The iGEM competition, as a premier international event in the field of synthetic biology, is not only a global stage for young students to showcase their technological innovation capabilities but also a cradle for cultivating future leaders in the life sciences. Amid the rapid wave of global scientific and technological advancement, the life sciences undoubtedly shine the brightest. Among them, the disciplines of biochemistry and molecular biology can be considered the core drivers of life science development. Focusing on the chemical characteristics of biological macromolecules, with particular attention to the physicochemical properties of protein molecules, they have propelled the progress of the entire life science field. With the elucidation of the DNA double helix structure and the deciphering of the genetic code, molecular biology rapidly emerged as an independent discipline. After nearly half a century of development, biochemistry and molecular biology have become the cornerstones of the life sciences. Correspondingly, their experimental techniques are the essential tools for transforming innovative ideas into tangible results.

However, for iGEM team members new to the laboratory, systematically and standardly mastering these key techniques often poses the primary challenge.To address this, our team has compiled this iGEM Essential Protocols based on years of practical experience and accumulated pedagogical insights from participating in the iGEM competition. This book aims to systematically outline the core experimental techniques involved in iGEM projects, comprising two main parts: Fundamental Experimental Protocols and Comprehensive Experimental Projects. The content ranges from the most basic RNA extraction and plasmid construction to complex protein analysis, gene editing, and cell experiments, striving to build a complete knowledge and skill system. The significance of this book lies in its hope to help iGEMers quickly bridge the gap between theory and practice, solidify their basic experimental skills, avoid common errors, and thus advance their projects more efficiently. We sincerely hope this book becomes a practical guide frequently kept at hand by every participant, aiming to provide students aspiring to work in the life sciences with a solid knowledge foundation and skill training.

This section forms the foundation and core of the entire book, meticulously selecting and detailing the 20 most commonly used and crucial fundamental experimental techniques in the iGEM competition. The content is organized according to the classic workflow of molecular biology research: from gene cloning and acquisition (e.g., RNA extraction, PCR, gel extraction), to vector construction and transformation (e.g., plasmid extraction, chemical and electrotransformation), and further to protein expression and analysis (e.g., SDS-PAGE, protein concentration determination, enzyme activity assay). Furthermore, keeping pace with technological advancements, this part specially includes computational biology methods such as protein interaction prediction based on AlphaFold3 and protein rational design based on FoldX, reflecting the "dry-wet integrated" paradigm of modern life science research. Each protocol strives for clear steps, highlighted key points, and includes a brief principle description and precautions, aiming to help readers build a solid experimental skill tree and lay a robust foundation for subsequent complex comprehensive projects.

This section aims to integrate the discrete fundamental techniques from Part I. Through four carefully designed comprehensive experimental projects, it simulates and restores real-world research scenarios. Comprehensive Experiment I, using "Heterologous Expression, Purification, and Identification of Fumarase" as the main thread, completely demonstrates the classic process of protein expression in a prokaryotic system. Comprehensive Experiment II, using "Expression and Characterization Analysis of Recombinant Acetylcholinesterase in a Pichia pastoris System" as an example, introduces key techniques in eukaryotic expression systems. Comprehensive Experiment III focuses on the cutting-edge CRISPR/Cas9 technology, detailing the strategy and steps for E. coli gene knockout. Comprehensive Experiment IV delves into the cellular level, using "Hep G2 Cell Hexokinase siRNA Interference and Its Metabolic Impact" as a case study, explaining a series of cellular and molecular biology techniques such as cell culture, gene silencing, and functional validation. Through these four major projects, readers will learn how to plan experiments comprehensively, connect different technical modules, and ultimately solve a complete scientific problem, thereby holistically enhancing their comprehensive scientific research literacy.

The Appendix is the laboratory's "treasure chest," compiling indispensable practical information for experimental work. It significantly enhances the portability and utility of this book, allowing readers to quickly look up key data during experiments and avoid interrupting their workflow with trivial information searches. The content covers everything from preparation recipes for common solutions and unit conversions, to antibiotic working concentrations, electrophoresis gel preparation schemes, and even data on cell culture vessels and symbol writing standards. It is especially worth mentioning that the Appendix also provides an experiment report template, designed to guide readers in developing good habits of standardized record-keeping and scientific analysis, which is particularly important for knowledge transfer and project review within iGEM teams.