iGEM 2024 — Team hamburg

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block page\_content %}

# Notebook

Welcome to our projects’ brain

# Resilin

# 05

# 06

# 07

# 08

# 09

# 10

### May

* 06.05.2024
* 07.05.2024
* 14.05.2024
* 15.05.2024
* 17.05.2024
* 18.05.2024
* 27.05.2024

##### 06.05.2024

### Preparing competent DH5α-cells

**Starting culture**

* Inoculate 5 mL of LB-medium with DH5α
* Incubate at 37 °C, 160 rpm overnight

**Aim:** Obtaining competent cells to transform with our plasmid.

##### 07.05.2024

### Harvesting of competent DH5α cells

* competent cells where prepared following protocol 1 [Preparing Competent Cells](experiments#zymogen-header) with the overnight culture from 06.05.2024
* 50/100 µL Aliquots of the cell suspension where stored in sterile 1,5 mL tubes and frozen at -80 °C

##### 14.05.2024

### Testing and transformation of competent DH5α cells

* A tansformation of the competent DH5α cells with pMK1 was carried out following protocol 2 [Transformation](experiments#transformation)
* The transformed DHF5α cells where plated on LB agar plates containing ampicilin (amp.; final concentration = 100 µg/mL) as pMK1 contains an amp. resistance incubated at 37 °C over night
* As positive control, cells were plated on LB agar plates without amp., as negative control DH5α cells without the plasmid on LB + amp. plates

**Interpretation:**- If colonies grow on the positive control, we have living bacteria
- If colonies grow on the LB + amp. plates with the non-transformed cells, the antibiotic is not working
- If no colonies grow on the LB + amp. plates with the transformed bacteria, the transformation was not successful
- If colonies grow on the LP + amp. plates with the transformed DH5α cells, the transformation was successful

##### 15.05.2024

### Competent DH5α cells

Colonies were growing on both the positive control and the LB + amp. plates with the transformed cells!

* This means we have successfully produced competent cells and could transform them with a plasmid.

##### 17.05.2024

### Top10 and BL21DE3 competent cells for the hyaloronic acid and resilin expression

**Starting culture**

* 5mL of LB-Medium + Top10 and BL21DE3 were incubated at 37°C, 160rpm over night

##### 18.05.2024

### Competent cells

* competent cells were made using the protocol 1. (Competent cells (Zymogen))
* cells were harvested at an OD as shown in the table below

|  |  |  |
| --- | --- | --- |
|  | BL21 | Top10 |
| OD | 0,03 | 0,033 |

##### 27.05.2024

### Cloning of RE4 into pMK1 with SacI and NdeI

Cloning of the pMK1 vector with a restriction ligation using enzymes SacI and NdeI to incorporate the fourfold resilin repeat (RE4).

Protocol 3 [Digest with Restriction Enzymes](experiments#restriction) was used for the double digestion of pMK1.

**Restriction mix contained:**

|  |  |
| --- | --- |
| Component | Volume |
| DNA | 1 µg |
| 10X rCutSmart Buffer | 5 µL |
| NdeI | 0.5 µL |
| SacI-HF | 0.5 µL |
| Nuclease-free Water | ad 50 µL |

Incubation: 37 °C for 1 h

### June

* 01.06.2024
* 05.06.2024
* 06.06.2024
* 07.06.2024
* 10.06.2024
* 20.06.2024
* 25.06.2024

##### 01.06.2024

### T4 Ligation of RE4 into pMK1

* The ligation was perfomed using protocol 4 [Ligation (T4 Ligase).](experiments#ligation-header) with three different insert:vector ratios: 1:5, 1:3 and 1:1

| Components | Volume |
| --- | --- |
| 10X T4 DNA Ligase Buffer | 2 µL |
| Vector (digested pMK1) | 0.56 µL |
| Insert (digested RE4) | 0.6 (1:5); 0.32 (1:3); 0.13(1:1) |
| T4 DNA Ligase | 1 µL |
| Nuclease-free water | ad 20 µL |

##### 05.06.2024

### Transforming pMK1\_RE4 into DHF5α + Amp.

* A Transformation of DHF5α with pMK1\_RE4 was made using protocol 2 [Transformation](experiments#tranformation-header)

##### 06.06.2024

### Over night culture

* Here grew a few colonies on the 1:3 and 1:5 ratio pMK1\_RE4 plates
* Making an overnight culture of 4 colonies for each plate for a restriction digestion

**Restriction digestion**

* Analytic digestion of the 8 pMK1\_RE4 mini preps were made with SacI and BsaI using the Protocol 3. (Restriction digestion (NEB)).

expected bonds:

* pMK1:
* pMK1\_RE4:

![](https://static.igem.wiki/teams/5077/aaa-hyaluronic-acid/resilin-6-6.svg)

**Analytic restriction digestion of pMK1\_RE4 colonies.** The restriction digestion was used to analyse which colonies contain the pMK1\_RE4 Plasmid.

##### 07.06.2024

### Mini prep of colony 1:3\_3 and 1:3\_ containing pMK1\_RE4

A miniprep of 9 colonies transformed at the 05.06.2024 was performed following the Miniprep protocol 7.

##### 10.06.2024

### Restriction digestion of pMK1\_RE4 and gel purifiaction

* Analytic digestion of pMK1\_RE4 with SacI and NdeI was performed at 37 °C for 1 h using the Protocol 3 [Digestion with Restriction Enzymes](experiments#restriction) with NdeI and SacI.

![](https://static.igem.wiki/teams/5077/notebook-pics/double-digest-pmk1-re4.svg)

**1% agarose gel with NdeI, SacI digested pMK1\_RE4.** pMK1(Left) and pMK1\_RE4(right); pMK1 was used as positive control; P Ladder: 1 kb Plus. The two restriction sites are functional, but no pMK1\_RE4 fragment at 3338bp.

##### 20.06.2024

### Site directed mutagenesis pMK1\_RE4 to remove the DpnI restricition side in the ampicillin sequence

* Site directed mutagenesis was done using the protocol 5.1 [PCR - Q5 Polymerase from NEB](experiments#pcr-header) with following primers and templates:

|  |  |
| --- | --- |
| **Primer** | FW\_pMK1\_RE4\_G->C |
|  | RW\_pMK1\_RE4\_G->C |
| **Template DNA** | pMK1\_RE4\_1:5\_2 |
|  | pMK1\_RE4\_1:5\_3 |
|  | pMK1\_RE4\_1:3\_2 |
|  | pMK1\_RE4\_1:3\_4 |

![](https://static.igem.wiki/teams/5077/notebook-pics/sitedirectedmutagenesis.png)

**Site directed mutagenesis of pMK1\_RE4..** The Site directed mutagenesis was performed on the plasmids from the four colonies pMK1\_RE4-1:5\_2, 1:5\_3, 1:5\_4 and 1:3\_4 shown in the red boxes; Ladder: 1 kb Plus; Agarose gel (1 %). In the gel only the band of the fragment is visible.

##### 25.06.2024

### Quick change mutagenesis PCR ([Q5 Polymerase Protokoll](experiments#pcr-header) with pMK1\_RE4

Templates used:

* (1)pMK1\_RE4 5:1\_3
* (2)pMK1\_RE4 5:1\_4
* (3)pMK1\_RE4 3:1\_3
* (4)pMK1\_RE4 3:1\_4

Thermocycling conditions:

| Step | Temperature [°C] | Time [s] | Cycles |
| --- | --- | --- | --- |
| annealing | 69 | 30 | 30 |
| elongation | 72 | >100 | 30 |

### July

* 01.07.2024
* 02.07.2024
* 04.07.2024
* 09.07.2024
* 10.07.2024
* 19.07.2024
* 24.07.2024
* 28.07.2024
* 31.07.2024

##### 01.07.2024

### TOP10 & BL21\* Competent cell testing

* Transformation of pMK1 to TOP10 & BL21 competent cells folllowing protocol 2.
* Neg control: plate with 100µL just bacteria + water

##### 02.07.2024

### Competence Test

Results of the competence test: Transformation of pMK1 into TOP10 was successful, but BL21 bacteria did not grow

##### 04.07.2024

### Doing a gradient PCR for Quick Change mutagenesis

Repeating the Quick Change mutagenesis using a longer elongation time and a temperature gradient to find the perfect annealing temperature.

template:

| Primer | Amount |
| --- | --- |
| FW\_pMK1\_RE4\_G->C | 0.5 µl |
| RW\_pMK1\_RE4\_G->C | 0.5 µl |
| **Template DNA:** |  |
| pMK1\_RE4\_1:3\_4 | 2 ng |
| Nuclease-Free Water | add to 10 µl |

Thermocycling Conditions:

|  |  |  |  |
| --- | --- | --- | --- |
| STEP | Temp | Time | Cyles |
| annealing (gradient) | 50°C | 30 seconds | 30 |
| elongation | 72°C | 120 seconds | 30 |

For annealing temperature:

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| lane | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| sample | DNA Ladder | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | DNA Ladder |
| annealing temp. | - | 50 °C | 53 °C | 56 °C | 59 °C | 63 °C | 66 °C | 69 °C | 72 °C | - |

Elongation time was 120 seconds

**Results:**
The lids popped of, so there was only one PCR to be seen in the gel (lane3).

![](https://static.igem.wiki/teams/5077/aaa-hyaluronic-acid/resilin-4-7.webp)

**Site directed mutagenesis of pMK1\_RE4.**
A new PCR with a gradient of different Annealing temperatures (see table above) and a longer elongation time. The Site directed mutagenesis was performed on the plasmid pMK1\_RE4-1:3\_4; Ladder: 1 kb Plus; Agarose gel (1 %).

**Problem:**
The lids were not closed properly

**Solution:**

* we ordered new primers which should bind better
* we also ordered primes to remove the second BsmBI-Binding sequence behind the poly-A tale
* we would also try a new method to generate repeats called the rolling circle method where circular DNA containing only 4 repeats of resilin is generated. Than a phi29 polymerase will circulate around this circular DNA generating more repeats.

##### 09.07.2024

### Trying a new approach Generating a resilin Fragment with four repeats for the rolling circle method.

* Double digestion with BsmBI and BsaI was performed on pMK1\_RE4 according to Protocol 3. (Restriction digestion (NEB)).
* Heat inactivation of the resticiton enzyme by heating it to 80 °C for 20 min

##### 10.07.2024

### Restriction digestion

* Double digestion with BsmBI and BsaI was performed on pMK1\_RE4 according to Protocol 3. (Restriction digestion (NEB))
* an agarose gel was made follwing the 8. protocol (Agarose gel)

expected bonds: 1775 bp, 1382 bp, 252 bp and 180 bp (the fragment with the four resilin repeats)

![](https://static.igem.wiki/teams/5077/aaa-hyaluronic-acid/resilin-10-7.svg)

**Restriction digestion of pMK1\_RE4.** To obtain the RE4 Fragment, a double digestion of pMK1\_RE4 with BsmBI and BsaI, 2% agarose gel.

**Problem:**
1775 bp and the 1382 bp can be seen in the gel, but not the two small ones.

##### 19.07.2024

### Checking the activity of XhoI restriction enzyme

Performing a restriction with XhoI with pMK1 during 1 h at 37 °C

pMK1\_RE4 5:1\_3 =212 ng/µL

so 2,5 µL are about 0,5 ng

| Component | 25 µl Reaction |
| --- | --- |
| DNA | 0.5 µg |
| 10X rCutSmart Buffer | 2.5 µl (1X) |
| XhoI | 0.25 µl (10 units)† |
| Nuclease-free water | to 25 µl = 21.75 |

##### 24.07.2024

### Rolling circle method

Make PCR with new Primers and at the same time try to amplify the RE4 Fragment for the rolling circle method, before cutting it with BsmBI and BsaI.

The PCRs was performed using the 5.1 protocol (Q5 Mastermix from NEB).

The reactions containd the Primers and templates as shown in the Table below and the elontation time was 185 seconds

| Reaction | Template | Primer |
| --- | --- | --- |
| BsaI | pMK1\_RE4\_1:3\_4 | FW\_Mut2\_BsaI\_pMK1\_RE4 |
| BsaI | pMK1\_RE4\_1:3\_4 | RW\_Mut2\_BsaI\_pMK1\_RE4 |
| BsmBI | pMK1-RE4 DNA | FW\_Mut2\_BsmBI\_pMK1\_RE4 |
| BsmBI | pMK1-RE4 DNA | RW\_Mut2\_BsmBI\_pMK1\_RE4 |
| RE4 Amplification | pMK1-RE4 | FW\_pMK1\_RE4\_ColPCR |
| RE4 Amplification | pMK1-RE4 | rev\_pMK1\_RE4\_ColPCR |

* The PCR tubes were put in a Thermocycler with a temperature gadient show in the Table below

![](https://static.igem.wiki/teams/5077/aaa-hyaluronic-acid/resilin-24-7.webp)

**PCR Amplification of pMK1.** Ladder: 1 kb Plus; Agarose gel (1%).

| Temperature [°C] | Probe | Probe | Probe | Probe |
| --- | --- | --- | --- | --- |
| 65 | Bsal (1a) |  |  |  |
| 63.4 | Bsal (1b) |  |  |  |
| 60.9 | Bsal (1c) |  |  |  |
| 57.1 | Bsal (1d) |  | PCR RE4 Amplification (4a) | PCR RE4 Amplification (4c) |
| 52.6 |  | BsmBI (2a) | PCR RE4 Amplification (4b) | PCR RE4 Amplification (4d) |
| 48.8 |  | BsmBI (2b) |  |  |
| 46.4 |  | BsmBI (2c) |  |  |
| 45 |  | BsmBI (2d) |  |  |

##### 28.07.2024

### Resilin production with the Reaptigo method

**DNA - concentrations**
Oligo 1 fw: concentration 1,3876 g/L

* for 4 µg we need 2,883 µL

Oligo 2 rev: concentration 1,3865 g/L

* for 4 µg we need 2,885 µL

 **Ndel fw - ratios**
NdeI fw concentration: 0,6436 g/L

* 32:1 Ratio: 0.0584 µg
  + we take 9.1 µL from the dilution (1:100, c=0.006436 g/L)
* 64:1 Ratio: 0.0292 µg
  + 4.56 µL from the 1:100 dilution
* 124:1 Ratio: 0.0151 µg
  + 2.36 µL from the 1:100 dilution

**Ndel fw - ratios**
NdeI rev concentration: 1,3308 g/L → dilution 1:100 c=0,01333 g/L

* 32:1 Ratio: 0.1219 µg
  + 9,16 µL from the 1:100 dilution
* 64:1 Ratio: 0.0609 µg
  + 4,75 µL from the 1:100 dilution
* 124:1 Ratio: 0.0315 µg
  + 2,37 µL from the 1:100 dilution

**Sac1 fw - ratios**
Sac1 fw concentration: 0,72424 g/L 1 :100 dilution c=0,00724 g/L

* 32:1 Ratio: 0.0723 µg
  + 9,98 µL from the 1:100 dilution
* 64:1 Ratio: 0.0361 µg
  + 4,98 µL from the 1:100 dilution
* 124:1 Ratio: 0.0187 µg
  + 2,58 µL from the 1:100 dilution

**Sac1 rev- ratios**
Sac1 rev concentration:  1,40187 g/L 1:100 dilution c=0,01402 g/L

* 32:1 Ratio: 0.1333 µg
  + 9,51 µL from 1:100 dilution
* 64:1 Ratio: 0.0667 µg
  + 4,76 µL from 1:100 dilution
* 124:1 Ratio: 0.0344 µg
  + 2,46 µL from 1:100 dilution

**DNA-Annealing:**
1. Prepare Oligos:

* Centrifuge dried DNA
* Dilution of dried DNA oligos to 100µM with Dnase, Rnase free water and vortexed for 20 sec
* After incuation at RT for 30 min centrifuge it for 1 min

2. Annealing:

* Prepare annealing mixture with the desired ratio of the same Oligos
  1. NdeI start fw + NdeI start rev
  2. Oligo 1 fw + Oligo 2 rev
  3. SacI end fw + SacI end rev
* Heat the mixtures to 90 °C for 5 min
* Gradually cool the mixture to room temperature over 12 min in the PCR block

##### 31.07.2024

### Resilin production with the Reaptigo method II

**Ligation of annealed Oligos:**

* Start & Middle A-Oligos will be ligated first
* 31:1 Start 16,26 µL + Middle 5,76 µL
* 64:1 Start 9,13 µL + Middle 5,76 µL
* 128:1 Start 4,73 µL + Middle 5,76 µL
* Add 2 μl T4 Ligase puffer
* Fill up to 19µL with Rizvee water (Dnase rnase free)
* Add 1µL T4 ligase
* Pipette up & down & centrifuge shortly
* incubate at 16 °C overnight

### August

* 01.08.2024
* 02.08.2024
* 05.08.2024
* 07.08.2024
* 08.08.2024
* 12.08.2024
* 14.08.2024

##### 01.08.2024

### Checking and repeating Repeatigo Resilin production

**Ligation of annealed Oligos:**

* Add end oligo (31:1 = 19,49 µL , 64:1 = 9,74 µL, 128:1 = 5,05 µL)
* Incubate at RT for 1h
* Heat inactivate at 65 °C for 10 min
* put on ice

Made a 1% agarose Gel to check the fragment lenghts of resilin see picture following protocol 8.

![](https://static.igem.wiki/teams/5077/aaa-hyaluronic-acid/resilin-1-8.svg)

**Repeatigo production of Resilin, with a 31:1, 61:1 and 128:1 ratio of start and middle oligos.**
Ladder: 1 kb Plus; Agarose gel (1 %)

**Result**

* The longest fragments on the 128:1 ratio sample
* The biggest fragment is about 300bp long
* For a 32 repat fragment one would expect about 1,5kb fragments

**Second try Repeatigo resilin production**

* We made two different concentration of the middle Oligos using the same volume
  + One with 2 µg and a volume of 10 µL
  + One with 4 µg and a volume of 2,883 µL

DNA - concentrations
**Tube 1**
Oligo 1 fw: concentration 1,3876 g/L

* For 4 µg we need 2,883µL

Oligo 2 rev: concentration 1,3865 g/L

* For 4 µg we need 2,885µL

NdeI fw concentration: 0,6436 g/L

* **Tube 3** 64:1 ratio: 0.0292 µg
  + 4,56 µL from the 1:100 dilution
* **Tube 4** 128:1 ratio: 0.0151 µg
  + 2,36 µL from the 1:100 dilution
* **Tube 5** 248:1 ratio: 0.0078 µg
  + 1,18 µL from the 1:100 dilution

NdeI rev concentration: 1,3308 g/L → 1 zu 100 verdünnen also c = 0,01333 g/L

* **Tube 3** 64:1 ratio: 0.0609 µg
  + 4,75 µL from the 1:100 dilution
* **Tube 4** 128:1 ratio: 0.0315 µg
  + 2,37 µL from the 1:100 dilution
* **Tube 5** 256:1 ratio: 0.0158 µg
  + 1,19 µL from the 1:100 dilution

Sac1 fw concentration: 0,72424 g/L 1:100 dilution c = 0,00724 g/L

* **Tube 6** 64:1 Ratio: 0.0361 µg
  + 4,98 µL from the 1:100 dilution
* **Tube 7** 128:1 Ratio: 0.0187 µg
  + 2,58 µL from the 1:100 dilution
* **Tube 8** 256:1 Ratio: 0.0935 µg
  + 1,29 µL from the 1:100 dilution

Sac1 rev concentration:  1,40187 g/L 1:100 dilution c = 0,01402 g/L

* **Tube 6** 64:1 Ratio: 0.0667 µg
  + 4,76 µL from the 1:100 dilution
* **Tube 7** 128:1 Ratio: 0.0344 µg
  + 2,46 µL from the 1:100 dilution
* **Tube 8** 256:1 Ratio: 0.0172 µg
  + 1,23 µL  from the 1:100 dilution

##### 02.08.2024

### Ligation of annealed Oligos

With 2 µg or 4 µg middle oligos:

* -> Start & middle A-Oligos will be ligated first
* 64:1 start 9,13 µL + middle 5,76 µL → 2,17 H2O
* 128:1 start 4,73 µL + middle 5,76 µL → 6,57 H2O (we only made one of the middle parts, so that we choose the 128:1 ratio since it workt out the best last time)
* 256:1 start 2,36 µL + middle 5,76 µL → 9 H2O

1. Add 2 μl T4 Ligase buffer
2. Fill up to 19 µL with ddH2O
3. Add 1 µL T4 ligase
4. Pipette up and down and centrifuge shortly
5. Incubate at 16 °C overnight

* add end oligo
* 31:1 ends 19,49 µL
* 64:1 ends 9,74 µL
* 128:1 ends 5,05 µL

1. Incubate at RT for 1 h
2. Heat inactivate at 65 °C for 10 minutes
3. Put on ice

Made a 1 % agarose Gel to check the fragment lenghts of resilin see picture following protocol 8.

