiple Gibson assemblies with various molar excess ratios and vectors. We tried to verify our desired plasmid sequences through Sanger sequencing but failed to verify certain sequences. Especially the His4 gene sequence required for genome integration and selection of desired Pichia pastoris clones.

We tried Biobrick assemblies as well, but discontinued them due to the perceived low probability of obtaining positive colonies.

As another alternative to Gibson assembly, we tried linkage PCRs which did not produce the correct products.

From these results, we conclude that the cloning strategy must be changed. The assemblies with 3-4 fragments per assembly are inefficient and can be optimized by choosing a vector that already contains the His4 sequence.

## Enzymes results

Our first goal was to generate overhangs on our ordered fragments: Dextranase expression cassette fragment 1 (Dex1), Dextranase expression cassette fragment 2 (Dex2), Mutanase expression cassette (Mut), Dispersin B expression cassette (Dis), His4 and a part of plasmid BBF10K\_000181 from the distribution kit (pBB).

The PCRs of pBB, Dex2, Mut and the His4 gene were successful without any further troubleshooting or modifications (Figures 1-3).

![pBB Gel](https://static.igem.wiki/teams/4575/wiki/pichia-vektor-gel.jpg)

![Dex2 Gel](https://static.igem.wiki/teams/4575/wiki/results-figure2.png)

***Figure 2: Succesful PCR to introduce overhangs for Gibson assembly to the Dextranase expression cassette fragment 2.*** Gel was stained with MIDORIGreen advance DNA stain (NIPPON Genetics). M1/M2 = 1 kb Plus DNA Ladder (New England Biolabs).

![Mut Gel](https://static.igem.wiki/teams/4575/wiki/gel-mut-ohne-beschriftung.jpg)

***Figure 3: Succesful PCRs to introduce overhangs for Gibson assembly to the Mutanase expression cassette and His4 gene.***  The gel was stained with MIDORIGreen advance DNA stain (NIPPON Genetics). M = 1 kb Plus DNA Ladder (New England Biolabs), Mut = Mutanase expression casette, His4 = His4 gene.

However, the following gel-extraction for Mut and His4 did not provide the needed yield and the PCRs needed to be repeated. For Mut, the repetition and pooled gel extraction, of several PCR samples, worked. For the His4 gene, the PCR did suddenly not work anymore under the initially successful conditions, and some further troubleshooting had to be done.

Different primer annealing temperatures, the addition of DMSO in various concentrations, GC-enhancer in different concentrations and more cycles as well as longer elongation times were tried. In addition, the integrity of our His4-template was also verified through a PCR with primers with the same annealing sequence but different overhangs. Eventually, new primers with a 3 bp longer annealing sequence were ordered, which resulted in the amplification of the correct product again (Figure 4).

![His4 Gel](https://static.igem.wiki/teams/4575/wiki/gel-his4.jpg)

***Figure 4: Succesful PCR to introduce overhangs for Gibson assembly to the His4 gene.***  The gel was stained with MIDORIGreen advance DNA stain (NIPPON Genetics). M = 1 kb Plus DNA Ladder (New England Biolabs), His4 = His4 gene, new = new His4-primer set, old = old His4-primer set.

This time, PCR clean-up was performed instead of gel extraction and provided sufficient yields.

The PCRs for the Dex1 and Dis fragments were accomplished by exchanging forward and reverse primer of the Dis-PCR as well as the forward primer of the Dex1-PCR with Mut primers, which have a similar annealing sequence (Figures 5-6).

![Dis Gel](https://static.igem.wiki/teams/4575/wiki/gel-dis.jpg)

***Figure 5: Succesful PCR to introduce overhangs for Gibson assembly to the Dispersin B expression cassette.***  The gel was stained with MIDORIGreen advance DNA stain (NIPPON Genetics). M = 1 kb Plus DNA Ladder (New England Biolabs), Dis = Dispersin B expression casette.

![Dis Gel](https://static.igem.wiki/teams/4575/wiki/gel-dex1.jpg)

***Figure 6: Succesful PCRs to introduce overhangs for Gibson assembly to the Dextranase expression cassette fragment 1.***  PCRs were carried out with Mut forward or Dex1 forward primer at different primer annealing temperatures. Additionally, a sample of the IDT stock of Dex1 was loaded on the gel. The gel was stained with MIDORIGreen advance DNA stain (NIPPON Genetics). M = 1 kb Plus DNA Ladder (New England Biolabs), Dex = Dextranase expression cassette fragment 1.

