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Site-saturation mutagenesis is a powerful tool for protein optimization due to its efficiency and simplicity. A degenerate codon NNN or NNS (K) is often used to encode the 20 standard amino acids, but this will produce redundant codons and cause uneven distribution of amino acids in the constructed library. Here we present a novel “small-intelligent” strategy to construct mutagenesis libraries that have a minimal gene library size without inherent amino acid biases, stop codons, or rare codons of Escherichia coli by coupling well-designed combinatorial degenerate primers with suitable PCR-based mutagenesis methods. The designed primer mixture contains exactly one codon per amino acid and thus allows the construction of small-intelligent mutagenesis libraries with one gene per protein. In addition, the software tool DC-Analyzer was developed to assist in primer design according to the user-defined randomization scheme for library construction. This small-intelligent strategy was successfully applied to the randomization of halohydrin dehalogenases with one or two randomized sites. With the help of DC-Analyzer, the strategy was proven to be as simple as NNS randomization and could serve as a general tool to efficiently randomize target genes at positions of interest.

Libraries are occupying spaces that are no longer dedicated solely as book repositories with stacks, reference services, and story times. Starting with an examination of how libraries are coping with digital and virtual competitors, this article presents two explorations of expansions of the Linebaugh Public Library in Rutherford Country, Tennessee (Murfreesboro) to nontraditional sites. One library is sited in a recreational center with computers in the main room and books in the backroom; the other is a new building located on the grounds of an elementary school that contains no books at all, only e-books with computers throughout. These moves provide a model for initiating such a venture in other public library systems. The role of the librarian in these new spaces is included. The primary investigator will use the history of the library, the minutes of board meetings, interviews with library personnel, study of the spaces, and review of the literature to document the process that led to the changes. Patron observations and interviews will be used to document the use of the facility that is already in place (in the recreational center). [ABSTRACT FROM AUTHOR]

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, V H Hs, nanobodies) are being increasingly utilized as viable immunotherapeutic modalities. Currently there are multiple V H H-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 V H H, a bivalent humanized V H H (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 V H H-based CAR-T cell product in 2022 (ciltacabtagene autoleucel; CARVYKTI™) and more recently a trivalent antitumor necrosis factor alpha-based V H H 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 V H Hs including llama immunization, construction of phage-displayed libraries, phage panning and screening of the soluble V H Hs 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 V H Hs. This includes protocols for expression, biotinylation, purification, and characterization of the isolated V H Hs. To demonstrate the feasibility of the entire strategy, we present examples of V H Hs previously isolated and characterized in our laboratory., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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 × 10 11 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-β1)., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Copyright of Journal of Southern Agriculture is the property of Journal of Southern Agriculture and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Huanglongbing (HLB) is a devastating citrus disease worldwide, without known cure. Since this disease shows visible symptoms on newly developed canopies, remote sensing methods based on spectral principles are considered as an effective tool. To explore a fast way to monitor HLB in large citrus groves, a satellite multispectral (MS) image with a 2-m resolution acquired by WorldView-2 was studied to evaluate its capability on HLB detection. Ground truth was conducted and two spectral libraries were constructed. Library 1 was based on RTK GPS locations, and Library 2 was combined with prior knowledge from the ground spectral features. To compare with the classification accuracy of previous work using airborne spectral images, the same classification methods were carried out. In the satellite MS image, Library 2 had higher average overall accuracy, higher Kappa coefficient, and higher producer's and user's accuracies than Library 1, indicating Library 2 which combined with prior knowledge was more effective. Mahalanobis distance had the highest overall accuracy of 81% and a Kappa coefficient of 0.464 with Library 2. The accuracy comparison between the satellite MS image and the airborne spectral images showed that the satellite MS image had the highest average producer's accuracy with Library 2, followed by the airborne MS image and the satellite MS image with Library 1, indicating that construction of an effective library was the most important step, and that the satellite MS image with a proper spatial resolution showed good potential to be a better choice than the airborne spectral image. [ABSTRACT FROM AUTHOR]