![](https://static.igem.wiki/teams/5077/aaa-hyaluronic-acid/resilin-2-8.svg)

**Repitigo production of Resilin and fragment lengths, with 4 µg and 2 µg of middle oligos.**
Ladder: 1 kb Plus; Agarose gel (1 %).

We have the longest fragments in the mixture with the 2 µL of middle oligos. Also the fragments are on average larger than from the first experiment.
**Gel purification:**
Small peaces from the gel of the desired length of the repeating fragements will be cut out of the gel and purified using the protocol 6.1.

##### 05.08.2024

### Gradient PCR of purified gel to check the right annealing temperature for oligos

PCR done according to protocol 5 with a temperature range of 48-56 degrees Celsius during annealing.
Tube 1, 2, 3 and 4: DNA concentration of 0,8 ng/µl
Tube 5, 6, 7 and 8: DNA concentration of 1.3 ng/µl

![](https://static.igem.wiki/teams/5077/aaa-hyaluronic-acid/resilin-5-8.webp)

**Repitigo production of Resilin and fragment lengths, after a gradient PCR from tubes 1-8.**
Ladder: 1 kb Plus; Agarose gel (1 %).

**Results:**
There are no bonds in the gel. This means that either a pipetting error or the endoligos were involved.
A reason could be that the end Oligos didn't bind because there was not addet additianly T4 Ligase or ATPs, which at this point could be degradet or inactive.
**Next step:** The next step is to repeat the oligo annealing and ligation step to obtain larger fragments and correctly ligate the end and start oligos.

##### 07.08.2024

### Second try Repeatigo resilin production

* We use 4 µg of mid Oligos and up to 20 µL with water, so we have the same concentration as with the experiment that worked out well.
* Then we use two different times for the Oligos to anneals while cooling down in the PCR machine. Since changing the time changed the length of the Fragments from about 300 bp to about 1-2 kb Fragments,
* we will use the old concentration (2 µg in 10 µL → 4µg in 20 µL) and we will use a smaller concentration, as reducing the concentration also has made a big difference:
  + sample 1: 4 µg of mid Oligos and up to 20 µL (previous concentration), shorter time = 38 min
  + sample 2: 4 µg of mid Oligos and up to 20 µL, longer time = 68 min
  + sample 3: 2 µg of mid Oligos and up to 20 µL, longer time = 68 min
  + sample 4: 2 µg of mid oligos and up to 20 µL, shorter time = 38 min
* additionally we will use 3 different ratios of middle oligo to start- and end-oligos:
  + 1:64
  + 1:128
  + 1:256

Protocol:

* Middle-Start and End Oligos where pipetted into PCR tubes as shown in the "Resilin\_Oligo\_Pipettier\_volumina"-Excel document.

![](https://static.igem.wiki/teams/5077/aaa-hyaluronic-acid/resilin-8-8.webp)

* for the dimerization the Oligos where heated up to 90 °C and then slowly cooled down to 20 °C in a PCR machine as shown in the table below

| Temperature (°C) | 90°C | 80°C | 75°C | 70°C | 65°C | 60°C | 50°C | 40°C | 30°C | 20°C |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample 1 and 4 | 5 min | 5 min | 3 min | 5 min | 3 min | 5 min | 3 min | 3 min | 3 min | 3 min |
| Sample 2 and 3 | 8 min | 8 min | 5 min | 8 min | 5 min | 8 min | 5 min | 5 min | 5 min | 5 min |

* the start Oligos where then mixed in differnt ratios with the two different concentrations of Mid Oligos. See "Resilin\_Oligo\_Pipettier\_volumina"-Excel document.
* 2 μl T4 Ligase puffer where addet
* 1 µL of T4 Ligase where addet.
* fill up to 20 µL with DNAse free water
* pipette up and down and centrifuge shortly
* the tubes where incubate at 16 °C overnight

→ this resulted in a total of 12 tubes

**The next steps:**
add end oligos and run a gel to investiagte the product

##### 08.08.2024

### Ligation of end oligos

add the same as shown in the "Resilin\_Oligo\_Pipettier\_volumina":

![](https://static.igem.wiki/teams/5077/aaa-hyaluronic-acid/resilin-8-8.webp)

* incubate at RT for 2 h
* Heat inactivate at 65 °C for 10 minutes
* investigate product on a gel

**Results:**
The results can be seen in the gel image. We got some bands close to 3 kb. Also, using 2 µg of the middle olgios and using a longer cooling down time in the annealing step resulted in larger fragments on average.
**Next steps:**

In the next steps, 10 fragments from the gel will be isoalted in the range of 1.5 to 3 kb and the oligos extracted from the gel.
**Gel:**

![](https://static.igem.wiki/teams/5077/aaa-hyaluronic-acid/resilin-8-8-2.webp)

**Gel electrophoresis of annealed oligos in varying concentrations and ratios.**
1 kb Plus DNA ladder, 1 % agarose gel.

##### 12.08.2024

### Repeatigo

Repetition of the oligo annealing
**Concentration after gel extraction:**

| Sample | Concentration [ng/µl] | 260/280 | 260/230 | Start/Stop Oligo Ratio |
| --- | --- | --- | --- | --- |
| Tube 1, 1.5 kb | 8.7 | 1.7 | 0.47 | 1:64 |
| Tube 2, 1.5 kb | 9.3 | 1.59 | 0.42 | 1:64 |
| Tube 3, 1.5 kb | 9 | 1.56 | 0.43 | 1:128 |
| Tube 4, 1.5 kb | 8.6 | 1.72 | 0.43 | 1:128 |
| Tube 5, 1.5 kb | 7.8 | 1.71 | 0.37 | 1:256 |
| Tube 6, 1.5 kb | 10.09 | 1.4 | 0.53 | 1:256 |
| Tube 1, 2 kb | 9 | 1.43 | 0.43 | 1:64 |
| Tube 2, 2 kb | 8 | 1.65 | 0.42 | 1:64 |
| Tube 3, 2 kb | 7.6 | 1.58 | 0.49 | 1:128 |
| Tube 4, 2 kb | 5.5 | 1.71 | 0.37 | 1:128 |
| Tube 5, 2 kb | 12.7 | 1.38 | 0.56 | 1:256 |
| Tube 6, 2 kb | 7 | 1.69 | 0.45 | 1:256 |
| Tube 1, 3 kb | 6.8 | 1.66 | 0.47 | 1:64 |
| Tube 2, 3 kb | 6 | 1.65 | 0.4 | 1:64 |
| Tube 3, 3 kb | 0 | 0 | 0 | 1:128 |
| Tube 4, 3 kb | 7.6 | 1.63 | 0.37 | 1:128 |
| Tube 5, 3 kb | 7.1 | 1.51 | 0.44 | 1:256 |
| Tube 6, 3 kb | 7 | 1.71 | 0.42 | 1:256 |

##### 14.08.2024

### Agarose gel for the Resilin-Oligo-PCR

1 % agarose gel according to protocol 8.

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1kb Plus Ladder | negativ control | tube1 | 10ng of template for tube 1 | tube5 | 10ng of template for tube 5 | tube6 | 10ng of template for tube 6 | 1kb Plus ladder |

![](https://static.igem.wiki/teams/5077/aaa-hyaluronic-acid/resilin-14-8.webp)

**Agarose gel for the Resilin-Oligo-PCR.**
1 kb Plus DNA ladder, 1 % agarose gel.

**Results:**

* the Fragments where exprected at 3 kb.
* as one can see only the primers can bee seen in the gel
* the negativ control cannot be seen, use more than 10 ng

**Trouble shooting:**

* use more than 10 ng of template next time
* One reason that the PCR did not work could be, that
  the T4 ligase has not properly ligated start and end oligos to mid fragments, also it could be that the mid Oligos did not ligate properly

**Next steps:**

* add end and start oligos in ratio 64:1 to the Oligo-Fragments isolated from the PCR-gel and add T4 ligase with ATP buffer.
* test T4 ligase by:
  + resticiton digestion of a Plasmid
  + religation of the fragment using T4 ligase.
  + heat to 95 °C
  + test ligation on a gel

### September

* 05.09.2024
* 07.09.2024
* 18.09.2024
* 19.09.2024
* 20.09.2024
* 25.09.2024
* 26.09.2024
* 27.09.2024
* 28.09.2024
* 29.09.2024
* 30.09.2024

##### 05.09.2024

### Re-Ligation of Start & End Oligos to middle oligos extracted from gel over 2 days

* gel extracted oligo samples were used

+ 2 and 6 from 1,5 kb
+ 1 and 5 from 2 kb
+ 4 and 5 from 3 kb

From each tube we transferred 9 µL. The first 2 µL of SacI\_ rev 1:100 and 2 µL SacI\_fw 1:100 were added and ligated for 1.5 hour at 37 °C. 1 µL T4 Ligase and 2 µL buffer (total volume 20 µL) were added. Then 2µL of NdeI\_fw 1:100 and 2µL NdeI\_rev 1:100 Oligos were added to the ligation mix. The total ligation lasted for 48 h (total volume now 20 µL).

##### 07.09.2024

### PCR with the ligation from 05.09.

All 6 tubes and a positive control prepared.
Mastermix according to protocol 5.1 with 8.75 μl FW\_RE\_Oligo\_amp-Primer and RV\_RE\_Oligo\_amp-Primer each and a total volume of 105 μl. In each PCR tube, 15 μl and 10 μl Template were pipetted.
A positive control was made as follows:

* Q5 High-Fidelity 2X Master Mix: 12,5 μl
* FW\_EGFP\_SLiCE\_PCR: 1,25 μl
* RV\_EGFP\_SLiCE\_PCR: 1,25 μl
* EGFP-Linker: 10 μl

The annealing temperature was 56 °C and the elongation time was 3 min for 30 cycles.
we numbered the PCR tubes from 1-6 and a +

| Number | Description |
| --- | --- |
| 1 | 1,5 kb Religation from 05.09 for 48h |
| 2 | 1,5 kb Religation from 05.09 for 48h |
| 3 | 2 kb Religation from 05.09 for 48h |
| 4 | 2 kb Religation from 05.09 for 48h |
| 5 | 3 kb Religation from 05.09 for 48h |
| 6 | 3 kb Religation from 05.09 for 48h |
| + | Positive control with EGFP+Linker and the respective primers |

The PCR was checked on a 1 % agarose gel (protocol 8).

![](https://static.igem.wiki/teams/5077/aaa-hyaluronic-acid/resilin-7-9.webp)

**Gel electrophoresis of PCR religation of oligos. 1 kb Plus DNA Ladder, 1 % agarose gel.**

##### 18.09.2024

### DNA-Annealing for Repeatigo

1. Calculate Oligos:

* Oligo 1: ca 70 µg DNA --> 50 μl
* Oligo 2: ca 70 µg DNA --> 50 μl
  + Ansatz An-Oligos: 100 μl mit 140 µg DNA von Oligo 1+2
* NdeI fw: ca 40 µg DNA --> V=m/c = 0.00004 g/ 0.6436 g/ L = 62.1 μl
* NdeI rev: ca 40 µg DNA --> V=m/c = 0.00004 g/ 1.3308 g/ L = 30.06 μl
  + Ansatz An-NdeI: 60 μl fw + 30 μl rev = 90 μl mit ca 80 µg DNA von NdeI fw+rev
* SacI fw: a 40 µg DNA --> V=m/c = 0.00004 g/ 0.72424 g/ L = 55.23 μl
* Sac I rev: a 40 µg DNA --> V=m/c = 0.00004 g/ 1.40187 g/ L = 28.53 μl
  + Ansatz An-SacI: 55 μl fw + 29 μl rev = 84 μl mit ca 80 µg DNA aus SacI fw+rev

2. Annealing:

* Prepare annealing mixture with the desired ratio of the same Oligos (Volume, conc, mass above)

1. An-Oligos: Oligo 1 fw + Oligo 2 rev
2. An-NdeI: NdeI start fw + NdeI start rev
3. An-SacI: SacI ende fw + SacI ende rev

* Heat the mixtures to 90°C for 5 minutes.
* Gradually cool the mixture to room temperature over 12 minutes in the PCR block

3. Concentrations:
An-Oligos: 139 µg/ 100 μl = 1.39 µg/ μl
An-NdeI: 80 µg/92.16 μl = 0.868µg/ μl
An-SacI: 80 µg/83.76 μl = 0.955µg/ μl
4. Calculate:

* 3 Ansätze:
  + Lig-1: 64:1 --> 6 µg An-Oligos + 0.09 µg Enden jeweils
  + Lig-2: 32:1 --> 4 µg An-Oligos + 0.12 µg Enden jeweils
  + Lig-3: 10:1 --> 4 µg An-Oligos + 0.4 µg Enden jeweils
  + Lig-4: 2:1 --> 4 µg An-Oligos + 2 µg Enden jeweils

5. Dilute:

* An-NdeI 1:10 --> 0.0868 µg/ μl
* An-SacI 1:10 --> 0.0955 µg/ μl
* Ligation 1: Oligos + NdeI jeweils 20 μl Ansätze
  + Lig-1: 4.32 μl von An-Oligos + 1.03 μl von An-NdeI 1:10 + 11.65 μl nuclease-free water 2 μl Ligase buffer + 1 μl Ligase
  + Lig-2: 2.88 µL von An-Oligos + 1.38 µL von An-NdeI 1:10 + 12.74 µL nuclease-free water + 2 µL Ligase buffer + 1 µL Ligase
  + Lig-3: 2.88 µL von An-Oligos + 4.61 µL von An-NdeI 1:10 + 9.51 µL nuclease-free water + 2 µL Ligase buffer + 1 µL Ligase
  + Lig-4: 2.88 µL von An-Oligos + 2.30 µL von An-NdeI + 11.82 µL nuclease-free water + 2 µL Ligase buffer + 1 µL Ligase

Pipette together + mix gently, Centrifuge shortly, incubate at 25 °C for 2 h

* Ligation 2: add An-SacI jeweils zu den 20 µL Erstansätzen
  + Lig-1: add 0.94 µL von An-SacI 1:10
  + Lig-2: add 1.26 µL von An-SacI 1:10
  + Lig-3: add 4.2 µL von An-SacI 1:10
  + Lig-4: add 2.1 µL von An-SacI

Pipette together + mix gently, incubate at RT over night (18.5 h)

1. Heat inactivate at 65 °C for 10 min
2. put on ice

##### 19.09.2024

### Gel and Gel Clean-up

Ligation 1 to 4 were loaded into a 1% gel with the NEB 1kb plus ladder.

| 1 | 2 | 3 | 4 | 5 | 6 |
| --- | --- | --- | --- | --- | --- |
| Ladder 10 μl | Ligation 1 (24 μl) | Ligation 2 (24 μl) | Ligation 3 (24 μl) | Ligation 4 (24 μl) | Ladder 10 μl |

![](https://static.igem.wiki/teams/5077/aaa-hyaluronic-acid/image-6.webp)

Bands were cut out of the gel and cleaned up according to protocol 6.2.

##### 20.09.2024

### Amplification of the Oligo Fragments

PCR for amplification of the Oligo fragments that have been cleaned the day before and a checkup with a 1 % agarose gel.
A mastermix (10x) was created (protocol 5.2):
In each PCR Tube 48 μl mastermix and 2 μl Template from each cleanuped tubes were pipetted. For the negative control, 2 μl water were used.
PCR was done with 60 °C annealing temperature and 3 min elongation.
The PCR was checked with a 1 % agarose gel. 20 μl PCR-product were mixed with 4 μl loading dye.
The 10 pockets were loaded as followed:

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Lig1 3 kb | Lig1 2 kb | Lig1 1,5 kb | Lig1 1 kb | ladder 1kb | Lig2 3 kb | Lig2 2 kb | Lig2 1,5 kb | Lig4 1 kb | negative control |

![](https://static.igem.wiki/teams/5077/aaa-hyaluronic-acid/resilin-20-9.webp)

Unfortunately the PCR didn´t work.

##### 25.09.2024

### Annealing approach for Resilin via Reapetigo Method

We used the following Oligo reactions:

1. Middle Oligo fw + rev 30 µg
2. Middle Oligo fw + rev 15 µg
3. Middle Oligo fw + rev 0,3 µg
4. Middle Oligo fw + rev & NdeI fw+ rev 30 µg
5. Middle Oligo fw + rev & NdeI fw + rev 15 µg
6. Middle Oligo fw + rev & NedI fw + rev 0,3 µg
7. NdeI fw + rev 1:10. 10X solution
8. SacI fw + rev 1:10. 10X solution

Sample 1-6 were prepared with 40 μl volume:
Sample 1(30 μg):

* Standard Ligation Buffer, 10x conc.: 4 μl
* T4 DNA Ligase: 0.8 μl\*
* Oligo\_1\_repeat\_fw (1:10): 10.8 μl
* Oligo\_2\_repeat\_fw (1:10): 10.8 μl
* Nuclease-free water: 13.6 μl

Sample 2 (15 μg):

* Standard Ligation Buffer, 10x conc.: 4 μl
* T4 DNA Ligase: 0.8 μl\*
* Oligo\_1\_repeat\_fw (1:10): 5.4 μl
* Oligo\_2\_repeat\_fw (1:10): 5.4 μl
* Nuclease-free water: 23.79 μl

Sample 3 (0.3 μg):

* Standard Ligation Buffer, 10x conc.: 4 μl
* T4 DNA Ligase: 0.8 μl\*
* Oligo\_1\_repeat\_fw (1:1000): 10.8 μl
* Oligo\_2\_repeat\_fw (1:1000): 10.8 μl
* Nuclease-free water: 13.6 μl

Sample 4 (30 μg):

* Standard Ligation Buffer, 10x conc.: 4 μl
* T4 DNA Ligase: 0.8 μl\*
* Oligo\_1\_repeat\_fw (1:10): 10.8 μl
* Oligo\_2\_repeat\_fw (1:10): 10.8 μl
* NdeI\_fw (1:10): 1.7 μl
* NdeI\_rev (1:10): 1.67 μl
* Nuclease-free water: 10.23 μl

Sample 5 (15 μg):

* Standard Ligation Buffer, 10x conc.: 4 μl
* T4 DNA Ligase: 0.8 μl\*
* Oligo\_1\_repeat\_fw (1:10): 5.4 μl
* Oligo\_2\_repeat\_fw (1:10): 5.4 μl
* NdeI\_fw (1:10): 0.85 μl
* NdeI\_rev (1:10): 0.84 μl
* Nuclease-free water: 22.1 μl

Sample 6 (0.3 μg):

* Standard Ligation Buffer, 10x conc.: 4 μl
* T4 DNA Ligase: 0.8 μl\*
* Oligo\_1\_repeat\_fw (1:1000): 10.8 μl
* Oligo\_2\_repeat\_fw (1:1000): 10.8 μl
* NdeI\_fw (1:1000): 1.7 μl
* NdeI\_rev (1:1000):1.67 μl
* Nuclease-free water: 10.23 μl
* added after the 55 min annealing time

The samples were put into the thermocycler and heated up to 95 °C. Over 55 min they were cooled down to 20 °C to anneal correctly. After that, the T4 DNA Ligase was added to the samples and incubated overnight at 16 °C.

