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Contribution

Why our project?

Lung cancer is the leading cause of cancer-related deaths worldwide, and non-small cell lung cancer (NSCLC) accounts for about 85% of all cases. Despite advances in treatment, most NSCLC cases are diagnosed late, when survival rates are drastically reduced. Early detection of driver mutations such as EGFR can enable targeted therapies and significantly improve patient outcomes.

However, current diagnostic methods—including PCR and next-generation sequencing—are expensive, time-consuming, and equipment-intensive, making them impractical for low-resource or decentralized settings. This global gap in accessibility motivated us to design a low-cost, rapid, and user-friendly diagnostic tool that could bring early molecular screening to more people.

Our project evolved from identifying this unmet need for accessible NSCLC screening to developing a portable diagnostic system capable of detecting EGFR exon 19 deletion mutations in circulating tumor DNA (ctDNA).

We aimed to address two major technical challenges in ctDNA-based screening:

  • Low DNA concentration in blood samples
  • High specificity required to distinguish between wildtype and mutant sequences

New documented parts

As part of the project, we have contributed multiple new documented parts as follows:

New analyte sequences

These analyte sequences have been adapted from Genome Data Viewer of the National Library of Medicine as a template sequence. They could be used by future iGEM teams in the study of the same mutation.

RPA primers

These primers are specifically designed for recombinase polymerase amplification (RPA) and have shown to be successful at amplifying the exon 19 of EGFR gene — either the wildtype or the mutation variant.

  • RPA Forward Primer for EGFR exon 19 23 bp deletion mutation: BBa_25ZSP4AQ This was referred to as “5bp Overlap Primer” in documentation.
  • Reverse primer: BBa_250CQDQM
  • RPA forward primer EGFR exon 19: BBa_25KPUU24

PCR primers

These primers have been designed and tested for PCR amplification of the template sequences above, which could be directly used by future iGEM teams for amplification and purification of the templates.

These parts have been collated in a part collection for future iGEM teams to directly implement and further develop for NSCLC detection.

Education

For education, our team has created a board game to raise awareness about synthetic biology and potentially educate future generations about its potential applications, while gradually introducing the concept to them, thereby helping to create a future generation that is more interested in synthetic biology.

In the process, we have contacted numerous high schools and specialists, ensuring that those who play the game receive the best experience. To ensure the project’s successful launch, we created a Kickstarter campaign to help fund our mission to educate the world about synthetic biology.

Besides the board game, our team has organized multiple educational activities in the school and regional communities to raise awareness about the synthetic biology fields, its potential, and also its ethics, including debates and weekly presentations.

RPA troubleshooting tips

Due to sensitivity of the RPA, multiple issues could arise in the process. Here are some tips that could possibly help overcome potential issues.

Using PCR purification kit for gel analysis

To avoid smeared bands on the gel during the analysis of RPA products, before loading the samples onto the gel, the samples should be run through PCR purification to avoid protein-DNA complexes producing unexpected bands. This will improve the reliability of the analysis. However, this does not have to be implemented into the workflow of diagnostic kits but only for analysis.

Avoiding contamination

Due to extremely high sensitivity of RPA to contaminants, workspace in which the RPA is carried out should be kept separate from gel electrophoresis space to avoid DNA contamination. Additionally, new water aliquot is recommended for each new experiment if contamination is suspected.

Improving specificity

To improve the specificity of RPA, higher temperature could be used. At 45°C, samples are shown to have higher specificity due to the temperature being closer to the melting temperature of the primer. Additionally, incubating in a shaker at 300 rpm could improve the homogenous nature of the reaction.