The need for dynamic, elastomeric polymeric biomaterials remains high, with few options with tunable control of mechanical properties, and environmental responses. Yet the diversity of these types of protein polymers pursued for biomaterials-related needs remains limited. Robust high-throughput synthesis and characterization methods will address the need to expand options for protein-polymers for a range of applications. Here, a combinatorial library approach with high throughput screening is used to select specific examples of dynamic protein silk-elastin-like polypeptides (SELPs) with unique stimuli responsive features, including tensile strength and adhesion. Using this approach, 64 different SELPs with different sequences and molecular weights are selected out of over 2000 recombinant E. coli colonies. New understanding of sequence-function relationships with this family of proteins is gained through this combinatorial-screening approach and can provide a guide to future library designs. Further, this approach yields new families of SELPs to match specific material functions. [ABSTRACT FROM AUTHOR]

In this study, we developed an efficient and practical procedure for the synthesis of tetra-substituted Δ5-2-oxopiperazine that mimics the bioactive β-turn structural motif of proteins. This synthetic route is robust and modular enough to accommodate four different substituents to obtain a high level of molecular diversity without any deterioration in stereochemical enrichment of the natural and unnatural amino acids. Through the in silico studies, including a distance calculation of side chains and a conformational overlapping of our model compound with a native β-turn structure, we successfully demonstrated the conformational similarity of tetra-substituted Δ5-2-oxopiperazine to the β-turn motif. For the library construction in a high-throughput manner, the fluorous tag technology was adopted with the use of a solution-phase parallel synthesis platform. A 140-membered pilot library of tetra-substituted Δ5-2-oxopiperazines was achieved with an average purity of 90% without further purification. [ABSTRACT FROM AUTHOR]

Three new lipolytic genes were isolated from a forest soil metagenomic library by functional screening on tributyrin agar plates. The genes SBLip1, SBLip2 and SBLip5.1 respectively encode polypeptides of 445, 346 and 316 amino acids. Phylogenetic analyses revealed that SBLip2 and SBLip5.1 belong to bacterial esterase/lipase family IV, whereas SBLip1 shows similarity to class C β-lactamases and is thus related to esterase family VIII. The corresponding genes were overexpressed and their products purified by affinity chromatography for characterization. Analyses of substrate specificity with different p-nitrophenyl esters showed that all three enzymes have a preference for short-acyl-chain p-nitrophenyl esters, a feature of carboxylesterases as opposed to lipases. The β-lactamase activity of SBLip1, measured with the chromogenic substrate nitrocefin, was very low. The three esterases have the same optimal pH (pH 10) and remain active across a relatively broad pH range, displaying more than 60 % activity between pH 6 and 10. The temperature optima determined were 35 °C for SBLip1, 45 °C for SBLip2 and 50 °C for SBLip5.1. The three esterases displayed different levels of tolerance to salts, solvents and detergents, SBLip2 being overall more tolerant to high concentrations of solvent and SBLip5.1 less affected by detergents. [ABSTRACT FROM AUTHOR]

Abstract: Site-directed mutagenesis is routinely performed in protein engineering experiments. One method, termed Kunkel mutagenesis, is frequently used for constructing libraries of peptide or protein variants in M13 bacteriophage, followed by affinity selection of phage particles. To make this method more efficient, the following two modifications were introduced: culture was incubated at 25°C for phage replication, which yielded two- to sevenfold more single-stranded DNA template compared to growth at 37°C, and restriction endonuclease recognition sites were used to remove non-recombinants. With both of the improvements, we could construct primary libraries of high complexity and that were 99–100% recombinant. Finally, with a third modification to the standard protocol of Kunkel mutagenesis, two secondary (mutagenic) libraries of a fibronectin type III (FN3) monobody were constructed with DNA segments that were amplified by error-prone and asymmetric PCR. Two advantages of this modification are that it bypasses the lengthy steps of restriction enzyme digestion and ligation, and that the pool of phage clones, recovered after affinity selection, can be used directly to generate a secondary library. Screening one of the two mutagenic libraries yielded variants that bound two- to fourfold tighter to human Pak1 kinase than the starting clone. The protocols described in this study should accelerate the discovery of phage-displayed recombinant affinity reagents. [Copyright &y& Elsevier]