The generated fragments with overhangs should be assembled into three distinct plasmids, one for each enzyme, using Gibson assembly (Figure 7).

![](https://static.igem.wiki/teams/4575/wiki/a.jpg)
![](https://static.igem.wiki/teams/4575/wiki/b.jpg)
![](https://static.igem.wiki/teams/4575/wiki/c.jpg)

***Figure 7: Cloning strategy of Dis-plasmid (A), Mut-plasmid (B) and Dex-plasmid (C).***

We carried out assemblies with different molar ratios which often produced colonies (Figure 8).

![Gibson assemblies](https://static.igem.wiki/teams/4575/wiki/gibson-assemblies-neu.jpg)

***Figure 8: Colonies on LB-agar plates after several Gibson assembly approaches that were transformed in NEB stable E. coli.***  LB-agar plates were supplemented with 50 µg/ml ampicillin. NEB stable E. coli without plasmids were employed as negative control whereas NEB stable E. coli transformed with the pSB1C3 vector were employed as positive control.

Figure 8 shows an exemplary outcome of a Gibson assembly experiment with subsequent transformation. It also demonstrates that in several instances a plasmid with ampicillin resistance was assembled in the Gibson assembly reactions.

These plasmids were investigated to find out if they contain all desired sequences as well as the correct sequences by colony PCR and Sanger sequencing. The colony PCRs rarely displayed bands with the expected size (Figure 9).

![Colony PCR](https://static.igem.wiki/teams/4575/wiki/results-figure9-new.png)

***Figure 9: Colony PCR of Mut- and Dis-plasmids.***  Plasmids contain a part of the pBAD18 vector instead of pBB. + = positive control (normal PCR with DNA fragment from IDT stock). The gel was stained with GelRed Nucleic Acid Gel Stain (Biotium). M = 1 kb Plus DNA Ladder (New England Biolabs).

Figure 9 shows an exemplary colony PCR of Mut- and Dis-plasmids. Only one amplicon (Dis colony 5) displays the correct size. This colony also displayed the correct Dis sequence when sent to Sanger sequencing (Figure 10).

![Seq](https://static.igem.wiki/teams/4575/wiki/dis-col-5-seq.jpg)

***Figure 10: Sanger sequencing of the Dis sequence with forward and reverse primer.***  The red area indicates the accordance of sequencing base calls with the desired sequence.

However, the His4 gene, important for the selection of positive clones and genome integration of the linearized plasmid in Pichia pastoris, could not be detected through Sanger sequencing. The absence of His4 in the plasmid was further validated through a restriction digest with SalI-HF that cleaves specifically at one site in the His4 gene.

As an alternative to Gibson assembly, linkage PCRs were performed. Linkage PCRs are PCR reactions where two DNA fragments with overlapping sequence overhangs can be linked together by using the forward primer of one DNA fragment and the reverse primer of the other DNA fragment.

A linkage PCR was, for example, carried out to link Dex1 and Dex2 together and reduce the four-fragment assembly to a three-fragment assembly. However, the agarose gel did not show a defined band at 3954 bp (Figure 11).

![linkage](https://static.igem.wiki/teams/4575/wiki/dex-linkage.jpg)

***Figure 11: Agarose gel electrophoreses of linkage PCR of Dex1 and Dex2 to link the two DNA fragments together.***  The gel was stained with MIDORIGreen advance DNA stain (NIPPON Genetics). M = 1 kb Plus DNA Ladder (New England Biolabs).

Biobrick assemblies were performed as well, especially to solve the problem of unsuccessful His4 overhang PCRs (see above) that we would have needed for a Gibson assembly. However, this was discontinued due to the perceived low probability of obtaining positive colonies and the eventual success of the His4 overhang PCR.

Moreover, we also transformed promising plasmids into our expression strain Pichia pastoris (Figure 12) and obtained colonies on selective minimal dextrose agar.

![Pichia trafo](https://static.igem.wiki/teams/4575/wiki/pichia-trafo.jpg)

***Figure 12: Colonies on selective minimal dextrose agar plates after transformation with a Dis-plasmid.***  Minimal dextrose agar does not contain Histidin and is therefore selective for His4 deficient Pichia pastoris, which have integrated the linearized plasmid into their genome.

Unfortunately, sequencing did not verify that the sequence of the transformed plasmid was concordant to our desired sequence.

