Introduction

Project overview

Our project focuses on optimizing the expression of ScFv-LR, a recombinant antibody fragment previously developed by Riaño-Umbarila et al. through directed evolution and phage display techniques (Riaño-Umbarila et al., 2011; Riaño-Umbarila et al., 2019). ScFv-LR is a human-derived single-chain variable fragment that specifically targets Cn2 toxin, one of the most clinically relevant components in Mexican scorpion venom from Centruroides species (Riaño-Umbarila et al., 2011; Riaño-Umbarila et al., 2019). This antibody fragment represents a simplified yet highly effective version of complete antibodies, retaining specific antigen recognition capabilities while offering advantages in production and stability (Gomez-Ramirez et al., 2023).

The ScFv-LR fragment demonstrates highly specific neutralization activity against Cn2 and Css2 toxins, with its VH3-Vκ3 domain combination providing complementary activity to existing antibody therapeutics (Riaño-Umbarila et al., 2011; Riaño-Umbarila et al., 2019). Unlike broader-spectrum alternatives, ScFv-LR's targeted specificity makes it an ideal component for combination therapies, where it can work synergistically with other antibody fragments like 10FG2 to achieve comprehensive venom neutralization (Riaño-Umbarila et al., 2021). Previous studies have demonstrated that the combination of both scFvs recognizing different epitopes on toxins enables complete neutralization of scorpion venoms at lower molar ratios than when using individual antibodies (Riaño-Umbarila et al., 2021)

Project aim

Our project specifically aims to enhance ScFv-LR production yields in Pichia pastoris by introducing the novel HpFMD (Hypocrea pseudokoningii formate dehydrogenase) promoter in combination with the optimized OPENPichia pep4 yps1 strain.

For expression and production, we employ the OpenPichia platform using the optimized OPENPichia pep4 yps1 strain of Komagataella phaffii. This license-free expression system features reduced protease activity through targeted gene deletions, significantly improving recombinant protein yield and stability. Our vector design utilizes the pPICZ family, specifically the pPICZα vector for ScFv-LR expression under AOX1 promoter control, ensuring efficient secreted protein production. This integrated approach combining advanced expression systems with targeted antibody design provides a robust platform for developing next-generation antivenoms.

Experimental workflow

Cloning in Escherichia coli

Chemically competent Escherichia coli cells will be prepared and transformed with the pPICZα vector containing the gene of interest. Transformed colonies will be selected on antibiotic-containing media. Plasmid DNA will then be extracted from confirmed clones and linearized through enzymatic digestion to prepare the genetic construct for integration into the yeast expression system.

Expression in Pichia pastoris

Following plasmid verification, competent Pichia pastoris cells will be transformed via electroporation with the linearized construct. Successful genomic integration will be confirmed through DNA extraction and PCR analysis of transformant colonies. Verified strains will subsequently be cultured in selective media and induced with methanol to express the ScFv-LR protein.

Purification and functional validation of ScFv-LR

The secreted protein will be recovered from the culture supernatant and purified using affinity chromatography. Protein purity and identity will be confirmed through analytical techniques, and the biological activity will be assessed by evaluating the ScFv-LR’s binding specificity to the Cn2 toxin using ELISA.

Issues and troubleshooting in the lab

This is our first time participating in iGEM, and we embarked on this journey with limited knowledge of what lay ahead. As a team of undergraduate students from Mexico, we approached this competition with excitement and determination, and discovered that conducting synthetic biology research in our context presented unique challenges that required innovative approaches.

When we started this journey, we were driven by passion for science and the opportunity to represent Mexico on an international stage. As we progressed, we discovered that our research environment presented unique characteristics and learning opportunities that shaped our iGEM experience in unexpected ways. This document reflects our honest experience as we navigated new systems, developed creative solutions, and learned about both iGEM and synthetic biology simultaneously.

We hope our story helps future Mexican teams prepare better and provides the iGEM community with insight into the realities faced by teams from developing countries.