##### 26.09.2024

### Adding the end and start oligos to the Ligation from 25.09.2024

The start and end oligos were diluted by the factor 100 to create a 1:1000 dilution.
Tube 9: NdeI fw + rev 1:1000
Tube 10: SacI fw + rev 1:1000
The Start and End Oligos were added to the tubes 1-6 as followed:

| tube number | Start Oligo (NdeI fw + rev) | End Oligo (SacI fw + rev) |
| --- | --- | --- |
| 1 | 3.37 μl (1:10 dilution (tube 7)) | 4.1 μl (1:10 dilution (tube 8)) |
| 2 | 1.69 μl (1:10 dilution (tube 7)) | 2.05 μl (1:10 dilution (tube 8)) |
| 3 | 3.37 μl (1:1000 dilution (tube 9)) | 4.1 μl (1:1000 dilution (tube 10)) |
| 4 | - | 4.1 μl (1:10 dilution (tube 8)) |
| 5 | - | 2.05 μl (1:10 dilution (tube 8)) |
| 6 | - | 4.1 μl (1:1000 dilution (tube 10)) |

Then 0.8 μl of Ligase was added again in Tube 1-6 and the tubes were put back into the thermoycycler at 16 degrees for about 5 hours.
**GEL of the Oligos + PCR of the good Oligo bands**
Making a 1% Agarose Gels with a total Volume of 75 μl (2 µL of Midori Green pro Gel)
Loading the samples: 20 μl of each sample + 4 μl Loading dye
Ladder: 7 μl

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Ladder | Sample 1 | Sample 2 | Sample 3 | Ladder | Sample 4 | Sample 5 | Sample 6 | Ladder |

Run the Gel at 120 V

![](https://static.igem.wiki/teams/5077/aaa-hyaluronic-acid/240926-last-repeatigo-1.webp)

**Oligo Fragments of different length.** Different concentrations of mid Oligos (see above the lanes) resulted in different length distributions of resilin fragments. Three different Fragmentlength were isolated from the gel (see red boxes); 1% agarose gel; DNA Ladder: 1 kb Plus.

**Clean up of the three resilin Gel pieces according to protocol 6.2**
with the additional ethanol wash step before the elution. The evaporation period of Ethanol is 15-20 min
Weight of the gel pieces and amount of QB buffer.

|  |  |  |
| --- | --- | --- |
| Resilin 2 3 kb | 320 mg | 960 μl |
| Resilin 2 1 kb | 347 mg | 1041 μl |
| Resilin 5 1 kb | 162 mg | 486 μl |

Nanodrop measurement:

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| Resilin 2 3 kb | Dilution to 1 ng/µL | 4.55 µL DNA + 15.45 µL water | 4.4 ng/µL | 2.26 | 0.24 |
| Resilin 2 1 kb | Dilution to 1 ng/µL | 11.1 µL DNA + 18.9 µL water | 18.2 ng/µL | 1.98 | 0.47 |
| Resilin 5 1 kb | Dilution to 1 ng/µL | 1.7 µL DNA + 18.3 µL water | 11.8 ng/µL | 1.81 | 0.58 |

**PCR of the purified Resilin Oligos**
PCR according to protocol 5.2 with the thermofischer Phusion. Each Resilin sample should get 3 reactions with different amounts of template. (2 ng, 1 ng, 0.5 ng). Therefore 3 x 150 μl were created with 7.5 μl of the Oligo\_Amp\_fw and Oligo\_Amp\_rev primers, 3 μl dNTPs, 15 μl Phusion Buffer and 1.5 μl Phusion Polymerase. For each amount of template a different water amount was added:
Mastermix 2 (2 ng): 113.5 μl water
Mastermix 1 (1 ng): 114.5 μl water
Mastermix 0.5 (0.5 ng): 115 μl water
PCR Tube 1

* 48 μl Mastermix 2
* 2 μl 2, 3 kb

PCR Tube 2

* 48 μl Mastermix 2
* 2 μl 2, 1 kb

PCR Tube 3

* 48 μl Mastermix 2
* 2 μl 5, 1 kb

PCR Tube 4

* 49 μl Mastermix 1
* 1 μl 2, 3 kb

PCR Tube 5

* 49 μl Mastermix 1
* 1 μl 2, 1 kb

PCR Tube 6

* 49 μl Mastermix 1
* 1 μl 5, 1 kb

PCR Tube 7

* 5 μl Mastermix 0.5
* 5 μl 2, 3 kb

PCR Tube 8

* 5 μl Mastermix 0.5
* 5 μl 2, 1 kb

PCR Tube 9

* 5 μl Mastermix 0.5
* 5 μl 5, 1 kb

PCR with 60 °C annealing temperature and 20 s elongation time  for 30 cycles.

##### 27.09.2024

### Transformation of pET28-c in TOP10

Transformation following protocol 2.
**Gel of the PCR from 26.09.2024**
Make a 1 % 75 mL Agarose gel (according to protocol 8)
NEW STEPS:
Let 20 µL of the Samples 1-9 evaporate in a 1.5 mL Eppi for 10 min
Add 4 μl Loading Dye and let it evaporate for 3 min
Load the samples on the Gel:
1 Lane Ladder
2-10 Lane:   Samples 1-9

![Gelelectrophoresis of PCR of purified Resilin Oligos](https://static.igem.wiki/teams/5077/aaa-hyaluronic-acid/image-7.webp)

**Gelelectrophoresis of PCR of purified Resilin Oligos.** 1 kb Plus DNA Ladder, 1 % agarose gel.

* Cut bands of  samples 2, 5,7  out of the gel they are good
* Cut band from sample 1 (Is ok)
* Clean the gel pieces according to protocol 6.2 with the extra ethanol wash step before the elution.

Measurement with Nanodrop:

| Sample | ng / μl | 260/280 | 260/230 |
| --- | --- | --- | --- |
| Sample 1 | 2.5 | 2.43 | 0.12 |
| Sample 2 | 10.5 | 1.76 | 0.21 |
| Sample 5 | 1.7 | 2.47 | 0.07 |
| Sample 7 | 1.7 | 2.27 | 0.10 |

**Repeat the Gel with the rest of the PCR-product**
repeating the same steps as before
1. Ladder 2. Sample 2 3. Sample 5 3. Sample 7
120 mV

![](https://static.igem.wiki/teams/5077/aaa-hyaluronic-acid/resilin-27-9-2.webp)

Cut a band from every sample since they were all good.

##### 28.09.2024

### Overnight culture of transformed TOP10 with pET28-c

Four colonies from the transformation of pET28-c in TOP10 performed on the 27.09.24 were picked and placed in four tubes with 5 mL of LB-medium.

* Additionally, 5 µL of ampicillin (50 mg/mL stock solution) were added
* Finally, the cell suspensions were incubated overnight at 37 °C and 150 rpm on a shaker

**Gel clean-up of the gel bands from the 27.09.24**
Clean the gel cut out according to protocol 6.2 with the additional ethanol wash step before the elution.
Nanodrop measurements:

| Sample | ng / μl | 260/280 | 260/230 |
| --- | --- | --- | --- |
| Sample 2 | 1.1 | 1.88 | 0.08 |
| Sample 5 | 1.2 | 1.79 | 0.05 |
| Sample 7 | 1.6 | 1.35 | 0.05 |

**Do a PCR to amplify the cleaned repeats again from today (2,5,7) and yesterday (1,2,5,7) because we need higher concentrations for the cloning into the plasmid**
PCR according to protocol 5.2 with the thermofischer Phusion (50 μl)
25 μl  sample + 5 μl loading dye for the Agarose gel

|  |  |
| --- | --- |
| 1 | Ladder |
| 2 | Sample 1 from gel clean up (27.09.24) |
| 3 | Sample 2 from gel clean up (27.09.24) |
| 4 | Sample 5 from gel clean up (27.09.24) |
| 5 | Sample 7 from gel clean up (27.09.24) |
| 6 | Ladder |
| 7 | Sample 2 from gel clean up (28.09.24) |
| 8 | Sample 5 from gel clean up (28.09.24) |
| 9 | Sample 7 from gel clean up (28.09.24) |

![](https://static.igem.wiki/teams/5077/aaa-hyaluronic-acid/grafik-2.webp)

Cutting bands
The gel pieces were cleaned according to protocol 6.2 with the additional ethanol wash step before the elution.

##### 29.09.2024

### Restriction ligation of pET28-c and resilin

Isolation of peT28-c from TOP10 following the Miniprep protocol from Qiagen.

| Clone | 1.1 | 1.2 | 2.1 | 2.2 |
| --- | --- | --- | --- | --- |
| Con. (ng/µL) | 28.8 | 24.9 | 21.8 | 28.4 |

The purified resilin samples from the 27.09.24 were pooled (From now on listed as resilin 1) and purified following the Qiagen gel clean up protocol stating at step 5. Before that, the equal amount of sample volume of buffer QB was added. The same steps were repeated with the purified resilin samples from the 28.09.24 (From now on listed as resilin 2).

| Sample | Resilin 1 | Resilin 2 |
| --- | --- | --- |
| Con. (ng/µL) | 51 | 46,7 |

First, the restriction of pET28-c and resilin was carried out with SacI and NdlI for 1 h at 37 °C. Next the samples were heat inactivated for 20 min at 65 °C.

| pET28-c | Clone 1.1 | Clone 1.2 | Clone 2.2 | Clone 2.1 | Negative control |
| --- | --- | --- | --- | --- | --- |
| 1 ng template (µL) | 35 | 40 | 35 | 45 | - |
| Buffer (µL) | 5 | 5 | 5 | 5 | - |
| SacI (µL) | 1 | 1 | 1 | - | - |
| NdeI (µL) | 1 | 1 | 1 | - | - |
| ddH2O (µL) | 8 | 3 | 8 | - | - |
| **The samples had a total volume of 50 µL** | | | | |

|  | Resilin 1 | Resilin 2 | Resilin 3\* | Negative control |
| --- | --- | --- | --- | --- |
| Template (µL) | 19 (962 ng) | 19 (887,3 ng) | 10 (109 ng) | 7 (76,3 ng) |
| Buffer (µL) | 5 | 5 | 1,8 | 1,2 |
| SacI (µL) | 1 | 1 | 0,1 | - |
| NdeI (µL) | 1 | 1 | 0,1 | - |
| ddH2O (µL) | 24 | 24 | - | 40 |

* Sample 3 is sample 2 from the 27.09.2024
* The restricted pET28-c plasmid and the resilin sequence were ligated for 1h at RT using the following 50 µL reactions.

|  | Ligation 1 | Ligation 2 | Ligation 3 | Ligation 4Negative control |
| --- | --- | --- | --- | --- |
| Ligase buffer (µL) | 4 | 4 | 4 | 4 |
| T4 Ligase (µL) | 0,4 | 0,4 | 0,4 | 0 |
| pET28 (µL) | 20 (400 ng) | 20 (400 ng) | 20 (400 ng) | 20 (400 ng) |
| Resilin (µL) | 20 (384 ng) | 20 (384 ng) | 10 (90,8 ng) | 20 (N/A) |
| ddH2O (µL) | 5,6 | 5,6 | 15,6 | 5,6 |

As a control the ligation 1 of was restricted with Sac1.

|  | Ligation 1 |
| --- | --- |
| Template (µL) | 5 |
| Buffer (µL) | 1 |
| SacI (µL) | 0,5 |
| ddH2O (µL) | 3,5 |

A 1% agarose gel of 75 mL with 4 µL Midori green was made to verify the restriction ligation of pEt28-c and resilin. 10 µL of each recombination sample were mixed with 2 µL loading dye.

![](https://static.igem.wiki/teams/5077/aaa-hyaluronic-acid/resilin-29-9.webp)

**pET28-c\_Resilin PCR. pET28-c not digested (negative control) and digested \*pET28-c 1-3 triplicates as well as digested \*pET28-c\_resilin. Additionally, the resilin sequence, as well as the undigested pET28-c\_resilin 1-3 are shown.**
Ladder: 1 kb Plus; Agarose gel: 1 %.

Transformation of the recombination samples into DH5α and TOP10 using LB-Kan plates following the transformation protocol 2.

|  | DH5α | TOP10 |
| --- | --- | --- |
| 1 | 10 µL | 38 µL |
| 2 | 10 µL | 38 µL |
| 3 | 10 µL | 38 µL |
| 1 | 10 µL | 38 µL |
| 3 | 10 µL | 38 µL |

Due to too high concentrations of the pET28-c\_resilin plasmid no bacteria grew on the plates.

##### 30.09.2024

### Repetition of the ligation and transformation of pET28-C\_resilin in DH5α

The remaining restriction reactions 1 and 2 from the 29.09.2024 were pooled and ligated for 1 h at RT and then heat inactivated for 10 min at 65 °C

|  | Ligation |
| --- | --- |
| Ligase buffer (µL) | 5 |
| T4 Ligase (µL) | 0,8 |
| pET28 (µL) | 20 |
| Resilin (µL) | 20 |
| ddH2O (µL) | 4.2 |

**Purification of the ligation reactions**
The purified ligation samples from the 29.09.24 were pooled as well as the ligation samples from today and purified following the Qiagen gel clean up protocol stating at step 5. Before that, the equal amount of sample volume of buffer QB was added.

| Resilin Ligation Pool | Concentration (ng/µL) |
| --- | --- |
| Resilin ligation pool 29.9 | 27,7 ng/µL |
| Resilin ligation pool 30.09 | 87,2 ng/µL |

Both ligation pools from the 29.09 and 30.09 were sent for a whole plasmid sequencing
**Transformation of pET28-c-resilin**

* Additionally, a transformation of the two purified pET28-c\_resilin plasmid pools was made into DH5α following an optimized version of the transformation protocol 2 using LB-Kan plates.
* Incubate 30 min on ice.
* Incubate for 45 seconds at 42 °C.
* Put on ice for 5 min.
* Add 200 µl prewarmed (37°C) SOC medium to the cells.
* Incubate the cells for 90 min at 37 °C and 500 rpm.
* Plate all on LB-Agar plates with ampicillin.
* Incubate on prewarmed plates at 37 °C overnight.

| Ligation reaction |  | ddH2O | DH5α |
| --- | --- | --- | --- |
| Negative control | / | 2 µL | 33 µL |
| Resilin ligation pool 29.9 | 1 µL | / | 33 µL |
| Resilin ligation pool 30.09 | 0,2 µL | / | 33 µL |

**Colony PCR of pET28-c-resilin in TOP10**
A colony PCR was performed from the four colonies that grew on the plates.

|  | Colonies 1-4 | Positive control | Negative control |
| --- | --- | --- | --- |
| Taq 2x Master Mix | 25 µL | 25 µL | 25 µL |
| Oligo\_1repeat\_fw\_primer | 1 µL | 1 µL | 1 µL |
| Oligo\_2repeat\_fw\_primer | 1 µL | 1 µL | 1 µL |
| Template | Colony 1, 2, 3 or 4 | pET-28-c\_resilin 1 µL (1:10) | / |
| ddH2O | 23 µL | 22 µL | 23 µL |

![](https://static.igem.wiki/teams/5077/aaa-hyaluronic-acid/image-8.webp)

**pET28-c\_Resilin colony PCR. Four reactions were made with different clones from a transformation of TOP10 with the pET28-c-Resilin. As a positive control the untransformed pET28-c-Resilin was used and as a negative control water was used as a template.**
Ladder: 1 kb Plus; Agarose gel: 1%.

The production of pET28-c\_resilin plasmid and it’s transformation into TOP10 was a success, since a band at about a height of 1,3 kbp, representing resilin, can be detected in colony PCR the clones 1-4.
**Overnight culture of transformed TOP10 with pET28-c\_resilin**
Four colonies from the transformation of pET28-c\_resilin in TOP10 performed on the 29.09.24 that were also picked for the colony PCR were placed in four tubes with 5 mL of LB-medium.

* Additionally, 5 µL of ampicillin (50 mg/mL stock solution) were added to generate.
* Finally, the cell suspensions were incubated overnight at 37 °C and 150 rpm on a shaker

### October

* 03.10.2024
* 04.10.2024
* 10.10.2024
* 16.10.2024
* 17.10.2024
* 18.10.2024
* 19.10.2024
* 20.10.2024
* 21.10.2024

##### 03.10.2024

### Overnight cultures for Sequencing and long term storage

* Discard overnight culture of DH5a with pET28(c)+\_resilin from tuesday
* Make 4 new overnight cultures from each clones picked on 01.10.24 and 03.10.24
* 5 mL LB-medium and Kanamycin

##### 04.10.2024

### Sequencing and long term storage of DH5a with pET28(c)+\_resilin

* Send clones 1 and 2 from the 01.10.24 overnight culture and clones 3 and 4 from the 03.10.24 overnight culture to sequencing with e.coli
  nightseq
* Made glycerol stocks with the overnight cultures for storage with 500 µL of the overnight culture and 500µL of 50% glycerol
* • Overnight cultures were stored in the fridge at 4 °C after incubation

##### 10.10.2024

### Miniprep pET28c(+)

* • Miniprep following protocol 7.1 with the overnight cultures from the 04.10.2024 from the clones 1-4.

|  |  |  |  |
| --- | --- | --- | --- |
| Clone | Con. (ng/µL) | 260/280 | 260/230 |
| 1.10 1 | 73.7 | 1.83 | 1.55 |
| 1.10 2 | 42.2 | 1.9 | 2.58 |
| 1.10 3 | 81.2 | 1.92 | 2.47 |
| 1.10 4 | 54.4 | 1.85 | 1.61 |
| 3.10 1 | 51.5 | 1.92 | 2.57 |
| 3.10 2 | 64 | 1.82 | 1.26 |
| 3.10 3 | 69 | 1.84 | 1.30 |
| 3.10 4 | 44.7 | 1.8 | 1.28 |

**Restriction of pET28c(+)\_resilin**
Made a Restriction reaction with a total volume of 0.5 µg.

* 1µL XhoI
* 1µL NdhI
* 5µL 10× rcutsmart buffer
* 0.5 µg template
* add to 50 µL ddH2O

Each of the 8 plasmids from above were cutted with the reaction from above. Additionally, there was a reaction with just pET28c(+) as a template.
They were incubated for 1h at 37 °C.
Afterwards they were checked with a 1% agarose gel. For this 20 µl restriction reaction was mixed with 5 µl loading dye and loaded onto the gel, as well as 8 µl of the ladder.

|  |  |
| --- | --- |
| 1 | Ladder |
| 2 | pET28(c)+ digested |
| 3 | pET28(c)+\_resilin 3.10 1 digested |
| 4 | pET28(c)+\_resilin 3.10 2 digested |
| 5 | pET28(c)+\_resilin 3.10 3 digested |
| 6 | pET28(c)+\_resilin 3.10 4 digested |
| 7 | pET28(c)+\_resilin 1.10 1 digested |
| 8 | pET28(c)+\_resilin 1.10 2 digested |
| 9 | pET28(c)+\_resilin 1.10 3 digested |
| 10 | pET28(c)+\_resilin 1.10 4 digested |
| 11 | pET28(c) undigested |

Unfortunately no resilin insertion was observed in the plasmid from the DH5α bacteria.

##### 16.10.2024

### Competent cells JM109

The E. coli strain JM109 was made competent following the protocol 1. 50 mL LB-Medium with 2 mL overnight culture of JM109 was put into a 500 mL culture flask and put into an incubator at 37 °C. The OD600 was measured and as the OD600 = 0,570 was reached the whole media was transferred into a 50 mL falcon.
Afterwards 100 µl of cell culture was put into 1,5 mL tubes and freezed at -80 °C.

##### 17.10.2024

### Transformation of pET28c(+)\_resilin into JM109

Transforming pET28c(+)\_resilin into JM109 bacteria following protocol 2.
The bacteria were plated on LB-agar plates spiked with kanamycin.
As a negative control JM109 bacteria were transformed with only pET28c(+).
As a second negative control JM109 were plated out without being transformed.

We transformed the JM109 bacteria cells with:

* 5 µl pET28c(+)\_resilin2 (1)
* 5 µl pET28c(+)\_resilin2 (1)
* 5 µL of pET28(c)+\_resilin1, 2, 3
* 5 µl pET28c(+)

##### 18.10.2024

### Making overnight cultures from the transformed bacteria from the 17.10.24

Add one clone in 5 mL LB-Media with 10 µL Kanamycin (Stock: 50 mg/mL).
Incubate them on a shaker at 37 °C and 180 rpm overnight.

