A promoter-RBS library for fine-tuning gene expression in Methanosarcina acetivorans.

Methanogens are the main biological producers of methane on Earth. Methanosarcina acetivorans is one of the best characterized methanogens that has powerful genetic tools for genome editing. To study the physiology of this methanogen in further detail as well as to effectively balance the flux of their engineered metabolic pathways in expansive project undertakings, there is the need for controlled gene expression, which then requires the availability of well-characterized promoters and ribosome-binding sites (RBS). In this study, we constructed a library of 33 promoter-RBS combinations that includes 13 wild-type and 14 hybrid combinations, as well as six combination variants in which the 5'-untranslated region (5'UTR) was rationally engineered. The expression strength for each combination was calculated by inducing the expression of the ß-glucuronidase reporter gene in M. acetivorans cells in the presence of the two most used growth substrates, either methanol (MeOH) or trimethyl amine (TMA). In this study, the constructed library covers a relatively wide range (140-fold) between the weakest and strongest promoter-RBS combination as well as shows a steady increase and allows different levels of gene expression. Effects on the gene expression strength were also assessed by making measurements at three distinct growth phases for all 33 promoter-RBS combinations. Our promoter-RBS library is effective in enabling the fine-tuning of gene expression in M. acetivorans for physiological studies and the design of metabolic engineering projects that, e.g., aim for the biotechnological valorization of one-carbon compounds. IMPORTANCE: Methanogenic archaea are potent producers of the greenhouse gas methane and thus contribute substantially to global warming. Under controlled conditions, these microbes can catalyze the production of biogas, which is a renewable fuel, and might help counter global warming and its effects. Engineering the primary metabolism of Methanosarcina acetivorans to render it better and more useful requires controllable gene expression, yet only a few well-characterized promoters and RBSs are presently available. Our study rectifies this situation by providing a library of 33 different promoter-RBS combinations with a 140-fold dynamic range in expression strength. Future metabolic engineering projects can take advantage of this library by using these promoter-RBS combinations as an efficient and tunable gene expression system for M. acetivorans. Furthermore, the methodologies we developed in this study could also be utilized to construct promoter libraries for other types of methanogens.

Recent Innovations and Technical Advances in High-Throughput Parallel Single-Cell Whole-Genome Sequencing Methods.

Single-cell whole-genome sequencing (scWGS) detects cell heterogeneity at the aspect of genomic variations, which are inheritable and play an important role in life processes such as aging and cancer progression. The recent explosive development of high-throughput single-cell sequencing methods has enabled high-performance heterogeneity detection through a vast number of novel strategies. Despite the limitation on total cost, technical advances in high-throughput single-cell whole-genome sequencing methods are made for higher genome coverage, parallel throughput, and level of integration. This review highlights the technical advancements in high-throughput scWGS in the aspects of strategies design, data efficiency, parallel handling platforms, and their applications on human genome. The experimental innovations, remaining challenges, and perspectives are summarized and discussed.

Thorough molecular configuration analysis of noncanonical AAV genomes in AAV vector preparations.

The unique palindromic inverted terminal repeats (ITRs) and single-stranded nature of adeno-associated virus (AAV) DNA are major hurdles to current sequencing technologies. Due to these characteristics, sequencing noncanonical AAV genomes present in AAV vector preparations remains challenging. To address this limitation, we developed thorough molecule configuration analysis of noncanonical AAV genomes (TMCA-AAV-seq). TMCA-AAV-seq takes advantage of the documented AAV packaging mechanism in which encapsidation initiates from its 3' ITR, for AAV-seq library construction. Any AAV genome with a 3' ITR is converted to a template suitable to adapter addition by a Bst DNA polymerase-mediated extension reaction. This extension reaction helps fix ITR heterogeneity in the AAV population and allows efficient adapter addition to even noncanonical AAV genomes. The resulting library maintains the original AAV genome configurations without introducing undesired changes. Subsequently, long-read sequencing can be performed by the Pacific Biosciences (PacBio) single-molecule, real-time (SMRT) sequencing technology platform. Finally, through comprehensive data analysis, we can recover canonical, noncanonical AAV DNA, and non-AAV vector DNA sequences, along with their molecular configurations. Our method is a robust tool for profiling thorough AAV-population genomes. TMCA-AAVseq can be further extended to all parvoviruses and their derivative vectors.

