iGEM 2025 — Team patras


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![](https://static.igem.wiki/teams/5836/engineering/gear-left.svg)
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# ENGINEERING

## Overview

In order to bring our biological system to reality, we followed the Design–Build–Test–Learn (DBTL) cycle. By applying this engineering approach through multiple iterations, we not only implemented our system successfully but also significantly optimized its performance, both in silico and in the laboratory.

To view different cycles, first click on any of the Design, Build, Test, or Learn sections. Once a section is open, use the arrows that appear to move forward or backward between the cycles. There are a total of 4 Wet Lab DBTL cycles and an additional Dry Lab cycle.

Click sections to see our progress ↴

DESIGN

BUILD

TEST

LEARN

### 4th engineering cycle

↑
↓

Based on what we learned from the first cycle, we redesigned hairpin probe H0 to achieve higher thermodynamic stability while retaining a poly-uracil loop as the collateral cleavage site for Cas13a. The goal is the initiation of CHA only in the presence of biomarkers.

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DESIGN

BUILD

TEST

LEARN

### 1st Engineering Cycle

Before validating our full system, we first wanted to assess whether the chosen hairpin sequences would be effective for the Catalytic Hairpin Assembly (CHA) reaction.

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Sections

[Protocols](#protocols)
[Notebook](#notebook)

![Bottle 1](https://static.igem.wiki/teams/5836/experiments/bottle1.svg)

# EXPERIMENTS

![Bottle 2](https://static.igem.wiki/teams/5836/experiments/bottle2.svg)

## Protocols

### Buffers

LYSIS BUFFER X5 STOCK

### LYSIS BUFFER X5 STOCK

* 250 mM Tris-HCl
* 500 mM NaCl
* 5% v/v Glycerol
* 1 mM DTT

Stored at 4 ℃ until use.

WASHING BUFFER

### WASHING BUFFER

* 50 mM Tris-HCl
* 500 mM NaCl
* 5% v/v Glycerol
* 1 mM DTT
* 25 mM Imidazole

Stored at 4 ℃ until use.

ELUTION BUFFER

### ELUTION BUFFER

* 50 mM Tris-HCl
* 500 mM NaCl
* 5% v/v Glycerol
* 250 mM Imidazole

Stored at 4 ℃ until use.

GEL FILTRATION

### GEL FILTRATION

* 20 mM Tris-HCl
* 200 mM KCl
* 5% v/v Glycerol

Stored at 4 ℃ until use.

REACTION BUFFER

### REACTION BUFFER

* 10 mM Tris-HCl
* 50 mM KCl
* 1.5 mM MgCl₂

### Plates and Media

SOC MEDIUM (1 L)

### SOC MEDIUM (1 L)

* 20 g tryptone
* 1.25 g yeast extract
* 0.125 g sodium chloride (NaCl)
* 0.046 g potassium chloride (KCl)
* Adjust pH to 7.5 with NaOH
* 1 M sterile-filtered glucose
* 2 M magnesium chloride (MgCl₂)

LB BROTH

### LB BROTH

Dissolve 25g LB Broth in one liter deionized water and stir until clumps are gone.

Transfer to an Erlenmeyer flask that is at least twice the volume of the liquid.

Autoclave the mixture.

LB – AGAR PLATES

### LB – AGAR PLATES

1) For the LB – Agar mixture:

* 1 L distilled water
* 10 g/L NaCl
* 10 g/L Tryptone
* 5 g/L Yeast Extract
* 15 g/L Agar

And mix until everything is dissolved.

2) Autoclave

3) Allow to cool at approximately 55 degrees and add 25mg/mL of Chloramphenicol while swirling

4) Pour 20mL of LB – agar per petri dish while working in flame for sterile conditions

5) Allow the plates to cool for 30-60min until the solidify

6) Seal the plates using parafilm and store in -4 conditions

### Cloning

TRANSFORMATION

### TRANSFORMATION

1) Thaw the competent cells on ice for 30 minutes.

