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Filamentous fungi of the genus Aspergillus include producers of industrially important organic acids, enzymes, and secondary metabolites, as well as pathogens of many plants and animals. Novel genes in the Aspergillus genome are potentially crucial for the fermentation and drug industries (e.g., agrochemicals and antifungal drugs). A research approach based on classical genetics is effective for identifying functionally unknown genes. During analyses based on classical genetics, mutations must be identified easily and quickly. Herein, we report the development of a cosmid-based plasmid pTOCK1 and the use of a genomic library of Aspergillus nidulans constructed using pTOCK1. The cosmid-based genomic library was used for convenient auxotrophic mutants (pyroA and pabaB), as well as mutants with abnormal colony morphology (gfsA) and yellow conidia (yA), to obtain library clones complementary to these phenotypes. The complementary strain could be obtained through a single transformation, and the cosmid could be rescued. Thus, our cosmid library system can be used to identify the causative gene in a mutant strain.

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Saccharomyces cerevisiae is a powerful system for the expression of genome-wide or combinatorial libraries for diverse types of screening. However, expressing large libraries in yeast requires high-efficiency transformation and controlled expression. Transformation of yeast using electroporation methods is more efficient than chemical methods; however, protocols described for electroporation require large amounts of linearized plasmid DNA and often yield approximately 106 cfu/µg of plasmid DNA. We optimized the electroporation of yeast cells for the expression of whole-genome libraries to yield up to 108 cfu/µg plasmid DNA. The protocol generates sufficient transformants for 10-100× coverage of diverse genome libraries with small amounts of genomic libraries (0.1 µg of DNA per reaction) and provides guidance on calculations to estimate library size coverage and transformation efficiency. It describes the preparation of electrocompetent yeast cells with lithium acetate and dithiothreitol conditioning step and the transformation of cells by electroporation with carrier DNA. We validated the protocol using three yeast surface display libraries and demonstrated using nanopore sequencing that libraries' size and diversity are preserved. Moreover, expression analysis confirmed library functionality and the method's efficacy. Hence, this protocol yields a sufficient representation of the genome of interest for downstream screening purposes while limiting the amount of the genomic library required.

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The lack of genetic tools to manipulate protozoan pathogens has limited the use of genome-wide approaches to identify drug or vaccine targets and understand these organisms' biology. We have developed an efficient method to construct genome-wide libraries for yeast surface display (YSD) and developed a YSD fitness screen (YSD-FS) to identify drug targets. We show the efficacy of our method by generating genome-wide libraries for Trypanosoma brucei, Trypanosoma cruzi, and Giardia lamblia parasites. Each library has a diversity of ∼105 to 106 clones, representing ∼6- to 30-fold of the parasite's genome. Nanopore sequencing confirmed the libraries' genome coverage with multiple clones for each parasite gene. Western blot and imaging analysis confirmed surface expression of the G. lamblia library proteins in yeast. Using the YSD-FS assay, we identified bonafide interactors of metronidazole, a drug used to treat protozoan and bacterial infections. We also found enrichment in nucleotide-binding domain sequences associated with yeast increased fitness to metronidazole, indicating that this drug might target multiple enzymes containing nucleotide-binding domains. The libraries are valuable biological resources for discovering drug or vaccine targets, ligand receptors, protein-protein interactions, and pathogen-host interactions. The library assembly approach can be applied to other organisms or expression systems, and the YSD-FS assay might help identify new drug targets in protozoan pathogens.

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INTRODUCTION: Species A rotavirus (RVA) infections are a major cause of severe gastroenteritis in children of <5 years worldwide. In Brazil, before vaccination, RVA was associated with 3.5 million episodes of acute diarrheal disease per year. Due to the segmented nature of their genomes, rotaviruses can exchange genes during co-infections, and generate new virus strains and new reinfections. OBJECTIVE: To evaluate the genomic diversity of RVA isolated in Brazil in 30 years, between 1986 to 2016, to investigate possible changes in the frequency of genotype constellations before and after the implementation of the vaccine. METHODS: In total, 4,474 nucleotide sequences were obtained from the Virus Variation Database. Genomic constellation was compared, and the proportion of rotavirus genotypes was analyzed by time and geographic region. RESULTS: Our results showed that major known genotypes were circulating in the country during the period under analysis, with a prevalence of the G1P[8] Wa-like genotype, decreasing only in the period immediately after the introduction of the vaccine. Regarding the geographical distribution, most of our constellations, 62 (39.2%), and 50 (31.6%) were concentrated in the North and Northeast regions. Our analysis also showed the circulation of multiple strains during the periods when the DS-1-like and AU-1-like genotypes were co-circulating with the Wa-like genotype. CONCLUSION: Therefore, it is likely that inter-genogroup reassortments are still occurring in Brazil and so it is important to establish an efficient surveillance system to follow the emergence of novel reassorted strains that might not be targeted by the vaccine.