Setup and Applications of Modular Protein Expression Toolboxes (MoPET) for Mammalian Systems.

The design and generation of an optimal protein expression construct is the first and essential step in the characterization of any protein of interest. However, the exchange and modification of the coding and/or noncoding elements to analyze their effect on protein function or generating the optimal result can be a tedious and time-consuming process using standard molecular biology cloning methods. To streamline the process to generate defined expression constructs or libraries of otherwise difficult to express proteins, the Modular Protein Expression Toolbox (MoPET) has been developed (Weber E, PloS One 12(5):e0176314, 2017). The system applies Golden Gate cloning as an assembly method and follows the standardized modular cloning (MoClo) principle (Weber E, PloS One 6(2):e16765, 2011). This cloning platform allows highly efficient DNA assembly of pre-defined, standardized functional DNA modules effecting protein expression with a focus on minimizing the cloning burden in coding regions. The original MoPET system consists of 53 defined DNA modules divided into eight functional main classes and can be flexibly expanded dependent on the need of the experimenter and expression host. However, already with a limited set of only 53 modules, 792,000 different constructs can be rationally designed or used to generate combinatorial expression optimization libraries. We provide here a detailed protocol for the (1) design and generation of level 0 basic parts, (2) generation of defined expressions constructs, and (3) generation of combinatorial expression libraries.

Design and Construction of a Designed Ankyrin Repeat Protein (DARPin) Display Library.

Protein display systems are powerful techniques used to identify protein molecules that bind with high affinity to target proteins of interest. The initial challenge in implementing a display system is the construction of a high-diversity naïve library. Here, we describe the methods to generate a designed ankyrin repeat protein (DARPin) display library using degenerate oligonucleotides. Specifically described is the construction of a single DARPin repeat module by overlap extension PCR, concatenation of the module by restriction enzyme digestion and ligation, and incorporation of the concatenated modules into a full-length DARPin sequence in a bacterial cloning or display vector containing the hydrophilic N- and C-terminal capping domains. Protocols for PCR amplification of DARPin sequences to estimate diversity of naïve and enriched libraries via next-generation sequencing are included, as is a simple Linux-based program for analysis of naïve and enriched sequences. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of a single DARPin repeat by overlap extension PCR Basic Protocol 2: Concatenation of DARPin repeats Basic Protocol 3: Ligation of internal repeats into cloning/display vector containing N- and C-terminal capping repeats Basic Protocol 4: Estimation of library size and diversity by next-generation sequencing (NGS) Basic Protocol 5: NGS analysis of naïve and enriched libraries.

Quality assessment of enzymatic methyl-seq library constructed using crude cell lysate.

Enzymatic methyl-seq (EM-seq), an enzyme-based method, identifies genome-wide DNA methylation, which enables us to obtain reliable methylome data from purified genomic DNA by avoiding bisulfite-induced DNA damage. However, the loss of DNA during purification hinders the methylome analysis of limited samples. The crude DNA extraction method is the quickest and minimal sample loss approach for obtaining useable DNA without requiring additional dissolution and purification. However, it remains unclear whether crude DNA can be used directly for EM-seq library construction. In this study, we aimed to assess the quality of EM-seq libraries prepared directly using crude DNA. The crude DNA-derived libraries provided appropriate fragment sizes and concentrations for sequencing similar to those of the purified DNA-derived libraries. However, the sequencing results of crude samples exhibited lower reference sequence mapping efficiencies than those of the purified samples. Additionally, the lower-input crude DNA-derived sample exhibited a marginally lower cytosine-to-thymine conversion efficiency and hypermethylated pattern around gene regulatory elements than the higher-input crude DNA- or purified DNA-derived samples. In contrast, the methylation profiles of the crude and purified samples exhibited a significant correlation. Our findings indicate that crude DNA can be used as a raw material for EM-seq library construction.

[Advances in the application of yeast surface display technology].