In the last two decades, several phage display-selected monoclonal antibodies (mAbs) have been described in the literature and a few of them have managed to reach the clinics. Among these, the anti-respiratory syncytial virus (RSV) Palivizumab, a phage-display optimized mAb, is the only marketed mAb directed against microbial pathogens. Palivizumab is a clear example of the importance of choosing the most appropriate strategy when selecting or optimizing an anti-infectious mAb. From this perspective, the extreme versatility of phage-display technology makes it a useful tool when setting up different strategies for the selection of mAbs directed against human pathogens, especially when their possible clinical use is considered. In this paper, we review the principal phage display strategies used to select anti-infectious mAbs, with particular attention focused on those used against hypervariable pathogens, such as HCV and influenza viruses [ABSTRACT FROM AUTHOR]

Techniques for microdissection and microcloning were established using chromosome 1 of triploid Lilium tigrinum. Chromosome 1 was dissected from a membrane slide using a microbeam system. Digestion with proteinase K was done before PCR amplification for more than 24 hours. The dissected chromosomes were then amplified by degenerate oligonucleotide primed PCR (DOP-PCR) and linker adaptor-mediated PCR (LA-PCR). Successful PCR amplification relied on critical concentrations of both MgCl and Taq polymerase. The optimum concentration of MgCl and Taq polymerase was 2.5 mM and 0.5 U, respectively. Amplification of the dissected chromosome using DOP-PCR had more sensitivity dependent upon PCR factors, but LA-PCR was more dependent on the linker ligation. Amplified DNA products ranged from 100 to 2500 bp both for DOP- and LA-PCR. Evaluated clones only ranged from 100 to 1700 bp for DOP-PCR and 100 to 900 bp for LA-PCR. Based on the sequence results, most of the sequences from the DOP-PCR and LA-PCR showed no significant similarity with known data in NCBI database. However, about 2% of the sequence data was partially matched with plant microsatellites with low similarity. The results derived from the microdissection of a large genome organism such as Lilium showed informative and useful for the development of microsatellite repeat markers. Sequence data from the chromosome specific DNA library was considered for the development of microsatellite markers. [ABSTRACT FROM AUTHOR]

The objective of this approach was to identify new CHO endogenous gene regulatory elements that are capable of regulating foreign gene expression in recombinant CHO host cells. The standard technology for the production of many biopharmaceutical products is frequently based on expression vectors that utilize strong mammalian viral promoters like SV40 or CMV which allow for very high expression rates but this may lead to constitutive over-expression resulting in a permanent stress for the cell. In addition, some heterologous promoters are cell-cycle dependent and can be subject to gene silencing generating heterogeneity within the cell population. Here, we describe the construction of a genomic CHO library and the subsequent identification and isolation of selected target sequences that are believed to be responsible for high level expression of the associated genes. The method that was used to isolate these regions of interest relies on gene specific amplification with primer pairs binding on different genes and the vector sequence. Flanking regions of these fragments were identified through Inverse PCR from fragmented and self-ligated genomic DNA. Expression levels of both the initially derived and the mapped fragments were determined through a luciferase reporter assay. [ABSTRACT FROM AUTHOR]

The University of Wyoming Libraries are currently experiencing a major expansion and renovation of the William Robertson Coe Library, the flagship library. From the standpoint of security, the challenges of creating a new addition adjacent to the existing structure are minor in comparison to the difficulties of renovating the existing building in several phases while providing a safe working environment for the library employees, providing patrons with access to the collections and technological infrastructure of a research library, and enabling construction personnel to work safely in various parts of the building at all hours. Communication is key to coordinating security activities and ensuring a safe workplace. [ABSTRACT FROM AUTHOR]

