iGEM 2025 — Team isbicskis-bkk

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[Open PDF in new tab](https://drive.google.com/file/d/1mWWnU5K5_nZHSdl_5FzZGCXNRosdGjhY/view)

[Open PDF in new tab](https://drive.google.com/file/d/1kifxgW9kTqMRc2D6VW1_19CbH_ss05Uq/view)

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Our team aimed to design and optimize a CRISPR-Cas9 system to target the mraY and dnaG genes of Staphylococcus aureus, two essential genes involved in cell wall synthesis and DNA replication, respectively. The engineering process was iterative and guided by the Design–Build–Test–Learn (DBTL) framework, allowing us to refine each stage from DNA extraction to in vitro gene cleavage.

## **CYCLE 1: Genomic DNA Extraction and PCR Amplification**

#### Design

We began by planning to amplify the mraY and dnaG genes from S. aureus genomic DNA for downstream CRISPR-Cas9 assays. Primers were designed to flank the target regions and optimized for GC content and melting temperature.

#### Build

Genomic DNA was extracted from S. aureus cultures and used as a template for PCR amplification of mraY (~2.5 kb) and dnaG (~4.9 kb).

#### Test

Initial PCR reactions failed due to insufficient or degraded genomic DNA. After improving extraction protocols, faint or smeared bands appeared across temperature gradients (59.6°C–60.9°C).

* mraY: Weak non-specific amplification
* dnaG: No amplification, likely due to secondary structure or long fragment size

#### Learn

We discovered that:

* The DNA template quality was critical for successful amplification.
* Large fragments such as dnaG may require preheating steps to denature secondary structures.
* Annealing temperature optimization was not enough; DNA concentration and primer specificity also needed refinement.

## **CYCLE 2: Optimization of PCR and In Vitro CRISPR-Cas9 Cleavage**

#### Design

Based on previous results, we redesigned the PCR workflow to include DNA precipitation and purification steps using sodium acetate and isopropanol. In parallel, we planned in vitro Cas9–gRNA cleavage assays to validate targeting efficiency.

#### Build

Purified genomic DNA was used for re-amplification:

* mraY successfully yielded a ~2.5 kb band after optimization.
* dnaG amplification was achieved after re-amplification, though the band appeared larger (~4.9 kb), suggesting non-specific amplification.

For in vitro cleavage, Cas9 and gRNA concentrations were optimized across multiple ratios:

* Initial ratio: 50 nM gRNA + 12.5 nM Cas9
* Modified ratio: 10 nM Cas9 + 20 nM gRNA (1:2 and 1:5 tested)

#### Test

Cleavage efficiency varied with different Cas9:gRNA ratios.Results indicated that:

* Lower DNA concentrations and sufficient Cas9 levels were essential for complete cleavage.
* Excess gRNA could lead to dimerization, reducing binding efficiency with Cas9.
* High DNA input required more Cas9 to achieve full cleavage.

#### Learn

We concluded that:

* The ratio between Cas9, gRNA, and DNA must be carefully balanced.
* Optimal cleavage occurred at low DNA, high Cas9, and moderate gRNA levels.
* Our modified protocol (10 nM Cas9 + 20 nM gRNA, ratio 1:2) provided the most consistent cleavage pattern.

[Open PDF in new tab](https://drive.google.com/file/d/1kifxgW9kTqMRc2D6VW1_19CbH_ss05Uq/view)

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[Open full PDF in new tab](https://drive.google.com/file/d/1SaWZu3TqUsKwnMaD0SdWAk_jlgyT4vgK/view)

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## What Should this Page Contain?

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* Clearly and objectively describe the results of your experiments and research.
* Use tables, graphs, and images to visualize your data.
* Discuss the implications of your findings in the context of your project goals.
* Highlight any trends, patterns, or significant observations.
* Include an analysis summary section to synthesize your findings and discuss their overall significance.
* Outline your future plans for the project, including potential applications, further research, and development.
* Explain what went wrong and what you learned from these experiences.
* Discuss potential improvements or alternative approaches.
* Emphasize the importance of scientific honesty and transparency.

## Inspirations

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* [2024 Heidelberg](https://2024.igem.wiki/heidelberg/results)
* [2024 Marburg](https://2024.igem.wiki/marburg/results)
* [2024 EPFL](https://2024.igem.wiki/epfl/results)
* [2024 UToronto](https://2024.igem.wiki/utoronto/results)
* [2024 Marburg](https://2024.igem.wiki/marburg/results)
* [2024 Sydney-Australia](https://2024.igem.wiki/sydney-australia/results)
* [2024 Toulouse-INSA-UPS](https://2024.igem.wiki/toulouse-insa-ups/results)
* [2024 BNDS-China](https://2024.igem.wiki/bnds-china/results)
* [2024 Vilnius-Lithuania](https://2024.igem.wiki/vilnius-lithuania/results)
* [2024 XMU-China](https://2024.igem.wiki/xmu-china/results)
* [2023 Estonia-TUIT](https://2023.igem.wiki/estonia-tuit/results)

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