INTRODUÇÃO: As infecções por rotavírus A (RVA) são uma das principais causas de gastroenterite grave em crianças <5 anos em todo o mundo. No Brasil, antes da vacinação, o RVA estava associado a 3,5 milhões de episódios de diarreia aguda por ano. Devido à natureza segmentada de seus genomas, os rotavírus podem trocar genes durante as coinfecções, gerar novas cepas de vírus e novas reinfecções. OBJETIVO: Avaliar a diversidade genômica de RVA isolados no Brasil entre 1986 a 2016 para investigar possíveis alterações na frequência das constelações de genótipos antes e após a implantação da vacina. MÉTODOS: No total, 4.474 sequências de nucleotídeos foram obtidas do Banco de Dados de Variação de Vírus. A constelação genômica foi comparada e a proporção dos genótipos de rotavírus foi analisada por tempo e região geográfica. RESULTADOS: Nossos resultados mostraram que os principais genótipos conhecidos circulavam no país no período em análise, com prevalência do genótipo G1P[8] Wa-like, diminuindo apenas no período imediatamente após a introdução da vacina. Em relação à distribuição geográfica, a maioria das nossas constelações, 62 (39,2%) e 50 (31,6%), concentrava-se nas regiões Norte e Nordeste. Nossa análise também mostrou a circulação de cepas múltiplas durante os períodos em que os genótipos DS-1-like e AU-1-like estavam co-circulando com o genótipo Wa-like. CONCLUSÃO: Portanto, é provável que rearranjos inter-genogrupos ainda estejam ocorrendo no Brasil e por isso é importante estabelecer um sistema de vigilância eficiente para acompanhar o surgimento de novas cepas rearranjadas que podem não ser protegidas pela vacina.

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Sublineages (SLs) within microbial species can differ widely in their ecology and pathogenicity, and their precise definition is important in basic research and for industrial or public health applications. Widely accepted strategies to define SLs are currently missing, which confuses communication in population biology and epidemiological surveillance. Here, we propose a broadly applicable genomic classification and nomenclature approach for bacterial strains, using the prominent public health threat Klebsiella pneumoniae as a model. Based on a 629-gene core genome multilocus sequence typing (cgMLST) scheme, we devised a dual barcoding system that combines multilevel single linkage (MLSL) clustering and life identification numbers (LINs). Phylogenetic and clustering analyses of >7,000 genome sequences captured population structure discontinuities, which were used to guide the definition of 10 infraspecific genetic dissimilarity thresholds. The widely used 7-gene multilocus sequence typing (MLST) nomenclature was mapped onto MLSL SLs (threshold: 190 allelic mismatches) and clonal group (threshold: 43) identifiers for backwards nomenclature compatibility. The taxonomy is publicly accessible through a community-curated platform (https://bigsdb.pasteur.fr/klebsiella), which also enables external users' genomic sequences identification. The proposed strain taxonomy combines two phylogenetically informative barcode systems that provide full stability (LIN codes) and nomenclatural continuity with previous nomenclature (MLSL). This species-specific dual barcoding strategy for the genomic taxonomy of microbial strains is broadly applicable and should contribute to unify global and cross-sector collaborative knowledge on the emergence and microevolution of bacterial pathogens.

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The development of antibody therapies against SARS-CoV-2 remains a challenging task during the ongoing COVID-19 pandemic. All approved therapeutic antibodies are directed against the receptor binding domain (RBD) of the spike, and therefore lose neutralization efficacy against emerging SARS-CoV-2 variants, which frequently mutate in the RBD region. Previously, phage display has been used to identify epitopes of antibody responses against several diseases. Such epitopes have been applied to design vaccines or neutralize antibodies. Here, we constructed an ORFeome phage display library for the SARS-CoV-2 genome. Open reading frames (ORFs) representing the SARS-CoV-2 genome were displayed on the surface of phage particles in order to identify enriched immunogenic epitopes from COVID-19 patients. Library quality was assessed by both NGS and epitope mapping of a monoclonal antibody with a known binding site. The most prominent epitope captured represented parts of the fusion peptide (FP) of the spike. It is associated with the cell entry mechanism of SARS-CoV-2 into the host cell; the serine protease TMPRSS2 cleaves the spike within this sequence. Blocking this mechanism could be a potential target for non-RBD binding therapeutic anti-SARS-CoV-2 antibodies. As mutations within the FP amino acid sequence have been rather rare among SARS-CoV-2 variants so far, this may provide an advantage in the fight against future virus variants.