2) In an Eppendorf tube, gently mix 50 µL of Rosetta 2(DE3)pLysS Competent Cells with 1–5 µg of DNA.

3) Incubate the mixture on ice for 2 minutes.

4) Heat shock the cells at 42 °C for 30 seconds.

5) Immediately place the tube on ice for 5 minutes.

6) Add 950 µL of SOC medium under sterile conditions.

7) Incubate at 30 °C, 250 rpm, for 1 hour.

8) Plate 50–500 µL of the transformed cells onto selective agar plates using a sterile glass pipette.

9) Incubate the plates at 37 °C for 24 hours.

FRAGMENT PCR PROTOCOL

### FRAGMENT PCR PROTOCOL

PCR (Polymerase Chain Reaction) is a technique used to amplify a template DNA by cycling through denaturation, annealing, and extension phases with a thermostable DNA polymerase.

**Master mix for total volume = 25 µL**

| Component | Amount / Conc. |
| --- | --- |
| Q5 High Fidelity 2X Master Mix | 12.5 µL |
| Forward primer | 10 µM |
| Reverse primer | 10 µM |
| Template DNA | 1 ng – 1 µg |
| Nuclease-free water | Up to 25 µL |

**PCR cycling temperatures**

| Step | Temperature (°C) | Time | Number of cycles |
| --- | --- | --- | --- |
| Initial denaturation | 98 | 30 s | 1 |
| Denaturation | 98 | 10 s | 25–40 |
| Annealing | 68–72(gradient) | 30 s | 25–40 |
| Extension | 72 | 30 s/kb → ~1 min 30 s | 25–40 |
| Final extension | 72 | 2 min | 1 |
| Hold | 4 | ∞ | 1 |

AGAROSE GEL PREPARATION AND ELECTOPHORESIS

### AGAROSE GEL PREPARATION AND ELECTOPHORESIS

Agarose gel electrophoresis is used to separate DNA fragments based on their size. The percentage of agarose gel is chosen according to the length of the DNA fragments to achieve optimal resolution.

| Linear DNA (bp) | % Gel |
| --- | --- |
| 1,000 – 30,000 bp | 0.5% |
| 800 – 12,000 bp | 0.7% |
| 500 – 10,000 bp | 1.0% |
| 400 – 7,000 bp | 1.2% |
| 200 – 3,000 bp | 1.5% |

**Making the agarose gel**

| Gel % | Agarose | TAE buffer |
| --- | --- | --- |
| 0.7% | 0.7 g | 100 mL |
| 1.5% | 1.5 g | 100 mL |
| 3% | 3 g | 100 mL |

1) Mix the desired amount of agarose in 100 mL of TAE buffer.

2) Microwave for 1–3 minutes until fully dissolved.

3) Allow the solution to cool to a temperature safe to handle (can also cool under running tap water).

4) Add 1 μL of Gel Red per 100 mL of agarose solution and mix gently.

5) Pour the solution into a gel casting tray with the appropriate comb and let it set for 20–30 minutes.

**Running the gel**

1) Add 5 uL of 100 bp DNA ladder to the first well.

2) Add 3 uL of 6x Orange G loading dye into all the wells and then add 5 uL of the DNA sample to each well (in our case we had 6 samples – 3 of each fragment).

3) Run at 100 V for 30–45 minutes.

**Visualizing the gel**

View the gel under a UV light or a blue light transilluminator

GEL EXTRACTION FROM AGAROSE GEL

### GEL EXTRACTION FROM AGAROSE GEL

For gel extraction from our gel we used the NucleoSpin® Gel and PCR Clean- up kit from Macherey – Nagel.

[NucleoSpin Gel and PCR Clean-up — Instructions (PDF)](https://www.mn-net.com/media/pdf/02/1a/74/Instruction-NucleoSpin-Gel-and-PCR-Clean-up.pdf)

VECTOR OVERHANGS PCR

### VECTOR OVERHANGS PCR

For this PCR, we used specific primers designed to linearize the DNA and generate the desired overhangs. This procedure prepares our vector for Gibson assembly.