* 2 overnight cultures with pET28(c)+
* 3 overnight cultures with pET28(c)+\_resilin from ligation 2
* 3 overnight cultures with pET28(c)+\_resilin from ligation pool1+2+3

##### 19.10.2024

### IPTG induction of resilin expression

6 asseys from transformation (see 17.10.2024) with:

* 50 μl kanamycin
* 1000 μl from
  + "pET28(c)+\_resilin1, 2, 3" → three assays
  + "pET28(c)+\_resilin2" → two assays
  + "pET28(c)+" without resilin → one assay (negative control)
* 49 ml LB media each
* incubated at 37 ° C and RPM 200 until OD 0.5 was reached

Induction of 6 asseys with IPTG:

* added 30 μl IPTG to each assay to induce resilin expression
* sample of each taken immediately afterwards and frozen at -20 ° C → "Ex0" (negative control)
* incubated rest of IPTG assays over night at 37 ° C and RPM 200 for expression of resilin

##### 20.10.2024

### SDS-PAGE for resilin detection

21 hours after the IPTG induction the cells were harvested. 20 mL of each culture was transferred into two 50 mL falcon tubes and centrifuged for 15 min at 3400g. The cell pellet of one half of the culture was resuspended with 3 mL lysis buffer and the other half was resuspended in LB-Medium.

* pET28(c)+
* pET28(c)+\_resilin ligation 1+2+3 1
* pET28(c)+\_resilin ligation 1+2+3 2
* pET28(c)+\_resilin ligation 1+2+3 3
* pET28(c)+\_resilin ligation 2.1
* pET28(c)+\_resilin ligation 2.2

Then two different lysis approaches were performed. First we did Heat Lysis with each of the 6 expression cultures in Lysis buffer and LB-Medium. This totals 12 eppis, each with 1 mL culture. They were heated for 10 min at 90 °C and then centrifuged for 25 min at 16.000g.
1 mL of each of the 6 expression cultures were used for sonification lysis. They were sonified for 10 s with a 30 s break for a total of 5 repeats. After that they were centrifuged for 25 min at 16.000g.
50 µl of the supernatant of the heated and sonified samples were mixed with 50 µl reducing sample buffer.
The samples without lysis were also mixed with 50 µl reducing sample buffer.
Three 10% SDS-Gels were prepared following the protocol 9. On Gel 1 the samples before and after the induction were put onto. Gel 2 contained the heat lysis samples and Gel 3 the sonified samples of all six expression cultures.
The gels were stained over night with Coomassie.

##### 21.10.2024

### SDS-PAGE for resilin detection- destaining

Destaining with water for 3 hours. Waterchange and again destaining for 4 hours. After that the pictures were taken.

![](https://static.igem.wiki/teams/5077/aaa-hyaluronic-acid/resilin-21-10-induction.webp)

**SDS page before (b.i.) and after (a.i.) induction of E. coli strain JM109 with IPTG for resilin expression.**
The bacteria were transformed with the pET28c(+) plasmid with (R) and without ((pET28c(+))) our resilin insert. Two different resilin samples were used. Resilin 1,2,3 was used three times and resilin 2 twice. Samples were mixed 1:1 with SDS loading buffer. 10% gel, ladder: PageRuler unstained protein ladder.

![](https://static.igem.wiki/teams/5077/aaa-hyaluronic-acid/resilin-21-10-soni.webp)

**SDS page after induction of E. coli strain JM109 with IPTG to detect resilin expression.**
The bacteria were transformed with the pET28c(+) plasmid with (R) and without (pET28c(+)) our resilin insert and two different resilin samples were used. The resilin 1,2,3 was used three times and the resilin 2 was used twice. Lysis was performed by sonication and each sample was lysed once in LB medium and once in lysis buffer (lysis). 10% gel, ladder: PageRuler unstained protein ladder.

![](https://static.igem.wiki/teams/5077/aaa-hyaluronic-acid/resilin-21-10-heat.webp)

**SDS page after induction of E. coli strain JM109 with IPTG to detect resilin expression.**
The bacteria were transformed with the pET28c(+) plasmid with (R) and without (pET28c(+)) our resilin insert. Two different resilin samples were used. Resilin 1,2,3 was used three times and Resilin 2 twice. Lysis was performed by heating the cells at 95°C and each sample was lysed once in LB medium and once in lysis buffer (lysis). 10% gel, ladder: PageRuler unstained protein ladder.

# Hyaluronic Acid

# 07

# 08

# 09

# 10

### July

* 01.07.2024
* 03.07.2024
* 04.07.2024
* 05.07.2024
* 16.07.2024
* 17.07.2024
* 18.07.2024
* 19.07.2024
* 24.07.2024

##### 01.07.2024

### Testing competent cells with pMK1

Transformation of pMK1 into TOP10 and BL21\* competent cells following protocol 2 [Transformation.](experiments#transformation-header)

Changes:

* After adding the pre-warmed SOC medium the cells were incubated for 60 min on the shaker at 37 °C and 300 rpm
* 100 µL of the bacteria suspension were plated on the LB-Agar-Amp. Plates
* As a negative control the competent cells were incubated in 100 µl water and plated on the LB-Agar-Amp

##### 03.07.2024

### Transformation of DH5α with pUC\_HasA

* Wet the dry pUC\_HasA plasmid DNA
* Transformation of pUC\_HasA into DH5α following protocol 2 [Transformation.](experiments#transformation-header)

Changes:

* After adding just 200 µL pre-warmed SOC medium the cells were incubated for 60 min on the shaker at 37 °C and 300 rpm
* 250 µL were plated on LB-Agar-Amp. Plates

##### 04.07.2024

### Overnight culture of transformed DH5α with pUC\_HasA for expression of hyaluronic acid synthase

* Two colonies (pUC\_HasA\_1 and pUC\_HasA\_2) from the transformation of pUC\_HasA in DH5α from 03.07.24 were picked and transferred in two tubes with 3 mL of LB-medium.
* Additionally 3 µL of ampicillin (100 mg/mL stock solution) were added for a 1:1000 dilution.
* Finally the cell suspensions were incubated over night at 37 °C, 150 rpm on a shaker

##### 05.07.2024

### Miniprep of pUC\_HasA for expression of hyaluronic acid synthase

A miniprep was carried out with the over night cultures from pUC\_HasA\_1 and pUC\_HasA\_2 from 04.07.24 following the protocol 7.1 [Plasmid Mini-Prep - NEB.](experiments#pmp-header)

##### 16.07.2024

### Transformation of pUC\_HasA in TOP10 for expression of hyaluronic acid synthase

* Transformation of pUC\_HasA\_1 and pUC\_HasA\_2, purified with a miniprep on 04.07.24, into competent TOP10 bacteria following the protocol 2 [Transformation.](experiments#transformation-header).
* A total of 100 µL of bacteria suspension with pUC\_HasA were plated on the LB-Agar-Amp. Plates.
* As a negative control water was added to the competent cells.

##### 17.07.2024

### Overnight culture of transformed TOP10 with pUC\_HasA for expression of hyaluronic acid synthase

* Three colonies from the tranformation of pUC\_HasA\_2 in TOP10 performed on 17.07.24 were picked and placed in three tubes with 3 mL of LB-medium each.
* Additionally 3 µL of ampicillin (100 mg/mL stock solution) were added to achieve a 1:1000 dilution.
* Finally the cell suspensions were incubated over night at 37 °C, 150 rpm on a shaker.

##### 18.07.2024

### Induction of hyaluronic acid synthase expression with arabinose

* For the initiation of the hyaluronic acid synthase expression 50 mL LB-Medium with 50 µL of ampicilin (100 mg/mL stock solution) were added to the 2 mL of overnight cultures 2 and 3 from 17.07.2024.
* The cell cultures were incubated at 37 °C, 130 rpm on the shaker until they reached an OD600 of 0.6.
* As a control 1 mL of the cell culture from clone 2 and 3 were taken at an OD600 of 0,6 and stored at 4 °C until further analysis.
* To start the synthetisation, 500 µL of arabinose (10 g/L stock solution; final concentration = 1 µg/mL) were added to the culture of clone 2 and 1000 µL of arabinose (final concentration = 2 µg/mL) were added to the culture of clone 3.
* The cell cultures of clones 2 and 3 were then incubated for 20 h at 37 °C, 150 rpm on a shaker.

##### 19.07.2024

### Analysis of the hyaluronic acid synthase expression with an SDS-PAGE

After 20 h expression of Hyaluronic acid through induction with arabinose the OD600 of the cultures from clones 2 and 3 were measured.

| Culture (Arabinose conc.) | OD600 (20 h after induction) |
| --- | --- |
| Clone 2 (1 µg/mL) | 2,750 |
| Clone 3 (2 µg/mL) | N/A |

300 µL of Clone 2 and 3 were diluted with 700 µL LB medium to reach an OD600 of 1. Even tho clone 3 was not measurable undiluted, it had an OD600 of 1 after adding the LB medium.

To analyze the hyaluronic acid expression an SDS-Page with an 12% gel was performed following the protocol 9 [SDS-PAGE](experiments#sds-header).

To prepare the samples under reducing conditions for the SDS-PAGE 50 µL of clones 2 and 3, both pre and post induction, were mixed with 50 µL of reducing sample buffer and a total of 15 µL were then loaded onto the gel. As a ladder the PageRuler Unstained Protein Ladder (Thermo Scientific, Cat. no.: 26614) was used.

After the SDS-PAGE the gel was stained with Coomassie for 1 h and destained with ddH2O over night.

Expected bands: 52 kDa (HasA)

![](https://static.igem.wiki/teams/5077/notebook-pics/sds-page-hasa.png)

**SDS-PAGE for validation of HasA expression in TOP10.** From left to right is the PageRuler, Clone 2 before and after induction and Clone 3 before and after induction. Bands were visible slightly above 50 kDa after induction.
Legend: bef. = before; aft. = after; Ind = Induction

**Results:**
We were able to see bands at a height of about 52 kDa, which is consistent with the molecular weight of the hyaluronic synthase.

In the next steps, we choose to prove and observe the hyaluronic acid expression with an HasA-eGFP fusion protein.

##### 24.07.2024

### Cloning of a HasA-eGFP fusion protein for the detection of HasA Expression

To insert the eGFP+Linker behind the HasA gene a SLiCE reaction will be used.
For the SLiCE the two fragments eGFP+Linker and pUC\_HasA are linearized with a PCR reaction containing homologue overhangs.

The PCRs were performed following the protocol 5.1  [PCR - Q5 Mastermix from NEB](experiments#pcr-header) with following templates, primers and annealing temperatures:

| Reaction | Template | Primer | Annealing Temperature [°C] | Elongation [s] |
| --- | --- | --- | --- | --- |
| HasA\_SLiCE\_1 | pUC\_HasA | pUC\_IDT\_hasA\_fw\_SLICE & pUC\_IDT\_hasA\_rev\_SLICE | 57.1 | 185 |
| HasA\_SLiCE\_2 | pUC\_HasA | pUC\_IDT\_hasA\_fw\_SLICE & pUC\_IDT\_hasA\_rev\_SLICE | 60.9 | 185 |
| eGFP\_SLiCE\_1 | eGFP\_Linker | GFP\_fw\_SLICE & GFP\_rev\_SLICE | 55 | 40 |
| eGFP\_SLiCE\_2 | eGFP\_Linker | GFP\_fw\_SLICE & GFP\_rev\_SLICE | 56.4 | 40 |
| eGFP\_SLiCE\_3 | eGFP\_Linker | GFP\_fw\_SLICE & GFP\_rev\_SLICE | 57.7 | 40 |
| eGFP\_SLiCE\_4 | eGFP\_Linker | GFP\_fw\_SLICE & GFP\_rev\_SLICE | 59.1 | 40 |

To determine if the PCRs were successful, we ran agarose gels produced after protocol 8 [Agarose gel](experiments#ag-header).

expected bands:- 5250 bp (HasA\_SLiCE)
- 762 bp (eGFP\_SLiCE)

![](https://static.igem.wiki/teams/5077/notebook-pics/slice-final-final.png)

**1% agarose gel elektrophoresis following pUC\_HasA and eGFP\_Linker gradient PCR.** A) shows the products of the HasA\_SLiCE gradient PCR with clearly visible bands at 500 bp, 600 bp and 900 bp. B) shows the products of the eGFP\_SLiCE gradient PCR with visible bands at 700-800 bp. As negative contol water was used as template for all PCRs; Ladder: 1 kb Plus.

**Results:** With bands being between 700-800 bp and the size of our desired product being 762 bp, eGFP\_SLiCE PCR can be considered successful as suggested by band size

**Problem:** there were no bands at the expected 5250 bp. Retry with lower annealing temperature.

### August

* 03.08.2024
* 08.08.2024
* 12.08.2024
* 14.08.2024
* 15.08.2024
* 16.08.2024
* 17.08.2024
* 18.08.2024
* 26.08.2024
* 28.08.2024

##### 03.08.2024

### PCR to amplify HasA\_SLiCE fragment

* PCR according to protocol 5.1 [PCR - Q5 Mastermix from NEB](experiments#pcr-header) with pUC\_IDT\_hasA\_fw\_SLICE and pUC\_IDT\_hasA\_rev\_SLICE as primers, 55 °C annealing temperature and 3:30 min elongation time.
* 2.52 µL of pUC\_HasA template were used.

To determine if the PCR was successful, we ran an agarose gel produced after protocol 8 [Agarose gel](experiments#ag-header).

expected bands: 5250 bp (HasA\_SLiCE)

![](https://static.igem.wiki/teams/5077/notebook-pics/hasa-slice.png)

**1% agarose gel elektrophoresis following pUC\_HasA SLiCE-PCR.** The gel shows clearly visible bands between 5,000 and 6,000 bp, right where the PCR product is expected. Ladder: 1 kb Plus.

**Result:** With expected bands at 5250 bp, this result suggests that the PCR was successful and our next step is the SLiCE recombination with both the HasA\_SLiCE and the eGFP\_SLiCE.

##### 08.08.2024

### Gel Clean up of HasA\_SLiCE and eGFP\_SLiCE

* To retrieve the PCR product of the successful HasA\_SLiCE and eGFP\_SLiCE PCRs from the gel, we did a Gel clean up according to protocol 6.1 [Gel clean up - Promega gel and PCR clean up.](experiments#gcu-header)
* The PCR products can now be used in the SLiCE recombination to obtain the pUC\_HasA\_eGFP fragment

##### 12.08.2024

### SLiCE recombination with HasA\_SLiCE & eGFP\_SLiCE

* Recombination of purified pUC\_HasA (see 03.08.24) and eGFP\_SLiCE (see 24.07.24) with SLiCE according to protocol 10.1.  [Homologous Recombination - SLiCE.](experiments#hr-header)
* 8 samples were prepared, of which 4 were prepared in duplicates, differing in incubation times:

| Sample | Vector [ng] | Ratio [insert to vector] | Volume [µL] | Incubation time |
| --- | --- | --- | --- | --- |
| 1 | 48 | 1:7 | 20 | 1x 1 h, 1x over night |
| 2 | 67 | 1:3 | 20 | 1x 1 h, 1x over night |
| 3 | 96 | 1:7 | 40 | 1x 1 h, 1x over night |
| 4 | 134 | 1:3 | 40 | 1x 1 h, 1x over night |

* Transformation of pUC\_HasA-eGFP\_SLiCE into TOP10 according to protocol 2 [Transformation](experiments#transformation) after 1h and over night incubation, respectively.

##### 14.08.2024

### Colony PCR of TOP10 - pUC\_HasA\_eGFP\_SLiCE

* Preparation of the PCR according to protocol 5.3 [PCR - DreamTaq Mastermix](experiments#pcr-header) with the following primers, annealing and extension temperatures:

| Step | Temperature [°C] | Time |
| --- | --- | --- |
| Annealing | 54 | 30 s |
| Extension | 72 | 80 s |
| Final extension | 72 | 3 min |

* Primers: Rev\_ColPCR\_HasA\_EGFP and FW\_ColPCR\_HasA\_EGFP
* Template: Picked 6 pUC\_HasA\_eGFP\_SLiCE colonies from two plates after 1h incubation as well as 6 from two plates after incubation over night.
* Positive control with 0.5µL of pUC\_HasA in a 25µL sample

##### 15.08.2024

### Agarose Gel for the Colony PCR of TOP10 - pUC\_HasA\_eGFP\_SLiCE

* Colony PCR samples from the day prior (14.08.2024) were run on a gel, prepared as described in protocol 8 [Agarose gel](experiments#ag-header).

![](https://static.igem.wiki/teams/5077/notebook-pics/top10hasaegfpslice-colony-pcr.png)

**Results for the Colony PCR of TOP10 - pUC\_HasA\_eGFP\_SLiCE.** 11 of the 12 picked colonies beginning from left to right with the positive control, 1 kb ladder, samples in descending order beginning with 11 - 8 incubated over night, 1kb ladder, sample 7 incubated over night, and 6 - 1 incubated for 1h. The positive control and all other included samples display a visible fragment at around 500 bp.
Legend: o. n. inc. = over night incubation

##### 16.08.2024

### DpnI digestion and Repetition of the SLiCE recombination from 12.08.2024 with HasA\_SLiCE & eGFP\_SLiCE

DpnI Digestion:

* The two pUC\_HasA\_SLiCE fragments and four eGFP\_SLiCE fragments were pooled, respectively.
* Concentrations were measured:

+ pUC\_HasA\_SLiCE: 28.6 ng/μL
+ eGFP\_SLiCE: 20.7 ng/µL

* Pooled samples were digested with DpnI according to protocol 11  [DpnI digestion.](experiments#dpn-header)

+ Incubation with DpnI was reduced to 1 h

SLiCE recombination:

* Recombination was preformed similar to the previous attempt of 12.08.2024 with the pooled and digested fragments pUC\_HasA and eGFP\_SLiCE according to protocol 10.1. [Homologous Recombination - SLiCE.](experiments#hr-header)
* Insert to vector ratios were adjusted, total volume was reduced and 2 samples were prepared, of which each was prepared in duplicates, differing in incubation times:

| Sample | Vector [ng] | Ratio [insert:vector] | Volume [µL] | Incubation time |
| --- | --- | --- | --- | --- |
| 1 | 85.8 [ng] | 1:1 | 10 | 1 h / over night |
| 2 | 28.6 [ng] | 1:10 | 10 | 1 h / over night |

##### 17.08.2024

### Transformation of pUC\_HasA-eGFP\_SLiCE-1 and -2 in TOP10

* Transformation of new Plasmids into TOP10 according to 2 [Transformation](experiments#transformation-header)

+ DNA used, optimized for SLiCE to 2µL of the recombined product

* four were Plates prepared:

+ two for pUC\_HasA-eGFP\_SLiCE-1, incubated for 1h and over night
+ two for pUC\_HasA-eGFP\_SLiCE-2, incubated for 1h and over night

##### 18.08.2024

### Homolog recombination of the HasA and EGFP fragment using NEBuilder

* New approach for the recombination of pUC\_HasA\_SLiCE and eGFP\_SLiCE according to protocol 10.2  [Homologous Recombination - NEB-builder](experiments#hr-header)

+ Total volume was reduced to 10 µL

| Sample | Vector [ng] | Ratio [insert:vector] | Volume [µL] |
| --- | --- | --- | --- |
| 1 | 129.7 | 1:2 | 10 |
| 2 | 37 | 1:4 | 10 |

##### 26.08.2024

### Transformation with the recombination products from 18.08.2024

* Transformation of pUC\_HasA\_eGFP\_HR-1 and -2
* Transformation according to protocol 2 Transformation

+ Transformed into TOP10
+ 3µL recombination product was used

##### 28.08.2024

### Colony PCR of pUC\_HasA\_eGFP\_HR

* 6 colonies were picked from pUC\_HasA\_eGFP\_HR-1 and pUC\_HasA\_eGFP\_HR-2, resulting in a total of 12 samples
* Colony PCR was performed as described in protocol 5.3
  [PCR - DreamTaq Mastermix](experiments#pcr-header)
* Primers: Rev\_ColPCR\_HasA\_EGFP and FW\_ColPCR\_HasA\_EGFP
* Positive control: was added consisting of

+ 12.5 μl DreamTaq Green PCR Master Mix (2x)
+ 1.25 μl of each primer (pUC\_IDT\_hasA\_fw\_SLICE, Rev\_ColPCR\_HasA\_EGFP)
+ 0.5 μl pUC\_HasA\_eGFP
+ 25 μl nuclease free water

All samples were negative!