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Vibrio parahaemolyticus is a shellfish-borne pathogen that is a highly prevalent causative agent of inflammatory gastroenteritis in humans. Genomic libraries have proven useful for the identification of novel gene functions in many bacterial species. In this study we prepared a library containing 40 kb fragments of randomly sheared V. parahaemolyticus genomic DNA and introduced this into Escherichia coli HB101 using a commercially available low copy cosmid system. In order to estimate coverage and suitability of the library and potentially identify novel antimicrobial resistance determinants, we screened for the acquisition of resistance to the fluoroquinolone norfloxacin - a phenotype exhibited by V. parahaemolyticus but not the heterologous E. coli host. Upon selection on solid medium containing norfloxacin, 0.52% of the library population was resistant, consistent with the selection of a single resistance locus. End-sequencing identified six distinct insert fragments. All clones displayed fourfold increased norfloxacin MIC compared with E. coli HB101 carrying an empty vector. The common locus contained within resistant clones included qnr, a previously described quinolone resistance gene. These results indicate that the library was unbiased, of sufficient coverage and that heterologous expression was possible. While we hope that this library proves useful for identifying the genetic determinants of complex phenotypes such as those related to virulence, not all norfloxacin resistance genes were detected in our screen. As such, we discuss the benefits and limitations of this approach for identifying the genetic basis of uncharacterized bacterial phenotypes.

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Infections due to Pseudomonas aeruginosa (PA) are becoming a serious threat to patients in intensive care units. A PA vaccine is a practical and economical solution to solve the problems caused by PA infection successfully. In recent years, several antigen candidates have been tested in animal and human clinical trials, but none of them has been approved to date. An alternative strategy for antigen screening and protective antigens is in urgent demand. In this study, we generated a genome-wide library of PA protein fragments tagged with maltose-binding protein (MBP). Using sera from patients who recovered after PA infection, we identified a novel protective antigen, FlgE, which is the structural component of the flagella hook. Vaccination with recombinant FlgE (reFlgE) induced a Th2-predominant immune response and reduced bacterial load and inflammation in PA-infected mice. Anti-reFlgE antibodies recognized native FlgE on the bacterial membrane in vitro and conferred protection in mice, which may be due to the mediation of opsonophagocytic killing and inhibition of bacterial motility. In addition, the combination of reFlgE with rePcrVNH, an engineered antigen we reported previously, provided elevated protection against PA infection. Our data demonstrate that FlgE is a promising vaccine candidate for PA and provide a new strategy for the efficient screening of antigens of other pathogens.

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Genomic variants libraries are conducive to obtain dominant strains with desirable phenotypic traits. The non-homologous end joining (NHEJ), which enables foreign DNA fragments to be randomly integrated into different chromosomal sites, shows prominent capability in genomic libraries construction. In this study, we established an efficient NHEJ-mediated genomic library technology in Yarrowia lipolytica through regulation of NHEJ repair process, employment of defective Ura marker and optimization of iterative transformations, which enhanced genes integration efficiency by 4.67, 22.74 and 1.87 times, respectively. We further applied this technology to create high lycopene producing strains by multi-integration of heterologous genes of CrtE, CrtB and CrtI, with 23.8 times higher production than rDNA integration through homologous recombination (HR). The NHEJ-mediated genomic library technology also achieved random and scattered integration of loxP and vox sites, with the copy number up to 65 and 53, respectively, creating potential for further application of recombinase mediated genome rearrangement in Y. lipolytica. This work provides a high-efficient NHEJ-mediated genomic library technology, which enables random and scattered genomic integration of multiple heterologous fragments and rapid generation of diverse strains with superior phenotypes within 96 h. This novel technology also lays an excellent foundation for the development of other genetic technologies in Y. lipolytica.

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High-copy rescue genetic screening is a powerful strategy for the identification of suppression genetic interactions in the model eukaryotic organism Saccharomyces cerevisiae (budding yeast). The strain carrying the mutant allele of interest is transformed with a genomic library cloned in a high-copy plasmid. Each clone carries a genomic fragment insertion of around 10 kb, typically containing one to three complete genes under their own promoters. The high-copy vector favors the accumulation of high levels of the corresponding protein, aimed at suppressing the mutant phenotype. Typically, high-copy genetic screens select for viable clones under conditions restrictive or lethal for the query mutant strain. Here, we describe in detail the procedure to generate a high-copy genomic library and a protocol for rescue genetic screening and identification of the suppressor clones.