A bacterial artificial chromosome (BAC) library of the genomic DNA ofCoprinus cinereusstrain MP#2 was constructed using the BAC vector pBACTZ, which carries theC. cinereus trp1gene. The library consists of 1536 clones. Analysis of inserts in some of the clones suggested that the library covers five times theC. cinereusgenome. Screening of the BAC clones using ten markers mapped on nine different chromosomes also indicated that the library is likely to cover the whole length of the genomic DNA. We show an example of transformation ofC. cinereuswith BACs containing inserts of longer than 170?kb. [ABSTRACT FROM AUTHOR]

Cardiac fibrosis (CF) and heart failure (HF) are common heart diseases, and severe CF can lead to HF. In this study, we tried to find their common potential molecular markers, which may help the diagnosis and treatment of CF and HF. RNA library construction and high-throughput sequencing were performed. The DESeq2 package in R was used to screen differentially expressed mRNAs (DEmRNAs), differentially expressed lncRNA (DElncRNAs) and differentially expressed miRNA (DEmiRNAs) between different samples. The common DEmRNAs, DElncRNAs and DEmiRNAs for the two diseases were obtained. The ConsensusPathDB (CPDB) was used to perform biological function enrichment for common DEmRNAs. Gene interaction network was constructed to screen out key genes. Subsequently, real-time polymerase chain reaction (RT-PCR) verification was performed. Lastly, GSE104150 and GSE21125 data sets were utilized for expression validation and diagnostic analysis. There were 1477 DEmRNAs, 502 DElncRNAs and 36 DEmiRNAs between CF and healthy control group. There were 607 DEmRNAs, 379DElncRNAs,s and 42 DEmiRNAs between HF and healthy control group. CH and FH shared 146 DEmRNAs, 80 DElncRNAs, and 6 DEmiRNAs. Hsa-miR-144-3p, CCNE2, C9orf72, MAP3K20-AS1, LEF1-AS1, AC243772.2, FLJ46284, and AC239798.2 were key molecules in lncRNA-miRNA-mRNA network. In addition, hsa-miR-144-3p and CCNE2 may be considered as potential diagnostic gene biomarkers in HF. In this study, the identification of common biomarkers of CF and HF may help prevent CF to HF transition as early as possible.

Over the last two decades, yeast display methodology has served as a popular tool for discovery, humanization, stability improvement, and affinity maturation of antibodies and antibody fragments, but also for development of diverse non-antibody protein scaffolds towards the ability of antigen recognition. Yeast display is particularly well suited for multiparametric analysis of properties of derivatized proteins, allowing the evolution of most diverse protein structures into antigen binding entities with favorable expression, stability, and folding properties. Here we present the methodological basics of a novel yeast display-based approach for the functionalization of the large extracellular loop of CD81 into a de novo antigen binding unit. CD81 is intrinsically overrepresented on the surface of extracellular vesicles (EVs), naturally occurring nanoparticle units that act as cell-to-cell messengers by delivering their intracellular cargo from the source cell into a recipient cell. This amazing feature makes them of highest biotechnological interest, yet methods for their targeted delivery are still in their infancy. As a novel approach for introducing EV surface modifications enabling specific target cell recognition and internalization, we have prepared yeast display libraries of CD81 large extracellular loop mutants, which are selected towards specific antigen binding and resulting mutants conveniently clicked into the full-length EV surface protein. Resulting EVs display wild-type-like characteristics regarding the expression level and distribution of recombinant proteins and are hence promising therapeutic tools., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Abbreviations: CDR: complementarity determining region; FACS: fluorescence-activated cell sorting; k a : association rate; k d : dissociation rate; K D : dissociation constant; scFv: single-chain variable fragment; SPR: surface plasmon resonance.

