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    31 October 2023, Volume 4 Issue 5
    Invited Review
    Applications of automated synthetic biotechnology in DNA assembly and microbial chassis manipulation
    Yongcan CHEN, Tong SI, Jianzhi ZHANG
    2023, 4(5):  857-876.  doi:10.12211/2096-8280.2023-049
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    The construction of microbial cell factories plays a critical role in green biomanufacturing. The production of chemicals can be achieved using microbial cell factories, instead of traditional production methods that rely on plants and land resources. In recent years, synthetic biology has made significant advancements in biological manufacturing, including the successful biosynthesis and commercialization of natural products derived from plants, such as artemisinin and cannabidiol. However, the construction of engineered microbial cell factories still faces challenges due to the high complexity of living systems, the iterative nature of experimental processes, and low experimental throughput. To overcome these obstacles, the application of high-throughput, automated, and intelligent hardware, and software platforms, as well as standard, modular libraries of synthetic biology parts and processes, has led to the emergence of automated synthetic biotechnology. This breakthrough allows for low-cost, high-throughput, rapid, and iterative experimentation to efficiently construct large-scale engineering prototypes of microbial cell factories, thereby facilitating related research and applications in this field. To this end, dozens of automated biofoundries have been built around the world. This article primarily focuses on the application of automated synthetic biotechnology in the most critical and time-consuming step of "build" in the "design-build-test-learn" cycle of synthetic biology. The automated building of microbial cell factory demands both the automated construction of engineered DNA and the automated manipulation of microbial chassis, including gene synthesis, PCR amplification, enzymatic digestion, DNA assembly, transformation and plating, colony picking, cell lysis, nucleotide purification, and sequencing. In this review, we summarized the state-of-the-art automated processes and platforms for DNA assembly and microbial chassis manipulation, provided recent advances of automated synthetic biotechnology in the mining and characterization of biosynthetic gene clusters, the combinatorial optimization of metabolic pathways, and the engineering of microbial chassis. We also discussed the challenges and opportunities in automating the construction of microbial cell factories, putting forward to one of most important future directions in full process automation, including the construction of non-model microbial cell factories. The localization and independent research and development of automated biofoundry would make significant contributions to this process.

    Establishment of iBioFoundry for synthetic biology applications
    Hui LU, Fangli ZHANG, Lei HUANG
    2023, 4(5):  877-891.  doi:10.12211/2096-8280.2023-027
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    Synthetic biology is an interdisciplinary field that focuses on designing, modifying, and synthesizing biosystems. It uses engineering principles to design and modify biosystems and even create novel "artificial life" to provide biological functions that meet human needs. Due to the high complexity of biosystems, extensive trial and error experiments are often required to gradually realize the desired engineering goals, resulting in high research costs and slow progress. With the continuous development of automated synthetic biology technology, multiple automated synthetic biology facilities have been established or are under construction worldwide. Biofoundry is an integrated automated scientific facility that combines the principles of intelligent manufacturing with the theoretical foundation of synthetic biology. It enables fast construction and testing of reprogrammed living organisms through the integration of intelligent and automated high-throughput devices. This article introduces the background, design, operation, and application aspects of iBioFoundry, an automated synthetic biology facility established at ZJU-Hangzhou Global Scientific and Technological Innovation Center. The iBioFoundry adopts a modular design concept and integrates various peripheral devices with three track-mounted robotic arms to build four functional modules: sample library, DNA assembly, cell screening and cultivation, and analysis. To meet the requirements of different research tasks and maximize the utilization of the device, iBioFoundry has a multi-task process management function, which is able to track and record all the process handling information of the entire experimental process and achieve traceability of all samples. Through a brief description and analysis of the high throughput construction of engineered E. coli strains and the enzyme directed evolution and screening, the article discusses the formulation of experimental automation schemes, programming of experimental processes, and on-machine operation. The article also shares some thoughts on the allocation of consumable storage space, parallel execution of multiple experimental tasks, and standardization of experimental processes. Automated synthetic biology facilities can help researchers significantly improve experimental efficiency. By combining the large amount of high-quality data generated by the facility with information technology, the automated "design-build-test-learn (DBTL)" engineering cycle in synthetic biology research can be achieved in a high-throughput, low-cost, and multi-cycle manner, accelerating the research efficiency of synthetic biology in both fundamental and many biotechnological application fields.