## Summary of Peptide Results

To clone our fragments into the pBAD18 empty vector, it was necessary to create Gibson overhangs on the fragments, which was achieved through PCR. After successful PCR, we conducted Gibson assembly at a vector-to-insert ratio of 1:2.

After cloning, we selected colonies and examined them using colony PCR, restriction digestion analysis, and Sanger sequencing to confirm that they carried the desired DNA construct. Cloning was successful. Subsequently, we transformed the desired DNA construct into L-arabinose-deficient E. coli strain BW25113 and induced expression.

Unfortunately, we encountered significant difficulties with expression and were unable to express our AMPs or AMP variants, as evident in the SDS-PAGE. Unfortunately, we did not have enough time to conduct further expression tests and determine when our products would be expressed.

From these experiences, we learned that we need to change our expression strategy. This includes positive control for expression, which also did not work (BW25113 E. coli strain with a plasmid construct expressing a protein previously expressed by a former graduate student).

## Peptide Results

## PCR for Gibson Assembly

To perform the Gibson assembly, we first had to generate fragments with specific overhangs. For this purpose, we conducted PCR with the AMP fragments and the 3'- and 5'-AMP overhang primers, as well as with the empty pBAD18 vector and the 3'- and 5'-pBAD18 overhang primers. Figure 13 shows the successful PCR results for AMP-0, and Figure 14 shows the successful PCR results for AMP-1 and AMP-2.

![BB41 Gel](https://static.igem.wiki/teams/4575/wiki/20230803-bb41.png)

***Figure 13: Agarose gel electrophoresis showing the desired AMP-0 band with overhangs using the AMP overhang primer and the His4 overhang primer.***  The gel was stained with MIDORIGreen advance DNA stain (NIPPON Genetics). M = 1 kb Plus DNA ladder (New England Biolabs), S1 = BB41 with AMP primer. S2 = BB41 with His4 Primer.

![AMP1undAMo2 Gel](https://static.igem.wiki/teams/4575/wiki/results-figure14.png)

***Figure 14: Agarose gel electrophoresis showing the desired AMP-1 and AMP-2 bands with overhangs using the AMP overhang primer.***  The gel was stained with MIDORIGreen advance DNA stain (NIPPON Genetics). M = 1 kb Plus DNA ladder (New England Biolabs).

A volume of 10 µl of the PCR products was loaded onto the gel, and the rest was used for PCR purification. We consistently obtained 2-3 ng/µl PCR-DNA per 1 µl PCR product with almost always excellent A260/A280- and A260/A230-purity values.

After successful amplification, we proceeded with the cloning. For cloning, we chose the Gibson Assembly method, starting with a vector-to-insert ratio of 1:2, as shown in Table 1, listing the required DNA concentrations for the various constructs. The transformation was successful, and numerous colonies were observed on ampicillin-containing plates.

Gibson Assembly with NEBuilder for AMP-0, AMP-1, AMP-2 in pBAD18

| Sample | Amount for Gibson [ng] | Volume Sample [µl] | Water | NEBuilder |
| --- | --- | --- | --- | --- |
| AMP0 | 18.77 | x0 | 10-x0 | 10 |
| AMP1 | 19.63 | x1 | 10-x1 | 10 |
||  |  |  |  |  |
| --- | --- | --- | --- | --- |
| AMP2 | 45.47 x2 | 10-x2 | 10 | |
| pBAD18 Vektor | 75 | 0.80 |  |  |

From these colonies, we examined some using colony PCR with NaOH, and the presumably correct clones were submitted for sequencing. As seen in Figures 16 and 17, the cloning was successful for AMP-0, AMP-1, and AMP-2.

![Restriction AMP Gel](https://static.igem.wiki/teams/4575/wiki/results-figure16.png)

***Figure 16: Restriction digestion analysis of pBAD18-AMP-0 plasmids.***  Two approaches with two different NEBuilder versions were used, one marked with Jana and the other with Sumo. The gel was stained with MIDORIGreen advance DNA stain (NIPPON Genetics). M = 1 kb Plus DNA ladder (New England Biolabs).

Figure 16: Restriction digestion analysis of pBAD18-AMP-0 plasmids. Two approaches with two different NEBuilder versions were used, one marked with Jana and the other with Sumo. The gel was stained with MIDORIGreen advance DNA stain (NIPPON Genetics). M = 1 kb Plus DNA ladder (New England Biolabs).