### September

* 04.09.2024
* 07.09.2024
* 08.09.2024
* 09.09.2024
* 10.09.2024
* 12.09.2024
* 13.09.2024
* 17.09.2024
* 18.09.2024
* 19.09.2024
* 20.09.2024
* 23.09.2024
* 26.09.2024
* 27.09.2024
* 29.09.2024
* 30.09.2024

##### 04.09.2024

### Colony PCR of the transformed SLiCE pUC\_HasA\_eGFP into Top10 bacteria

to test the colonies from the NEBuilder homolog recombination with pUC\_HasA-  and eGFP-SLiCE-Fragments a Colony PCR was performed. 18 colonies were tested. 9 colonies were picked from the plate with a 1:2 vector: insert ratio. The other 9 colonies were picked from the plate with a 1:4 vector: insert ratio.
The Colony PCR Mastermix was done with 100 μl DreamTaq Green PCR Master Mix (2x), 10 μl of each primer Rev\_ColPCR\_HasA\_eGFP and FW\_ColPCR\_HasA\_eGFP and 80 μl nuclease-free water. The colonies were picked with a toothpick or a pipette tip and added into 10 μl PCR Mastermix. 12 colonies were tested. 6 colonies from the plates where the Top10 cells were transformed with the pUC\_HasA-eGFP\_SLiCE extract that was incubated for 1 hour and 6 from the plates where the extract was incubated overnight. A positive control was added consisting of 12.5 μl DreamTaq Green PCR Master Mix (2x), 1.25 μl of each primer (pUC\_IDT\_hasA\_fw\_SLICE, Rev\_ColPCR\_HasA\_eGFP), 0.5 μl pUC\_HasA\_eGFP as Template and 25 μl nuclease free water.

The PCR was done as follows:

| Step | Temperature, °C | Time | Number of cycles |
| --- | --- | --- | --- |
| Initial denaturation | 95 | 3 min | 1 |
| Denaturation | 95 | 30 s | 30 |
| Annealing | 54 | 30 s | 30 |
| Extension | 72 | 1:20 min | 30 |
| Final Extension | 72 | 3 min | 1 |
| Stop | 10 | infinite | 1 |

The PCR was checked on a 1 % agarose gel (protocol 8).

##### 07.09.2024

### Second try to Transform the Homolog recombination of the HasA and eGFP fragment using NEBuilder

Transformation was done according to protocol 2 with 2 μl SLiCE-product (1:2 vector:insert ratio and 1:4 vector:insert ratio) and positive control and 1 μl negative control.

##### 08.09.2024

### Colony PCR after Transformation

The Colony PCR Mastermix was done with 50 μl DreamTaq Green PCR Master Mix (2x), 5 μl of each primer Rev\_ColPCR\_HasA\_eGFP and FW\_ColPCR\_HasA\_eGFP and 40 μl nuclease-free water. The colonies were picked with a toothpick or a pipette tip and added into 10 μl PCR Mastermix. 12 colonies were tested. 6 colonies from the plates where the Top10 cells were transformed with the pUC\_HasA-eGFP\_SLiCE extract that was incubated for 1 hour and 6 from the plates where the extract was incubated overnight. A positive control was added consisting of 12.5 μl DreamTaq Green PCR Master Mix (2x), 1.25 μl of each primer (pUC\_IDT\_hasA\_fw\_SLICE, Rev\_ColPCR\_HasA\_eGFP), 0.5 μl pUC\_HasA\_eGFP as Template and 25 μl nuclease free water.

The PCR was done as follows:

| Step | Temperature, °C | Time | Number of cycles |
| --- | --- | --- | --- |
| Initial denaturation | 95 | 3 min | 1 |
| Denaturation | 95 | 30 s | 30 |
| Annealing | 54 | 30 s | 30 |
| Extension | 72 | 1:20 min | 30 |
| Final Extension | 72 | 3 min | 1 |
| Stop | 10 | infinite | 1 |

From each plate 5 colonies were picked which resulted in 10 PCR tubes plus the positive control.

1: Colony of the 1:2 ratio plate
2: Colony of the 1:2 ratio plate
3: Colony of the 1:2 ratio plate
4: Colony of the 1:2 ratio plate
5: Colony of the 1:2 ratio plate
6: Colony of the 1:4 ratio plate
7: Colony of the 1:4 ratio plate
8: Colony of the 1:4 ratio plate
9: Colony of the 1:4 ratio plate
10: Colony of the 1:4 ratio plate

The PCR was performed with 60 °C annealing temperature and 2:20 elongation time for 30 cycles.

![](https://static.igem.wiki/teams/5077/notebook-pics/08-09-hyaluron1.png)

Colony PCR from the 08.09. results

![](https://static.igem.wiki/teams/5077/notebook-pics/08-09-hyaluron2.png)

Colony PCR from the 08.09. max. contrast results

##### 09.09.2024

### Overnight culture

An overnight culture was prepared from the following samples: 5, 6, 7, 8, 9, 10. These showed bands at the right length and therefore should be checked via sequencing. The picked colonies were added to LB-Medium and put on the shaker at 37 °C overnight.

##### 10.09.2024

### Sequencing

The overnight cultures grew well and were sent in for sequencing.
They came all back negative for the insert.

##### 12.09.2024

### Isolate the pUC-HasA by performing a miniprep

For the isolation protocol 7.2 was followed.

##### 13.09.2024

### Add overhangs at the puC-HasA for the homolog recombination

PCR according to protocol 5.1 with 30 cycles, annealing temperature 98 °C, and elongation for 3:20 min. Primers pUC\_IDT\_HasA\_rev and pUC\_IDT\_HasA\_fw were used.
Do a Dpn1 digestion to get rid of the template without overhangs and clean it up.
DpnI digest following protocol 3. 25 μl of PCR-product and 1 μl DpnI (NEB) were added to the reaction. Incubation for 2 hours at 37 °C, followed by a heat inactivation for 20 min at 80 °C. After that, a PCR purification was carried out according to protocol 6.1. The cleaned PCR product was analyzed on a 1 % agarose gel (protocol 8, figure 1.). There was no plasmid, so the HasA-PCR was repeated on the same day (figure2).

![](https://static.igem.wiki/teams/5077/notebook-pics/13-09-hyaluron1.png)

figure 1

![](https://static.igem.wiki/teams/5077/notebook-pics/13-09-hyaluron2.png)

figure 2

##### 17.09.2024

### pUC-HasA PCR

The pUC-HasA PCR from the 13.09.2024 was repeated.

##### 18.09.2024

### pUC-HasA and eGFP-SLiCE-PCR

pUC-HasA PCR according to protocol 5.2, with pUC\_IDT\_HasA\_rev and pUC\_IDT\_HasA\_fw for primers, 55 °C annealing temperature and 3:20 min elongation time. eGFP\_SLiCE-PCR was also performed according to protocol 5.2 with GFP\_fw\_SLiCE and GFP\_rev\_SLiCE as primers. The elongation time was 1:20 min and the annealing temperature was 67 °C.

##### 19.09.2024

### DpnI digest and control via agarose gel

DpnI digest according to protocol 3 with 40 µl template, incubation for 2 hours at 37 °C, and heat inactivation for 20 min at 80 °C. The digest was put onto a 1 % agarose gel (according to protocol 8).

Gel:

1: Ladder 10 µl

2: HasA 2 (13.09.) undigested 9 µl

3: HasA  3.1 (18.09) 20 µl

4: HasA  3.2 (18.09) 20 µl

5: HasA  3.13(18.09) 20 µl

6: Ladder 10 µl
7: HasA  4.1 (18.09) 20 µl

8: HasA  4.2 (18.09) 20 µl

9: HasA  4.3 (18.09) 20 µl

10: Ladder 10 µl

![](https://static.igem.wiki/teams/5077/notebook-pics/19-09-hyaluron.png)

blubbberrrrr

After that a Gel purification was performed according to protocol 6.2, and the concentrations were measured.

| Sample | Conc. | 280/260 | 260/230 |
| --- | --- | --- | --- |
| HasA CU1 | 16.5 | 1.73 | 0.12 |
| HasA CU3 | 9.7 | 1.61 | 0.05 |
| HasA CU4 | 11.2 | 1.53 | 0.13 |
| HasA CU5 | 14 | 1.59 | 0.14 |
| HasA CU6 | 18.2 | 1.66 | 0.15 |
| HasA CU7 | 10.7 | 1.64 | 0.09 |

##### 20.09.2024

### NEBuilder Recombination

A 1 % agarose gel (according to protocol 8) was loaded with the eGFP-SLiCE-PCR product from the 18.09.

1: Ladder 1 kb

2: eGFP 1

3: eGFP 2

![](https://static.igem.wiki/teams/5077/notebook-pics/20-09-hyaluron-1.png)

figure 1

There were no bands (figure 1), so we started another eGFP-SLiCE-PCR according to protocol 5.2  but and used 69 ° degrees as a annealing temperature and 1:30 for elongation.
The we loaded yet another 1 % agarose gel (according to protocol 8) and filled the pockets with the new eGFP-PCR product just like above.

This time the PCR did work at least for eGFP 1 (figure 2).

![](https://static.igem.wiki/teams/5077/notebook-pics/20-08-hyaluron-2.png)

figure 2

##### 23.09.2024

### Amplification of the pUC\_HasA\_SLiCE Fragments (second try)

For the recombination of pUC\_HasA, the fragment pUC\_HasA will be amplified with primers containing the homolog regions in the overhang.

pUC\_hasA-PCR (Protocol 5.2)
annealing temp 55°C

elongation: 3:20min

pUC\_hasA from the overnight colony from 07.09.2024 diluted to 1ng/µl as template and we followed the protocol 5.2 with the following primers 10 µM pUC\_HasA\_fw\_SLICE and 10 µM pUC\_Has\_rev\_SLICE.

The PCR product was analysed on a 1% agarose gel (following protocol 8).

![](https://static.igem.wiki/teams/5077/notebook-pics/screenshot-2024-10-01-193625.png)

uhjnjdbhbh

Afterwards, the Fragments were isolated from the Gel (protocol 6.2), adding a wash step with 750µL 70% ethanol right before the elution. Additionally, the ethanol was evaporated for 6min before the elution step. This extra step  was only done with pUC\_HasA1 and pMK1\_1.

pUC\_HasA1: 53.6ng/ µL; 260/280 1.77; 260/230 1.25

pUC\_HasA2: 64.8ng/µL; 260/280 1.72; 260/230 0.13

pMK1\_1: 16.4ng/µL; 260/280 1.47; 260/230 0.75

pMK1\_2: 31ng/µL; 260/280 1.42; 260/230 0.15

As one can see, the extra step resulted in a way better 260/230 ratio. So we decided to keep that step for any other Gel Clean Ups.

##### 26.09.2024

### GEL of eGFP from 27.09.24 + Isolation of good Bands

Making a 1% Agarose Gels with atotal Volume of 75 µL (2 µL of Midori Green pro Gel)

Loading the samples: 20 µL of each sample + 4 µL Loading dye

Ladder: 7 µL

Gel:

1. Ladder

2. eGFP 1 from 20.09

3. eGFP 2 from 20.09

Run the Gels at 120 V

![](https://static.igem.wiki/teams/5077/screenshot-2024-10-01-184710.png)

nunfnknk

cut out the band at eGFP 1

Clean up of eGFP 1

According to protocol 6.2 with the additional ethanol wash step before the elution

Weight of the gel pieces and amount of QB buffer

| eGFP | 229,6mg | 688, 8 µL |

Nanodrop measurment:

eGFP

19.9 ng/µL

260/280 1.87

260/230 0.21

(Dilution to 1 ng/µL   1 µL DNA + 19 µL water)

##### 27.09.2024

### Titel

##### 29.09.2024

### Homologue recombination of pUC\_HasA and eGFP

* Homologous recombination of puC\_HasA and eGFP following the NEB-builder

![](https://static.igem.wiki/teams/5077/notebook-pics/29-09-homoreco-puc-hasa-egfp.png)

**pUC\_HasA\_eGFP PCR.**

##### 30.09.2024

### Transformation of puC\_HasA\_eGFP into DH5α

Clean up of the pooled remaining recombination samples from the 29.09.2024 following the Qiagen gel clean up protocol stating at step 5. Before that, the equal amount of sample volume of buffer QB was added.
**Konz: 42,3 ng/ µL**
Additionally a transformation of the purified pUC\_HasA\_eGFP plasmid was made into DH5α following an optimized version of the transformation protocol 2 using LB-Amp plates.

* Incubate 30 min on ice.
* Incubate for 45 seconds at 42 °C.
* Put on ice for 5 min.
* Add 200 µl prewarmed (37°C) SOC medium to the cells.
* Incubate the cells for 90 min at 37 °C and 500 rpm
* Plate all on LB-Agar plates with ampicillin
* Incubate on prewarmed plates at 37 °C overnight.

| Homologous recombination |  | TOP10 |
| --- | --- | --- |
| Positive control | 1 µL | 33 µL |
| Negative control | 10 µL (ddH2O) | 33 µL |
| Pooled Recombination | 0,5 µL | 33 µL |

### October

* 01.10.2024

##### 01.10.2024

### Transformation results of pUC\_HasA\_eGFP

No bacteria grew on the plates. We will try the transformation again.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering{% endblock %}
{% block page\_content %}

# Engineering

Over the past year, we have overcome many challenges in the lab. Here, we present you some of them.

![](https://static.igem.wiki/teams/5077/igem-eng-cycle.png)

# Cloning of Resilin Repeats

## Cycle 1 - Restriction/Ligation Approach

#### Design

Our first attempt on cloning the 64 repeats of resilin was to apply a classical restriction and ligation approach. Specifically, we aimed to apply directed PCR-free engineering to obtain these highly repetitive DNA sequences [1].

This method is based on a sophisticated arrangement of recognition sites for four type IIS restriction enzymes. In particular the enzymes NdeI, SacI, BsaI and BsmBI are used. It is an approach for a stepwise and seamless elongation of repetitive sequences. We would like to demonstrate that method using our project as an example.

To use this approach, we needed two parts.

The first part is the backbone. Here, we used the pMK1 backbone that was mutated to only have the restriction recognition sites for NdeI and SacI once at the desired position of the backbone and nowhere else. In figure 1 we depict a dummy vector. Note, that this figure is only for demonstration purposes and does not reflect the sequence for the actual pMK1 vector!

![](https://static.igem.wiki/teams/5077/vector-map.svg)

Fig. 1: Map of the vector having one restriction recognition site for NdeI and one for SacI. Note that this is a simplified vector for demonstration purposes and that it does not depict the true pMK1 plasmid map.

The second part is an initial sequence for the gene that is supposed to be repeated - in our case resilin. To be able to skip a few steps we modified the sequence of resilin using different bases for the same amino acid to get some variance between repeats. Thus, we were able to synthesize a gene fragment containing four repeats of resilin which were flanked with restriction recognition sites (RS) for NdeI, BsaI, BsmBI and SacI (Fig.2). This fragment will be referred to as R4 (Fig. 2).

![](https://static.igem.wiki/teams/5077/r4-map.svg)

Fig. 2: Map of the R4 sequence. Four different sequences representing one resilin repeat each are seamless connected with each other. The repeats are flanked with restriction recognition sites (RS) for NdeI, BsaI, BsmBI and SacI.

Both the vector and R4 are being cut NdeI and SacI to create complementary overhangs for a following ligation. The predicted result is depicted in figure 3 and is from now on addressed as PR4 (plasmid with 4 repeats).

![](https://static.igem.wiki/teams/5077/pr4-map.svg)

Fig. 3: Simplified plasmid map for PR4 resulting from cutting vector and R4 with NdeI and SacI followed up with a ligation of the insert into the backbone.

Now the first combination of repeats takes place. PR4 is now used in two different restriction reactions before combining them again. One part is cut in a double digest using BsmBI and SacI to open the plasmid directly behind repeat 4. This is the accepting vector. The other part is cut in a double digest using BsaI and SacI to cut out the four repeats including the BsmBI RS. This is the donor part provding the new insert. The insert now can be incorporated into the accepting vector by ligating the SacI overhangs and the overhangs from BsmBI and BsaI which conveniently have complementary overhangs (Fig. 4). Thus, now a new vector containing 8 repeats is obtained (PR8, Fig. 5).

![](https://static.igem.wiki/teams/5077/overhangs-map.svg)

Fig. 4: Depiction of the resulting overhangs after cutting the PR4 vector in two different tubes. BsmBI and BsaI form complementary overhangs that can be ligated for obtaining the PR8 plasmid.

![](https://static.igem.wiki/teams/5077/pr8-map.svg)

Fig. 5: Simplified plasmid map of PR8 containing 8 consecutive seamless repeats of resilin.

This procedure is repeated for the PR8 vector to obtain PR16 containing 16 repeats. As seen, there is a linear increase in repeats. The procedure can be repeated to eventually get a vector containing all 64 repeats (Fig. 6).

![](https://static.igem.wiki/teams/5077/pr64-map.svg)

Fig. 6: Simplified plasmid map of PR64 containing all of the 64 repeats.

To now express the resilin, the repeats can be cut using BsmBI and BsaI to be inserted into an expression vector. In our case we planned to use the pET-19b to include our 64 repeats to finally have the expression system at our hands (Fig. 7).

![](https://static.igem.wiki/teams/5077/pet-19b-r64-map.svg)

Fig. 7: Plasmid map of the pET-19b\_R64. This poses an expression system for the resilin protein polymer.

Using this method it would be easy to express different lengths of resilin (16 repeats, 32 repeats or 64 repeats).

#### Build

We ordered the R4 sequence and started the procedure by inserting it into the pMK1 plasmid was kindly provided by the group of Elke Deuerling. We then continued proceeding with trials to combine two pET-19b\_R4 with each other to obtain the pET-19b\_R8 plasmid.

#### Test

When attempting the insertion of R4 into pMK1 we promptly encountered difficulties. After transforming the supposedly pMK1\_R4 into \*Escherichia coli\* and obtaining the plasmids afterwards, we conducted an analytical digest using BsaI and SacI. As expected, the lanes containing the alleged pMK1\_R4, two bands were visible, representing the shorter insert (lower band) and the bigger vector backbone (higher band) (Fig. 8).

![](https://static.igem.wiki/teams/5077/resilin-240506-engineerinh.svg)

Fig. 8: 1 % agarose gel after double digest of pMK1 and pMK1\_R4 with SacI and BsaI. Lane 2: pMK1, lane 3-5: pMK1\_R4 from different colonies after transformation.

What was peculiar was the control lane with the empty pMK1 vector. According to the literature there was supposed to be no BsaI restriction recognition site in the empty pMK1 [1]. Thus, when cutting with BsaI and SacI we would only have expected SacI to cut, thus only one band to be seen in the gel. Since the ladder was not optimal and to investigate the abnormality we attempted another double digest.

For the second double digest we used SacI and NdeI. This time, we expected one band at around 3800 kb representing the backbone of pMK1. The fragment between the SacI site and the NdeI site should be too small to be seen on the gel. Against our expectations, two bands appeared for the empty pMK1 plasmid (Fig. 9).

![](https://static.igem.wiki/teams/5077/resilin-240610.svg)

Fig. 9: 1 % agarose gel after double digest of pMK1 and pMK1\_R4 with SacI and NdeI. Lane 2: pMK1, lane 3: pMK1\_R4.