    Biofoundry and its industrial application
    Guomiao ZHAO, Xin YANG, Yuan ZHANG, Jing WANG, Jian TAN, Chao WEI, Nana ZHOU, Fan LI, Xiaoyan WANG
    2023, 4(5):  892-903.  doi:10.12211/2096-8280.2023-024
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    Traditional methods for strain isolation and improvement can be cumbersome, time-consuming, error-prone, and difficult to scale up, leading to inefficiencies in research and development workflows. With the integration of automation and robotics technology, biofoundry can achieve automated operations through guide rails and robotic arms, leading to improved stability and precision of experimental operations. Additionally, by utilizing smaller cultivation volumes, such as microplates or droplets, the cultivation and screening throughput can be increased, addressing the currently existing issues of traditional methods. This can greatly improve research and development efficiency, allowing for the testing and optimization of large numbers of microbial strains or genetic variants in a high-throughput manner. The biofoundry encompasses interdisciplinary fields such as mechanical engineering, automation, computer science, and life sciences. The collaboration among these fields is crucial for the development and advancement of laboratory automation. By leveraging automation and high-throughput technologies, the field of strain isolation and improvement can benefit from increased efficiency, improved reliability, and scalability. These advancements can accelerate the progress of microbial strain engineering for various applications in biotechnology, medicine, agriculture, and energy production. This paper briefly introduces the composition and classification of the biofoundry, the high-throughput detection method, and focuses on the high-throughput screening platform built by the research team from Nutrition & Health Research Institute, COFCO. In combination with the projects carried out, it introduces the application of the high-throughput screening platform in the fields of biofuel strain development, traditional brewing strain screening, feed substitute antimicrobial screening, directed evolution and screening of enzymes, etc.

    The recent progresses and applications of in-parallel fermentation technology
    Zhonghu BAI, He REN, Jianqi NIE, Yang SUN
    2023, 4(5):  904-915.  doi:10.12211/2096-8280.2023-026
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    At the beginning of this century, high-throughput in-parallel fermentation (cell culture) technology and its related apparatus based on microbioreactor and miniature bioreactor were developed and widely applied. This was to solve the challenges encountered by biopharmaceutical process researches in terms of cultivation throughput, R&D efficiency, and the continuous rising on the cost for microbial fermentation and mammalian cell cultures of multiple clones, and more importantly, the urgent needs for conducting design of experiment (DoE) driven by the principle of quality by design (QbD). In the recent years, the rapid advance of microbial metabolic engineering and synthetic biology has put forward a rather strong demands for the high-throughput screening on high-performance strain libraries and the early evaluation of strain phenotypic potential performance under industrial cultivation conditions. This has further expanded the application range of in-parallel fermentation technology with various culture volumes in the field of modern industrial biotechnology. Up to now, integrated systems containing in-parallel fermentation setups with multiple microbioreactors, online probes, operating software, and data processing units, which can simulate industrial cultivation conditions and implement accurate control of process parameters, have become a powerful tool for accelerating bioprocesses engineering R&D. They play a central role in transforming basic research achievements, such as drug discovery, metabolic engineering, and synthetic biology, into industrial technologies. Especially in the field of synthetic biology, based on the principle of "industrial similarity", in-parallel fermentation technology addresses key limitations of the conventional microplate and shake flask based high throughput screening, which cannot characterize the significant impact of culture conditions on the phenotypic performance of the selected clones at a large scale. This enables a process-oriented, high throughput, and efficient screening and evaluation if microbial strain libraries. This review provides an overview on the recent development in high throughput in-parallel fermentation & cell culture technology and its application scenarios of synthetic biology researches. In particular, it emphasizes the value of in-parallel fermentation technology in high-throughput strain screening complying with the three-stage strategy, and how in-parallel fermentation technology makes the implementation of the industrial similarity principle of strain screening possible, and how the technology combines DoE experimental tactic to significantly improve the efficiency of bioprocess development. It is also discussed the general procedure to build up a multivariate batch model of bioprocess based on in-parallel fermentation technology. In the end the approach of using in-parallel culture to establish a process scale down model is also explored.

