Part Collection

The HpBusters Part Collection provides a comprehensive and modular platform for the detection and sensing of biomarkers (especially human molecule analogs), rapid multi-gene knockout, and the expression of key proteins and functional domains in Saccharomyces cerevisiae. This collection is designed to offer researchers in synthetic biology and bioengineering, as well as future iGEM teams, a flexible and user-friendly toolkit to promote the application of yeast as a synthetic biology platform. Our Parts Collection is highly flexible and reusable, adaptable to different research needs and experimental designs. At the same time, the HpBusters Part Collection has great potential for future development, allowing for further expansion and optimization to meet a wider range of application scenarios.

Specifically, to realize and validate our project design, we developed a set of modified GPCRs based on the concept of chimerizing the extracellular domain of human GPCRs with the intracellular domain of the yeast mating factor signaling pathway GPCRs^[1]. We also developed another set of modified GPCRs based on the yeastlized-human GPCR approach^[2]. We also designed and validated a reporter system to detect and quantify GPCR-mediated signal transduction. Furthermore, to enhance the sensitivity and specificity of the signal transduction system, we knocked out two key genes in the yeast signaling pathway, Ste2 and Sst2. To this end, we designed a single-transcript, multi-sgRNA vector system based on the XXXX approach^[3] for rapid, single-vector, multi-gene knockout in yeast. Finally, to implement the therapeutic and adhesion systems, we designed and implemented an efficient yeast protein expression system, successfully expressing our key proteins, fusion proteins, and domains in yeast.

It is worth noting that all these parts have undergone multiple rounds of the design-build-test-learn engineering cycle to ensure their functional reliability and stability. We believe that these parts not only meet the needs of our project but also provide a solid foundation for future research.

HpBusters Part Collection consists of the following three parts:

Sensory System: This includes two sets of chimeric GPCR receptors designed and validated based on two different approaches, as well as a reporter system for detecting and quantifying GPCR-mediated signal transduction. These parts enable the sensing and detection of specific biomarkers, are highly flexible and reusable, and are suitable for further development to meet broader application needs.

Knock-out Strategy: This includes a single-transcript, multi-sgRNA vector knockout system we developed and validated for rapid, single-vector, multi-gene knockout in yeast. These parts enable efficient knockout of multiple genes in yeast, improving the efficiency and precision of gene editing, and have great development potential.

Key Proteins & Domains: This includes an efficient yeast protein expression system we designed and implemented for expressing key proteins, fusion proteins, and domains in yeast. These parts enable high-level expression of target proteins, meeting the functional requirements of the therapeutic and adhesion systems, and are highly flexible and reusable.

Sensory System

These parts enable the sensing and detection of specific biomarkers and the quantification of signal transduction.

Chimeric GPCR

This set of parts is a chimeric GPCR receptor developed based on the idea of chimerizing the extracellular domain of a human GPCR receptor with the intracellular domain of a yeast mating factor signaling pathway GPCR receptor.

Chimeric GPCR

This set of parts is a chimeric GPCR receptor developed based on the idea of yeastlizing a human GPCR receptor in yeast.

Reporters

This set of parts is a reporter system we designed and validated to detect and quantify GPCR-mediated signal transduction.

Knock-out Strategy

This set of parts is a multi-gene knockout system we developed for rapid, single-vector, multi-gene knockout in yeast.

Key Proteins & Domains

This set of parts is an efficient yeast protein expression system designed and implemented to express key proteins, domains, and fusion proteins in yeast.

References

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• [1] Schulz, R., Korkut-Demirbaş, M., Venturino, A. et al. Chimeric GPCRs mimic distinct signaling pathways and modulate microglia responses. Nat Commun 13, 4728 (2022). https://doi.org/10.1038/s41467-022-32390-1