We figured, there must be something inaccurate with the arrangement of restriction recognition sites in pMK1, so we sent the plasmid to sequencing. The sequencing report confirmed our suggestion. The plasmid contains two more additional restriction recognition sites, compromising our plan: one for BsmBI and one for BsaI (Fig. 10)

![](https://static.igem.wiki/teams/5077/pmk1-map.png)

Fig. 10: Plasmid map of pMK1 after sequencing. The sequence contains restriction recognition sites for BsaI and BsmBI that are hindering the success of the planned method.

#### Learn

At this point of our story line we could include several more engineering cycles on the trials of removing the restriction sites by mutagenesis.

In summary, removing the unwanted sites of BsmBI and BsaI took longer than expected and the method itself, even if it would work at the first attempt, would have taken several weeks as well. Due to time constraints we decided that we should look around for new, faster methods and to sadly let this cloning approach down.

## Cycle 2 - Rolling Circle Amplification

#### Design

The Rolling Circle Amplification (RCA), was one of the recommendations provided by Daniel Wedemeyer ([Link](https://2024.igem.wiki/hamburg/human-practices)) which makes use of unique polymerases, capable of isothermal DNA replication of circular DNA (cDNA) molecules. Their products consist of multiple repeats of the given cDNA template through a continuous addition of nucleotides to the primer, making it an ideal candidate for our goal of producing a molecule with up to 64 repeats of the resilin exon [2].

Ideally this method would consist of two steps:

1. Circularization of our DNA template by using a ligation template and the T4-ligase followed by a purification.
2. Replication by the phi29 polymerase which adds complementary nucleotides continuously to one primer until aborted.

![](https://static.igem.wiki/teams/5077/captura-de-pantalla-2024-10-02-a-las-0-54-32.png)
![](https://static.igem.wiki/teams/5077/captura-de-pantalla-2024-10-02-a-las-0-54-03.png)

Fig. 11: Schematic illustration of the planned RCA. (A) Steps for the circularization of an DNA template. (B) Linear RCA reaction for the synthesis of multiple repeats of the template cDNA through the binding of a polymerase to a primer bound to the template that incorporates nucleotides continuously [2].

For the use in our project, we planned a double digestion with BsaI and BsmBI after the amplification of pMK1-RE4. The samples would be run on an 2 % agarose gel from which the 180 bp RE4 fragment would have been cut out and purified for further usage.

![](https://static.igem.wiki/teams/5077/pmk1-re4-mutagen2.png)

Fig. 12: Simulation of pMK1-RE4.

![](https://static.igem.wiki/teams/5077/image.png)

Fig. 13: Simulation of an 2 % agarose gel after the double digestion with BsaI and BsmBI.

The T4-Ligase could then be used to ligate our fragment to form the cDNA template. A complementary sequence to the individual repeats was planned to be used as the primer for the phi29 polymerase, which would continuously add nucleotides until terminated.

With this approach, we would have to terminate the reaction after varying durations in our attempt to obtain molecules comprised of 16, 32, and 64 repeats.

Finally, to insert our fragment into an expression vector, we considered blunt end ligation and SLiCE cloning.

![](https://static.igem.wiki/teams/5077/rca-re4-circular-map.png)

Fig. 14: Circular four resilin repeats for rolling circle amplification.

A complementary sequence to the individual repeats was planned to be used as the primer for the phi29 polymerase, which would continuously add nucleotides until terminated.

---

With this approach, we would have to terminate the reaction after varying durations in our attempt to obtain molecules comprised of 16, 32, and 64 repeats.

Finally, to insert our fragment into an expression vector, we considered blunt end ligation and SLiCE cloning. However, this method was abandoned due to expected complications and the further development of another more promising approach.

#### Build

The previously constructed pMK1\_RE4 plasmid was used as our basis for this approach. The phi29 polymerase expression construct was included in the current distribution kit and the primer was ordered from IDT.

#### Test

We encountered issues as early as our agarose gels after digesting our pMK1\_RE4 plasmid with BsaI and BsmBI. The 180 bp fragment including our four resilin repeats that we intended to isolate was not visible (Fig. 13).

![](https://static.igem.wiki/teams/5077/resilin-240710-png.svg)

Fig. 13: Double digest of pMK1\_RE4 with BsmBI and BsaI. Both samples show bands at the height of 2000 bp and 1500 bp. 2 % agarose gel.

#### Learn

Simultaneously to planning the RCA method we also started to work on the following Repeatigo method approach.

After comparing both methods, we decided to go for the Repeatigo method, since we were facing diffuculties with the RCA approach.

For Repeatigo we knew how to design our parts, how to build experiments in the lab, and who to contact in case of problems (see our Human Practices page).

Thus, we discarded the RCA approach and focused on the Repeatigo method.

## Cycle 3 - Introducing the Repeatigo Method

#### Design

Our final approach for the cloning of the resilin repeats was a method based on the assembly of oligos. Thus, we developed the Repeatigo method. Read more about the concept of the method on our Project Description page ([Link](https://2024.igem.wiki/hamburg/description)) or Parts Page ([Link](https://2024.igem.wiki/hamburg/parts)).

The concept of this method is to synthesize single stranded oligos with a 5’-phosphorylization, that will assemble while repetitively creating overhangs for further assembly. Finally, double-stranded DNA will be generated containing several consecutive repeats of the same sequence. The start and end oligos do not allow further assembly and stop the extension process.

The first ligation is seperated by size in agarosegel and the desired length of the insert can be cut out and cleaned up form the gel.

We chose to use pET28a(+) as a vector because it is a simple standard expression vector with a C-terminal His-tag which we wanted to use to purify our resilin proteinpolymer. The His-tag is removed with thrombin which we already had in the lab.

The start and end oligos incorporate a restriction recognition side. The start oligos have a NdeI restriction recognition side and the end oligos have a SacI restriction site. This allows specific restriction and ligation into a vector. The ligated resilin oligos and pET28a(+) are cutted with NdeI and SacI and ligated.

![](https://static.igem.wiki/teams/5077/cloning-pet28a-re32.png)

Fig. 17: Restriction and Ligation of pET-28a(+) with the ligated 32 resilin repeats.

To express our vector we can not use normal E. coli strains. Standard strains do not like repetitive sequences and tend to recombine them, cut or even degrade them. Therefore we looked for a recombinase A deficient E. coli strain.

#### Build

We ordered the six needed oligos and the E. coli strain JM109 for expression as it is recA-deficient.

![](https://static.igem.wiki/teams/5077/blank-resilin-repeats.png)

Fig. 18: Schematic sequence map for n resilin repeats flanked by restriction recognition sites for NdeI and SacI.

![](https://static.igem.wiki/teams/5077/start-oligos.svg)

Fig 19: In silico annealing of the start-oligos to the desired sequence.

![](https://static.igem.wiki/teams/5077/middle-oligos.svg)

Fig 20: In silico annealing of the middle-oligos to the desired sequence.

![](https://static.igem.wiki/teams/5077/end-oligos.svg)

Fig 21: In silico annealing of the end-oligos to the desired sequence. They also insert the recognition sites for the SacI restriction enzyme.

#### Test

We used different ratios of start- and middle-oligos (Fig. 18) and different amounts of middle-oligos during the ligation step (Fig. 19). The gel showed DNA hinting to the fact that at least some oligos

![](https://static.igem.wiki/teams/5077/first-oligo-try.svg)

Fig. 22: The first trial of the Repeatigo method to produce repeat sequences of resilin. Lane 2-4 show results of different ratios of start- and middle-oligos. The gel shows smear bands in every sample lane centering around 100 bp. 1 % agarose gel.

![](https://static.igem.wiki/teams/5077/second-oligo-try.svg)

Fig. 23: The second trial of the Repeatigo method to produce repeat sequences of resilin. Lane 2 and 3 show results of the ligation using either 4 µg (lane 2) or 2 µg (lane 3) of middle-oligos for the ligation. 1 % agarose gel.

#### Learn

Our newly developed method did not work out at the first attempt. We decided to give it more chances and try different concentrations ratios, annealing and ligation times.

## Cycle 4 - Optimizing the Repeatigo Method

#### Design & Build

In a few more trials we had a lot of new realizations on how we could improve the Repeatigo method. Here a short overview on why and how we improved the process.

1. At first we did not see any bands in the gel. To try out a new approach we decided to change up the times for the annealing of the start and end oligos. The largest middle part is formed in this time, so we gave it 55 min to anneal instead of only 12. This made our repeats significantly longer.
2. We noticed that eventhough we clearly could see that the Repeatigo step worked, we got no PCR product visible on gels after the PCR amplification. We figured, this might be due to the fact that the primers of the PCR, which were exclusively binding to the start- and end-oligos, could not bind because these start and end oligos simply did not ligate to the middle part during the ligation step. Before, the start oligos were added and ligated to the middle part overnight before the end-oligos were added for a ligation over 2 h. Now we added the start-oligos and only waited 2 h for them to ligate before adding the end-oligos for a ligation overnight. This resulted in better products of the reaction as all oligos have enough time to be ligated and also the ends can bind.
3. The amount of DNA to obtain after the annealing and ligation is not very high. So after doing a gel clean up from the Repeatigo products we decided to run a PCR to amplify it.

#### Test

![](https://static.igem.wiki/teams/5077/240926-last-repeatigo.png)

Fig. 24:

The annealing and ligation times were improved so longer resilin fragments can form. We used 55 min for complementary oligo annealing and ligated all oligos over night with a T4 ligase.

Different concentrations of mid oligos were tried and the differences are visible in the gel (Fig. 24).

![](https://static.igem.wiki/teams/5077/second-oligo-try.svg)

Fig. 24: Oligo fragments of different lenghts. Different concentrations of mid oligos (see above the lanes) resulted in different length distributions of resilin fragments. Three different Fragmentlength were isolated from the gel (see red boxes); 1% agarose gel; DNA Ladder: 1 kb Plus.

Gel slices were extracted from 3, 1.5 and 1 kb and cleaned with the Quiagen gel clean up kit.

The following PCR amplification and gel (Fig. 25) worked as well after optimizing the ligation, DNA concentrations and the gel clean up, showing us a fragment at approximately 1400 bp. This means we were able to synthesize 30 resilin repeats.

![](https://static.igem.wiki/teams/5077/image-1.png)

Fig. 25: Oligo fragments of different lenghts. Different concentrations of template from gel clean up used (see above the lanes) Three fragments from lane 2, 5 and 6 were isolated from the gel; 1% agarose gel; DNA Ladder: 1 kb Plus.

After repeating the PCR some more times and saving the positive probes, we did a double digest of our resilin repeat sequence and pET28c(+) . We used pET28c(+) instead of pET28a(+) because it was available in our lab and has the same sites and vector properties.

![](https://static.igem.wiki/teams/5077/image-2.png)

Fig.26: pET28-c\_Resilin PCR. pET28-c not digested (negative control) and digested \*pET28-c 1-3 triplicates as well as digested \*pET28-c\_resilin. Additionally, the resilin sequence, as well as the undigested pET28-c\_resilin 1-3 are shown. Ladder: 1 kb Plus; Agarsoe gel: 1%

The restricted pET28-c and resilin sequences were effectively ligated into the pET28-c\_resilin plasmid. In comparison to the negative controls of just the undigested pET28-c plasmid lacking the resilin insert, the plasmids containing the resilin insert demonstrate a larger size, which corresponds to an approximate size of over 1 kb. As previously demonstrated, the size of the resilin fragment, synthesised using the repeatigo method, was approximately 1,4 kb.

Our plasmid pET-28c(+) was successfully transformed into E. coli DH5a strain. The following Colony PCR with primers binding in the resilin insert was positive for all testes colonies.

![](https://static.igem.wiki/teams/5077/image-3.png)

Fig. 27: pET28-c\_Resilin colony PCR. Four reactions were made with different clones from a transformation of DH5a with the pET28-c\_resilin. As a positive control the untransformed pET28-c\_resilin was used and as a negative control water was used as a template. Ladder: 1 kb Plus; Agarose gel: 1%.

Our insert is still 1,4 kb long so we successfully cloned about 30 resilin repeats into E. coli.

#### Engineering Success!

By using the Repeatigo method we were finally able to clone consecutive repeats of resilin. We had positive results in the gel after the ligation of the oligos (Fig. 24), were able to amplify the product in a PCR (Fig. 25), and finally could clone the insert into pET28c(+) followed by a positive result in a colony PCR (Fig. 27), we were able to confirm the presence of the resilin repeats in the vector. Our plasmid is currently at sequencing and we can not wait to see the results!

## Cycle 5 - Optimizing the gel extraction procedure

#### Design

We relied on gel extraction kits for experiments on both resilin and hyaluronic acid. For example for resilin we used our Repeatigo method to generate many resilin fragments of different lengths, but only wanted to work with fragments that were approx. 1 kb in size. We cut out these bands at the appropriate height and extracted them up. In the course of the process, we used three different gel clean-up kits and encountered two significant problems with all of them:
Despite working carefully and strictly following the protocol instructions, our samples were heavily contaminated with proteins, probably salts from the gel and TAE buffer, and organic solvents from the clean-up, as indicated by the 260/280 and 260/230 ratios. Furthermore, when we amplified the samples by PCR after the gel clean-up and then tried to load them onto a gel again, the samples did not sink to the bottom as usual but flowed out of the gel pocket. As a result, we didn't get any bands on the gel and could not progress with our experiments from this point on.

#### Build

We, therefore, thought about how we could optimize the Quiagen MinElute Gel Extraction Kit ourselves and came up with the following new approaches:

* additional washing step with 70 % ethanol
* let the alcoholic components evaporate by letting the tubes stand for 15-20 min with an open lid
* after the evaporation transfer the spin column in a new tube
* elute with 20 µL Elution Buffer
* run PCR as usual
* Before loading the PCR samples, let the samples again stand with an open lid for about 10-15 min
* add the loading dye pipette up and down and let it stand again for 5 min
* Pipette up and down before loading the samples until loading dye and sample are well mixed

The working group of Prof. Landau, also located at the CSSB, kindly provided us with 70 % ethanol, enabling us to test our new approaches to improving the gel clean-up. We had enough of the other necessary materials, such as tubes, which accelerated the implementation of our ideas.

#### Test

As we received the 70 % ethanol we tested our newly designed protocol. The instruction of Quiagen are marked in black and our additional steps are marked in green.

1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
2. Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg gel ~ 100 µL). The maximum amount of gel slice per spin column is 400 mg. For >2% agarose gels, add 6 volumes Buffer QG.
3. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). Vortex the tube every 2-3 min during incubation to help dissolve the gel.
4. Alter the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 pl 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn to yellow.
5. Add 1 gel volume of isopropanol to the sample and mix by inverting
6. Place a MinElute spin column • in a provided 2 mL collection tube or A into a vacuum manifold. For information about set up, see the MinElute Handbook.
7. Apply sample to the MinElute column and • centrifuge for 1 min or A apply vacuum until the entire sample has passed through the column. • Discard flow-through and place the MinElute column back into the same collection tube. For sample volumes of more than 800 µL, simply load and spin again.
8. Add 500 µL Buffer QG to the MinElute column and • centrifuge for 1 min or A apply vacuum. • Discard flow-through and place the MinElute column back into the same collection tube.
9. Add 750 µL Buffer PE to MinElute column and centrifuge for 1 min or A apply vacuum. • Discard flow-through and place the MinElute column back into the same collection tube. Note: If the DNA will be used for salt-sensitive applications, such as direct sequencing and blunt-ended ligation
10. Add 750 µL 70% ethanol to MinElute column and centrifuge for 1 min
11. Let the alcoholic components evaporate by letting the tubes stand for 15-20 min with an open lid
12. Transfer the MinElute column after the evaporation in a new 2 mL tube
13. Centrifuge the column in a 2 mL collection tube (provided) for 1 min. Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation.
14. Place each MinElute column into a clean 1.5 ml microcentrifuge tube. To elute DNA, add 20 µL instead of 10 µL Buffer EB (10 mM Tris-Cl, pH 8.5) or water to the center of the MinElute membrane. (Ensure that the elution buffer is dispensed directly onto the membrane for complete elution of bound DNA.) Let the column stand for 1 min, and then centrifuge the column for 1 min.
15. Measure concentration and 260/280 and 260/230 ratios using a NanoDrop
16. run PCR as normal
17. Let the samples stand with an open lid for about 10-15 min before loading the PCR samples
18. Add the loading dye and pipette up and down to suspend
19. Let it stand again with an open lid for 5 min
20. Pipette up and down before loading the samples until loading dye and sample are well mixed

First, we tested the optimized clean-up protocol using pUC\_HasA and pMK1 samples, which we amplified by PCR and then loaded onto a 1% agarose gel. We can show that we increased the purity regarding contamination with salts and organic solvent.

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| additional washing step with 70% ethanol | Sample | Concentration ng/µL | 260/280 | 260/230 |
| yes | pUC\_HasA 1 | 53.6 | 1.77 | 1.25 |
| no | pUC\_HasA 2 | 64.8 | 1.72 | 0.13 |
| yes | pMK1 1 | 16.4 | 1.47 | 0.75 |
| no | pMK1 2 | 30.9 | 1.42 | 0.15 |

After following our optimized protocol, we were first able to load our beforehand extracted and then amplified resilin DNA sequence on an agarose gel (Fig. 25) and then move on to the next steps in cloning resilin, which was extracting the amplified resilin sequence and starting a restriction-ligation reaction with pET28c(+), followed by transformation into *E. coli*.

#### Engineering Success

We learned that the elongated, additional evaporation steps and the 5-min incubation with the loading dye before loading the samples on the agarose gel were significant in avoiding the samples floating out of the gel pockets. We are really proud of our engineering success because it allows us to move on with our project and especially bring the cloning of our resilin repeats forward.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block page\_content %}

# Experiments

Caution! Experimentation Unleashed!

# +

# 1. Preparing Competent Cells

**Adapted from Zymo Research "Mix and Go E. coli Transformation Kit & Buffer Set" for generation of chemically
competent E. coli**
[Mix and Go E. coli Transformation Kit & Buffer Set](https://files.zymoresearch.com/protocols/_t3001_t3002_mix_go_e._coli_transformation_kit_buffer_set.pdf)

**Protocol**

**Preparation:**
Cells:
Day 1

* Prepare an overnight culture of the respective E. coli stem in 0.5 mL LB-medium.

Day 2

* Inoculate 50 mL SOB medium in a 500 mL culture flask with the 0.5 mL overnight culture.
* Incubate at 20-33 °C and 150-250 rpm until an OD of 0.4-0.6 is reached.

Buffer:
Zymo Wash and Competent Buffers are provided as 2X solutions. Dilute each to 1X by adding an equal amount of
Dilution Buffer.

1X buffers can be stored for up to 2 days at 4 °C.

Keep buffers on ice!

Further Preparation:

* Get Ice and liquid nitrogen.
* Keep empty 50 mL falcons on ice and prepare centrifuge to be at 4 °C.
* Bring equipment under the clean bench.
* Prepare 10 tubes for each 50 mL culture and store at -20 °C.

**Procedure:**
Work on ice all times!

1. After reaching an OD of 0.4-0.6, transfer the cell suspension into 50 mL Falcons and incubate it on ice for 10
   min.
2. Pellet the cells by centrifuging at 300-3700 rpm (1600-2500 x g) for 10 min at 4 °C.
3. Remove supernatant and resuspend cells gently in 5 mL 1X Wash Buffer.

   Adapt the volume if the measured OD was below 0.4 e.g. with an OD of 0.2, use 1 mL or with OD of 0.3 use 2 mL
4. Re-pellet cells as in step 2.
5. Completely remove supernatant and gently resuspend cells in 5 mL 1X Competent Buffer. If volume was adapted in
   step 3 adapt the volume here as well.
6. Aliquot 50-100 µL of the cell suspension into sterile 1.5 mL tubes.
7. Shock freeze cells in liquid nitrogen and store them at -80 °C until further usage.

