Synthetic Biology Atlas
Catalogue
1. Introduction
2. Synthetic Biology Overview
3. Synthetic Biology Ideology
4. Synthetic Biology Technology
5. Interdisciplinary study of synthetic biology
6. Application of Synthetic Biology
7. Synthetic Biology Perspectives
8. Reference
Introduction
Synthetic biology is an emerging cross-cutting and convergent discipline concerned with the design and resynthesis of life. Since Stéphane Leduc first introduced the concept of synthetic biology in 1910, its impact has become increasingly widespread. Especially in the 21 century, developments in bioengineering technology and the rise of bioinformatics have led to unprecedented boosts in synthetic biology. Synthetic flesh and artificial life, which were once fantasies of mankind, are gradually becoming a reality with synthetic biology. There is no doubt that synthetic biology is shaping our lives. However, during our human practice, we have found that many people have little knowledge of synthetic biology. As iGEM participants, we recognize that it is our responsibility to encourage more people to understand, participate in and even shape the development of synthetic biology. Therefore, seven teams: ZJUintl-China iGEM, CPU_China iGEM, NMU China iGEM, TJU-SLS, ZJUT-China, Hi-ZJU, ZJU-iGEM, have launched a project to build an atlas of synthetic biology. This Synthetic Biology Knowledge Atlas includes an overview, ideas, technologies, interdisciplinary intersections, specific applications, and perspectives of synthetic biology. We sincerely hope that through the promotion of this atlas, the public will have an initial understanding of the contours of synthetic biology, thus enabling more people to participate in the construction of synthetic biology.
Synthetic Biology Overview
Synthetic biology is a subdiscipline of biological sciences that has just emerged in the 21st century, and research on synthetic biological substances has progressed rapidly in recent years. Synthetic biology is different from traditional biology, which is to study the inner structure of living organisms by dissecting them, but the research direction of synthetic biology is completely opposite, it starts from the most basic elements and builds components step by step. Unlike genetic engineering, which perpetuates, alters, and transfers genes from one species to another, synthetic biology aims to create artificial biological systems that operate like electrical circuits.
Synthetic Biology Ideology
2.1 Engineering thinking
The advent of the industrial age is due to engineering thinking. Modularity, hierarchy, quantification, and even DBTL cycle are all characteristics of engineering thinking. Computers, electrical appliances, automobiles, and buildings in life are all like this, while synthetic biology aims to dismantle living organisms and use engineering thinking to discover, modify, and create.
Unlike other biological disciplines, synthetic biology has a distinct engineering character. Synthetic biologists expect to introduce engineering ideas and concepts into life science research, so that synthetic biology research can be standardized, modularized and systematized, and further promote the rapid development of cell factories, artificial life and other scientific research while exploring the existing biological phenomena in nature.
For most non-living systems, the “bottom-up” engineering approach is well established, but living systems are highly complex and the details of their operation are still uncertain, so the main challenge to apply modern engineering principles to biological systems is the inherent complexity of living phenomena.
2.1.1 Modularity
Modular design is the basis of modern engineering, with the advantage of being able to build and maintain complex systems quickly, efficiently, and reproducibly, while modularity in synthetic biology aims to break down biological systems into structurally and functionally independent components.
In order to achieve “plug-and-play” of biological modules and save time and cost, synthetic biologists have creatively proposed the concept of BioBriok, a standardized biological module. By standardizing the definitions of different BioBrioks, researchers can flexibly apply BioBrioks for various operations. BioBrioks include gene modules, subcellular modules, biosynthetic gene networks, metabolic pathways, signal transduction pathways, and operational mechanisms. For example, a piece of DNA with certain functions can constitute a biological component, i.e., a small bioblock.
2.1.2 Hierarchy
Hierarchy refers to the ability to break down biological systems layer by layer, e.g., to subdivide larger-scale unit blocks into many smaller-scale biological components.
2.1.2.1 Element
Components are the “Lego blocks” of life and are the basic functional units of DNA. Biological components can be classified into promoters, protein-coding genes, terminators, reporter components and other categories according to their functional differences, and each type of biological component is assigned a different standard code for users to easily find.
Four common biological components are described below.
2.1.2.1.1 Operon
Operons are expression regulators of bacterial genes, consisting of promoters and other cis-acting element and multiple genes in tandem, and regulated by trans-acting factors. Operons in the genome generally consist of two or more coding sequences, promoter sequences, operon sequences and other regulatory sequences in clusters in tandem.
2.1.2.1.2 Promoter
A promoter is a component of a manipulator that, when bound specifically to RNA polymerase, determines the location of gene transcription initiation and controls the timing and intensity of gene expression. The promoter and transcription factors (proteins that bind to specific nucleotide sequences upstream of a gene and regulate gene transcription) act together to regulate gene activity.
2.1.2.1.3 Ribosome binding site
The ribosome binding site is an untranslated sequence in the mRNA molecule immediately downstream of the promoter and upstream of the start codon that binds the ribosome in order to start transcription. This binding can alter the secondary structure of the 5' end of the mRNA, thus affecting the free energy required for the ribosomal 30S subunit to bind to the mRNA, resulting in differences in the efficiency of protein synthesis.
2.1.2.1.4 Terminators
Terminators are specific nucleotide sequences that have the function of terminating gene transcription. Some terminators can completely stop gene transcription, while others can only partially stop it. Some RNA polymerases can cross over such weak termination sequences and continue to move along the DNA and carry out transcription.
2.1.2.2 Devices
Biological components can be standardized and assembled into more complex biological modules and further assembled into biological devices. Biological devices can perform one or more biochemical reactions, including transcription, translation, protein phosphorylation, metamorphic regulation, ligand-receptor binding, and enzymatic reactions, etc. Synthetic biologists need to rationalize the structure of biological devices like regulating electrical circuits to make them function smoothly.
2.1.2.2.1 Switch
A gene switch refers to one of two possible states in which a gene will be in the presence or absence of a chemical inducer or in response to two independent exogenous stimuli.
In the design of synthetic biology, there are five main switch gene pathways.
● Conversion Switch
A conversion switch is similar to a “non-” gate logic gate operation, where the output is a conversion functions of the input. In natural systems, there are many conversion switchs, such as positive control deterrent systems, where high concentrations of effector molecules are present, activating proteins are inactive and transcription cannot take place, and vice versa, gene expression takes place and the system outputs effector proteins.
● Bilateral Switch
Bilateral switches have both positive and negative regulatory effects on gene transcription. For example, a bilateral manipulator of λ phage, PRM, is regulated by the binding of the repressor protein CI and the anti-repressor protein Cro to three adjacent binding sites, OR1, OR2 and OR3. OR1 has a high affinity, and low concentrations of CI protein first bind to OR1 and promote their own transcription and binding to OR2, and as the concentration of CI protein gradually increases, OR1, OR2 and OR3 sites are bound, the transcription of CI gene is inhibited, and the CI protein concentration gradually decreases.
In summary, due to the regulation of the bilateral switch, low CI protein concentration will promote gene transcription, i.e., positive regulation, while high CI protein concentration will inhibit gene transcriptional expression, i.e., negative regulation.
●Nucleic Acid Switch
Nucleic acid switches function mainly through conformational changes in ribonucleic acid (RNA) to achieve the switch. Most nucleic acid switches have only one ligand binding site or aptamer, which can bind directly to small molecule ligands. When the aptamer binds to a metabolite, the switch will change its structure and perform gene regulation at the transcriptional or translational level by forming a repressive conformation to terminate transcription or inhibit the initiation of translation.
● RNA Switch
RNA molecules are structurally flexible, can change conformation depending on the environment, and have metastable properties. an RNA switch usually connects an input domain (RNA aptamer) and an output domain (RNA gene regulatory component), and the control element that regulates gene expression binds to a ligand such as a protein or small molecule for regulation. Synthetic RNA switches responsive to exogenous small molecules can be used in multiple output domains in different host cells. When the input domain of an RNA switch differs from the output domain, the new input domain will be selected for resynthesis and quickly fused with the existing switch.