**Master mix**

| Component | Total volume = 50 µL | Master mix for ×4 samples |
| --- | --- | --- |
| Q5 Master Mix | 25 µL | 100 µL |
| 10 µM forward primer | 2.5 µL | 10 µL |
| 10 µM reverse primer | 2.5 µL | 10 µL |
| DNA template | 5 µL | 20 µL |
| DMSO | 3% | 3% |
| Nuclease-free water | Up to 50 µL | Up to 200 µL |

**PCR cycling temperatures**

| Step | Temperature (°C) | Time | Number of cycles |
| --- | --- | --- | --- |
| Initial denaturation | 98 | 30 s | 1 |
| Denaturation | 98 | 10 s | 25–40 |
| Annealing | 68–72(gradient) | 30 s | 25–40 |
| Extension | 72 | 2 min 30 s | 25–40 |
| Final extension | 72 | 5 min | 1 |
| Hold | 4 | ∞ | 1 |

GIBSON ASSEMBLY

### GIBSON ASSEMBLY

Gibson Assembly is a molecular cloning method used to join multiple DNA fragments that have overlapping ends. Unlike traditional restriction enzyme-based cloning, it does not require specific restriction sites for fragment assembly.

**Master mix for 1 fragment insertion**

| Component | Amount |
| --- | --- |
| DNA | 0.03–0.2 pmol |
| Gibson Assembly Master Mix (2×) | 10 µL |
| Nuclease-free water | Up to 20 µL |

Incubate the reaction at 50 °C for 15–60 minutes.

DIGESTION

### DIGESTION

Restriction endonucleases are commonly used to clone DNA fragments into a plasmid backbone.

**For Vtotal = 20 µL**

| Component | Amount |
| --- | --- |
| EcoRV | 0.5 µL |
| SmartCut Buffer | 2 µL |
| DNA | 5 µL |
| Nuclease-free water | 12.5 µL |

Incubate the mixture at 37 °C for 1 hour.

NATIVE-PAGE 10%

### NATIVE-PAGE 10%

**Gel preparation**

* 2.5mL 38:2 Acrylamide:Bis Acrylamide
* 5mL of 1.5M Tris-HCl (pH=8.8)
* 7.5ml TBE 1x
* 40μL 10% w/v APS
* 10μL TEMED

**Procedure**

1) Mix all components except TEMED by vortexing.

2) Add TEMED and vortex.

3) Pour on top of polymerized separating gel.

4) Insert comb carefully to form wells.

5) Allow polymerization 20–30 min at room temperature.

6) Run the Gel with TBE buffer 1x, float constant voltage 5 V/cm (50 V) for 5 min to allow the DNA to enter gel, then increase the voltage to 10 V/cm (100 V) for the duration of the run.

### Protein expression and purification

PROTEIN EXPRESSION

### PROTEIN EXPRESSION

* Plasmid containing the CRISPR/Cas13a protein
* Rosetta 2(DE3)pLysS Competent Cells
* Chloramphenicol for selection

**Procedure**

**Plasmid Transformation & Overnight Culture:**

1. Transform the plasmid into Rosetta 2(DE3)pLysS Competent Cells.

2. Plate on chloramphenicol-containing agar and incubate overnight at 37°C.

3. Pick a single colony and inoculate 5 mL LB + chloramphenicol; incubate overnight at 37°C with shaking.

4. Transfer the overnight culture into 500 mL LB broth; incubate at 37°C with shaking.

**Protein Expression:**

* Using the constitutive promoter BBa\_J23100, protein expression occurs during cell growth.
* Harvest cells when OD₆₀₀ ≈ 3.

**Protein Harvesting:**

* Centrifuge at 14,000 rpm for 15 min to collect the cell pellet.
* Store at –80°C with glycerol.

