Introduction
This work is a timely rain in the field of synthetic biology, providing a powerful tool for synthetic biology to leap from single cells to complex biological communities, tissues and organs.
Meng Fankang| Author
In multicellular organisms, contact cell communication, short-distance (autocrine), mid-distance (paracrine) and long-distance (endocrine) signal communication between cells control the division of labor and spatiotemporal development of the multicellular system. The key to immune response and maintaining physiological homeostasis. Similar to the electronic circuit that coordinates a large number of computing units in a computer, cell-to-cell communication—this chemical circuit guides the process of collaboration between different cells in a multi-cell system.
In the field of synthetic biology, we have developed a variety of synthetic artificial communication systems, such as synthetic quorum sensing, scalable peptide-GPCR signalling, synthetic morphogen system, etc. However, the existing high-quality intercellular communication tools are still limited. There are two main aspects that limit the utility of such tools:
versatility: The ideal cell-cell communication system should work in a modular manner, and it needs to meet The communication scenes in the biological world (bacteria, fungi, animals, etc.) can be compatible or migrate to multiple cell types. However, existing systems either need to artificially add exogenous precursors to synthesize signal molecules, or cannot be transferred from one species to another, and their compatibility in different biological chassis is very poor.
Orthogonality: The ideal cell-cell communication system relies on a series of well-insulated channels for correct signal transmission. In electronic products, the insulation of different channels can be achieved through spatial isolation, and in biological systems, the most feasible way to achieve insulation is through "chemical orthogonality". However, the existing intercellular communication tools are orthogonal and interfere with each other when used in the same cell or population.
In order to solve the above-mentioned problems, the research team from Chinese Academy of Sciences, Peking University and Kyanite Microbes designed a complete set of high versatility, strong orthogonality, and cross-biological communication based on the Quorum Sensing system. Synthetic Biology Toolbox, related articles were published on Nature Communications.
has developed a total of 10 sets of new or optimized cell communication tools through component mining, rational design and directed evolution. The comprehensive performance of these tools far exceeds traditional quorum sensing signal systems. This set of tools will greatly expand the capabilities of synthetic biology in multi-cell bioengineering, provide a solid foundation for large-scale biocomputing in cells, and pave the way for complex multi-cell engineering including artificial ecosystems and intelligent tissues. Ping the road
Paper title:
De novo design of an intercellular signaling toolbox for multi-channel cell-cell communication and biological computation
Paper address:
1. Solving versatility: Starting from general cell metabolites
in order to design beyond the current level A key aspect of cell communication tools is versatility. After all, we can always optimize in the same biological chassis and tune a set of communication tools to higher performance, but this still cannot meet the needs of future applications in heterogeneous multicellular systems.
In order to solve the versatility of the intercellular communication system, the researchers proposed a new development strategy: select "conserved cell metabolites" as the precursor molecules of the signal molecule, and design a small signal molecule biosynthesis for the precursor molecule. way. In this way, even if the biosynthetic pathway is introduced into a different biological chassis, the same precursor molecule can be used to synthesize the final signal molecule, which greatly expands the versatility between different cell systems.
researcher in Kyoto Encyclopedia of Genes and Genomes (KEGG) dataThe secondary metabolite data and corresponding transcriptional regulatory factors in the library and literature were screened according to the following criteria:
(i) signal molecules can be biosynthesized and can freely diffuse across cell membranes;
(ii) signal molecules should be specific Strong transcription factor perception;
(iii) signal molecule precursor must be a common intracellular metabolite in prokaryotic and eukaryotic cells;
(iv) biological pathway to synthesize this signal molecule contains the least total number of enzymes.
Screening potential cell-cell communication systems starting from general cell metabolites
The researchers finally screened out 10 candidate cell-cell signal communication systems from a variety of species, including Pseudomonas, Rhodobacter, Streptomyces, Photorhabdus, Bradyrhizobium, Yersinia and higher plants. In subsequent tests, 6 out of 10 candidate systems achieved a relatively ideal signal response effect.
At the same time, the researchers also added four classic QS communication systems to the toolbox. Therefore, a total of 10 sets including 6 new designs and 4 optimized cell communication systems were obtained in this work. Subsequent researchers optimized related elements through rational design of promoters and directed evolution of transcription factors, making these 10 systems reach a high level of performance.