● Bistable Switches
Most biochemical reactions regarding cellular signal transduction are reversible, such as phosphorylation and dephosphorylation of proteins, synthesis and degradation or release and separation of secondary properties, and entry and exit of proteins into and out of the nucleus. However, many biological transitions are irreversible, such as the cell cycle switch, and no cells have been seen to return from the M-phase to the G2-phase. The bistable switch, also known as the “fluctuation switch”, can be artificially regulated to switch the gene route between two different stable states. The classical transcriptional bistable switch consists of two promoters, each with a distinct boundary between the on and off states, which can only be switched by a transient change in the inducing factor and can be maintained after the removal of that input stimulus.
2.1.2.2.2 Logic Gates
Logic gates are the basic concept of digital circuits, the fundamental component and the most basic arithmetic unit of various modern highly sophisticated digital instruments, and logic circuits based on Boolean algebra are an important cornerstone of computer manufacturing. Since logic circuits can be described very clearly and simply with truth tables, they are widely used in a variety of fields for the description of inputs and outputs. The logic gate gene circuit in synthetic biology originates from the logic operation in digital circuits, and draws on its control theory and the design rules of logic circuits to study the logical relationships and regulation methods of gene circuits, simulating various logical relationships and genetic routes of digital components. So far, complex biology has been abstracted into mapping relationships in {0, 1} space, which helps to better understand the functions of genetic circuits in depth.
Many logic gates are constructed based on DNA-binding proteins. A variety of gene regulatory proteins exist in organisms that restrict the binding or advancement of RNA polymerases by binding to promoters or manipulators. Such deterrents are often constructed from zinc finger proteins, transcriptional activator effectors, TetR homologs, phage deterrents and LacI homologs. DNA binding proteins can be used in circuit design for synthetic biology systems or as activators to increase the flux of RNA polymerase on DNA.
2.1.2.2.3 Gene circuits
People use biological components to design and construct gene routes such as gene switches, oscillators, amplifiers, logic gates and counters to realize the re-editing of living systems and the execution of special functions.
The functions of gene routes can be mainly divided into logical gene routes and other functional genetic routes.
Logical genetic routes originate from the idea of logical operations in digital circuits, mainly drawing on control theory and the design rules of logical circuits to study the logical relationships and regulation methods of genetic routes, simulating logical relationships and digital components of genetic routes, similar to computer programming, a programming language for living organisms, and eventually synthesizing DNA sequences to operate in cells in the form of genetic routes.
Other functional genetic lines are genetic lines with specific biological functions, mainly using the original functions of gene modules, designing completely new genetic lines and modifying existing biological systems by means of genetic manipulation such as gene recombination and gene cloning to endow the original systems with specific desired functions.
2.1.2.2.4 Metabolic pathways
Metabolic pathways refer to a sequence of chemical reactions that occur within a cell, catalyzed by enzymes and requiring elaborate regulation, through which an organism modifies a substrate into a desired chemical structure, leading to the synthesis or breakdown of a metabolite.
The purpose of industrial fermentation is to accumulate as many microbial metabolites as possible for social life and to artificially break the metabolic control system of microorganisms so that the intracellular metabolism proceeds in the desired direction. The current artificial control methods include: changing the genetic characteristics of microorganisms (genetic method), controlling fermentation conditions (biochemical method), and changing cell membrane permeability.
2.1.2.3 Systems
A biological system is a group of biological devices with interconnected functions that can perform complex tasks, designed to connect biological devices in series, feedback, feedforward, etc. to form more complex cascade lines or regulatory networks. Cascade routes are commonly found in natural biological systems, such as transcriptional regulatory networks, protein signaling pathways, and metabolic networks, which employ cascade processes to regulate their activities.
2.1.2.4 Community, organization
Communities of cells are interconnected like a networked world, with close interactions and divisions of labor, and the limited capacity of a single cell often makes it difficult to achieve the complex biological functions expected by synthetic biologists.
Community sensing is an important way for bacteria to communicate within or between species, often using the secretory transmission of signaling molecules to make bacterial communities respond to their environment, thereby changing cell density and colony composition. Because of its relatively simple structure and clear mechanism, this system can be applied as a gene module for the characterization of complex cellular response mechanisms and the study of interactions between different bacteria, which has far-reaching implications for future exploration in the field of synthetic biology.
At present, synthetic biologists have mainly coordinated the population-sensing behavior among cells by controlling intercellular communication to build engineered cell communities. The construction of artificial cellular communication among multiple cells enables multi-cellular interaction, improves the functionality of biological systems, and builds mutually coordinated and cooperative multi-cellular systems to overcome the lack of reliability of a single individual.
For example, in a bacterial community sensing system, bacteria produce and release a chemical signal molecule called autoinducer into the environment, and when stimulated by the autoinducer in the environment, the bacteria will change their gene expression pattern according to the difference in concentration of the signal molecule to achieve a response.
2.1.3. Quantification
Standardized functional modules can be used as the hardware for carrying gene functions, while standardized system quantification platforms and abstract conceptual signals can be used as the software for carrying functions.
An idea to quantify biological design emerged as synthetic biologists integrated expertise from multiple domains including biology, physics and engineering. As more and more biological components and even systems complete quantitative data collection, researchers can build models based on available data to design and modify biological systems with quantitative principles, just like sizing a house before it is built.
For example, the iGEM Registery provides a set of input and output signals — PoPS and RIPS — on its website, where PoPS (RNA polymerase per second) is used to measure the level of transcription of a gene, and for For each RNA copy, the number of RNA polymerase molecules passing through a point on the DNA molecule per second is PoPS, similar to the current flow through a particular location on a wire.
RIPS (ribosomal initiations per second) is a measure of the level of translation of mRNA and, for a single mRNA, is the number of ribosomal molecules that pass through a point on the mRNA molecule per second.
Molecules are the basic units of biological systems, and synthetic biology requires the analysis, investigation, and estimation of the dynamic interactions between genes and proteins in biological systems. Since living systems exhibit a surprising complexity at different scales of measurement, modeling and simulation are difficult to achieve, and model design can only be simplified by making various assumptions (e.g., assuming homogeneity within cells and within cell populations), and then comparing observations from subsequent experiments with the model, and if inconsistent, the assumptions must be modified.
2.1.4 DBTL
DBTL refers to Design (design)-Build (build)-Test (test)-Learn (learn) .
On the leading website of synthetic biology (http://syntheticbiology.org), the main research of synthetic biology is summarized as “the design and construction of novel biological components or systems and the redesign of existing biological systems in nature”. The cycle of “design, build, test, and learn” is an important feature of synthetic biological systems, and the four phases are complementary and recurring throughout the process of synthetic biology research, which is the way to finally realize the predefined functions of synthetic biological systems.
“Design” is the core of this cycle, which includes the selection of chassis cells, the exploration of required components and applications, and computer-aided design analysis. “After the “design” of synthetic biology is completed, the synthesis and assembly of a synthetic DNA fragment begins with a single transcription unit and continues with the lengthening of the synthesized DNA fragment. Finally, the “construction” of the whole synthetic biological system is realized. “With the development of DNA sequencing technology, the emerging rapid testing technology can effectively reduce the time required for further learning and system optimization. System learning and optimization includes single gene optimization, multiple gene pathway combination optimization and genome simplification and reconstruction, analysis and evaluation of functional modules and chassis fitness to reach the optimal solution for the operational efficiency of the artificial system, and the process is often complemented by directed evolution and other techniques to improve the system.
2.2 Artificial Life
The “bottom-up” engineering approach of synthetic biology aims to create molecular assemblies that replicate the organization and function of living organisms and integrate them in a modular and hierarchical manner into cells, the basic units of life, and beyond.
Artificial life is the process of extracting genes from other living organisms, creating new chromosomes, and embedding them in cells that have had their genetic code removed, and ultimately having these artificial chromosomes control cell development and form new living organisms, symbolizing that new life can be “created” in the laboratory, not necessarily through This symbolizes that new life can be “created” in the laboratory, not necessarily through “evolution”.