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., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Microbes employ sophisticated cellular networks encoded by complex genomes to rapidly adapt to changing environments. High-throughput genome engineering methods are valuable tools for functionally profiling genotype-phenotype relationships and understanding the complexity of cellular networks. However, current methods either rely on special homologous recombination systems and are thus applicable in only limited bacterial species or can generate only nonspecific mutations and thus require extensive subsequent screening. Here, we report a site-specific transposon-assisted genome engineering (STAGE) method that allows high-throughput Cas12k-guided mutagenesis in various microorganisms, such as Pseudomonas aeruginosa and Klebsiella pneumoniae. Exploiting the powerful STAGE technique, we construct a site-specific transposon mutant library that focuses on all possible transcription factors (TFs) in P. aeruginosa , enabling the comprehensive identification of essential genes and antibiotic-resistance-related factors. Given its broad host range activity and easy programmability, this method can be widely adapted to diverse microbial species for rapid genome engineering and strain evolution. [Display omitted] • Cas12k-guided transposase mediates site-specific transposition in microorganisms • Development of a genome engineering method STAGE using Cas12k-guided transposase • Generation of a STAGE-based transcription factor mutant library • Identification of essential genes and antibiotic-resistance-related factors Chen et al. develop a site-specific transposon-assisted genome engineering (STAGE) method using a Cas12k-guided transposase. This method enables rapid and efficient gene disruption of an entire genome or a subset of genes of interest, providing a promising tool for rapid high-throughput genome engineering in diverse microbial species. [ABSTRACT FROM AUTHOR]

With the advent of combinatorial chemistry a new paradigm is evolving in the field of drug discovery. The approach is based on an integration of chemistry, high-throughput screening and automation engineering. The chemistry arm is usually based on solid-phase synthesis technology as the preferred approach to library construction. One of the most powerful of the solid-phase methods is encoded split synthesis, in which the reaction history experience by each polymeric bead is unambiguously recorded. This split-and-pool approach, employing chemically robust tags, was used to construct a 85 000-membered dihydrobenzopyran library. [ABSTRACT FROM AUTHOR]

Standardisation of genetic parts has become a topic of increasing interest over the last decades. The promise of simplifying molecular cloning procedures, while at the same time making them more predictable and reproducible has led to the design of several biological standards, one of which is modular cloning (MoClo). The Yeast MoClo toolkit provides a large library of characterised genetic parts combined with a comprehensive and flexible assembly strategy. Here we aimed to (1) simplify the adoption of the standard by providing a simple design tool for including new parts in the MoClo library, (2) characterise the toolkit further by demonstrating the impact of a BglII site in promoter parts on protein expression, and (3) expand the toolkit to enable efficient construction of gRNA arrays, marker-less integration cassettes and combinatorial libraries. These additions make the toolkit more applicable for common engineering tasks and will further promote its adoption in the yeast biological engineering community.

A new and expedient assembly of an interesting class of amido-substituted furo[4,5-c]coumarins has been successfully achieved through a one-pot sequential coupling and cyclization strategy starting from 4-hydroxycoumarin, aryl glyoxals and benzamide. All the reactions showed selectivity towards furo[4,5-c]coumarin, instead of the corresponding isoxazolo-substituted coumarins. [ABSTRACT FROM AUTHOR]

Discovering therapeutic agents: New bioactive agents, either as sole or combinational agents, have been constructed through the synthetic manipulation of the intermediates within the total synthesis of the uvaretin class of natural products. It was found that increasing the hydrophobic character of the phenolic core correlates to a decrease in sole agent cytotoxicity. The synthesis of new, small chemical screening libraries (CSL) constructed from the intermediates of our total synthesis route of the uvaretin class of natural products is demonstrated herein. Numerous chalcone-based CSLs with various substitution on the phenolic groups within the chalcone core were assembled. Through cytotoxicity investigations, it was found that the level of hydrophobicity of the phenolic core of the chalcones gives biases: less cytotoxicity with more hydrophobic cores. In addition, it was observed that the potentiation, evaluated with 6-thiopurine in the pancreatic cancer cell line MIA PaCa-2, is tunable by the inclusion of less-hydrophobic character on the phenolic core. The role of the o-hydroxybenzyl group, present within the uvaretin family, was revealed to be cytotoxic in character. Merging all of the structure-activity relationship studies performed on the CSLs constructed in this effort led to the construction of a new chalcone hybrid possessing both a cytotoxic enone group and a small-molecule-potentiating, reduced enone group., (© 2021 Wiley-VCH GmbH.)