The first author of the article
6 de novo-designed intercellular communication systems, Dr. Du Pei, an associate researcher of the Institute of Microbiology, Chinese Academy of Sciences, answered the question "How do rational design, directed evolution, and component mining work together in this work?" These three are reflected in different degrees in the overall design. The results of component mining and component characterization are the premise of rational design. According to the preliminary characterization results, we have targeted rational design of some quorum sensing system promoters. , Replacing the natural promoters to make it have better performance parameters (dynamic range, background, sensitivity, etc.). Some systems originally did not have natural promoters, so we designed several through rational design from the beginning Version of the synthetic promoter, and then select the optimal design according to the test results, such as the IV and pC system in the article. There is also a system for synthesizing signal molecules: we mine enzymes from different species, and then artificially build a large The synthesis pathway of some signal molecules. We must ensure that enough small molecules can be produced in E. coli without adding any nutrients other than the basic medium. This system requires a lot of debugging, which also leads to the design of the cell communication system The biggest factor of failure. Directed evolution is a relatively late strategy adopted to improve the performance of partially constructed communication systems, such as increasing the sensitivity of signal receiving systems."
2. Universal design breaks the gap between different biological worlds Obstacles to communication
In order to achieve cross-species and cross-biological communication, the signal sending module and the receiving module need to be able to play a role in various cell models. The "Universal Metabolic Molecules" design strategy provides the most necessary guarantee for breaking the barriers of communication between different biological communities.
By replacing E. coli promoters with species-specific promoters, researchers have established a cross-biological cell communication system, including (i) DAPG-PhlF channels from E. coli to yeast, and (ii) from yeast to E. coli Sal-NahR channel, and (iii) pC-RpaR channel from HEK-293T human cell line to E. coli. All communication systems are successfully activated. This fully demonstrates the versatility of these communication tools and their ability to communicate across biological circles. The
communication tool has the ability to communicate across the biological world.
Du Pei mentioned in an interview: "The meaning of versatility is to maximize the application scenarios of the cell communication system and make our work more useful. The system we designed is in E. coli However, our purpose is not just to use it in E. coli, so that the meaning and usage scenarios of cell communication are greatly reduced. For example, for automatic control of fermentation, the target host that needs to be considered is often bacteria, Prokaryotes such as nematodes and streptomyces may also be eukaryotic single-celled organisms such as yeast; modeling or artificial control of multi-cell behavior is not limited to E. coli. Because of usSuccessfully completed the migration of cell communication in mammalian cells, opening up more possibilities. For example, based on the communication between mammalian cells, it is possible to study cell differentiation, tissue development, and even the construction of artificial organs in the future. These are ultimately inseparable from universal communication tools between cells. Of course, when a system is transferred to a new species, a lot of adjustments and modifications are required, but its signal molecules remain unchanged, and the basic signal molecule synthesis pathways and sensing principles are also unchanged. "Z2z
3. Solving orthogonality: using the diversity of biosynthetic molecules and transcription factors
When multiple communication channels are integrated in the same gene circuit, interference may occur at the two levels of "signal induction" and "promoter response" . Traditionally, they were defined as "signal cross interference" and "promoter cross interference". Zhao Huiwei, the co-first author of the
article and an assistant researcher at the Institute of Microbiology, Chinese Academy of Sciences, said in an interview: "The modularization and orthogonalization of biological components is a basic scientific issue in synthetic biology, especially in the process of designing complex gene circuits. Modularization Orthogonalization is a necessary condition for the predictable assembly of components. In intercellular communication tools, orthogonalization can ensure that multi-channel communication does not interfere with each other. "Z2z
Among the 10 communication tools developed, four classic QS The channels (C4, 3OC6, C8, and 3OC12, highlighted in the yellow box) show obvious cross-interference, but the six newly designed channels (highlighted in the red box) are either at the signal sensing level or the promoter The response level has significantly lower cross-interference. This remarkable orthogonality is precisely due to the highly different compositional structures of these signal systems-the chemical diversity of biosynthetic small molecules and the rich element library of small molecule sensing transcription factors have brought huge design exploration space. The cross-interference of
communication tool at the signal sensing level (upper) or promoter response (lower) level
"The success of this design emphasizes a general principle," the article further explains in the discussion section, "natural biochemical mechanism Did not explore all possible solutions-"enough is enough", although it is naturally "lazy", but this leaves almost unlimited design space for synthetic biology design. "Z2z
Synthetic Biology extends the research of biological systems to the unexisting field of life
(this figure is modified from Nature review: Build life to understand it)
4. High-performance orthogonality tools bring more complex cell designs
currently The engineering of complex biological calculations in cells is not progressing smoothly. This is largely due to our very limited ability to program large gene circuits: not only is there a lack of tools, but also complex gene circuits are easy in cells. Excessive use of cell resources, and evolutionary instability.