HIS TAG PROTEIN PURIFICATION

### HIS TAG PROTEIN PURIFICATION

For the protein purification we used Ni-NTA

**1. Cell Lysis:**

* Resuspend the harvested cell pellet in lysis buffer
* Lyse cells via sonication and centrifuge at ~14,000 × g for 20–30 min at 4°C. Collect the clarified lysate.

**2. Column Preparation:**

* Equilibrate the Ni-NTA column with 5–10 column volumes of binding buffer.

**3. Protein Binding:**

* Load the clarified lysate onto the column. His-tagged protein binds the resin.

**4. Wash:**

* Wash the column with 5–10 column volumes of wash buffer to remove non-specifically bound proteins.

**5. Elution:**

* Elute the protein with elution buffer containing 250–500 mM imidazole.

**6. Storage:**

* –80°C with glycerol for long-term.

### CRISPR/Cas – Catalytic Hairpin Assembly with miRNAs

CRISPR/Cas13a AND CHA REACTION

### CRISPR/Cas13a REACTION

All the hairpin probes were formed by heating the sample to 95°C for 5 min, and then slowly cooling to 25°C

**(total volume = 20 μL):**

* 1 μL Cas13a protein (0.4 μM)
* 1 μL crRNA (0.4 μM)
* 4 μL H0 hairpin (5 μM)
* 2 μL of miRNA at the desired concentration
* Add reaction buffer to bring the final volume to 20 μL

Incubate the reaction at 37°C for 1 hour.

### CHA REACTION

Transfer the reactants from each reaction tube to new tubes. Heat the hairpin probes to 95°C for 5 minutes, then allow them to slowly cool to 25°C to ensure proper formation.

**(total volume = 100μL)**

* 5 µL hairpin 1 (1 µM)
* 5 µL hairpin 2 (1 µM)
* 20 µL CRISPR/Cas13a reaction
* 70 µL PBS (10×)

The Catalytic Hairpin Assembly took place at 37 degrees for 3 hours.

### MicroRNAs isolation and quantification

MICRORNA ISOLATION FROM SERUM

### MICRORNA ISOLATION FROM SERUM

MicroRNAs were isolated using the miRNeasy Serum/Plasma Advanced Kit (Qiagen), following the manufacturer's protocol "Purification of Total RNA, Including miRNA, From Serum and Plasma."

[miRNeasy Serum/Plasma Advanced Kit (Qiagen) (PDF)](https://static.igem.wiki/teams/5836/experiments/hb-2390-003-hb-mirneasy-serum-plasma-0421-ww.pdf)

REVERSE TRANSCRIPTION PCR (RT-PCR)

### REVERSE TRANSCRIPTION PCR (RT-PCR)

Reverse transcription PCR (RT-PCR) combines the conversion of RNA into complementary DNA (cDNA) with the amplification of specific DNA targets by PCR. In this case the method was used to quantify microRNAs. For cDNA synthesis, we used the first-strand cDNA synthesis protocol provided in Qiagen's miRCURY® LNA® miRNA SYBR® Green PCR kit, along with specific miRNA primers from the same manufacturer.

[miRCURY® LNA® miRNA SYBR® Green PCR kit (Qiagen) (PDF)](https://static.igem.wiki/teams/5836/experiments/hb-2431-002-hb-mirc-lna-mirna-pcr-1019-ww.pdf)

REAL-TIME PCR (qPCR)

### REAL-TIME PCR (qPCR)

Quantitative PCR was performed using the miRCURY® LNA® miRNA SYBR® Green PCR kit (Qiagen), following the manufacturer's protocol "Quantitative, Real-Time PCR Using Individual miRCURY LNA miRNA PCR Assays."

[miRCURY® LNA® miRNA SYBR® Green PCR kit (Qiagen) (PDF)](https://static.igem.wiki/teams/5836/experiments/hb-2431-002-hb-mirc-lna-mirna-pcr-1019-ww.pdf)

## Notebook

Download our lab notebook here: [notebook.pdf](https://static.igem.wiki/teams/5836/experiments/igem-patras-notebook.pdf)

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