    Progress and prospect of ultrasonic liquid transfer and low-volume liquid transfer technology
    zhiqiang ZHANG, Yang ZHANG, Weibao QIU, Hairong ZHENG
    2023, 4(5):  916-931.  doi:10.12211/2096-8280.2023-036
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    In recent years, the rapid development of modern biological and medical technologies, such as synthetic biology, new drug research, and in vitro diagnosis, has put forward increasingly high requirements on the precision, accuracy, throughput and cost of low-volume liquid transfer technology as the number of liquid samples increases but the volume of liquid samples decreases greatly. Although the traditional piston-based pipetting technology can achieve automation and high throughput, the pipetting precision is limited to sub-microliter, and it consumes a large amount of disposable pipette tips. The liquid transfer technologies based on solenoid valve and piezoelectric actuator can improve liquid transfer precision greatly, however, the throughput of these technologies are lower than pipetting technology due to their complex structures. The liquid transfer technologies based on electric field, magnetic field, and light can achieve high transfer precision of nanoliter and picoliter, but these technologies are mainly based on the microfluidic platform for some special applications. In addition, the tips such as the pipette tip, tubing, or nozzle used in the aforementioned liquid transfer technologies are in direct contact with the liquid, leading to the risks of tip blockage, liquid residue and sample cross-contamination. Moreover, the tips are mostly disposable, resulting in high cost and environmental pollution. Non-contact ultrasonic liquid transfer technology, using acoustic radiation force of focused ultrasonic wave to eject droplets from liquid surface, does not need disposable tips, and the ejected droplets do not contact with any other media except the liquid containers during the transfer process. The size of ejected droplets can be accurately controlled by adjusting the focus size and acoustic energy of ultrasound beam. The liquid transfer volume can be adjusted over a large range from nanoliter to picoliter with high precision. Due to its characteristics of fully contact-free, high precision and high transfer speed, non-contact ultrasonic liquid transfer technology shows great potential in biological and medical applications. In this paper, we introduce the development and representative progress of low-volume liquid transfer technology, with emphasis on the development and progress of non-contact ultrasonic liquid transfer technology. Finally the future trends of low-volume liquid transfer technology are analyzed and discussed, such as non-contact ultrasonic liquid transfer technology with high throughput and good versatility, intelligent liquid handling workstation, and low-volume liquid handling technology based on microfluidic platform.

    Application of automated high-throughput technology in natural product biosynthesis
    Zhehui HU, Juan XU, Guangkai BIAN
    2023, 4(5):  932-946.  doi:10.12211/2096-8280.2023-035
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    Natural products are usually secondary metabolites derived from animals, plants, or microorganisms. They are closely related to people's daily lives and important sources of drugs, food and nutrition additives, pigments, and cosmetics. The exploration of novel natural products and the efficient synthesis of value-added products are key to enriching and improving people's living standards. Researchers have exploited a large number of natural products through strategies such as direct product isolation, homologous activation, and heterologous expression. However, low throughput and low titer have become the two major bottlenecks of this field. With the rapid development of synthetic biology, automated high-throughput technology has been developed to transform the low throughput, stochastic manual experimental process that relies on human resources into an automated, standardized, and efficient research process, which is regarded as a leading technology in natural product biosynthesis. This review compares and highlights the advantages of automated high-throughput technology over the traditional techniques within the context of natural products biosynthesis, provides an overview of the components and principles of automated high-throughput workstations, and summarizes the existing and upcoming automated high-throughput facilities. In addition, this review highlights the applications of automated high-throughput technology, including the batch mining of natural product biosynthetic genes and gene clusters, overproduction, and efficient detection of value-added natural products. These examples effectively illustrate the benefits of this technology in the field of natural product biosynthesis. In conclusion, the use of automated high-throughput technology in synthetic biology has shown promising results, particularly in the high-throughput discovery and biosynthesis of natural products. Finally, we discuss the existing shortcomings of the technology. Despite the cost and operational challenges associated with the automated high-throughput technology, it is expected to significantly advance both basic and applied research in synthetic biology, while also provide a sustainable source for the development of new functional products based on newly discovered natural products.