"Encapsulate different biological computing modules into different cells, then connect different cells, and adopt a divide-and-conquer strategy" is the potential to break through the bottleneck of complex gene circuit design Program. This strategy can theoretically achieve stability, programmability and computational complexity at the cellular level. The tools developed in this work are providing important support for this design concept.
To demonstrate more complex multi-cell biological computing functions, the researchers designed a complex three-input XOR-AND logic gate circuit. XOR-AND logic gates are deployed in seven different E. coli strains, coordinated by four communication channels. Each strain contains a NOR gate (cell-1 to cell-6) or a Buffer gate (cell-7). This is the first biological computing circuit known to utilize four communication channels simultaneously.
XOR-AND logic gate circuit
Zhao Huiwei said in an interview: "Through the sharing of four systems, we have implemented the "3 Input-8 Output" gene circuit design in E. coli, and the biological calculations between 7 cells can eventually be It is a big breakthrough to output the correct signal. Because there is no reason for cells to work according to your ideas, it is still difficult for them to work together in the same time and space. The first 6 cells are NOR gates, each cell must have a clear ON/OFF, and the production intensity of small molecules, the rate and concentration of diffusion, and the cellThe receiving rate, response interval and sensitivity of the inter-receiving system all need to be considered by the system.
5. What can a good cell communication system bring to synthetic biology?
Cell-cell communication is ubiquitous in nature. From an engineering perspective, these extensive natural communication systems provide a large reserve of synthetic communication elements, including signal molecules, highly specific receptors and transcriptional regulators. Starting from the discovery of general metabolic molecules with communication potential in nature, this research proposes a de novo design route for communication channels between cells, using rational design and directed evolution to develop 10 sets of new or optimized cell communication tools with comprehensive performance Far beyond the traditional quorum sensing signal system.
Regarding the impact that this work can bring to synthetic biology, Zhang Haoqian, the first author of the article, a PhD from Peking University, and co-founder and CEO of Blue Crystal Microorganism, said in an interview: "During the entire biological evolution process, intercellular Communication is a vital link for biology to move from single cell to multi-cell. Synthetic biology, as a discipline aimed at "recreating" life, has always lacked the elements for designing communication between cells. The existing elements not only have There is a strong mutual interference, and the number is extremely limited, and there are very few that can be used in human cells. But whether it is fermentation production, cancer treatment or artificial organs, cell communication is an engineering object that cannot be ignored. I He believes that this work is a timely rain in the field of synthetic biology and provides a powerful tool for synthetic biology to leap from single cells to complex biological communities, tissues and organs.” Z2z
Du Pei also added: “Talking about the significance of this work, Specifically, we should not talk about any single application scenario. The ultimate goal of synthetic biology-synthetic life, is not to synthesize single-cell life. Then, how to bridge the gap from single cell to multi-cell? The communication between cells is impossible. The ability to bypass. Although it is still far from synthetic life, we at least paved a floor on the road to the future. Specific to the application scenario, this set of tools we developed can support research from single cell to multi-cell Level: Whether it is basic research or applied research, and regardless of the model species currently used in this research. The versatility and orthogonality of this set of tools are a guarantee for this, and they complement each other and are indispensable. Automatic fermentation control , Multi-cell behavior modeling, cell differentiation, and even artificial organs are all applicable scenarios. However, as a supporting component, it may be applied in completely unexpected scenarios in the future."
, a famous synthetic biology researcher Chris Voigt's vision for the future development of synthetic biology: In the future, we will move from a single cell to a system.
After 2030, products will turn to "systems" instead of individual cells or systems. In these systems, designed biological cells can work together as a group or be integrated into non-living materials or electronic products. In agriculture, engineered plants and bacteria symbiotically cooperate with each other to control gene expression as a whole in response to different environmental conditions. The future hamburger patties can be produced using bacteria, fungi and livestock cell populations, and they can jointly build complex structures to synthesize molecules with unique nutrition and flavor. Building materials can be embedded in living modified cells to realize the function of self-repair or air pollution removal. The biological system included in the total paint project can prevent biological contamination of the hull, reduce pipeline corrosion, and stabilize the soil structure. Robots produced by coupling engineered living cells and electronic devices can utilize energy in the natural environment, use biosensors for navigation or achieve better brain-computer integration.
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