Yeast surface display (YSD) is a technology that fuses the exogenous target protein gene sequence with a specific vector gene sequence, followed by introduction into yeast cells. Subsequently, the target protein is expressed and localized on the yeast cell surface by using the intracellular protein transport mechanism of yeast cells, whereas the most widely used YSD system is the α-agglutinin expression system. Yeast cells possess the eukaryotic post-translational modification mechanism, which helps the target protein fold correctly. This mechanism could be used to display various eukaryotic proteins, including antibodies, receptors, enzymes, and antigenic peptides. YSD has become a powerful protein engineering tool in biotechnology and biomedicine, and has been used to improve a broad range of protein properties including affinity, specificity, enzymatic function, and stability. This review summarized recent advances in the application of YSD technology from the aspects of library construction and screening, antibody engineering, protein engineering, enzyme engineering and vaccine development.

[Application of deep mutational scanning technology in protein research].

As central players in cellular structure and function, proteins have long been central themes in life science research. Analyzing the impact of protein sequence variation on its structure and function is one of the important means to study proteins. In recent years, a technology called deep mutational scanning (DMS) has been widely used in the field of protein research. It introduces thousands of mutations in parallel in specific regions of proteins through high-abundance DNA libraries. After screening, high-throughput sequencing is employed to score each mutation, revealing sequence-function correlations. Due to its high-throughput, fast and easy, and labor-saving features, DMS has become an important method for protein function research and protein engineering. This review briefly summarizes the principle of DMS technology, highlighting its applications in mammalian cells. Moreover, this review analyzes the current technical bottlenecks, aiming to facilitate relevant research.

Isolation and Characterization of Single-Domain Antibodies from Immune Phage Display Libraries.

Naturally occurring heavy chain antibodies (HCAbs) in Camelidae species were a surprise discovery in 1993 by Hamers et al. Since that time, antibody fragments derived from HCAbs have garnered considerable attention by researchers and biotechnology companies. Due to their biophysico-chemical advantages over conventional antibody fragments, camelid single-domain antibodies (sdAbs, VHHs, nanobodies) are being increasingly utilized as viable immunotherapeutic modalities. Currently there are multiple VHH-based therapeutic agents in different phases of clinical trials in various formats such as bi- and multivalent, bi- and multi-specific, CAR-T, and antibody-drug conjugates. The first approved VHH, a bivalent humanized VHH (caplacizumab), was approved for treating rare blood clotting disorders in 2018 by the EMA and the FDA in 2019. This was followed by the approval of an anti-BCMA VHH-based CAR-T cell product in 2022 (ciltacabtagene autoleucel; CARVYKTI™) and more recently a trivalent antitumor necrosis factor alpha-based VHH drug (ozoralizumab; Nanozora®) in Japan for the treatment of rheumatoid arthritis. In this chapter we provide protocols describing the latest developments in isolating antigen-specific VHHs including llama immunization, construction of phage-displayed libraries, phage panning and screening of the soluble VHHs by ELISA, affinity measurements by surface plasmon resonance, functional cell binding by flow cytometry, and additional validation by immunoprecipitation. We present and discuss comprehensive, step-by-step methods for isolating and characterization of antigen-specific VHHs. This includes protocols for expression, biotinylation, purification, and characterization of the isolated VHHs. To demonstrate the feasibility of the entire strategy, we present examples of VHHs previously isolated and characterized in our laboratory.

Construction of an Ultra-Large Phage Display Library by Kunkel Mutagenesis and Rolling Circle Amplification.

An important contributor to the successful generation of recombinant affinity reagents via phage display is a large and diverse library. We describe, herein, the application of Kunkel mutagenesis and rolling circle amplification (RCA) to the construction of a 1.1 × 1011 member library, with only 26 electroporations, and isolation of low- to sub-nanomolar monobodies to a number of protein targets, including human COP9 signalosome subunit 5 (COPS5), HIV-1 Rev. binding protein-like protein (HRBL), X-ray repair cross-complementing 5/6 (Ku70/80) heterodimer, the receptor-binding domain (RBD) of SARS-CoV-2, and transforming growth factor beta 1 (TGF-ß1).

Cell-Free RNA Sequencing from Biofluid Samples.