    Fluorescence detection-based high-throughput screening systems and devices facilitate cell factories construction
    Mengchu SUN, Liangyu LU, Xiaolin SHEN, Xinxiao SUN, Jia WANG, Qipeng YUAN
    2023, 4(5):  947-965.  doi:10.12211/2096-8280.2023-017
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    Microbial industrial manufacturing focuses on construction of microbial cell factories using low-cost, renewable resources as materials to achieve sustainable production of value-added compounds. The "test" stage in the development of microbial factories that relies on the "Design-Built-Test-Learn" cycle has quickly become one of the bottlenecks restricting the development of synthetic biology and metabolic engineering. To accelerate DBTL cycling, high-throughput screening techniques need to match the size of the library during the testing phase. Microtiter plates (MTP), as a traditional screening method, uses the optical changes of metabolites in microliters of culture medium for detection and analysis, which can meet the repeated detection and accurate determination of mutants in the library, and also have the ability to screen high-yield strains of extracellular metabolites. However, this screening method is time-consuming and has low throughput. The automated platform is a good solution to the limitations of low screening throughput of microplates. However, the high cost of automation equipment and equipment maintenance makes this method not universal. At present, the main method of high-throughput screening is fluorescence-activated cell sorting (FACS), which can reach a screening throughput of about 100 000 cells per second. However, FACS is limited to detecting intracellular fluorescence signals associated with target products or metabolite fluorescence signals bound to membranes. This problem is well solved by droplet microfluidic technology, which embeds and cultures single cells in monodisperse and picoliter droplets; each droplet acts as a separate microreactor to achieve genotype and phenotype coupling. In the process of screening of huge mutant libraries by droplet microfluidic technology, the screening throughput can reach 107 per day, which effectively improves the work efficiency, and also shows great advantages in experimental cost, realizing the development of microbial cell factories with high screening throughput and low cost and the screening of highly active enzyme variants. In conclusion, based on the high-throughput automated screening platform using microtiter plates, the human labor investment of the high-throughput screening process is greatly reduced, and the development of FACS and droplet microfluidic technology further improves the throughput. In particular, the development of fluorescence-activated droplet sorting (FADS) high-throughput screening technology opens up the possibility for automated, high-throughput, and low-consumption screening. This paper reviews the main progress of the application of different high-throughput screening techniques in the field of synthetic biology. Emphasis will be put on the application of fluorescence-activated cell sorting and FADS in microbial cell factories and enzyme directed evolution in recent years, especially the common strategies of coupling the molecules to be tested with fluorescence signals. We also briefly introduce the current research and development of high-throughput screening equipment based on droplet microfluidic technology.

    Research and application progress of microdroplets high throughput screening methods
    Weitong QIN, Guangyu YANG
    2023, 4(5):  966-979.  doi:10.12211/2096-8280.2023-033
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    High throughput analysis and sorting of biological functions at the single-cell level is an important technology for optimizing the performance key genes, elements, pathways, and cell factories. The screening method based on microdroplet has been widely applied in various fields such as biology, medicine, food, and industry due to its advantages of low cost and ultra-high throughput. Traditional droplet sorting includes droplet generation, incubation, operation, and sorting. For the first three steps, there have been many technological advances in the last decade. The major limitation is in the sorting step, whose frequency and diversity restrict the sorting efficiency and target scope. According to the different principles of detection signals, droplet sorting technology is mainly divided into labeled and unlabeled sorting method. This article mainly reviews the progress of microdroplet screening equipment based on mainstream fluorescence-activated droplet sorting (FADS) to detect fluorescence signals, absorbance-activated droplet sorting (AADS) to detect UV/visible light absorption changes, and unlabeled droplet sorting, such as mass spectrometry, Raman spectrometry, nuclear magnetic resonance, electrochemistry, and image recognition. This article summarized the successful cases of microdroplet screening equipment applied in the fields of enzyme evolution and microbial breeding in the past 5 years. In addition, we also discussed the advantages and challenges faced by different microdroplet screening devices, and pointed out that the development of various new fluorescent probes and the further development of unlabeled detection methods such as mass spectrometry in the future will be the main development direction of microdroplet screening equipment. Although FADS remains the primary choice for cell sorting, the development of other microfluidic sorting devices has further expanded the application range of microfluidic sorting devices. Its high screening throughput and independent reaction environment provide a new technological platform for research in different fields, including protein engineering, antibody screening, sorting of different types of cells, and clinical directions.

