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About this book

This book describes advanced studies in cell-free synthetic biology, an emerging biotechnology that focuses on cell-free protein synthesis and cell-free systems for fundamental and industrial research in areas such as genetic circuit design, small-molecule synthesis, complicated-macromolecule synthesis, unnatural-macromolecule synthesis, high-throughput screening, artificial cells, and biomaterials. Cell-free synthetic biology is now an integral part of developing fields like nanotechnology, materials science, and personalized medicine.

The book discusses the main research directions in the development of cell-free systems, as well as a number of applications of cell-free synthetic biology, ranging from structural biology to the human health industry. It is intended for students and researchers in life sciences, synthetic biology, bioengineering, and chemical engineering.

Table of Contents

Frontmatter

Chapter 1. An Introduction to Cell-Free Synthetic Biology

Abstract
Synthetic biology, as a new life science discipline, involves structured construction of biological systems using modular and standardized engineering concepts. It enables engineering biological parts, devices, and systems for versatile applications such as medical therapeutics, medical diagnostics, bioenergy production, and understanding biology. Considering the complexity, variability, and redundancy of living cellular systems, a view comes from scientists who focus on the engineering of biosystems in vitro from the bottom up. It is like solution biochemistry for better applications and opens up a new understanding about biology. Therefore, an enabling technology called cell-free synthetic biology has been rapidly adopted and developed.
Yuan Lu

Chapter 2. Cell-Free Natural Protein Synthesis

Abstract
Because no living cells are involved, the open cell-free biosystems can be flexibly regulated to produce proteins in a few hours. In the open system, the transcription and translation process can be directly controlled on demand by adjusting the temperature, salts, and redox environment and adding nature or human-made materials. Therefore, cell-free biosystem could be well adopted to produce proteins which are difficult to synthesize in living cells, such as membrane proteins and toxic proteins.
Yuan Lu

Chapter 3. Cell-Free Unnatural Protein Synthesis

Abstract
Naturally, proteins generally consist of twenty natural amino acids (NAAs) as building blocks, which can form a nearly unlimited number of combinations by random combination to realize structural and functional diversity. Unfortunately, it is not enough using these natural blocks to generate proteins with specific characteristics. Therefore, incorporating unnatural amino acids (UNAAs) with some novel groups to expand the repertoire of structures and functions of proteins is becoming more and more popular [1]. Diverse bioreactivity from novel functional groups are unreachable to natural proteins, which opens gates for new protein engineering and provides new ways for fundamental research, therapeutics, and synthetic biology [2]. Incorporation of UNAA at a specific site has been used for studying protein structure and dynamics, characterizing protein-protein interactions, mimicking post-translational modifications of proteins like eukaryote, and synthesize novel products hard to be created by other methods, such as enzymes, biomaterials, and therapeutics [3].
Yuan Lu

Chapter 4. Cell-Free Biosensing

Abstract
Biosensing has precise sensitivity and high specificity, and the potential use of biosensor ranges from environmental monitoring, food safety, to disease diagnosis [1]. Biosensing is using the biological sensing element to detect some substances, include inorganic molecules, disease biomarkers, and others. A typical biosensor usually includes biological sensing elements and transducers. The transducer can convert the chemical information generated during the biochemical reaction process into corresponding physical signals, such as optical signals, magnetic signals, and electrical signals. In recent years, with the development of synthetic biology, a large number of cell-based biosensors have been developed, and their constructions become more complicated. Cell-based biosensors have a wide range of detection ability. However, there are still some issues that need to be addressed, like transmembrane transport limitations, the need to maintain cell activity, and a long time to assay [2]. Another problem is that cell-based biosensors make use of living, genetically modified microorganisms (GMOs). The application of these GMO biosensors is often not possible due to biosafety concerns, as it must be prevented that GMOs are released into the environment. To address these limitations of cell-based biosensors, cell-free synthesis system as a platform for biosensing has been developed [3].
Yuan Lu

Chapter 5. Artificial Life

Abstract
Cells are considered to be “life blocks” as the basic structure and functional unit of living things [1]. Research on cell biology has not been limited to its structural function. Many new fields, such as drug delivery, biosensors, bioremediation, drug preparation, and the origin of life, require broad studies on cells. However, with the development of cell biology, some shortcomings such as complexity and vulnerability of cells affect people’s exploration of new research fields. To solve these problems, artificial cells have been constructed to simulate biological cells. Artificial cells are controllable and more robust than natural cells [2].
Yuan Lu

Chapter 6. Other Emerging Development Trends

Abstract
Many proteins require post-translational modification (PTM) to maintain their biological activity. PTMs can significantly affect overall protein characteristics, including stability and solubility. In addition to changing the physical, chemical and structural characteristics of amino acid sequences, PTM is also a major determinant of successful protein synthesis. It should be noted that many eukaryotic proteins require multiple PTMs to achieve natural and bioactive conformation [1]. Although cell-free biosystems have been developed as a robust protein engineering and synthesis platform, one of its disadvantages is that PTM is not as good as cellular systems. To address these challenges, some cell-free approaches have been developed.
Yuan Lu

Chapter 7. Conclusions

Abstract
To address challenges cell-based synthetic biology faces, CFSB has been fast developed as a robust enabling technology to engineer biological parts and networks for versatile applications, such as biopharmaceuticals, medical diagnostics, and others. Without using living cells, the biological transcription, translation, metabolism, and network can be engineered with unprecedented freedom in defined or undefined cell-free biosystems.
Yuan Lu
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