# +

# 2. Transformation

* Thaw 100 µL of competent cells on ice for 15 min.
* Add 100 ng/µl of plasmid to aliquot and resuspend carefully (work on ice and under sterile conditions).
* Incubate DNA and cells for 20 min on ice.
* Incubate the solution for 45 seconds at 42 °C.
* Put the cells on ice for 5 min.
* Add 600-700 µl SOC medium to the cells.
* Incubate the cells for 45 min at 37 °C and 300 rpm.
* Centrifuge for 1 min at 14.000 x g.
* Discard all but 50 µL of the supernatant and resuspend the pellet in the remaining 50 µL.
* Plate the sample on LB-Agar plates containing 100 µg/mL of the respective antibiotic(s).
* Incubate plates at 37 °C overnight.

# +

# 3. Digest with Restriction Enzymes

We used the NEBcloner to find the specific protocol for the desired restriction enzymes: <https://nebcloner.neb.com/#!/redigest>

**Protocol**

* Set up reaction as follows:

|  |  |  |
| --- | --- | --- |
| Component | Volume for 50 µL | Volume for 20 µL |
| DNA | 1 µg | 0.5 µg |
| 10 XNEBuffer\* | 5 µL (1X) | 2 µl (1X) |
| restriction enzyme | 0.5 | 0.25 |
| Nuclease-free Water | up to 50 µL | up to 20 µl |

\* for all enzymes except BsmBI\_V2 the rCutSmart-buffer was used. for BsmBI\_V2 the r3.1-buffer was used

* All enzymes except BsmBI\_V2 were Incubate at 37 °C for 1h
* BsmBI\_V2 was Incubate at 55 °C for 1h

# +

# 4. Ligation (T4 Ligase)

### 4.1 T4 Ligase NEB

The [T4 Ligase from
NEB](https://www.neb.com/en/protocols/0001/01/01/dna-ligation-with-t4-dna-ligase-m0202) was used:

1. Set up the following reaction in a microcentrifuge tube. Work on ice! We used a molar ratio of 1:1 up to 1:7 of
   vector to insert.
   For the molar ratio calculation we used the [NEBioCalculator](http://nebiocalculator.neb.com/#!/#!/)

   |  |  |
   | --- | --- |
   | Component | Volume for 20 µL |
   | Vector DNA | 0.020 pmol |
   | Insert DNA | 0.06 pmol |
   | T4 DNA Ligase Buffer (10X) | 2 μl |
   | T4 DNA Ligase | 1 μl |
   | Nuclease-free water | up to 20 μl |
2. Gently mix the reaction by pipetting up and down and microfuge shortly.
3. For cohesive (sticky) ends, incubate at 16 °C overnight or room temperature (RT) for 10 minutes.
4. For blunt ends or single base overhangs, incubate at 16 °C overnight or at RT for 2 hours.
5. To inactivate the ligase, incubate at 65 °C for 10 minutes.
6. For further usage in a transformation, put the tube on ice and transform 1-5 μl of the reaction into 50 μl
   competent cells (see “2. Transformation”). Alternatively, DNA can be stored at -20 °C until further usage.

### 4.2 T4 DNA Ligase Jena Bioscience

The protocol was adapted from the T4 DNA Ligase-protocol from <https://www.jenabioscience.com/images/PDF/EN-149.0004.pdf>

**Protocol**

set up reaction as follows

Component | 20 µl Assay || Standard Ligation Buffer, 10x conc. | 2 µl |
| Vector/Insert DNA | 1 µg |
| T4 DNA Ligase | 0.4 µl |
| PCR-grade Water | Add up to 20 µl |

Incubate for 30 min at 16°C.

# +

# 5. PCR

### 5.1 Q5 Polymerase from NEB

Adapted protocol from the [NEB Q5®
High-Fidelity 2X Master Mix](https://www.neb.com/en/protocols/2012/12/07/protocol-for-q5-high-fidelity-2x-master-mix-m0492)

**Preparations:**

* Work on ice for mixing the components.
* Preheat the thermocycler to 98 °C.

**Procedure:**

* Mix the following components in PCR tubes.

  |  |  |  |
  | --- | --- | --- |
  | Component | Volume for a 50 µl Reaction | Final Concentration |
  | Q5 High-Fidelity 2X Master Mix | 25 µl | 1X |
  | 10 µM Forward Primer | 2.5 µl | 0.5 µM |
  | 10 µM Reverse Primer | 2.5 µl | 0.5 µM |
  | Template DNA | variable | 1-10 ng |
  | Nuclease-Free Water | to 50 µl |  |
* Gently mix the components by pipetting up and down.
* If necessary, collect all liquid at the bottom of the tubes by shortly centrifuging them.
* Transfer the PCR tubes to the thermocycler and begin thermocycling.

Thermocycling Conditions for a Routine PCR:

|  |  |  |
| --- | --- | --- |
| Step | Temperature | Time |
| Initial denaturation | 98 °C | 3 min |
| Denaturation | 98 °C | 30 sec |
| Annealing\* | TM-5 | Experiment dependendent |
| Elongation | Experiment dependendent | 30 seconds/kb |
| Final Extension | 72 °C | 3 min |
| Hold | 4-10°C | ∞ |

\* We either used SnapGene or the "Takara Primer design"-tool to calculate the annealing temperature.

### 5.2 Phusion from Thermo Fisher or Promega

Phusion PCR protocol adapted from Thermo Fisher and Promega original Protocol:
**Thermo Fisher:** <https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0012393_Phusion_HighFidelity_DNAPolymerase_UG.pdf>

**Promega:**  [https://www.promega.de/-/media/files/resources/protocols/product-information-sheets/g/pfu-dna-polymerase-protocol.pdf?rev=5680f3dffd8a41449c71d0e50ca27ad4≻\_lang=en](https://www.promega.de/-/media/files/resources/protocols/product-information-sheets/g/pfu-dna-polymerase-protocol.pdf?rev=5680f3dffd8a41449c71d0e50ca27ad4&sc_lang=en)

In a sterile, nuclease-free microcentrifuge tube, combine the following Components for a 50µL mix:

|  |  |
| --- | --- |
| Component | Volume |
| Primer 10 µM | 2,5 µL |
| Primer 10 µM | 2,5 µL |
| Template | 1-10 ng, e.g. 1 µL from a 5 ng/µL concentrated Template solution |
| dNTPs(200 µM) | 1 µL |
| 10X Phusion Buffer | 5 µL |
| Phusion Polymerase | 0.5 µL |
| H2O | Add to 50 µL |

**Thermocycler Program:**

|  |  |  |
| --- | --- | --- |
| Steps | Temperature | Time |
| Initial denaturation | 98 °C | 3 minutes |
| denaturation | 98 °C | 30 seconds |
| Annealing\* | TM-5 | Experiment dependendent |
| Extension | 72 °C | Thermo Fisher: 15-30 seconds/kb Promega: 2 minutes/kb |
| Final extention | 72 °C | 3 min |
| Hold | 6 °C | ∞ |

\* We either used Snapgene or the “Takara Primer design”-tool to calculate the annealing temperature.

### 5.3 DreamTaq Mastermix from Thermo Fisher

DreamTaq PCR protocol adapted from Thermo Fisher [Link](https://static.igem.wiki/teams/5077/dreamtaq-dna-polymerases-labaid.pdf)
<https://assets.thermofisher.com/TFS-Assets/BID/Reference-Materials/dreamtaq-dna-polymerases-labaid.pdf>

In a sterile, nuclease-free microcentrifuge tube, combine the following Components for a 50 µL mix:

Components | Volume || Forward Primer (1 µM) | 2.5 |
| Reverse Primer (1 µM) | 2.5 |
| Template DNA | 10 pg–1 μg |
| DreamTaq DNA Polymerase/DreamTaq PCR Master Mix (2X) | 25 μL |
| H2O | Ad 50 μL |

**Thermocycler Program:**

Steps | Temperature | Time || Initial denaturation | 98 °C | 3 minutes |
| denaturation | 98 °C | 30 seconds |
| Annealing\* | TM-5 | 30 seconds |
| Extension | 72 °C | 1 min for products up to 2 kb. Longer extensions: 1 min/kb |
| Final extention | 72 °C | 3- 10 min |
| Hold | 4 °C | ∞ |

1 cycle for the Initial denaturation.
30 cycles for the steps up to the final extension.
For colony PCR: Pick colony with pipette tip and resuspend in PCR sample.

# +

# 6. Gel Clean Up

### 6.1 Promega gel and PCR clean up

#### Original “Wizard® SV Gel and PCR Clean-Up System” Protocol

Website: <https://www.promega.de/products/nucleic-acid-extraction/clean-up-and-concentration/wizard-sv-gel-and-pcr-clean-up-system/?catNum=A9281>

**A. Dissolving the Gel Slice**

1. Following electrophoresis, excise DNA band from gel and place gel slice in a 1.5ml microcentrifuge tube.
2. Add 10μl of Membrane Binding Solution per 10 mg of gel slice. Vortex and incubate at 65 °C until gel slice is
   completely dissolved.

**B. Processing PCR Amplifications**

1. Add an equal volume of Membrane Binding Solution to the PCR amplification.

**Binding of DNA**

1. Insert SV Minicolumn into Collection Tube.
2. Transfer dissolved gel mixture or prepared PCR product to the Minicolumn assembly. Incubate at room temperature
   for 1 minute.
3. Centrifuge at 16,000 × g for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube.

**Washing**

1. Add 700 μl of Membrane Wash Solution (ethanol added). Centrifuge at 16,000 × g for 1 minute. Discard flowthrough
   and
   reinsert Minicolumn into Collection Tube.
2. Repeat Step 4 with 500 μl of Membrane Wash Solution. Centrifuge at 16,000 × g for 5 minutes.
3. Empty the Collection Tube and recentrifuge the column assembly at top speed for 2 minute with the
   microcentrifuge lid open (or off) to allow evaporation of any residual ethanol.

**Elution**

1. Carefully transfer Minicolumn to a new 1.5 ml microcentrifuge tube.
2. Add 50 µl of Nuclease-Free Water to the Minicolumn. Incubate at room temperature for 1 minute.
   Centrifuge at 16,000 × g for 1 minute.
3. Discard Minicolumn and store DNA at 4 °C or -20 °C.

#### Adapted “Wizard® SV Gel and PCR Clean-Up System” Protocol

**A. Dissolving the Gel Slice**

1. Following electrophoresis, excise DNA band from gel and place gel slice in a 1.5ml microcentrifuge tube.
2. Add 10μl of Membrane Binding Solution per 10 mg of gel slice. Vortex and incubate at 50-65 °C until gel slice is
   completely dissolved.

**Processing PCR Amplifications**

1. Add an equal volume of Membrane Binding Solution to the PCR amplification.

**Binding of DNA**

1. Insert SV Minicolumn into Collection Tube.
2. Transfer dissolved gel mixture or prepared PCR product to the Minicolumn assembly. Incubate at room temperature
   for 1 minute.
3. Centrifuge at 9,000 × g for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube.

**Washing**

1. Add 700 μl of Membrane Wash Solution (ethanol added). Centrifuge at 9,000 × g for 1 minute. Discard flowthrough
   and
   reinsert Minicolumn into Collection Tube.
2. Repeat Step 4 with 500 μl of Membrane Wash Solution. Centrifuge at 9,000 × g for 5 minutes.
3. Empty the Collection Tube and recentrifuge the column assembly at top speed for 2 minute with the
   microcentrifuge lid open (or off) to allow evaporation of any residual ethanol.

**Elution**

1. Carefully transfer Minicolumn to a new 1.5 ml microcentrifuge tube.
2. Add 20-30μl of Nuclease-Free 65 °C Water to the Minicolumn. Incubate at room temperature for 5-10 minutes.
   Centrifuge at 16,000 × g for 1 minute.
3. Discard Minicolumn and store DNA at 4 °C or -20 °C.

### 6.2 Quiagen gel clean up

#### Original "MinElute® Gel Extraction Kit" protocol

[https://www.qiagen.com/de/resources/resourcedetail?id=0d0d73a7-e906-4a74-8f11-3b70311d7d66⟨=en](https://www.qiagen.com/de/resources/resourcedetail?id=0d0d73a7-e906-4a74-8f11-3b70311d7d66&lang=en)

Notes before starting

* This protocol is for cleanup of up to 5 pg DNA fragments (70 bp to 4 kb).
* The yellow color of Buffer QG indicates a pH of <7.5. Adsorption of DNA to the membrane is efficient only at
  pH <7.5.
* Add ethanol (96-100%) to Buffer PE concentrate before use (see bottle label for volume).
* Isopropanol {100%} and a heating block or water bath at 50 °C are required.
* All centrifugation steps are carried out at 17,900 x g (13,000 rpm) in a conventional tabletop microcentrifuge
  at room temperature (15-25 °C).
* Symbols: @ centrifuge processing; & vacuum processing.

1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
2. Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg gel ~ 100 pl).
   The maximum amount of gel slice per spin column is 400 mg. For >2% agarose gels, add 6 volumes Buffer QG.
3. Incubate at 50 °C for 10 min (or until the gel slice has completely dissolved). Vortex the tube every 2-3 min
   during incubation to help dissolve the gel.
4. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer
   QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 pl 3 M sodium acetate, pH
   5.0, and mix. The color of the mixture will turn to yellow.
5. Add 1 gel volume of isopropanol to the sample and mix by inverting.
6. Place a MinElute spin column in a provided 2 ml collection tube or & into a vacuum manifold. For information
   about set up, see the MinElute Handbook.
7. Apply sample to the MinElute column and centrifuge for 1 min or & apply vacuum until the entire sample has
   passed through the column. Discard flow-through and place the MinElute column back into the same collection
   tube. For sample volumes of more than 800 pl, simply load and spin again.
8. Add 500 pl Buffer QG to the MinElute column and centrifuge for 1 min or & apply vacuum. @ Discard flow-through
   and place the MinElute column back into the same collection tube.
9. Add 750 pl Buffer PE to MinElute column and centrifuge for 1 min or & apply vacuum. - Discard flow-through and
   place the MinElute column back into the same collection tube. Note: If the DNA will be used for saltsensitive
   applications, such as direct sequencing and bluntended ligation, let the column stand 2-5 min after addition of
   Buffer PE.
10. Centrifuge the column in a 2 ml collection tube (provided) for 1 min. Residual ethanol from Buffer PE will not
    be completely removed unless the flow-through is discarded before this additional centrifugation.
11. Place each MinElute column into a clean 1.5 ml microcentrifuge tube. To elute DNA, add 10 pl Buffer EB (10 mM
    Tris-Cl, pH 8.5) or water to the center of the MinElute membrane. (Ensure that the elution buffer is dispensed
    directly onto the membrane for complete elution of bound DNA) Let the column stand for 1 min, and then
    centrifuge the column for 1 min.
12. If purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA, and mix by
    pipetting up and down before loading the gel.

#### Adapted “MinElute® Gel Extraction Kit” protocol

1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
2. Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg gel ~ 100 pl).
   The maximum amount of gel slice per spin column is 400 mg. For >2% agarose gels, add 6 volumes Buffer QG.
3. Incubate at 50 °C for 10 min (or until the gel slice has completely dissolved). Vortex the tube every 2-3 min
   during incubation to help dissolve the gel.
4. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer
   QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 pl 3 M sodium acetate, pH
   5.0, and mix. The color of the mixture will turn to yellow.
5. Add 1 gel volume of isopropanol to the sample and mix by inverting.
6. Place a MinElute spin column in a provided 2 ml collection tube or & into a vacuum manifold. For information
   about set up, see the MinElute Handbook.
7. Apply sample to the MinElute column and centrifuge for 1 min or & apply vacuum until the entire sample has
   passed through the column. Discard flow-through and place the MinElute column back into the same collection
   tube. For sample volumes of more than 800 pl, simply load and spin again.
8. Add 500 pl Buffer QG to the MinElute column and centrifuge for 1 min or & apply vacuum. Discard flow-through and
   place the MinElute column back into the same collection tube.
9. Add 750 pl Buffer PE to MinElute column and centrifuge for 1 min or & apply vacuum. - Discard flow-through and
   place the MinElute column back into the same collection tube.
10. Add 750 µL 70% ethanol to MinElute column and centrifuge for 1 min. If the DNA will be used for saltsensitive
    applications, such as direct sequencing and bluntended ligation, let the column stand 5-10 min after addition of
    Buffer PE.
11. Centrifuge the column in a 2 ml collection tube (provided) for 1 min. Residual ethanol from Buffer PE will not
    be completely removed unless the flow-through is discarded before this additional centrifugation.
12. Place each MinElute column into a clean 1.5 ml microcentrifuge tube. To elute DNA, add 10 pl Buffer EB (10 mM
    Tris-Cl, pH 8.5) or water to the center of the MinElute membrane. (Ensure that the elution buffer is dispensed
    directly onto the membrane for complete elution of bound DNA) Let the column stand for 1 min, and then
    centrifuge the column for 1 min.
13. If purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA, and mix by
    pipetting up and down before loading the gel.

# +

# 7. Plasmid Mini-Prep

### 7.1 NEB

We used the original "Monarch® Plasmid DNA Miniprep Kit" Protocol from the webside:
<https://www.neb.com/en/protocols/2015/11/20/monarch-plasmid-dna-miniprep-kit-protocol-t1010>

### 7.2 Roth

Adapted from the original "ROTI®Prep Plasmid MINI-XL" protocol:
[https://www.carlroth.com/medias/BA-8456-EN.pdf](https://www.carlroth.com/medias/BA-8456-EN.pdf?context=bWFzdGVyfGluc3RydWN0aW9uc3w1MzEzMTd8YXBwbGljYXRpb24vcGRmfGluc3RydWN0aW9ucy9oNTUvaGZjLzkwMzc3MjI1ODMwNzAucGRmfGQ5MDgzNWU1Yzk0Y2U3MDhiNTg0MmJlNzgxMTQ1MjFmZGI2NzRjMWY3OTYzMzRjNWRiYzI1Njg5NWE3NmI5M2M)

1. Cell Harvest and Resuspension Harvest the bacterial cell culture (5-15 ml) by centrifugation for 10 mins at approx. 4.500 rpm (RT). Discard supernatant and remove remaining culture medium carefully. Resuspend pelleted bacterial cells completely in 550 μl Resuspension Buffer RB (incl. RNAse A). as the a original Resuspension Buffer has expired and was stored at RT and we didn't know how long it had been there, we took a different lysis buffer from the fridge.
2. Cell Lysis and Neutralization. Add 550 μl of Lysis Buffer LS. Mix gently by inverting the tube 10 times (Do not vortex!). Incubate for 5 mins at room temperature. Add 750 μl of Neutralization Buffer NB and immediately mix by inverting the tube 10x. Centrifuge at 12.000-14.000 rpm (or full speed) for 10 mins/RT.
3. Column Loading. Place each Spin Column into a 2 ml collection tube. Apply 750 μl of the clearified supernatant from step 2 into the spin column. Be sure to omit as much of the white precipitant as possible. Centrifuge at 10.000 g (or 12.000 rpm) for 1 min/RT and discard the flow-through\*. Apply the residual clearified supernatant from step 2 into the spin column. Be sure to omit as much of the white precipitant as possible. Centrifuge at 10.000 g (or 12.000 rpm) for 1 min/RT and discard the flow-through\*
4. Column Washing. Add 650 μl of Washing Buffer PA to the Spin Column. Centrifuge at 10.000 g (or 12.000 rpm) for 1 min/RT and discard the flow-through\*. Add 750 μl of Washing Solution PB to the Spin Column. Centrifuge at 10.000 g (or 12.000 rpm) for 1 min/RT and discard the flow-through\*. Again centrifuge columns at 12.000-14.000 rpm (or full speed) for 2 mins/RT in order to remove residual ethanol.
5. Elution. Place the Spin Column into a clean 1.5 ml elution tube. Add 100 μl Elution Buffer XEB or sterile, distilled water to the centre of the membrane\*\*. Incubate for 3 mins at room temperature. Centrifuge at 6.000 g (or 8.000 rpm) for 1 min/RT to elute DNA.

\*If the solution has not completely passed through the spin column, centrifuge again at higher speed or prolong the centrifugation time.

