Parts

Introduction:

As part of our project on sustaining antibody production in Chlamydomonas reinhardtii, we've developed new parts and added them to the iGEM Registry. We have designed these parts to make it easier to express, target, and use antibody-related proteins within the algae system. On this page, we'll show you the parts we've created and explain how they work within our project. You will also find a brief description of their functions here.

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Best Basic Part Special Prize

An important highlight of our collection is the humanized Cetuximab single-chain variable fragment (HumcetFv). The humanization of antibody fragments such as HumcetFv is a significant step toward making them suitable for patient-derived applications. Non-human antibodies such as those traditionally produced in mammalian cell cultures (e.g., CHO cells) often contain regions that the human immune system recognizes as foreign. This can lead to immunogenicity, in which the body triggers an immune response against the therapeutic protein. Such reactions can lead to the development of anti-drug antibodies (ADAs), rapid clearance from the bloodstream, lower therapeutic effectiveness, or in severe cases, hypersensitivity and unwanted immune reactions.

By modifying the original Cetuximab scFv into a humanized sequence, HumcetFv reduces the probability of these immune responses. Humanization involves carefully replacing the non- human framework regions with human antibody framework sequences, while preserving the complementarity-determining regions (CDRs) which are responsible for antigen recognition. In this way, HumcetFv maintains the strong binding specificity and affinity of Cetuximab to EGFR, but in a form that is more compatible with the human immune system.

That is why we decided to nominate HumcetFv for a Best Basic Part Special Prize. It demonstrates the viability of expressing a clinically relevant, humanized antibody fragment in C. reinhardtii and offers a sustainable and scalable alternative to conventional antibody production in mammalian cells. Furthermore, HumcetFv complements other EGFR-binding fragments in our SUSPACT library by targeting a specific epitope on EGFR, allowing for collaborative studies, combinatorial binding approaches, and potential synergistic applications in cancer research. In contrast, Trastuzumab, which is another therapeutic antibody fragment we reference, does not target EGFR but instead binds to HER2, a different receptor that is overexpressed in certain breast cancers. Including both EGFR- and HER2-targeting fragments in our collection highlights the versatility of our system and demonstrates how Chlamydomonas can be used to produce different clinically relevant antibodies for diverse cancer therapies. To learn more about how we tested these constructs and validated their performance, please visit our [Results section].

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Standardization with MoClo

Beyond the design of individual antibody fragments, we placed strong emphasis on standardization and compatibility. All our antibody-related coding sequences were generated as Level 0 parts following the Modular Cloning (MoClo) standard. MoClo uses Type IIS restriction enzymes and predefined overhangs to enable scarless and modular assembly of multiple genetic elements such as promoters, introns, secretion signals, and terminators in a single reaction. This ensures that our parts can be flexibly combined with other standardized components for rapid construction of expression-ready systems.

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Maximizing Expression in Chlamydomonas reinhardtii

To maximize expression in Chlamydomonas reinhardtii, we followed the most up-to-date design strategies for nuclear transgenes. This included reverse-translating our antibody fragments with optimal Chlamydomonas codon usage and inserting native RBCS2 introns approximately every 150–200 bp to improve transcript stability and mRNA processing. Furthermore, our constructs were built as Level 0 parts in the Modular Cloning (MoClo) system, which enabled us to flexibly assemble them into Level 2 expression cassettes under the control of strong promoters and efficient terminators. By combining codon optimization, intron insertion, and robust regulatory elements, we designed our constructs according to the latest gene architecture principles known to maximize nuclear expression in Chlamydomonas, ensuring that our antibody fragments could be expressed at the highest possible levels.