Background: Cholesterol gallstone (CG) is the most common gallstone disease, which is induced by biliary cholesterol supersaturation. The purpose of this study is to investigate the pathogenesis of CG., Methods: Sixteen mice were equally and randomly divided into model group and normal control group. The model group was fed with lithogenic diets to induce CG, and then gallbladder bile lipid analysis was performed. After RNA-seq library was constructed, differentially expressed mRNAs (DE-mRNAs) and differentially expressed lncRNAs (DE-lncRNAs) between model group and normal control group were analyzed by DESeq2 package. Using the cluster Profiler package, enrichment analysis for the DE-mRNAs was carried out. Based on Cytoscape software, the protein-protein interaction (PPI) network and competing endogenous RNA (ceRNA) network were built. Using quantitative real-time reverse transcription-PCR (qRT-PCR) analysis, the key RNAs were validated., Results: The mouse model of CG was suc cessfully established, and then 181 DE-mRNAs and 33 DE-lncRNAs between model and normal groups were obtained. Moreover, KDM4A was selected as a hub node in the PPI network, and lncRNA MEG3 was considered as a key lncRNA in the regulatory network. Additionally, the miR-107-5p/miR-149-3p/miR-346-3-MEG3 regulatory pairs and MEG3-PABPC4/CEP131/NUMB1 co-expression pairs existed in the regulatory network. The qRT-PCR analysis showed that KDM4A expression was increased, and the expressions of MEG3 , PABPC4 , CEP131 , and NUMB1 were downregulated., Conclusion: These RNAs might be related to the pathogenesis of CG., Competing Interests: The authors declare there are no competing interests., (©2021 Qian et al.)

Violacein is a naturally occurring anticancer therapeutic compound with deep purple color. In this work, we harnessed the modular and combinatorial feature of a Golden Gate assembly method to construct a library of violacein producing strains in the oleaginous yeast Yarrowia lipolytica , where each gene in the violacein pathway was controlled by three different promoters with varying transcriptional strength. After optimizing the linker sequence and the Golden Gate reaction, we achieved high transformation efficiency and obtained a panel of representative Y. lipolytica recombinant strains. By evaluating the gene expression profile of 21 yeast strains, we obtained three colorful compounds in the violacein pathway: green (proviolacein), purple (violacein), and pink (deoxyviolacein). Our results indicated that strong expression of VioB , VioC , and VioD favors violacein production with minimal byproduct deoxyvioalcein in Y. lipolytica , and high deoxyviolacein production was found strongly associated with the weak expression of VioD . By further optimizing the carbon to nitrogen ratio and cultivation pH, the maximum violacein reached 70.04 mg/L with 5.28 mg/L of deoxyviolacein in shake flasks. Taken together, the development of Golden Gate cloning protocols to build combinatorial pathway libraries, and the optimization of culture conditions set a new stage for accessing the violacein pathway intermediates and engineering violacein production in Y. lipolytica . This work further expands the toolbox to engineering Y. lipolytica as an industrially relevant host for plant or marine natural product biosynthesis.

Due to crucial roles in gene regulation, noncoding small RNAs (sRNAs) of 20-30 nucleotides (nt) have been intensively studied in mammals and plants and are implicated in significant diseases and metabolic disorders. Elucidation of biogenesis mechanisms and functional characterization of sRNAs is often achieved using tools such as separation of small-sized RNA and deep sequencing. Although RNA interference pathways, such as quelling and meiotic silencing, have been well-described in Neurospora crassa, knowledge of sRNAs in other filamentous fungi is still limited compared to other eukaryotes. As a prerequisite for study, isolation and sequence analysis of sRNAs is necessary. We developed a protocol for isolation and library construction of sRNAs of 20-30 nt for deep sequencing in two filamentous fungi, N. crassa and Fusarium oxysporum f.sp. lycopersici. Using 200-300 μg total RNA, sRNA was isolated by size-fractionation and ligated with adapters and amplified by RT-PCR for deep sequencing. Sequence analysis of several cDNA clones showed that the cloned sRNAs were not tRNAs and rRNAs and were fungal genome-specific. In order to validate fungal miRNAs that were imported into the host cell, we developed a straightforward method to isolate protoplasts from tomato roots infected by Fusarium oxysporum f.sp. lycopersici using enzymatic digestion.