On May 20, 2010, the Craig Venter Institute, a private research institute in the United States, announced the birth of the world’s first artificial life, a single-celled mycoplasma, named Cynthia, controlled entirely by artificial genes. The research on the ab initio design and artificial synthesis of five chromosomes, chromosomes 2, 5, 7, 10 and 12, of Saccharomyces cerevisiae is a landmark advance in synthetic biology.
2.2.1 Genomes
With the development of biotechnology, initially to synthesize individual genes, but with the eventual goal of editing or writing entire genomes on a large scale from scratch, synthetic biologists have developed a variety of techniques that have enabled the biosynthesis of genome-wide DNA at a technical level.
Synthetic genomic approaches can be divided into two types.
First, a more simplified genome is created “top-down” by eliminating or replacing the genome of a living organism. Knocking out redundant genes has no impact on the survival of living organisms and reduces unnecessary waste of energy and material, which is conducive to subsequent purposeful trait modification of living organisms.
Secondly, the “bottom-up” orderly assembly of non-living components to form an organism that can replicate the basic properties of natural cells requires researchers to compare the similarities and differences of gene functions among different species by means of bioconferencing, to obtain the core set of genes shared among different living organisms, and to construct a core set of genes based on the core set by synthetic genomics. This requires researchers to obtain the core set of genes shared among different living organisms by comparing the functional similarities and differences of genes among different species, and to construct the minimal genome necessary for the survival of life based on the core genes by synthetic genomics. However, due to the limitations of the current knowledge of genome function, no successful cases of this synthetic approach have been reported.
2.2.2 Cellular organelles
Compartmentalized compartments are one of the main features of living systems. Creating physically separated microenvironments allows for better control of biochemical processes and is a fundamental requirement for making organelles in living cells functional. Inspired by this phenomenon, researchers have developed a range of different artificial organelles designed to add new functions to living cells.
In symbiotic systems in the field of nitrogen fixation biology, nitrogen-fixing bacteria appear as organelles in the cytoplasm of the host plant, and nitrogen-fixing synthetic biologists hope to break the exclusivity of the host in this system and enable nitrogen-fixing bacteria to live in symbiosis with major cash crops or to achieve autonomous nitrogen fixation.
2.2.3 Prototype cells
The “bottom-up” synthesis of prototocells from inanimate molecules and materials is one of the major challenges of the synthetic biology field in this era. In the past decade, researchers have developed different prototocell models, designing genetic circuits that mimic one or more physiological activities of biological organisms, such as transcription and translation of genetic information, enzyme-mediated metabolism, etc. These single-cell models are capable of self-replication and evolution, and possess the basic characteristics of “life”, which can almost be called These single-cell models are capable of self-replication and evolution, and possess the basic characteristics of “life”, and can almost be called life.
2.3. Bio-chassis
Chassis are ubiquitous in contemporary industrialized production. While automobiles have chassis and manufacturers can add models with different appearance and functions to the basic chassis, in synthetic biology, cells also have chassis. On the chassis cells, synthetic biologists add genetic components and organize genetic circuits to make the system achieve specific functions and purposes.
At the beginning of carrying out the design of a synthetic biological system, the researcher needs to select a chassis organism with good traits according to the characteristics of the target product, i.e., the host used for the production of that product. Choosing the right chassis organism can achieve twice the result with half the effort, and a good chassis organism needs to have the following characteristics.
First, the assembly process of synthetic biological systems requires a lot of genetic manipulation and genetic modification to make the chassis organism an excellent cell factory, requiring cells with genetic operability and stability, and able to accept exogenous DNA under controlled conditions.
Secondly, chassis organisms need to have well-characterized and controllable metabolic engineering modules, such as promoters, terminators, transcriptional regulatory switches and other components with known and controllable strength and function, which facilitate targeted regulation of cell phenotypes.
Finally, to reduce production costs, chassis cells should have such features as far as possible.
● the ability to grow in basal media containing inexpensive carbon sources.
● short growth cycles and high metabolic efficiency.
● Simple fermentation process with strong environmental adaptability and tolerance to high concentrations of substrates and products.
2.3.1 Prokaryotes
One of the purposes of synthetic biology is to modify life so as to achieve the purpose of application, and prokaryotes are simpler and easier to modify life forms, such as E. coli, Bacillus subtilis, cyanobacteria and Corynebacterium glutamicum.
2.3.2 Eukaryotes
Eukaryotes such as brewer’s yeast, baker’s yeast, algae, fungi, plant and mammalian cells can be transformed into chassis, and different chassis have their own uniqueness and are suitable for different purposes.
2.3.3 Nucleic acid-free cells
Nucleic acid-free cells, or SimCells (simple cells), refer to bacteria after removal of chromosomes by double-strand cleavage activity of exogenous I-CeuI endonuclease or degradation activity of endogenous nucleases. In these chromosome-free SimCells, the cellular machinery still functions to process various inducible genetic circuits, complete the glycolytic pathway independently, and have the ability to regenerate ATP and NADH/NADPH. The current study shows that SimCells after removal of chromosomes are able to continuously express synthetic instructions for genetic circuits within ten days.
While the step of engineered bacteria from the laboratory to the clinic is often hindered by the potential risk of bacterial genetic instability and uncontrollable replication of engineered bacteria in patients, SimCells are simplified chassis controlled by artificially designed genetic pathways that eliminate the risk of uncontrolled host bacterial growth and can safely produce and deliver certain chemicals without interference from the host cell genome, with broad promising applications.
2.4 Cell-Free System
Cell-free systems, also known as cell-free in vitro transcription-translation systems, are synthetic biology systems that do not require living cells, but have all the materials needed to execute the central law.
The system does not require cellular synthesis, and three typical features exist.
1) No cell membrane, allowing direct regulation of intracellular biological activities, such as transcription, translation and metabolism.
2) No natural genomic DNA, which removes the need for cell growth and unnecessary gene regulation and reduces energy consumption, allowing all material and energy resources to be focused on the synthesis of target products or the achievement of target functions.
3) No substance transport barriers, the system has an open operating environment that allows real-time monitoring and rapid sampling and analysis during the reaction, facilitating the addition of substrates and extraction of products, and regulating the synthetic state of the system.
Currently, a wide range of applications for cell-free systems exist in structural biology, high-throughput screening, biocatalysis, biomedicine and disease diagnosis.
Synthetic Biology Technology
3.1 DNA Sequencing Technology
DNA sequencing enables scientists to determine the exact sequence of each DNA fragment. On the one hand, this allows scientists to know whether the DNA product is the desired target, enabling an assessment of the quality of the intermediate product. On the other hand, large-scale genome-level sequencing has facilitated the establishment of genomic databases, which serve as an encyclopaedia for the construction of standardised components and further facilitate the engineering of synthetic biology. Since Sanger’s determination of the first genomic bacteriophage, PhiX174, in 1997, DNA sequencing has advanced rapidly and has now entered the era of high-throughput sequencing. The spontaneous parallelism of a large number of small reactions in high-throughput sequencing has greatly reduced costs and made it possible to build large-scale databases. The emergence of Next generation sequencing based on the fluorescent dyes and third generation nanopore sequencing based on the ionic current are all indelible milestones of high throughput sequencing and leed to a unprecedently understanding of genomic element function.
3.2 DNA synthesis and DNA assembly Technology
3.2.1 DNA synthesis
‘The application of science, technology, and engineering to facilitate and accelerate the design, manufacture, and/or modification of genetic materials in living organisms. This is the introduction of synthetic biology from the European Commission’s synbio summit part one. The phosphoramidite method is one of the most widely used technology for oligonucleotide synthesis which includes the constant stepwise addition of nucleotide residues to the 5’ terminus of the newly growing oligonucleotide chain. Four steps, Detritylation, Coupling, Capping, and Oxidation on a solid support material allow the ligation of the oligonucleotide. Following the ligation methods for DNA synthesis, the array-based oligo synthesis with photolabile compounds and the semiconductor-based electrochemical method for oligo synthesis paved the way for the emergence of the DNA microarray synthesis industry.