As seen in Figure 16, there are several samples with the correct band patterns. Considering the numerous samples with correct band patterns, two samples were submitted for Sanger sequencing, and their results are shown in Figure 17.

![Seq AMP-0](https://static.igem.wiki/teams/4575/wiki/20230803-seq-bb41-neu.png)

***Figure 17: Sanger sequencing of the AMP-0 sequence with forward and reverse primers.***  The red area indicates the alignment of the sequencing bases with the desired sequence.

As seen in Figure 17, the cloning of AMP-0 into pBAD18 was successful.

## Cloning of AMP-1 and AMP-2 in pBAD18 was also successful, as indicated in Figures 18 and 19.

![Colony-PCR AMP-1 AMP-2](https://static.igem.wiki/teams/4575/wiki/results-figure18.png)

***Figure 18: Colony PCR of pBAD18-AMP-1 and -AMP-2 plasmids.***  + = positive control (normal PCR with DNA fragment from IDT strain). The gel was stained with GelRed Nucleic Acid Gel Stain (Biotium). M = 1 kb Plus DNA ladder (New England Biolabs).

As shown in Figure 18, almost all colonies on the AMP2 Gibson Assembly LB plates exhibited the desired construct, and the same was observed for the AMP1 plate. However, due to the similarity in size between the plasmid region removed by PCR and the AMP-1 fragment, it was difficult to directly determine the success of cloning for AMP1 from the gel. Three samples per AMP were sent for Sanger sequencing, confirming successful cloning. The results of the Sanger sequencing are presented in Figure 19.

![](https://static.igem.wiki/teams/4575/wiki/amp1-seq.png)
![](https://static.igem.wiki/teams/4575/wiki/seq-amp2-neu.png)

***Figure 19:
(A) Sanger sequencing of the AMP-1 sequence with forward and reverse primers.***  The red area indicates the alignment of the sequencing bases with the desired sequence.
 **(B) Sanger sequencing of the AMP-2 sequence with forward and reverse primers.** The red area shows the alignment of sequencing bases calls with the desired sequence.

Figure 19:
(A) Sanger sequencing of the AMP-1 sequence with forward and reverse primers. The red area indicates the alignment of sequencing bases with the desired sequence.
(B) Sanger sequencing of the AMP-2 sequence with forward and reverse primers. The red area shows the alignment of sequencing bases calls with the desired sequence.

For the AMP-1 samples, it appears that all examined colonies carry the desired construct. The same applies to the AMP-2 samples. In one colony of the AMP-2 samples, however, we identified a mutation in our GFP open frame, leading to the exclusion of this colony from subsequent experiments.

After successful cloning, we transformed and expressed two colonies of each construct in the BW25113 strains.

During expression, our cells grew well with the constructs, as shown in Figure 20.

![Expression AMP](https://static.igem.wiki/teams/4575/wiki/20230920-ampexpression-1.png)

***Figure 20: Growth curve of the expressed cells.***  The expression was performed in triplicate technical execution with AMP2 Colony 6, AMP1 Colony 7, wild-type BW25113 strain, and pSB1C3-GFP-BW25113. The optical density (OD600) of the samples was measured continuously during expression. The expression was induced by adding 5 ml of a 2% arabinose solution at an OD600 of 0.4-0.6. Expression started at 07:00 AM, induction occurred at 01:30 PM, and expression ended at 09:30 PM.

Fluorescence was also measured during expression. Unfortunately, the values remained constant throughout the experiment, leading us to believe that this expression was not successful. Therefore, another expression experiment was conducted with BW25113, BW25113-AMP-2, and BW25113-pSB1C3-GFP, using different L-arabinose concentrations to induce the cells. In this experiment, only fluorescence was measured, and the values are listed in Table 2. This experiment also investigated whether the use of E. coli strain BW25113 resulted in a difference in expression compared to stable NEB cells.

Table: Fluorescence values of the various expressed cells with different L-arabinose concentrations. B stands for BW25113; N for NEB Stable; 1 for 0.02% L-arabinose; 2 for 0.5% L-arabinose; 3 for 1% L-arabinose; 4 for 1.5% L-arabinose; 5 for 2% L-arabinose.