\*\* Elution with lower volumes of Elution Buffer increases the final concentration of DNA. In case DNA shall be eluted in 50 μl only, preheating of the elution buffer is obligatory. Using heated Elution Buffer (approx. 50 °C) will increase the DNA yield. Alternatively, DNA may be eluted in 2x 50 μl buffer. If so, use 50 μl preheated buffer for elution, centrifuge. Then repeat this elution process with another 50 μl and finally pool both fractions.

# +

# 8. Agarose gel

* for a 100 ml 1% agarose gel 100 ml TAE buffer was mixed with 1 g of agarose.
* 1 µL of "midori Green Advance" was addet per 50 µL of agarose gel.
* for the ladder "NEB Quick-Load Purple 1 kb Plus DNA Ladder" was used. here we used 8 µL for a 100 mL medium geltray.
* as loading dye "Gel Loading Dye, Purple (6X)" was used.

# +

# 9. SDS-Page

**Make the SDS-Gel**

1. Clean a big and a small glass plate with 70% ethanol.
   * leave them on the bench and wipe in one direction
2. Place the small glass plate on the big 1.5 mm glass plate (cleaned surfaces to each other).
3. Place the sandwich in the green clamps (small glass plate facing outwards).
4. Check for leakage with water (remove in the sink with paper tissues).
5. Acrylamide concentration depends on the molecular weight of the target protein.

Running gels. Volumes per 10 mL-gels.

|  |  |  |  |
| --- | --- | --- | --- |
|  | 7.5% | 10% | 15% |
| Acrylamide (30% w/v) !!shake | 2.5 mL | 3.33 mL | 5 mL |
| 4x running buffer | 2.5 mL | 2.5 mL | 2.5 mL |
| H2O | 4.89 mL | 4.06 mL | 2.39 mL |
| APS (10% w/v) | 100 µL | 100 µL | 100 µL |
| TEMED | 10 µL | 10 µL | 10 µL |

Stacking gel

|  |  |  |  |
| --- | --- | --- | --- |
|  | 10 mL (1 oder 2 Gele) | 20 mL | 30 mL |
| Acrylamide (30% w/v) !!shake | 1.16 mL | 2.32 mL | 3.48 mL |
| 4x stacking buffer | 2.5 mL | 5 mL | 7.5 mL |
| H2O | 6.23 mL | 12.46 mL | 18.69 mL |
| APS (10% w/v) | 100 µL | 200 µL | 300 µL |
| TEMED | 10 µL | 20 µL | 30 µL |

1. Label two 50 mL tubes (stacking and running gel).
2. Add Acrylamide, 4X running buffer, and H2O.
3. With the help of the comb, mark where the running buffer should be poured (5 mm below the end of the comb).
4. Take up the volume of TEMED with the pipette and set aside.
5. Pipette the volume of APS into the tube and quickly after that also TEMED.
6. Invert the tube 4 times and pour it between the glass plates until the mark is reached.
7. Add quickly a thin layer of isopropanol.
8. Let it polymerize for 15-30 min.
9. Pour off isopropanol and wash twice with ddHO (wipe with tissue paper).
10. Clean the comb from both sides with 70% ethanol.
11. Add Acrylamide, 4X stacking buffer, and H2O.
12. With the help of the comb, mark where the running buffer should be poured (5 mm below the end of the comb).
13. Take up the volume of TEMED with the pipette and set aside.
14. Pipette the volume of APS into the tube and quickly after that also TEMED.
15. Invert the tube 4 times and pour it till it overflows.
16. Insert the comb initially at a 45-degree angle.
17. Let it polymerize for 15-30 min.
18. Check if there is enough reservoir buffer (1800 mL ddHO and 200 mL 10x Resevoir buffer).

**Load & run SDS-PAGE**

1. Take the gels out of the fridge so they can warm up a bit if you prepared them the day before.
2. Prepare the running chamber.
3. Centrifuge the samples for 15 s (sort them in order).
4. Boil the REDUCING samples at 95 degrees for 5 minutes.
5. Unwrap the gels from plastic wrap & paper towels.
6. Clamp & position the gels in the electrophoresis tank (pay attention to the electrode color!).
   * the small glass plate should face the inner side
7. Label the gels with (NR/R) on the side of the tank with some tape.
8. Fill the running chamber with reservoir buffer by first filling the chamber between the gels and then overflowing and filling up to approx. 1 cm under the upper edge of the large glass plate.
9. Remove the comb by lifting it straight upwards.
10. Load the gels with the sample.
11. Close the lid, switch on the device, and plug in the cable (pay attention to colors!).
12. Set 200 V, press "run" & check if air bubbles are rising.
13. After 5 min, check whether the samples are migrating.
14. Allow running until the blue band has run out.

# +

# 10. Homologous Recombination

### 10.1 SLiCE

Adapted from the paper "Seamless Ligation Cloning Extract (SLiCE) Cloning Method"
doi: [10.1007/978-1-62703-764-8\_16](https://doi.org/10.1007/978-1-62703-764-8_16)

**SLiCE Reaction protocol**

1. Set up the SLiCE reaction in a 0.2 mL microcentrifuge tube and mix well by vortexing.

   SLiCE reaction conditions

   |  |  |
   | --- | --- |
   | Components | Standard cloning |
   | Vector DNA | Purified, 50-200 ng |
   | Insert DNA | Purified, 1:1-10:1\*\* |
   | 10× SLiCE buffer\* | 1 μL |
   | PPY SLiCE extract\* | 1 μL |
   | ddH2O | to 10 μL |
   | *Reaction conditions* |  |
   | Incubation temperature | 37 °C |
   | Incubation time | 55 min |

   *\* The SLiCE buffer and Extract was provided by the Kolbe Working Group*

   *\*\* Molar ratio of insert to vector*
2. Incubate the SLiCE reaction mix at 37 °C for 15 min to 1 h using a PCR machine or water bath.
3. Transfrom 1 μL SLiCE reaction mix into 50-100 μL Competent Cells (see 2. Transformation).
4. Plate the transformed cells onto agar plates containing appropriate antibiotics.

### 10.2 NEB-builder

The “NEBuilder® Protocol Calculator” was used to adapt the protocol.
Web: <https://nebuildercalculator.neb.com/>

**Protocol for homolog recombination**
Shown here using the example of a homologous recombination of EGFP with the pUC\_hasA vector (length and concentration may differ from the original).

|  |  |  |
| --- | --- | --- |
| Name | Length (basepairs) | Concentration (ng/µl) |
| pUC\_HasA | 5200 | 100 |
| EGFP | 750 | 35 |

* Set up the reaction on ice (see table).

  |  |  |  |
  | --- | --- | --- |
  | Component | Volume | Amount |
  | pUC\_HasA | 1.6 μl\* | 0.050 pmoles\* |
  | EGFP | 1.3 μl\* | 0.100 pmoles\* |
  | Deionized H2O | 7.1 μl | - |
  | NEBuilder HiFi DNA Assembly Master Mix | 10.0 μl | - |
  | Total | 20.0 μl | 0.150 pmoles |

  \* to calcualte the vector and insert volumes the "NEBuilder® Protocol Calculator" was used. The molar ratio varied between 1:1 and 1:10
* Incubate samples in a thermocycler at 50 °C for 15 minutes. Following incubation, store samples on ice or at -20 °C for subsequent transformation. Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases.
* Transform NEB® 5-alpha, NEB Stable or NEB 10-beta Competent *E. coli* cells (provided in the cloning kit, bundle or purchased separately from NEB) with 2.0 μl (10%) of the chilled assembled product, following the transformation protocol.

# +

# 11. DpnI digestion

Adapted from NEB.

Components | Volume || PCR Sample | 20 µL |
| 10x rCutSmart Buffer | 2.5 µL |
| DpnI | 0.5 μL |
| H2O | Ad. 25 µL |

* Incubation for at least 2h at 37°C
* Heat inactivation for 30 min at 80°C

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block page\_content %}

# Results

When the idea becomes reality.

# Resilin

## pMK1

## Repeatigo Method

**pMK1 with four Resilin repeats**
For the specific properties of our hydrogel, we needed to synthesize resilin consisting of 32–64 repeats to meet our requirements. A simple way to achieve this was by using the pMK1 vector, which was kindly provided by Elke Deuerling. By designing repeats flanked by two type IIS endonucleases, it is theoretically possible to generate as many repeats as needed for the intended application.

The design of our repeats (RE4) flanked by BsmBI and BsaI recognition sites was successfully completed. These restriction sites were additionally flanked by SacI and NdeI to facilitate ligation into the pMK1 vector. The pMK1 vector and the RE4 fragment, which was provided by IDT, were both digested with the appropriate restriction enzymes.

Subsequently, RE4 was successfully cloned into the pMK1 vector, resulting in the plasmid pMK1\_RE4. The results were confirmed through restriction digestion (see Fig. 1) and sequencing.

![](https://static.igem.wiki/teams/5077/result/figure-1.svg)

Fig. 1: 1 % agarose gel after double digest of pMK1 and pMK1\_R4 with SacI and BsaI. Lane 2 and 5 with digested pMK1 shows two bands at around 3000 bp and 600 bp. Lane 3-5 and 6-9 with digested pMK1\_R4 show two bands each at around 3000 bp and 200 bp.

Additionally the insertion was confirmed by Sanger sequencing (see Fig. 2). The sequencing also showed that there where no mutionas in the RE4 sequence.

![](https://static.igem.wiki/teams/5077/result/figure-2.webp)

Fig. 2: Sequencing result of the RE4 region in the pMK1\_RE4 Plasmid. On A is shown the first halve of the RE4 insertion and on B is shown the second half of the RE4 insertion.

**Repeatigo Method**
The method utilising the pMK1 vector presents a challenge in the generation of a fragment with numerous repeats. As the number of repeats increases, the consumption of resources and time also rises. For a fragment requiring 64 repeats, the estimated cloning time using this method is at least 17 days. However, our proposed approach could theoretically generate the 64 repeats and potentially many more within a two-day timeframe.

For the method we designed 6 Oligos. First, the middle primers and also the end and start oligos were cooled down from 90 degrees to RT within 12 min. The middle oligos were then combined with the start oligos and incubated overnight with a T4 ligase. The next day, the end oligos were ranigated for one hour to obtain fragments with a maximum size of 300-500 bp (see Fig. 3).

![](https://static.igem.wiki/teams/5077/result/figure-3.svg)

Fig. 3: The first trial of the Repeatigo method to produce repeat sequences of resilin. Lane 2-4 show results of different ratios of start- and middle-oligos. The gel shows smear bands in every sample lane centering around 100 bp. 1 % agarose gel.

![](https://static.igem.wiki/teams/5077/result/figure-4.svg)

Fig. 4 The second trial of the Repeatigo method to produce repeat sequences of resilin. Lane 2 and 3 show results of the ligation using either 4 µg (lane 2) or 2 µg (lane 3) of middle-oligos for the ligation. 1 % agarose gel.

Next, we tried to extend the time in which the oligos cool down from 90 °C to 20 °C and in which they anneal to approx. 35 min. In addition, we only used half the concentration of oligos with the same volume in one sample.

As can be seen, this allowed us to create much longer oligos. We were able to show that the length of the cooling time plays a role and that a lower concentration also has a positive effect on the length of the fragments (Fig. 4).

After this, we purified several different sizes of fragments from the gel, but none of the PCRs were successful (see Fig. 5).

After this we improved several steps:

1. We optimized the gel purification by purchasing a new gel purification kit and optimizing our purification protocol by adding an additional ethanol purification step with 70% ethanol and longer ethanol evaporation phases.
2. We tried to amplify only the middle oligos overnight in three out of six samples (see Fig. 6) without adding end or start oligos (these were added the next day). Doing this, we were able to generate long oligo fragments even though the concentrations were up to 10 times higher than usually. This also helps to increase the purity of the isolated fragments.
3. We switched to a more efficient T4 ligase. Our previous ligase could ligate 0.02 pmol of insert in a 20 µL reaction, while the new ligase was capable of catalyzing 1 nmol under the same conditions. This was important as we had large quantities of DNA that had to be aligned.

![](https://static.igem.wiki/teams/5077/result/figure-5.webp)

Fig. 5: Gradient PCR to verify the correct annealing temperature for the oligos. Ladder: 1 kb Plus; agarose gel (1 %).

![](https://static.igem.wiki/teams/5077/result/figure-6.webp)

Fig. 6: Oligo Fragments of different lengths. Different concentrations of middle-oligos (see above the lanes) resulted in different length distributions of resilin fragments. Three different fragment lengths were isolated from the gel (see red boxes); 1% agarose gel; DNA Ladder: 1 kb Plus.

Thanks to this, we finally were able to successfully amplify a fragment of approximately 1300 bp hinting to approximately 32 consecutive resilin repeats (Fig. 7).

To reach a higher concentration, which is essential for the restriction ligation of resilin and the pET28-c plasmid, we performed several PCR reactions to amplify our resilin sequence.

![](https://static.igem.wiki/teams/5077/result/figure-7.webp)

Fig. 7: PCR for amplification of resiilin oligos with a lenght of about 1,3 kb from two different resilin reactions. 1% agarose gel; DNA Ladder: 1 kb Plus.

**Restriction Ligation of pET28-c with resilin**

![](https://static.igem.wiki/teams/5077/result/figure-8.webp)

Fig.8: pET28-c\_Resilin PCR. pET28-c not digested (negative control) and digested \*pET28-c 1-3 triplicates as well as digested \*pET28-c\_resilin. Additionally, the resilin sequence, as well as the undigested pET28-c\_resilin 1-3 are shown. Ladder: 1 kb Plus; agarsoe gel: 1%

The restricted pET28-c and resilin sequences were effectively ligated into the pET28-c\_resilin plasmid. In comparison to the negative controls of undigested pET28-c plasmid lacking the resilin insert, the plasmids containing the resilin insert demonstrate a larger size, which corresponds to an approximate size of over 1 kb. As previously demonstrated, the size of the resilin fragment, synthesised using the repeatigo method, was approximately 1.3 kb.

**Colony PCR of TOP10 transformed with pET28-c\_resilin**
A colony PCR was conducted to confirm the restriction ligation reaction of pET28-c and resilin, as well as the capacity of the plasmid to be transformed into bacteria. The formation and subsequent transformation of pET28-c\_resilin in TOP10 and DH5-α bacteria was successful, as evidenced by the synthesis of an approximately 1.3- 1.5 kb resilin sequence, which was achieved through the binding of the resilin-specific oligo primer to the resilin sequence incorporated in the pET28-c plasmid.

![](https://static.igem.wiki/teams/5077/result/figure-9.webp)

Fig. 9: pET28-c\_Resilin colony PCR. Four reactions were made with different clones from a transformation of DH5a with the pET28-c\_resilin. As a positive control the untransformed pET28-c\_resilin was used and as a negative control water was used as a template. Ladder: 1 kb Plus; agarose gel: 1%.

**Sequencing**
In the Next step the colonies where sequenced by Sanger sequencing. Here the isnertion of the 1.3-1.5 resilin sequence into the pET28-c plasmid could not be confimred (See Fig. 10). As one can see, the sequenzing results could only be alligned to the vector pET28-c but not to the insertet repeats.

![](https://static.igem.wiki/teams/5077/result/figure-10.webp)

Fig. 10: Sequencing result of on of the colonies. On the lower figure the filled red line indicaeds the sequenced sequence matches the desired sequence. Where the red line is not filled, the sequenced sequence does not match the desired sequence. In the upper figures, the sequencing of both ends of the RE32 insertion was zoomed in on

**Detection of Resilin Expression**
Following the successful transformation of the pET28-c\_resilin plasmid into competent JM109 bacteria of the novel reCA-deficient expression strain, the expression of resilin was induced with IPTG. After 21 hours, the cells were either lysed with only SDS-sample buffer (Figure 11), sonification (Figure 12) or heat (Figure 13), and subsequently analysed by SDS-PAGE.

![](https://static.igem.wiki/teams/5077/result/figure-11.webp)

Fig. 11: SDS page before (b.i.) and after (a.i.) induction of E. coli strain JM109 with IPTG for resilin expression. The bacteria were transformed with the pET28c(+) plasmid with (R) and without ((pET28c(+))) our resilin insert. Two different resilin samples were used. Resilin 1,2,3 was used three times and resilin 2 twice. Samples were mixed 1:1 with SDS loading buffer. 10% gel, ladder: PageRuler unstained protein ladder.

![](https://static.igem.wiki/teams/5077/result/figure-12.webp)

Fig. 12: SDS page after induction of E. coli strain JM109 with IPTG to detect resilin expression. The bacteria were transformed with the pET28c(+) plasmid with (R) and without (pET28c(+)) our resilin insert and two different resilin samples were used. The resilin 1,2,3 was used three times and the resilin 2 was used twice. Lysis was performed by sonication and each sample was lysed once in LB medium and once in lysis buffer (lysis). 10% gel, ladder: PageRuler unstained protein ladder.

![](https://static.igem.wiki/teams/5077/result/figure-13.webp)

Fig. 13: SDS page after induction of E. coli strain JM109 with IPTG to detect resilin expression. The bacteria were transformed with the pET28c(+) plasmid with (R) and without (pET28c(+)) our resilin insert. Two different resilin samples were used. Resilin 1,2,3 was used three times and Resilin 2 twice. Lysis was performed by heating the cells at 95°C and each sample was lysed once in LB medium and once in lysis buffer (lysis). 10% gel, ladder: PageRuler unstained protein ladder.

The expressed resilin protein of the 30 resilin repeat sequence should exibit a molecular weight of approximately 50 kDa. Unfortunately, no bands at about 50 kDa were visible compared to the respective controls (Figures 11-13). To conclude, further research is required to repeat and optimise the expression of resilin.

# Hyaluronic Acid

## Hyaluronic Acid

## HasA\_eGFP

**Hyaluronic acid**
In order to synthesise hyaluronic acid, it is only necessary to incorporate the hyaluronic acid synthase into an E. coli strain, as these organisms already contain the majority of the enzymes required for the hyaluronic acid synthesis. The synthesis of hyaluronic acid synthase is enabled by the pUC\_HasA plasmid, which contains the sequence of the enzyme.

The expression of hyaluronic acid synthase was evaluated through SDS-PAGE analysis following an induction with arabinose (see Fig. 14). We could recognize more distinct bands in the samples after induction than in the samples before induction with a height of about 52 kDa, which is consistent with the molecular weight of hyaluronic acid synthase. This demonstrates the expression of hyaluronic acid synthase.

![](https://static.igem.wiki/teams/5077/result/figure-14.webp)

Fig. 14: SDS-PAGE for validation of HasA expression in TOP10. From left to right is the PageRuler, Clone 2 before and after induction and Clone 3 before and after induction. Bands were visible slightly above 50 kDa after induction. Legend: bef. = before; aft. = after; Ind = Induction

**HasA\_eGFP**
In the next steps we decide to detect and observe hyaluronic acid expression with a HasA-eGFP fusion protein. This allows us to visually track the induction of hyaluronic acid synthase with arabinose and helps us to optimize it.

**Homologous recombination SLiCE of pUC\_HasA and eGFP**
For the production of the hyaluronic acid synthesis fluorescence-labeled fusion protein, we have opted for labeling with eGFP. This fusion protein can be produced by SLiCE homologous recombination by attaching eGFP to the hyaluronic acid synthase gene using sequence-specific linker sequences. The results of a succssesful SLiCE homologous reaction (HR) is shown. The pUC\_HasA\_eGFP plasmid has a total lenght of 5982 bp. In all HR 1-4 bands of the pUC\_HasA\_eGFP plasmid are shown between the heights of 5,000 and 8,000 kb.

The follow-up steps will be an induction of the hyaluroic acid synthase expression with arabinose controlled by the fluorecence of eGFP. The resulting insight facilitates the determination of the optimal parameters for the subsequent synthesis of hyaluronic acid.

![](https://static.igem.wiki/teams/5077/result/figure-15.webp)

Fig. 15: pUC\_HasA\_eGFP PCR. The negative control lacked of the T4 ligase, \*pUC\_HasA represents the original restricted plasmid. Four homologous recombination reactions HR 1-4 are shown. Ladder: 1 kb Plus; agarose gel: 1%.

{% endblock %}