3.2.2 DNA assembly
3.2.2.1 Based on DNA Polymerase
The invention of PCR provided the basis for the development of DNA assembly techniques that relied on DNA polymerase. Polymerase cycling assembly (PCA) is one PCR-based technique. The basic idea of PCA assembly is to assemble chemically synthesizable oligonucleotides into long fragments by template-free PCR. In 1989, Pease et al proposed the overlap extension PCR technique (Overlap extension polymerase chain reaction, OE-PCR), a method that can be used to assemble longer fragments of DNA. DNA fragments with overlapping sequences at the ends are first denatured and annealed. Then let these DNAs serve as primers for each other, and under the catalyst of polymerase, strand extension is performed to obtain fused DNA. Generally, 2–6 DNA fragments can be mixed proportionally as templates, and the oligonucleotides at the outermost two ends are used as primers for overlapping extension, while a homologous overlap region of 20–40 bp between two adjacent DNA fragments should be ensured. Since the OE-PCR method has the unique advantages of simple operation and saving time and effort, it has been widely used nowadays.
3.2.2.2 Based on Endonuclease
BioBrick is a classical gene assembly technique in the field of synthetic biology. It is a class of restriction endonucleases that recognize different DNA sequences but cut the same sticky ends. However, the cut ends are interconnected and do not form the original cleavage site. This method uses a pair of homotrimeric enzymes and two non-homotrimeric enzymes to cut the vector and DNA components, making the original DNA sequence into spliceable bricks and standardizing the DNA components; the standardized components can be assembled sequentially by DNA ligase action.
3.2.2.3 Based on Exonuclease
Gibson assembly technology was first proposed by Dr. Daniel and colleagues in 2009. Gibson ligation is known for its ability to easily assemble multiple linear DNA fragments, and can also be used to insert a target fragment into a selected vector to complete a basic clone. Briefly, Gibson ligation is performed by first obtaining DNA fragments containing homologous regions at the ends, usually by PCR, and then co-incubating these fragments with an enzyme master mix to complete the ligation process. The entire ligation reaction consists of four parts. First, T5 exonuclease digests the DNA fragments in a 5' to 3' direction, and each DNA strand forms a single-stranded DNA protruding end. Second, the protruding ends of two adjacent fragments contain homologous regions, and the DNA fragments can be annealed and the complementary sequences paired. After pairing there is still a gap and DNA polymerase is used to fill the gap. Finally, DNA ligase forms a phosphodiester bond at the cut to complete the ligation. The advantages of this technique include: no specific restriction sites are required and almost any two fragments can be ligated regardless of order; it is a seamless clone with no seams between ligated fragments, and multiple DNA fragments can be ligated at once.
3.3 Genome editing
Since scientists discovered that bacteria use restriction enzymes to cut foreign DNA as a defense against bacteriophages in the 1970s, the concept of ‘gene editing’ gradually enter the scope of scientists. The gene editing technology also undergone extensive development. In short, this development marvelous trajectory contains five milestones.
3.3.1 Application homologous recombination in genome editing
In the 1980s, scientists first applied homologous recombination to integrate exogenous into the genome of living cells. This genome editing was achieved by the intrinsic homologous recombination (HR) with newly transfected exogenous DNA in mammalian cells. With this HR, the mutation of a specific gene can be introduced to the target cells for the elucidate the unique role of this gene in biological activity. However, the efficiency and accuracy of this method are limited and off-target events sometimes happen.
3.3.2 Meganuclease: the first genetic Scissors
Meganuclease is one type of endodeoxyribonucleases that recognizes a long DNA sequence (12 to 40 base pairs). Typically, the recognition site of one meganuclease appears only once in the whole genome, which enables precision cutting. In 1995, I-Scel endonuclease was utilized to induce the double strand break in embryonic stem cells, later interruption of chromosomal neomycin resistant gene and gene correction of HR. Nowadays, hundreds of meganuclease have been found in nature, including three widely used types: I-Sce I the mitochondria of Saccharomyces cerevisiae, I-Cre I and in I-Dmo in the chloroplasts of Chlamydomonas reinhardtii. Despite hundreds of meganuclease, each one has its unique recognition sequence, and its difficult to find one suitable for targeting a specific gene sequence. Thus, the engineered meganuclease appeared. Based on the structure of original meganuclease, such as I-CreI, scientists achieved the modification of this scaffold on different scales to further modify the recognition sites. What’s more, the development of computational biology enables the simulation of the meganuclease working process and later optimization of the meganuclease sequence.
3.3.3 Zinc finger nuclease: customisable gene editing tools
Zinc finger nuclease contains two parts: zinc finger protein and Fok I protein. Each zinc finger protein unit can identify the specific 3-base pair length DNA sequence. Thus, the combination of several zinc finger proteins can form the complex recognizing long DNA sequence. The restriction endonuclease Fok 1 has the unique DNA binding domain and cleavage domain. It homodimerizes at the target site and then cleaves the target DNA fragment. Considering the distinct function of these proteins, researchers fuse the DNA-binding domain of zinc finger protein to the DNA-cleavage domain of Fok 1 and get the artificial endonuclease Zinc finger nuclease (ZFN). To induce the break of each specific target, scientists were required to design two different zinc finger proteins targeting 2 adjacent sites. The zinc finger domain of the two individual fusing proteins will bind to the adjacent DNA sequence on opposite strands of DNA, then two cleavage domains will dimerize and then induce the double strand break at the target sites. Despite its highly customized property, ZFN also has some limitations. For instance, it’s difficult to design the zinc finger protein with high affinity to target sites considering the complicated zinc finger protein targeting 3 base pairs. And the off-target phenomenon is also annoying.
3.3.4 Transcription activator-like effector nucleases: Revolution in the flexibility of gene editing tools
Transcription activator-like effector nucleases (TALEN) is also a kind of restrict enzyme that consists of the DNA binding domain and DNA cleavage domain similar to ZFN. The DNA binding domain, TAL effectors are proteins secreted by Xanthomonas bacteria via their type III secretion system when they infect plants. Consist of about 30 amino acids, TAL effectors obtain the specific affinity to one single base pair with two adjacent amino acids inside. With this one to one specificity, it is much easier to build the TAL effector repeat targeting DNA sequence than zinc finger proteins. As for the DNA cleavage, Fok 1 also serves as the main choice for TALEN. Thus, after the binding of two individual TAL effector repeats on opposite DNA strands, the Fok 1 protein dimerize and then induce the DSB, creating the basis for HR. Except for the flexibility, TALEN also has other advantages beyond ZFN. First, it is much smaller than ZFN, which eases the stress of their delivery and expression. Second, the off-target events are rare in TALEN induced gene editing. Third, the target sites for ZFN are always less than 18 base pairs. But for TALEN, the length can be longer. Last but not least, for each base pair, there are sometimes more than one TAL effector is effective which helps to the optimization of the whole TALEN.