Liquid biopsy has obvious advantages over invasive sampling methods. Cell-free DNA is now widely used in prenatal and cancer diagnosis. Cell-free RNA is relatively unstable but could contain potential biomarkers, and some studies have demonstrated that cfRNA sequencing may be developed to be a promising diagnostic assay for disease detection. In this chapter, we introduce a modified protocol for the detection of cfRNA based on next-generation sequencing (NGS). This protocol has been specifically optimized for low-input, fragmented cfRNA samples.

Sample Collection, DNA Extraction, and Library Construction Protocols of the Human Microbiome Studies in the International Human Phenome Project.

The human microbiome plays a crucial role in human health. In the past decade, advances in high-throughput sequencing technologies and analytical software have significantly improved our knowledge of the human microbiome. However, most studies concerning the human microbiome did not provide repeatable protocols to guide the sample collection, handling, and processing procedures, which impedes obtaining valid and timely microbial taxonomic and functional results. This protocol provides detailed operation methods of human microbial sample collection, DNA extraction, and library construction for both the amplicon sequencing-based measurements of the microbial samples from the human nasal cavity, oral cavity, and skin, as well as the shotgun metagenomic sequencing-based measurements of the human stool samples among adult participants. This study intends to develop practical procedure standards to improve the reproducibility of microbiota profiling of human samples. Supplementary Information: The online version contains supplementary material available at 10.1007/s43657-023-00097-y.

Construction of high coverage whole-genome sequencing libraries from single colon crypts without DNA extraction or whole-genome amplification.

OBJECTIVE: Comprehensive and reliable genome-wide variant analysis of a small number of cells has been challenging due to genome coverage bias, PCR over-cycling, and the requirement of expensive technologies. To comprehensively identify genome alterations in single colon crypts that reflect genome heterogeneity of stem cells, we developed a method to construct whole-genome sequencing libraries from single colon crypts without DNA extraction, whole-genome amplification, or increased PCR enrichment cycles. RESULTS: We present post-alignment statistics of 81 single-crypts (each contains four- to eight-fold less DNA than the requirement of conventional methods) and 16 bulk-tissue libraries to demonstrate the consistent success in obtaining reliable coverage, both in depth (≥ 30X) and breadth (≥ 92% of the genome covered at ≥ 10X depth), of the human genome. These single-crypt libraries are of comparable quality as libraries generated with the conventional method using high quality and quantities of purified DNA. Conceivably, our method can be applied to small biopsy samples from many tissues and can be combined with single cell targeted sequencing to comprehensively profile cancer genomes and their evolution. The broad potential application of this method offers expanded possibilities in cost-effectively examining genome heterogeneity in small numbers of cells at high resolution.

Advances of bioorthogonal coupling reactions in drug development.

Currently, bioorthogonal coupling reactions have garnered considerable interest due to their high substrate selectivity and less restrictive reaction conditions. During recent decades, bioorthogonal coupling reactions have emerged as powerful tools in drug development. This review describes the current applications of bioorthogonal coupling reactions in compound library building mediated by the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction and in situ click chemistry or conjunction with other techniques; druggability optimization with 1,2,3-triazole groups; and intracellular self-assembly platforms with ring tension reactions, which are presented from the viewpoint of drug development. There is a reasonable prospect that bioorthogonal coupling reactions will accelerate the screening of lead compounds, the designing strategies of small molecules and expand the variety of designed compounds, which will be a new trend in drug development in the future.

Phage Display-Derived Peptides and Antibodies for Bacterial Infectious Diseases Therapy and Diagnosis.

The emergence of antibiotic-resistant-bacteria is a serious public health threat, which prompts us to speed up the discovery of novel antibacterial agents. Phage display technology has great potential to screen peptides or antibodies with high binding capacities for a wide range of targets. This property is significant in the rapid search for new antibacterial agents for the control of bacterial resistance. In this paper, we not only summarized the recent progress of phage display for the discovery of novel therapeutic agents, identification of action sites of bacterial target proteins, and rapid detection of different pathogens, but also discussed several problems of this technology that must be solved. Breakthrough in these problems may further promote the development and application of phage display technology in the biomedical field in the future.

