Abstract
Innovations in sustainable biomanufacturing are made possible by cell-free synthetic biology advances. Cell-free workflows frequently combine purified enzymes with cell extracts for cell-free protein synthesis processes to create and analyze biosynthetic pathways and synthesize therapeutics. The development of cell-free platforms, high-yielding, cell-free gene expression systems, and multiplexed strategies for quickly evaluating biological design have all been made possible by recent technological advances in cell-free platforms, such as microfluidics, machine learning, CRISPR-mediated genome editing, etc. Developments in cell-free platforms satisfy many of the requirements of preparatory biochemical research, clinical purposes, diagnostic devices, industrial application, biosensors, etc. that offer exciting opportunities to transform synthetic biology and biotechnology. Therefore, this chapter aims to elaborate on the rapidly advancing field of cell-free platforms.
TopIntroduction
Cell-free systems (CFS) have emerged as powerful tools in biotechnology, enabling the study and manipulation of biological processes outside the traditional boundaries of intact cells. By harnessing the essential components and machinery of cellular systems, cell-free systems offer unique advantages, including increased control, rapid prototyping, and simplified manipulation of biological processes (Figure 1). CFS offer several advantages for synthetic biology and biotechnology applications, including synthesis and production of vaccines, antibiotics, antibodies, proteins, enzymes, and other biomaterials. Therefore, this chapter focuses on the cell-free formats for protein synthesis and recent engineering and technological intervention advancing cell-free platforms, including CRISPR, microfluidics, and artificial cell. This chapter also highlights the necessary supplements for the cell-free synthesis of valuable proteins for their biomedical application.
Figure 1. Advantages of cell-free system over cell-based platforms
TopEngineering Cell-Free Synthetic Biology Reactions
Although, the use of CFS has been implemented as a method for synthesizing proteins, historically. However, CFS has been constrained by several issues despite many promising applications, such as synthesis of complex proteins, low production rate, short reaction duration, adequate energy supply, byproduct-inhibiting reaction, expensive reagents cost, chemically stable environment, etc. Recent technological, technical, or engineering advances have addressed such limitations of CFS approaches, resulting in reduced cost and high production rates at the manufacturing scale. Changes in extract preparation procedures in recent years have resulted in more durable extracts, the ability to activate central metabolism for cell-free protein synthesis (CFPS) energy supply, and the ability to improve production scale. Cell extract is the most critical component of the CFPS reaction because CFPS systems use a group of catalytic proteins derived from cell lysates. Cell extract composition is affected by the growing medium, lysis procedure, and processing conditions (Carlson et al., 2012). For CFPS, proteins in the crude lysate and the composition of cell extract significantly affect protein synthesis and productivity. The cell extract depends on the experimental procedure, such as growth media, lysis method, and homeostatic conditions. Therefore, modifying the composition of cell extract through emerging engineering and technological approaches.
Key Terms in this Chapter
Artificial Cell: An engineered particle that mimics one or many functions of a biological cell. Artificial cells are biological or polymeric membranes, enclosing biologically active materials from cell-free system. As such, nanoparticles, liposomes, polymersomes, microcapsules and a number of other particles have qualified as artificial cells.
Synthetic Biology: A multidisciplinary area of research that involves the application of engineering principles to biology.
Microfluidics: The science and technology of manipulating fluids and controlling their behavior at the microscale, typically in devices with channels and chambers on a small scale.
Genetic Circuit: A network of genes and regulatory elements that interact with each other to produce specific biological functions or behaviors, analogous to electronic circuits, enabling programmable control of gene expression and cellular behavior.
Machine Learning: A field of artificial intelligence that focuses on the development of algorithms and statistical models that enable computer systems to learn and improve from data without explicit programming.
Biomanufacturing: The production of valuable compounds, such as pharmaceuticals, biofuels, and industrial enzymes, using living organisms or cell-free systems.
CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats, a gene-editing technology that allows for precise modifications of DNA sequences, enabling targeted genetic modifications and potential therapeutic applications.
Cell-Free Protein Synthesis: CFPS, or in vitro protein synthesis, is the production of proteins using biological machinery in a cell-free system, i.e., without the use of living cells.