3.3.5 CRISPR-Cas9: the promising land of genome editing tools
ZFN and TALEN enable the customization of gene editing tools. However, the customized parts are always a protein, which means high cost and difficulty in the tool development. Thus, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems appeared. Shortly, CRISPR-Cas9 system consists DNA binding domain, CRISPR DNA and cleavage domain, and Cas9 protein. In nature, CRISPR-Cas9 serves the innate immune system of bacteria and archaea that against the infection of bacteriophages. CRISPR is conserved repeated sequences that are derived from the previously infected bacteriophages. It recognizes the similar DNA fragments in newly invaded foreign DNA and induces the subsequent cleavage. As a widespread acquired immunity mechanism, CRISPR exists in about 50% of known bacterial genomes and 90% of known archaea genomes. As a kind of endonuclease, Cas9 is guided by CRISPR. In normal conditions, CRISPR RNA is combined with the partially complementary TracrRNA which helps with the maturation of CRISPR RNA. In genome editing, scientists design the gRNA which contains the CRISPR RNA and TracrRNA simultaneously. The optional target site has specific features. Protospacer adjacent motif (PAM), a short DNA sequence that varies among bacterial species should be downstream to the cut site. With RNA-DNA hybrid structure formed by target sequence and the partially complementary sequence at 5 prime end of CRISPR RNA, the CAS9 protein was guided to the target site and made the double strand break at cutting site 3 base pair upstream to PAM. Without a homologous vector, non-homologous end joining (NHEJ), and homology-directed repair (HDR) mechanism may repair the double strand break and possibly induce the small insertion or deletion to the cleavage site and later frameshift mutation. To achieve precise editing, the vector containing the homologous regions in the cell can create the customization target gene insertion and gene deletion. Compared to the protein-guided ZFN and TALEN, CRISPR-Cas9 system obtains specificity by the gRNA partially complementary to the target sequence. With the convenience of RNA synthesis and base pair matching in the targeting mechanism, CRISPR-Cas9 exhibit unprecedently flexibility, and is lightweight beyond former genome tools, facilitating the use and dissemination of gene editing tools as well as the elucidation of specific genes in biological activity and disease progression. Despite various advantages, the CRISPR Cas9 system has a relatively high risk of off-target mutation and high dependence on the PAM sequence downstream of the target sequence.
4.Automatic-design tools in synthetic biology:
How to design reliable and complex biocomputing systems is the core problem of synthetic biology. As an important part of the discipline of bioengineering, synthetic biology has many similarities with electrical engineering. Genetic circuits and integrated circuits have great similarities. In the present time of highly developed computer technology, electrical engineers have started to use electronic design automation (EDA) software tools to design integrated circuits, which greatly facilitates the development of integrated circuits. Based on similar ideas, a wave of synthetic biology tools for computer-aided genetic circuits is emerging. For example, CELLO, is a tool suitable for efficient and reliable DNA-based logic circuit design in E. coli and Saccharomyces cerevisiae. In practice, the user simply programs the cell in Verilog programming language, specifying the cell type used to design the gene circuit and the function of the gene circuit, i.e., the corresponding inputs and outputs. The software then selects the already constructed gene components from the database, automatically calculates which components must be put together, constructs the complete gene line, and outputs the required nucleic acid sequence in a physical sense. The most complex part of the manual design of the gene circuit is the design of the complex logic gates between the body of the gene circuit, the inputs, and the outputs. Unlike integrated circuits, the intracellular environment is very complex, with a wide variety of interactions between various compounds. one of the greatest features of Cello is that many of its genetic components, which have been experimentally validated by the authors, are independent of each other. This ensures that the goal of modularity inline design is achieved. In addition to this, CELLO can simulate and evaluate the efficiency of the gene lines, thus making it possible to compare all potential alternative lines and finally select the optimal one. The advent of numerous gene circuit design tools has made it possible to program cells in the true sense of the word, using computer programming languages to assist synthetic biologists in designing specific gene circuits to enable cells to perform specific functions. They will greatly improve the efficiency of synthetic biologists’ research and make it possible to synthesize more complex and functionally diverse genetic circuits.
Interdisciplinary study of synthetic biology
4.1 Synthetic Biology and Immunology (Synthetic Immunology)
Synthetic immunology is the integration of synthetic biology and immunology. Synthetic immunology is a new scientific discipline, which is an intersection of immunology and synthetic biology. Synthetic immunology aims to use biotechnology to properly regulate and control immune responses for the benefit of patients. Immune responses need to be calibrated in the presence of immune system disorders, such as the use of immune cells to suppress proinflammatory mediators in rheumatoid arthritis (RA).
It is also possible to use immune cells to treat diseases, for example, by using their cytotoxic effects to eliminate tumors. And the immune system can be disrupted by using recombinant cytokines or reprogramming immune cells. It is worth mentioning that recombinant cytokines and growth factors or therapeutic antibodies have been applied to patients, but more therapeutic drugs and therapies based on the principle of synthetic immunology are still in the stage of preclinical testing or clinical research. At the molecular level, new therapeutic agents such as antibody derivatives and antibody mimics have been modified to improve the safety and efficacy of recombinant immune cell therapy. At the cellular level, people have engineered immune cells to alter the effector targets of recombinant immune cells. Synthetic immunology is a synthetic system that can reasonably design and construct complex immune response functions and can achieve the purpose of regulating immune response to the greatest extent. It mainly controls immune response through artificial synthetic components (such as nanomaterials, immune adjuvants, and vaccines, etc.).
Synthetic immunology will also drive the further development of immunotherapy. Studies have shown that NK cells may be ideal chassis cells for “general-purpose” synthetic immune cell therapies due to their unique properties. Through the loading of chimeric antigen receptors and intelligent responsive gene circuits that accurately identify tumors, functional enhancement of NK cells will be realized, and the “shelf” supply mode of synthetic immune cells will be realized under the support of large-scale expansion technology of NK cells and closed, automated and programmable “cell factories”. In addition to synthetic immune cell therapy, attenuated and synergistic synthetic immune molecules offer more possibilities for artificial manipulation of immune responses.
In the future, synthetic immunology-driven immune cell therapy will be complemented with novel synthetic immune molecules to further improve the efficacy and safety of antitumor immunotherapy.
4.2 Synthetic Biology and Molecular Biology
Molecular biology is a science that studies the structure and function of biological macromolecules at the molecular level, to clarify the nature of life phenomena. Biochemistry and biophysics use chemical and physical methods to study biological problems at the cellular level, the global level, and even the population level. In contrast, molecular biology focuses on studying the universal laws of life activities at the level of molecules (including multi-molecular systems).
The development of synthetic biology is deeply rooted in molecular biology, which aims to reprogram genetic wiring at the molecular level to (1) engineer natural biological systems or (2) design and build artificial organisms that do not exist in nature. In 1953, James Dewey Watson and Francis Harry Compton Crick discovered the double helix structure of DNA, ushering in the era of molecular biology. Since then, researchers have elucidated many of life’s mysteries from a molecular perspective and developed DNA recombination technology on this basis. DNA recombination technology is the first attempt to cross biology and engineering, which opens up a new field of life science and biotechnology. In 1972, Dr. Paul Berg, a biochemist at Stanford University, created the first recombinant DNA molecule by splicing the DNA of a bacterial virus into the simian virus SV40.1. In 1973, Cohen first linked DNA fragments to plasmids and transformed them into Escherichia coli. In 1974, scientists created the first genetically modified mammals by introducing foreign DNA into mouse embryos. In 1980, Hobom B began to describe genetic recombination techniques in terms of synthetic biology. Since then, the rapid development of polymerase chain reaction (PCR) technology has become an extremely important engineering technology in biological research, and gene sequencing technology has also been advanced. In the early 1990s, the development of sequencing technology and the introduction of information technology led to the application of automatic DNA sequencers in the Human Genome Project.
With the maturity of large-scale genome sequencing technology and sequence analysis methods, life science research has entered the genome era, and many research results have laid the foundation for the generation of synthetic biology. As Polish geneticist Waclaw Szybalski noted in 1974, “Molecular biology has always been described, but the real challenge begins when we enter the stage of synthetic biology. We will design new regulatory elements, add new molecules to the existing genome, or even construct an entirely new gene.” Szybalski believes that synthetic biology is a field with unlimited potential, that there are few limits to how scientists can build better genetic control circuits, and that synthetic organisms will eventually emerge. In 1978, he wrote in the journal Gene about the Nobel Prize in Physiology or Medicine awarded to Danien Nathans, Wemer Arber, and Hamilton O. Smith for the discovery of DNA-limiting enzymes: “Restriction enzyme technology will lead us into a new era of synthetic biology.” Using the restriction splicing method, molecular biologists can analyze the function of individual genes and record their observations as functional descriptions of each gene. Tens of thousands of scientists around the world are doing just that, accumulating knowledge about life and the genome.