    Advances and applications of ambient ionization mass spectrometry in screening of microbial strains
    Huan LIU, Qiu CUI
    2023, 4(5):  980-999.  doi:10.12211/2096-8280.2023-018
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    Mass spectrometry (MS) is a powerful analytical tool that provides information on the molecular weight and chemical structure of analytes. With the advantages of high specificity, sensitivity, speed, universality, minimal sample requirements, and label-free detection, MS holds great potential in the "Design-Build-Test-Learn" engineering strategy employed in synthetic biology. MS, particularly ambient ionization MS (AI-MS) technology, with the continuous development of MS instruments and their methodologies, enables the detection of intact cellular metabolic phenotypes of microbial cells. This makes it an essential tool for high-throughput screening and imaging in the "test" link. Matrix-assisted desorption/ionization MS (MALDI-MS) is a well-established platform for rapid screening of microbial strains, with a throughput of about one second per sample, by directly analyzing intact cells. AI-MS, a novel set of analytical techniques, allows for direct desorption and ionization of cellular metabolic phenotypes from intact cells under open atmospheric pressure without the need for sample preparation. Its real-time, surface, and in situ capabilities make AI-MS suitable for high-throughput analysis and imaging of microbial strains with a throughput of about ten seconds per sample. In this review, we first introduce the desorption and ionization mechanisms of MALDI-MS and AI-MS based on electrospray, plasma, and laser, and illuminate these MS methods' analytical processes and their uniqueness for different intact microbial strains. We then summarize important research progresses of MALDI-MS and AI-MS in high-throughput screening of microbial mutant libraries and in situ MS imaging of living microbial colonies. Finally, we outline the advantages and limitations of different AI-MS methods for screening microbial strains, and discuss the application of AI-MS in synthetic biology. Compared to time-consuming and labor-intensive liquid or gas chromatography-based cell phenotype detection methods, AI-MS offers low sample requirements, rapid analysis, in situ capabilities, and environmental friendliness, providing an efficient and cost-effective analytic biotechnology platform for strain engineering. MS will play an important role in the development of high-throughput screening equipment in the "test" phase of synthetic biology.

    Research progress of strain screening and quantitative analysis of key molecules based on high-throughput liquid chromatography and mass spectrometry
    Yujie WU, Xinxin LIU, Jianhui LIU, Kaiguang Yang, Zhigang SUI, Lihua ZHANG, Yukui ZHANG
    2023, 4(5):  1000-1019.  doi:10.12211/2096-8280.2023-031
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    Synthetic biology is an emerging interdisciplinary research area and has brought significant changes to fields such as medicine, chemical engineering, energy, food and agriculture. The construction and application of microbial cell factories (MCFs) is an important task of synthetic biology, which will lead the way to a sustainable industrial-scale manufacturing sector. With the progress of genome editing technology, the construction strategy of MCFs has evolved from random mutation to customized transformation at the whole genome level. Biologists can obtain 103~107 strain mutant libraries in a short time, creating an urgent need for the development of high-throughput screening methods. In addition, the demand for precise quantitative analysis of key molecules in metabolic pathways, such as metabolic enzymes and metabolites, is increasingly prominent in the process of strain transformation, evolution, and fermentation monitoring. Liquid chromatography (LC) offers excellent separation capability, allowing efficient separation of target molecules, while mass spectrometry (MS) provides strong detection ability due to its high specificity and sensitivity. Therefore, LC and MS techniques have been widely applied in the field of life sciences, especially for the qualitative and quantitative analyses of proteins and metabolites. With the development of high-throughput LC and MS technologies, these methods have become powerful tools for screening strains and quantitative research of key molecules in synthetic biology. Therefore, this work reviews the research progresses of LC and/or MS based strain screening and quantification of key molecules, focusing on the following aspects: sample preparation, chromatographic separation, mass spectrometric analysis and data processing. Finally, we briefly summarize the future prospects and challenges in this field. It is expected that with the continuous progress of LC and MS, the deep integration with automatic devices and data processing platforms, the advantages of LC and MS in high-throughput screening of synthetic biology strains and quantitative analysis of key molecules will become increasingly prominent, which will certainly promote the rapid development of biological intelligence based on synthetic biology.