RNA silencing plays a critical role in diverse biological processes in plants including growth, development, and responses to abiotic and biotic stresses. RNA silencing is guided by small non-coding RNAs (sRNAs) with the length of 21-24 nucleotides (nt) that are loaded into Argonaute (AGO) to repress expression of target loci and transcripts through transcriptional or posttranscriptional gene silencing mechanisms. Identification and quantitative characterization of sRNAs are crucial steps toward appreciation of their functions in biology. Here, we developed a step-by-step protocol to precisely illustrate the process of cloning of sRNA libraries and correspondingly computational analysis of the recovered sRNAs. This protocol can be used in all kinds of organisms, including Arabidopsis, and is compatible with various high-throughput sequence technologies such as Illumina Hiseq. Thus, we wish that this protocol represents an accurate way to identify and quantify sRNAs in vivo.

Phage display is a commonly used technology for the screening of large clonal libraries of proteins and peptides. The construction of peptide libraries containing very short sequences, however, poses certain problems for conventional restriction-based cloning procedures, which are rooted in the necessity to purify restricted library oligos. Herein, we present an alternative cloning method especially suitable for such very short sequences of about only 21 base pairs resulting in a 60 bp insert. The employed restriction-free hot fusion cloning strategy allows for facile library construction bypassing the need for purification of the small oligo. The library includes one well-defined disulfide bridge rendering the displayed macrocyclic peptide sequences as attractive scaffolds for novel active principles.

The status of T cell receptors (TCRs) repertoire is associated with the occurrence and progress of various diseases and can be used in monitoring the immune responses, predicting the prognosis of disease and other medical fields. High-throughput sequencing promotes the studying in TCR repertoire. The chapter focuses on the whole process of TCR profiling, including DNA extraction, library construction, high-throughput sequencing, and how to analyze data.

Since two decades, yeast display methodology is a popular tool for discovery, stability improvement, and affinity maturation of diverse protein scaffolds, intended for antigen recognition. Yeast display is particularly well suited for the selection of heterodimeric proteins, such as antibodies and T-cell receptors (TCRs), as it allows rapid library creation via gap-repair-driven homologous recombination and subsequent construction of a combinatorial library after mating of yeast of opposite mating types. Certain properties of the TCR scaffold, such as its stability, inferior to most antibody fragments, require modifications of traditional antigen selection strategies. Their selection can be monitored and guided upon staining with the soluble versions of their original antigen, peptide-major histocompatibility complex (MHC), or clonotypic antibodies, whose binding is critically dependent on the TCR structural integrity. Overall, this chapter underlines the importance of the versatile yeast display technique for the diversification of the TCR scaffold for antigen recognition and optimization of its stability.

At present, approaches to hybrid capture sequencing have many limitations, from their significant complexity and labor requirements, to their low enrichment efficiency, resulting in their limited utilization. In an effort to overcome these drawbacks, we have developed a novel method that relied upon direct genomic DNA hybridization and single-stranded DNA library preparation. Using this novel protocol, we were able to achieve a targeting efficiency as high as 75%, and we found this approach to overall be an efficient and simple approach to DNA library construction. • We report a new method for preparing hybrid capture library, which shows a dramatic improvement of targeting efficiency. • The novel method that relied upon direct genomic DNA hybridization and single-stranded DNA library preparation. • This approach is also an efficient and simple approach to DNA library construction. [ABSTRACT FROM AUTHOR]