In the foreseeable future, new synthetic or composite life forms may emerge. It is based on the great success in the field of molecular biology that the understanding and knowledge of the mechanisms of biological systems at the molecular level are continuously deepened. The process of “building genes — expressing specific proteins — forming specific functions” was further elucidated; The basic gene components, such as promoter, ribosome binding site and transcriptional repressor site, were found in a large number. Gene regulatory mechanisms at various stages of transcription, processing and translation have been discovered. Genome editing technologies like CRISPR are constantly evolving… All these have laid a solid theoretical and technical foundation for synthetic biologists to enhance the ability of rational design and the ability of genome synthesis modification. A variety of powerful regulatory elements and gene circuits, as well as modules capable of regulating gene expression, have been standardized to form the component library and toolbox of synthetic biology.
4.3 Synthetic Biology and Systems Biology
Systems biology studies the composition of all the components of a biological system and their interactions under specific conditions on a molecular basis. Systems biology to integration and quantitative characteristics, implementation from genes to cells, tissues, such as individuals at all levels of correlation information integration, focus on from the Angle of system to recognize and explain the basic principle of biological system, understand the system behavior, and application, its core task is to know structure and dynamics of biological systems finally realizes the control and design of life.
Classical molecular biology is a vertical study, while genomics, proteomics and other omics are horizontal studies. The characteristic of systems biology is to integrate horizontal research and vertical research, and to connect the behavior of each component of the system with the characteristics and functions of the system, becoming a three-dimensional study.
First, systems biology integrates the different components of a system (genes, mRNAs, proteins, small biological molecules, etc.). Secondly, for multicellular organisms, systems biology should realize the integration of all levels, from genes to cells, to tissues, to individuals. It systematically studies the composition of all components of a biological system (DNA, RNA, protein, etc.) and the relationships between these components under specific conditions and establishes mathematical models to quantitatively describe and predict biological functions, phenotypes, and behaviors.
The study of systems biology includes two aspects. One is to obtain experimental data; The second is to build biological models. So, scientists divide systems biology into a “Wet Lab” (research done in a laboratory) and a “Dry Lab” (computer simulations and theoretical analysis).
This “wet” and “dry” division of labor also exists in synthetic biology research. Systems biology methods, especially simulation, control, and design methods, are the methods adopted in the important “dry experiment” part of synthetic biology.
The DNA genetic code is often thought of as the software that commands and controls life, while cell membranes and all biological machines inside cells are thought of as hardware. The understanding of life system software and hardware with the aid of electronic engineering, the research method and basic techniques and tools, like technical personnel with standardization, off-the-shelf parts assembled into the computer, synthetic biology to borrow the design concept of the digital circuit in electronic engineering, USES the concept of abstraction, standardization of engineering, through the rational design, the components assembled into devices, Devices are integrated into modules, modules are built into complex systems, and finally encapsulated in the living organism to make it a biological machine that can perform its intended function.
The key to achieving this goal is to create cellular “circuits”, like electronic circuits, that respond to incoming signals and output accordingly. Logic gate in the cells for cell to achieve its core work is extremely critical, like the circuit of computer, the logic inside the cell door also included AND, NOR, OR, NAND, such as logic gate, they are very core AND foundation of synthetic biology tools, almost all synthetic biology applications which require the participation of logic gate in the cell, so it is very important to build genetic logic gates.
Gene bistable switches and gene oscillators are the first biological components synthesized under the guidance of synthetic biology. Their implementation demonstrates the feasibility of rational design of biological components. Later, more and more researchers to be discovered by standard components, use of these standardized biological components to achieve the basic logic gates, build a variety of genes, and further to assemble the many biological systems with new functions, such as e. coli ecological system with sensitive to population, can produce artemisinin yeast cells, only a single chromosome of yeast cells, etc. Synthetic biologists envision a future in which bioengineers can assemble fully characterized biological components into robust host organisms that perform specific biological functions in the same way that electrical engineers assemble computers.
4.4 Synthetic Biology and Electrical Engineering
Electronic engineering is an engineering oriented to the field of electronics. Its main research fields include circuits and systems, communications, electromagnetic fields and microwave technology, and digital signal processing. The ideas include decoupling, abstraction, and standardization. The core of it is the circuit operation among various logics, namely logic circuit. They are circuits that perform basic logic operations and are widely used in computers and other electronic devices.
In modern industrial production, equipment or device often exist multiple control loops to control it. Due to the increase of the control loop, often cause mutual influence, that is, the input signal of each control loop in the system will have an impact on the output of all the loops, and the output of each loop will be affected by all the inputs. It is almost impossible for one input to control only one output, which constitutes a “coupled” system. Because of this coupling relationship, the system is often difficult to control, resulting in poor performance. Decoupling is the way to solve this problem. By breaking a complex problem into several relatively simple and independent problems, and finally integrating into a unified whole with specific functions. Mathematical decoupling refers to the transformation of mathematical equations containing multiple variables into a set of equations that can be expressed by a single variable, that is, variables no longer directly affect the result of an equation at the same time, to simplify the analysis and calculation. By means of appropriate control quantity selection and coordinate transformation, a multivariable system is transformed into multiple independent mathematical models of univariate system, that is, the coupling between various variables is removed. In solving engineering coupling problems, the basic goal is to design a control device so that each output variable of the multivariable control system is completely controlled by only one input variable, and different outputs are controlled by different inputs. After decoupling is realized, a multi-INPUT multi-output control system can remove the cross-coupling between input and output variables, to realize autonomous control, that is, mutually independent control. This mutually independent control method has been successfully applied in the electronic engineering fields such as engine control and VLSI manufacturing.
Similarly, for complex biological systems, synthetic biology designs adopt similar engineering strategies, namely: (1) Standardization: methods for establishing basic biological components, defining and characterizing biological functions; Classification of genome sequences to define biological functions; (2) Decoupling: the complex biological system is decomposed into simple components with independent functions, and the design and assembly are separated, so that the whole system can be effectively assembled after the design is completed; (3) Extraction: hide the complexity of biological information and management, form simplified redesigned devices and modules, and establish a component library with recognizable interface in the field of bioengineering; (4) Modularity: Modularity design is an important aspect to ensure the robustness of the system. It ensures that the failure of one part of the system will not spread to the whole system. In engineering, each bottom module should be fully independent and encapsulated, and the change of the upper structure does not affect the internal dynamics of the lower module; (5) Hierarchical: establish the layers of components and modules, separate and limit the information exchange between the layers. Components are the basic units of the system. In biological systems, transcription products such as genes and proteins are components. Module is a set of devices with specific functions. Organelles and gene regulatory circuits in biological systems can be considered as modules.
4.5 Synthetic Biology and Genetic Engineering
In 1974, Polish geneticist Waclaw Szybalski called genetic recombination synthetic biology; In 1978, when the Nobel Prize in medicine was awarded to Daniel Nathans, Werner Arber, and Hamilton Smith for the discovery of DNA-limiting enzymes, Szybarski wrote in the journal Genes: ‘Restriction enzymes will lead us into a new era of synthetic biology; In 2000, synthetic biology was redefined as “genetic engineering based on the principles of systems biology”. The close relationship between synthetic biology and genetic engineering can be seen in the famous competition of synthetic biology, iGEM, literally translated as International Genetic Engineering Machine Competition.
Genetic engineering is based on the theory of molecular genetics and modern methods of molecular biology and microbiology. Recombinant DNA molecules are constructed in vitro with genes from different sources according to a pre-designed blueprint, and then introduced into host cells to change the original genetic characteristics of organisms. Obtain new varieties and produce new products. That is, genetic engineering research is mainly to obtain gene expression products, the preparation of a large number of useful proteins and peptides, or transgenic animals and plants or through gene editing to obtain new traits of individuals.
The core of synthetic biology is design, both forward and reverse design, which aims to achieve and continuously optimize the desired goals with standardized engineering methods, which are achieved through genetic engineering. Synthetic biology is not equal to genetic engineering, but the research of synthetic biology cannot be carried out without genetic engineering, which is the most important engineering platform of synthetic biology.