Small RNA Profiling by Next-Generation Sequencing Using High-Definition Adapters.

Next-generation sequencing (NGS) of small RNA (sRNA) cDNA libraries permits the identification and characterization of sRNA species de novo. However, the method through which these libraries are constructed can often introduce artifacts such as over- or underrepresentation of specific sequences or adapter oligonucleotides due to sequence biases held by the enzymes used. In this chapter we describe a protocol for sRNA library construction making use of high-definition (HD) adapters for the Illumina sequencing platform, which reduce ligation bias. This protocol leads to drastically reduced direct 5'/3' adapter ligation products and can be used for the synthesis of sRNA libraries from total RNA or sRNA of various plant, animal, and fungal samples. This protocol also includes a method for total RNA extraction from plant leaf and cultured cells or body fluids.

Recent progress in adaptive laboratory evolution of industrial microorganisms.

Adaptive laboratory evolution (ALE) is a technique for the selection of strains with better phenotypes by long-term culture under a specific selection pressure or growth environment. Because ALE does not require detailed knowledge of a variety of complex and interactive metabolic networks, and only needs to simulate natural environmental conditions in the laboratory to design a selection pressure, it has the advantages of broad adaptability, strong practicability, and more convenient transformation of strains. In addition, ALE provides a powerful method for studying the evolutionary forces that change the phenotype, performance, and stability of strains, resulting in more productive industrial strains with beneficial mutations. In recent years, ALE has been widely used in the activation of specific microbial metabolic pathways and phenotypic optimization, the efficient utilization of specific substrates, the optimization of tolerance to toxic substance, and the biosynthesis of target products, which is more conducive to the production of industrial strains with excellent phenotypic characteristics. In this paper, typical examples of ALE applications in the development of industrial strains and the research progress of this technology are reviewed, followed by a discussion of its development prospects.

Simultaneous affinity maturation and developability enhancement using natural liability-free CDRs.

ABBREVIATIONS: CDR: complementarity determining region; FACS: fluorescence-activated cell sorting; ka: association rate; kd: dissociation rate; KD: dissociation constant; scFv: single-chain variable fragment; SPR: surface plasmon resonance.

Antha-Guided Automation of Darwin Assembly for the Construction of Bespoke Gene Libraries.

Protein engineering through directed evolutison is facilitated by the screening and characterization of protein libraries. Efficient and effective methods for multiple site-saturation mutagenesis, such as Darwin Assembly, can accelerate the sampling of relevant sequence space and the identification of variants with desired functionalities. Here, we present the automation of the Darwin Assembly method, using a Gilson PIPETMAX™ liquid handling platform under the control of the Antha software platform, which resulted in the accelerated construction of complex, multiplexed gene libraries error-free and with minimal hands-on time, while maintaining flexibility over experimental parameters through a graphical user interface rather than requiring user-driven library-dependent programming of the liquid handling platform. We also present an approach for barcoding libraries that overcomes amplicon length limitations in next generation sequencing and enables fast reconstruction of library reads.

Construction of a Large Size Human Immunoglobulin Heavy Chain Variable (VH) Domain Library, Isolation and Characterization of Novel Human Antibody VH Domains Targeting PD-L1 and CD22.

Phage display is a well-established technology for in vitro selection of monoclonal antibodies (mAb), and more than 12 antibodies isolated from phage displayed libraries of different formats have been approved for therapy. We have constructed a large size (10^11) human antibody VH domain library based on thermo-stable, aggregation-resistant scaffolds. This diversity was obtained by grafting naturally occurring CDR2s and CDR3s from healthy donors with optimized primers into the VH library. This phage-displayed library was used for bio-panning against various antigens. So far, panels of binders have been isolated against different viral and tumor targets, including the SARS-CoV-2 RBD, HIV-1 ENV protein, mesothelin and FLT3. In the present study, we discuss domain library construction, characterize novel VH binders against human CD22 and PD-L1, and define our design process for antibody domain drug conjugation (DDC) as tumoricidal reagents. Our study provides examples for the potential applications of antibody domains derived from library screens in therapeutics and provides key information for large size human antibody domain library construction.