The Reality of Funding and Resources in Mexico

Fighting for funding

Securing funding became our first major reality check. Unlike established teams with institutional backing, we found ourselves:

  • Applying for small grants that barely covered basic materials.
  • Organizing fundraising events to buy reagents that cost 3-4 times more in Mexico than abroad.
  • Making difficult choices between essential experiments due to budget constraints.
  • Watching our limited funds evaporate due to peso devaluation against the dollar.

The Reagent Procurement Nightmare

What we thought would be a simple process of ordering materials turned into a months-long ordeal:

Distribution Challenges
  • Many reagents simply aren't available through Mexican distributors.
  • Waiting 6-8 weeks for basic materials that teams elsewhere receive in days.
  • Paying premium prices for items that had to be specially imported.
Vector Synthesis Communication Barriers
  • Language barriers complicated our vector synthesis orders with international companies.
  • Time zone differences meant waiting entire days for responses to critical questions.
  • Technical specifications got lost in translation, causing delays and confusion.
  • No local technical support that understood both our language and our science.
The Customs Nightmare
  • Our vectors got stuck in customs for weeks due to complex import regulations.
  • Customs officials unfamiliar with biological materials delayed our shipments.
  • Additional storage fees while materials sat in bureaucratic limbo.
  • Some materials arrived degraded after extended processing times.

Infrastructure Reality Check

Making Do Without Proper Facilities

Our biggest shock was discovering that our institution lacked specialized yeast research infrastructure:

  • No dedicated yeast cultivation equipment.
  • Limited biosafety facilities for genetically modified organisms.
  • Basic molecular biology equipment that couldn't handle our project's demands.
  • No local technical support for specialized equipment maintenance.

Learning Everything the Hard Way


iGEM is Still Unknown Here
  • Faculty members had never heard of iGEM and couldn't provide guidance.
  • No previous teams to learn from or consult.
  • Academic calendar misalignment that compressed our timeline.
  • Having to educate ourselves about competition requirements while executing the project.
Yeast Isn't E. coli
  • Designing genetic circuits for yeast requires different principles we had to learn from scratch.
  • Homologous recombination mechanisms that aren't covered in standard coursework.
  • Transformation protocols requiring expensive, specialized reagents we couldn't afford.
  • Troubleshooting integration issues without local expertise to guide us.

Conclusion

While our team was unable to complete the experimental phase of our iGEM project, this experience has provided invaluable insights into the realities of conducting synthetic biology research in Mexico. The obstacles we encountered (from funding limitations and reagent procurement difficulties to infrastructure gaps and regulatory hurdles) taught us resilience and creative problem-solving while representing an important milestone for synthetic biology education in our country.

We remain committed to advancing our project beyond the competition timeline and to establishing better support systems for future Mexican iGEM teams. The lessons learned from our first iGEM experience will serve as a foundation for future success and contribute to making synthetic biology education more accessible worldwide. We are proud to have taken this first step and look forward to building upon what we've learned for future competitions.

References:

  1. Riaño-Umbarila, L., Contreras-Ferrat, G., Olamendi-Portugal, T., Morelos-Juarez, C., Corzo, G., Possani, L.D., Becerril, B. (2011). Exploiting cross-reactivity to neutralize two different scorpion venoms with one single chain antibody fragment. Journal of Biological Chemistry, 286, 6143-6151.
  2. Riaño-Umbarila, L., Gomez-Ramírez, I.V., Ledezma-Candanoza, L.M., Olamendi-Portugal, T., Rodríguez-Rodríguez, E.R., Fernandez-Taboada, G., Possani, L.D., Becerril, B. (2019). Generation of a broadly cross-neutralizing antibody fragment against several Mexican scorpion venoms. Toxins, 11, 32.
  3. Gomez-Ramírez, I.V., Corrales-García, L.L., Possani, L.D., Riaño-Umbarila, L., Becerril, B. (2023). Expression in Pichia pastoris of human antibody fragments that neutralize venoms of Mexican scorpions. Toxicon, 223, 107012.
  4. Riaño-Umbarila, L., Romero-Moreno, J.A., Ledezma-Candanoza, L.M., Olamendi-Portugal, T., Possani, L.D., Becerril, B. (2021). Full neutralization of Centruroides sculpturatus scorpion venom by combining two human antibody fragments. Toxins, 13, 708.