    Advances and applications of single-cell Raman spectroscopy testing and sorting equipment
    Zhidian DIAO, Xixian WANG, Qing SUN, Jian XU, Bo MA
    2023, 4(5):  1020-1035.  doi:10.12211/2096-8280.2023-025
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    The advancement of synthetic biology depends on the breakthroughs in the "design", "build", "test", and "learn" (DBTL) stages. With the rapid methodological innovations in genome sequencing, editing, synthesis, and artificial intelligence, the industry has made remarkable progress in designing and building mutants and even artificial cell factories. However, the "unmanageable complexity of large systems" remains one of the ongoing challenges for synthetic biology. As the cell mutant libraries get larger, the process of testing becomes more tedious and even impossible. Hence, there is an urgent need to develop high throughput sorting platforms. SCRS (single-cell Raman spectrum)technology can identify panoramic information at the single-cell level in a non-labeled state, distinguishing complex functional phenotypes. It has advantages such as being fast, low-cost, and capable of being coupled with downstream omics research, thus making it a novel technology for single-cell phenotype identification. At present, based on the powerful ability of SCRS in phenotyping, a series of synthetic phenotypic testing and cell sorting equipment have been developed and a wide range of application demonstrations have been carried out, which demonstrates its enormous potential in accelerating the phenotypic testing and cell sorting in synthetic biology. In this review, we selected the self-developed Raman-activated cell sorting coupled sequencing system (RACS-Seq), single-cell microfluidic droplet sorting system (EasySort), and high-throughput flow cytometry Raman-activated cell sorting (FlowRACS) as the typical equipment, by introducing their technical principles, technical iterations and characteristic application cases. Despite the advances in SCRS-based synthetic phenotypic testing and cell sorting equipment, there are still challenges to overcome. For example, there is a need for improving automation and standardizing protocols to ensure reproducibility and scalability. The development of more powerful artificial intelligence algorithms for dissecting SCRS is also required to exploit more complicated phenometypes. Finally, the throughput and sensitivity still need to be improved significantly. In conclusion, in spite of some disadvantages, the SCRS-based equipment has shown great promise in accelerating the phenotypic testing and cell sorting in synthetic biology.

    Research Article
    Multifunction microplate reader for automated foundry platform
    Cui MA, Fan YANG, Juntai ZHANG, Kai HE
    2023, 4(5):  1036-1049.  doi:10.12211/2096-8280.2023-023
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    Bioanalytical technology is being developed towards the direction of high throughput, high sensitivity and multi-functionality. Microplate readers serve as fundamental instruments for high-throughput analysis. They need to meet the requirements of microanalysis, automation and integration. At present, domestic instruments have limitations in terms of functionality and automation. Although the single-function microplate reader has basically reached the level of oversea equivalents, there is still a gap in the multifunctional instruments. Most of the multi-function microplate reader used are imported instruments. They are usually expensive and some operating instructions are not fully disclosed. It is difficult to integrate them into the automated foundry platform. In this study, we will research key technologies of high-precision absorbance detection and high-sensitivity fluorescence detection, and develop a multifunction microplate reader. The control and soft system is also developed independently to access the automated foundry platform. In our system, the absorbance optical system includes light source, filters, fibers and lens array. Multiple channels of transmitted light are detected in parallel to improve the speed of detection. Combined with the one-dimensional movement of microplate, the whole 96 wells are measured. The fluorescence optical system mainly includes LED light source, filter cube (emission filter, excitation filter and dichroic mirror) and photodiode detector. Single channel of excitation light is detected and two-dimension scanning mechanism is realized for the whole microplate measurement. Besides the optical system, high precision data acquisition system is developed, including signal amplifying, conditioning and isolating circuit. This microplate reader is compact with independent absorbance and fluorescence modules, and scalable wavebands. According to experimental results, the reproducibility of absorbance measurement is high with a standard error of 0.3% and the total 96 micro-wells can be detected accurately within 10 seconds. The fluorescence detection has good linearity in the picomole concentration and the limit of detection for sodium fluorescein is about 3.9 pmol/L. For synthetic biology automated foundry platform, this microplate reader can be directly connected to the integration control system through serial port, and can also be remotely controlled by TCP communication.