Gene synthesis is a case in point
4.5.1 Definition of gene synthesis
Gene synthesis is a technique in which a synthetic oligonucleotide is splicing into a gene using biochemical methods. There are some differences between gene synthesis and oligonucleotide synthesis: (1) oligonucleotide is single-stranded, and the longest fragment can be synthesized is only about 100nt, while gene synthesis is double-stranded DNA molecule, and the length can be synthesized in the range of 50bp~ 12kb; (2) The raw material of gene synthesis is oligonucleotide, and oligonucleotide synthesis is by adding nucleotides one by one by the solid-phase phospholamide method.
Gene synthesis is the main content of current synthetic biology. Genes that do not exist in nature can be obtained through gene synthesis, which opens a completely new direction for human beings to transform biology, and its role in the fields of new energy, new materials, artificial life, nucleic acid vaccines, biomedicine and so on has been preliminarily manifested. But gene synthesis also has the potential to be exploited as a biological weapon, and this possibility has become more prominent since the synthesis of several viruses.
4.5.2 Advantages of gene synthesis
(1) High efficiency, short cycle of gene synthesis, can ensure the correctness of the sequence.
(2) When eukaryotic genes are cloned and expressed in prokaryotes, the codons preferred by eukaryotes should be changed to those preferred by prokaryotes to achieve efficient expression. The best way to solve this problem is to synthesize the gene after codon optimization according to the codon preference of corresponding cells.
(3) When only the amino acid sequence of a protein or peptide is known but the nucleotide sequence is not known, the best way is to clone and express the gene by artificial synthesis.
(4) Site-directed mutagenesis can be carried out according to the need to modify genes.
(5) Researchers can engineer genes that are hard to get or even don’t exist in nature.
4.5.3 Main pathways of gene synthesis
At present, the main way of gene synthesis is to customize the gene synthesis company. Only by providing the gene sequence or protein amino acid sequence and other information to the company, the company can receive the high-purity lyophilized plasmid DNA containing the target fragment, the strain containing the recombinant plasmid and the sequencing result file.
Information submitted to Gene synthesis companies includes gene sequences or protein amino acid sequences; For gene cloning, 5 ‘restriction site and 3’ restriction site information should also be provided. Whether codon optimization and the required carrier are selected according to the requirements; And whether you need to add a flanking sequence at the 5 ‘end or 3’ end of the sequence.
Synthetic immunology can also be captured in the work of the iGEM team. For example, in Whack A Mole, a project of NMU-China, a gene synthesis approach was used to construct a suicide circuit and CAR expression vector to control gene expression and element-factor interactions by design.
4.6 Synthetic Biology and Metabolic Engineering
Metabolic engineering, also known as metabolic pathway engineering, refers to the rational design of metabolic pathways based on known knowledge of cellular metabolic networks and the use of molecular biological methods. For example, DNA recombination technology is an application discipline that transforms the metabolic pathway and the regulation of metabolic pathway to produce and accumulate target products.
Metabolic engineering notice to enzymology, chemometrics and molecular reaction dynamics and the theory and technology as the research methods of modern mathematics, at the cellular level to elucidate metabolic pathways and metabolic network, partial and the whole relationship between intracellular metabolic process and extracellular material transfer coupling between metabolic flow and flow direction and control mechanism, On this basis, specific genetic manipulation and environmental condition control can be realized to enhance the yield and production capacity of biotechnology process, or the overall modification of cell properties can be achieved to optimize the cell performance through engineering and process operation.
After more than 20 years of development, with the completion of whole genome sequencing of a variety of microorganisms and the systematic understanding of microbial metabolic network, the scope of metabolic engineering transformation has developed to involve cross-species and multi-gene joint expression and regulation. By combining a variety of enzyme molecules from different sources to construct a new metabolic pathway, the purification of chemical synthetic intermediates can be omitted, and the synthesis of intermediates of biofuels, natural complex products and their derivatives can be realized more easily and more energy efficient.
Different from the traditional single gene expression of protein peptides targeted at the production of proteins or industrial enzymes, the genes encoding the key enzymes in the catalytic metabolic pathway do not need to be highly expressed in the process of metabolic pathway modification, because the overexpression of target genes will consume the metabolites available in the cell for the generation of target metabolites. At the same time, intermediates of exogenous metabolic pathways may also cause toxic effects on host cells, resulting in a reduction in the yield of end products. Therefore, in metabolic engineering, the expression of each enzyme in metabolic pathway must be limited to a certain range to achieve the joint and cooperative expression of multiple genes. Although there are many mature systems to control gene expression, such as LAC promoter subsystem, there are few systems that can meet the requirements of metabolic engineering with multiple genes and multiple levels of precise regulation. In addition, many high value-added products do not have natural metabolic pathways, that is, the lack of natural enzyme molecules to synthesize these products, which limits the feasibility of microbial production.
Synthetic biology aims to provide new technologies and tools for mankind to overcome the major challenges in social and economic development. Solving these problems in metabolic engineering has greatly promoted the rapid development of synthetic biology.
Synthetic biology elements, such as tunable promoters, riboswitches, intergenic regions, and other gene circuits, are more accurate than traditional molecular biology tools to regulate gene expression levels and can be used modularized in metabolic pathway construction work. At the same time, due to the standardized and detailed description of the physical and biological characteristics of biological elements in synthetic biology, the use of biological elements can greatly simplify the work of metabolic engineering and make the metabolic regulation of multi-enzyme systems easier and easier.
Application of Synthetic Biology
5.1 Food
Synthetic biology has already made it possible to replace barns and grasslands with bioreactors, allowing food to emerge from laboratories rather than farms. In the era of synthetic biology, engineered microbes have made it possible to produce food ingredients on a large scale rapidly. Here are some examples:
1. Artificial meat, including cell meat and biological fermentation meat.
2. Artificial milk.
3. Sweeteners, including the sweet protein monellin, the flavonoid sweetener aspartame, and the terpenoid sweetener xylitol.
Rare sugars are a class of monosaccharides and derivatives that exist in nature but have very little content. They have critical physiological functions and can be used as potential anti-diabetes and anti-obesity food additives. However, the production cost of rare sugars is often higher than traditional sugars, preventing their widespread use. The natural sweetener, Tagose, has less than half the calories of sucrose. Still, synthetic biology methods can reduce the cost of production, allowing the production of rare sugars to be commercialized on a large scale.
4. Flavoring agent: colorant, preservative, acid preparation agent
Applying synthetic biology to the food industry allows the production of high-value-added products. Also, it provides excellent opportunities to design new or improve existing foods and their manufacturing processes. In an increasingly health-conscious society, synthetic biology will find greater use in improving food nutrition, flavor, and safety. There is no doubt that synthetic biology is shaping the future of food.
5.2 Chemistry Materials
Biomanufacturing may be the optimal solution for producing and developing chemical materials. There are more than a large number of new molecules and new materials in natural organisms to be discovered and applied, and their diversity is far greater than that of the petrochemical industry. At the same time, there are many alternatives to chemical synthesis pathways in complex biological systems, such as the biosynthesis of small molecular precursors and the replacement of chemical catalytic processes with enzymes. Synthetic biology is bringing new historical opportunities to the research and development of molecules and materials.
5.2.1. Biofuels
At present, finding renewable clean alternative energy has become one of the most urgent tasks for human beings. Renewable biomass energy, such as fuel ethanol and biodiesel, can be performed through synthetic biology and can also solve the current problems of low conversion efficiency and high production cost. Microbial fermentation is often used to produce bulk chemicals and natural products. It can partially replace petrochemical refining and plant extraction. The development of synthetic biology technology has dramatically improved the ability to construct microbial cell factories to produce bulk chemicals and natural products.