As shown in Table 2, the fluorescence value of the wild-type BW25113 remained unchanged. In the GFP positive control, the value increased at the end and showed a 27-fold change after normalization, both in BW25113-pSB1C3-GFP cells and NEB-stable-pSB1C3-GFP cells. In contrast, the other samples, after normalization, showed about a 2.5-fold change compared to BW25113 wild-type in the BW25113 samples and about a 3.5-fold change in the stable NEB samples.

As we obtained such minor fold changes, we attempted to examine the expression of GFP, AMP-0, AMP-1, and AMP-2 by SDS-PAGE. The results of the SDS-PAGE are shown in Figure 21.

![SDS-PAGE1](https://static.igem.wiki/teams/4575/wiki/20231010-sdspage-1.png)
![SDS-PAGE2](https://static.igem.wiki/teams/4575/wiki/20231010-sdspage-2.png)

***Figure 21: SDS-PAGE.***  The schematic representation shows that 20 µg of protein was applied everywhere, and the marker used is Roth's Tricolor.

As seen in Figure 21(A) and Figure 21(B), neither overexpression was observed in the samples nor in the positive control. The positive control consists of BW25113 cells that received a pBAD18 plasmid successfully expressed by a former doctoral student at the Institute of Technical Biochemistry, University of Stuttgart.

Therefore, we believe that our expression protocol or approach could be incorrect, leading to the lack of expression. Unfortunately, we do not have the time to investigate this further and conduct additional expression experiments.

## Monolaurin results summary

We attempted to synthesize monolaurin (glycerol monolaurate) through a condensation reaction of lauric acid and glycerol catalysed by *Candida antarctica* lipase B. Due to the incompatibility of our analytical method with the experimental design, the experiment had to be stopped.

We conclude that the experimental design and the analytical method need to be made compatible. For instance, by finding a way to measure the decrease of lauric acid instead of glycerol or detecting monolaurin, for instance, via high-pressure liquid chromatography (HPLC).

## Monolaurin results

The synthesis of monolaurin was attempted by reverting the hydrolysis reaction that the Candida antarctica lipase B (CALB) and other lipases usually catalyse. We wanted to realize this by performing the reaction, without any water, in the non-polar solvent n-hexane, as it has been done before in literature [1]. Sadly, we were not able to perform our analytical method of choice, an HPLC, due to time limitations and safety concerns. Therefore, we searched for alternatives and thought we could utilize an enzymatic assay that measures glycerol concentrations through an enzymatic cascade that produces NADH. The absorption of NADH can be measured at 340 nm and thereby calculated back to the glycerol concentration. By measuring the decrease of the substrate, we wanted to show that the reaction had taken place as well as classify the product yield in terms of size.

Unfortunately, we did not think about the issue of glycerol insolubility in our solvent n-hexane. Thus, we observed the formation of insoluble complexes of glycerol and CALB (Figure 22).

![Insoluble glycerol](https://static.igem.wiki/teams/4575/wiki/results-glycerol-figure13.jpg)

***Figure 22: Insoluble complexes of glycerol and CALB in n-hexane.***

Moreover, the measured initial absorptions of NADH at 340 nm resulted in a negative glycerol concentration (Table 1).

***Table 1: Initial measurement of NADH absorption/glycerol concentration for the enzymatic conversion of lauric acid and glycerol to monolaurin.***  The blank consisted of enzyme assay kit buffer, enzyme assay kit enzymes and water. The absorption at 340 nm was measured after the pre-reaction (A1) and after completion of the whole enzymatic assay (A2). ΔA is defined as the difference between sample and blank whereas sample and blank values are the difference of A1 and A2. Glycerol concentration can be calculated through the following formula: 2.781/ε ΔA F.

![Glycerol assay table](https://static.igem.wiki/teams/4575/wiki/glycerol-assay-table.jpg)

## Future plans for the project

Regarding the enzyme section of our project, it would probably be reasonable to start from the beginning with a more efficient cloning strategy. If the new cloning strategy were successful, we would be able to continue with the expression and purification as well as the proof-of-concept part of the project.

Regarding the monolaurin section of the project, the experiment could be repeated when access to an HPLC or gas chromatography (GC) device is provided and monolaurin can then be detected through a chromatographic method. The solution could be utilized for proof-of-concept experiments.

## **References**

---

[1] Pereira, C.C.B., da Silva, M.A.P. & Langone, M.A.P. Enzymatic synthesis of monolaurin. Appl Biochem Biotechnol 114, 433–445 (2004). https://doi.org/10.1385/ABAB:114:1-3:433

{% endblock %}