5.2.2. Specialty chemicals
Specialty chemicals play an essential role in all aspects of human life. Special biomass chemicals have gradually been paid attention to with the gradual improvement of people’s environmental protection awareness. Some special chemicals that are difficult to prepare can be prepared by using components in the biomass structure, or the biomass structure can be separated and derived by small molecules of its constituent units, which significantly reduces the difficulty of preparation, broadens the scope of products, and reduces the problem of subsequent environmental decomposition. The application of synthetic biology technology in the production of special chemicals can help to make the production of special chemicals more efficient and safer. Some complex synthesis processes no longer need to be explored comprehensively but only need to find and cultivate suitable engineering bacteria or design reaction chassis to complete, significantly improving people’s production efficiency.
5.2.3 Materials development
Materials research using synthetic biology can improve the performance of traditional materials or create new environmentally friendly materials. The main products include three components: polymers, protein materials and living functional materials.
ZJU-China 2022’s project achieves innovation in the direction of inorganic production, using microbial mineralisation to restore stone artefacts and promote the development of synthetic biology.
5.2.3.1. Polymeric biomaterials
Biofactories, with their green and sustainable and low consumption and high output characteristics, have naturally become an important means of material preparation. The most familiar biocommunication strategies are the use of microorganisms and cells to produce medical pharmaceuticals, such as enzymes and insulin, and the production of plastic monomers through microorganisms; the second is the rational design of materials through modular genetic manipulation.
5.2.3.2. Inorganic nanomaterials
Natural or engineered microbial organelle structures can be used as templates for the preparation of nano-micron materials in different dimensions, the
Using synthetic biology through DNA as the main building material can produce DNA nanostructures with excellent characteristics such as high designability, precision, biophilicity and modular assemblability, which can lead to the orderly assembly and quantitative production of other biological macromolecules. In addition, organisms themselves can also be used as factories for the synthesis of inorganic nanomaterials.
5.2.3.3. In vivo functional materials
Borrowing from synthetic biology, functional modifications are introduced into the synthesis of target proteins using non-standard amino acid injections, which can also be used as materials from living organisms (including extracellular matrices, etc.).
5.2.3.4 Biosensors
The construction of biosensors in engineered bacteria or circuits in cell-like systems without a cellular chassis by synthetic biology methods can eliminate the purification and other steps involved in traditional assays, greatly improving the efficiency of substance detection.
5.2.3.5 Fluorescent materials and molecular probes
Synthetic biology can be applied to synthesise specific fluorescent materials and probes to specifically label, modify and modify biological target molecules, thus enabling the labelling, imaging and dynamic tracing of intracellular molecules, which is safer, more accurate and more efficient than traditional methods.
5.3 Agriculture
There is also scope for synthetic biology in agriculture, in fertiliser manufacture, pest control and crop production, to maximise yields and minimise adverse environmental impacts.
5.3.1. Improving crops
Synthetic biology for plant crops can be used to quantify known biological processes and modify existing plant signalling and metabolic pathways to increase nutritional value and improve the anabolism of plant growth, leading to breakthroughs in yield, stress resistance and quality improvement.
5.3.2. Agricultural pollution
Synthetic biology is used to create a particulate material that can adsorb microscopic chemical pollutants from pesticides, pharmaceuticals, and wastewater.
5.3.3. Insecticide
Whereas insecticides have previously had adverse effects on plant crops as well as beneficial insects, there is now research into the use of RNA genetic targets to kill pests without harming other insects. Scientists are also investigating microbial natural product pesticides that could play an important role in crop pest and disease control.
5.4 Art:
Thanks to its ability to produce chemicals with specific colours or flavours, synthetic biology also shines in the field of art
5.4.1. Smell
Scientists have used synthetic biology to recreate the smell of extinct flowers by restoring the genome sequence of an extinct species and putting the sequence into the yeast genome to produce odour molecules.
5.4.2. Colour
Using synthetic biology to modify engineered bacteria so that they can produce different colours of pigment for use in artistic creation.
5.4.3. Restoration of artworks
Selecting the right strain of microorganism can be adapted to meet the needs of artwork conservation. Microorganisms can clean some silicates and organic residues from artworks, and can also be used to remove casting moulds, glues, and oils from sculptures. Restoring artefacts and works of art back to their former glory.
5.5 Environment
Synthetic biology can make its contribution to environmental detection and environmental restoration.
- Using engineered microorganisms as sensors to detect substances in soil and wastewater.
2. Targeted design and modification of existing strains to construct engineered strains capable of degrading one or more pollutants to achieve degradation of harmful substances, and adsorption and recovery of heavy metals, etc.
3. For compound pollution, such as wastewater, based on the establishment of a modular library of genetic components related to the metabolism, regulation, and resistance of typical organic pollutants, artificial flora and other strategies are introduced to rationally design and assemble biological systems, constructing an efficient degradation flora of typical environmental pollutants.
5.6 Medical
Developments in the medical field of synthetic biology have played a huge role, for example in the improvement of traditional drug production and nucleic acid drug development, gene therapy, CAR-T, engineered bacteria, and phage therapy, to name but a few. The tools of synthetic biology are helping scientists to develop new therapeutic modalities and address some of the unmet needs in medical clinics today. Synthetic biology is leading the future of pharmaceutical manufacturing.
5.6.1. Drug manufacturing
The use of synthetic biology approaches allows the design of targeted engineered probiotics for the targeted treatment of major chronic diseases such as digestive disorders, metabolic diseases, and psychiatric disorders, greatly improving the controllability and stability of microecological therapies. Suicide switches can be set in engineered microorganisms to prevent engineered bacteria from leaking and thus causing harm to the environment and humans, such as in higher temperatures where mRNA with thermally inhibited RNA thermometers cannot be translated properly. Scientists can also design engineered phages that can target different strains, or different types of bacterial hosts, by genetically mutating the phage tail protein.
5.6.2 Diagnostics.
Through synthetic biology, new engineered modified bacteria are being developed to enable cheaper, simpler, and more sensitive diagnoses of various diseases such as bacterial infections, cancer, inflammatory diseases, and metabolic diseases. The main types include nucleic acid diagnostics and microbial smart response.
1. Nucleic acid testing
Nucleic acid testing through the method of synthetic biology, to achieve the purpose of disease diagnosis RNA is a marker that is used in medicine to sort out diseases and to detect the direction and severity of the disease, especially for the clinical treatment of high-risk groups and to predict their risk of development.
2. Response testing
Modified microorganisms used to diagnose certain diseases can provide a better, cost-effective, non-invasive alternative.
Synthetic Biology Perspectives
6.1. Synthetic biology becomes routine
Synthetic biology as an emerging frontier discipline has many technical tools that seem highbrow, and as time progresses, these tools will no longer be limited to the hands of synthetic biologists, but will become a fundamental technology coming to the forefront of all biologists. Synthetic biology as a discipline may not die out, but its actual content will be determined by its frontier research, evolving forward.
6.2. Explosive growth of the bio-industry
Various bio-industries driven by synthetic biology are already growing at a rate of 20% per year in Europe and the US. But this is actually just the beginning of an explosive growth that will come in the future. The bio-industry will also eventually spawn entirely new industries that will change human life in ways that we can hardly predict today.
6.3. The Wave of iGEMer
One of the obvious problems brought about by the booming bioindustry is the talent gap. iGEM is credited with the development of synthetic biology in the first 20 years, not only dramatically promoting the visibility of the entire field, but also nurturing a large number of talents in academia and industry. The industrial and academic networks assembled by iGEMers will influence the development of the bioindustry in China and the world in the next 20 years.
6.4. Development in combination with AI technology
AI and synthetic biology are both emerging sciences, and in the future, the combination of the two is sure to create more world-disrupting products.
6.5. A major force in dealing with future crises
Synthetic biology has been hailed as one of the “top ten technologies that will change the world in the future”. The National Development and Reform Commission has mentioned “synthetic biology” several times in the “14th Five-Year Plan” for the development of bio-economy. Synthetic biology is an important strategic development direction for China. To achieve carbon neutral carbon peak, synthetic biology is the underlying support for ‘green manufacturing’.
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