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Several microbes are polyploid, meaning they contain several copies of their chromosome. Cyanobacteria, while holding great potential as photosynthetic cell factories of various products, are found among them. In these clades the diversity of genetic elements that serve within the basic molecular toolbox is often limiting. To assist mining for the latter, we present here a method for the generation of fully segregated genomic libraries, specifically designed for polyploids. We provide proof-of-principle for this method by generating a fully segregated genomic promoter library in the cyanobacterium Synechocystis sp. PCC 6803. This new tool was first analyzed through fluorescence activated cell sorting (FACS) and then a fraction was further characterized regarding promoter sequence. The location of libraries on the chromosome provides a better reflection of the behavior of its elements. Our work presents the first method for constructing fully segregated genomic libraries in polyploids, which may facilitate their usage in synthetic biology applications.

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Understanding the antibody response is critical to developing vaccine and antibody-based therapies and has inspired the recent development of new methods to isolate antibodies. Methods to define the antibody-antigen interactions that determine specificity or allow escape have not kept pace. We developed Phage-DMS, a method that combines two powerful approaches-immunoprecipitation of phage peptide libraries and deep mutational scanning (DMS)-to enable high-throughput fine mapping of antibody epitopes. As an example, we designed sequences encoding all possible amino acid variants of HIV Envelope to create phage libraries. Using Phage-DMS, we identified sites of escape predicted using other approaches for four well-characterized HIV monoclonal antibodies with known linear epitopes. In some cases, the results of Phage-DMS refined the epitope beyond what was determined in previous studies. This method has the potential to rapidly and comprehensively screen many antibodies in a single experiment to define sites essential for binding interactions.

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Our limited understanding of the relationship of genotype to phenotype in the spirochete Leptospira interrogans stems from the inefficiency of the genetic tools available to manipulate the pathogen. The recent development of random transposon mutagenesis in L. interrogans has allowed the creation of large libraries of mutants, permitting the identification of several genes involved in certain functions such as virulence. However, the process of phenotypically screening individual mutants in the library remains time- and labor-intensive. Here, we describe a transposon sequencing technique (Tn-Seq), which combines random transposon mutagenesis with high-throughput sequencing for screening L. interrogans mutants more rapidly with fewer resources than traditional methods.

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Protein-protein interactions govern many cellular processes, and identifying binding interaction sites on proteins can facilitate the discovery of inhibitors to block such interactions. Here we identify peptides from a randomly fragmented plasmid encoding the ß-lactamase inhibitory protein (BLIP) and the Lac repressor (LacI) that represent regions of protein-protein interactions. We utilized a Jun-Fos-assisted phage display system that has previously been used to screen cDNA and genomic libraries to identify antibody antigens. Affinity selection with polyclonal antibodies against LacI or BLIP resulted in the rapid enrichment of in-frame peptides from various regions of the proteins. Further, affinity selection with ß-lactamase enriched peptides that encompass regions of BLIP previously shown to contribute strongly to the binding energy of the BLIP/ß-lactamase interaction, i.e., hotspot residues. Further, one of the regions enriched by affinity selection encompassed a disulfide-constrained region of BLIP that forms part of the BLIP interaction surface in the native complex that we show also binds to ß-lactamase as a disulfide-constrained macrocycle peptide with a KD of ∼1 µM. Fragmented open reading frame (ORF) libraries may efficiently identify such naturally constrained peptides at protein-protein interaction interfaces. With sufficiently deep coverage of ORFs by peptide-coding inserts, phage display and deep sequencing can provide detailed information on the domains or peptides that contribute to an interaction. Such information should enable the design of potentially therapeutic macrocycles or peptidomimetics that block the interaction.

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Di-(2-ethylhcxyl) phthalate (DEHP) is applied as plasticizer, which results in the pollution of environment. In this study, the effects of DEHP on soil microbial functions, structure and genetic diversity were investigated. The concentration of DEHP in the soil were 0, 0.1, 1, 10 and 50 mg/kg, and the experimental period were 28 days. DEHP reduced the quantity, abundance, species dominance and homogeneity of soil microbes during the first 14 days. In addition, microbial utilization efficiency of carbon (carbohydrates, aliphatics, amino acids, metabolites) was impacted after 28 days, though the effects gradually weakened. Based on denaturing gradient gel electrophoresis and clone library analysis, in the presence of DEHP, the dominant microbes in the DEHP-contaminated soil were Sphingomonas and Bacillus, which belonged to the Acidobacteria and Proteobacteriav, respectively. With 0.1 or 1 mg/kg of DEHP, the relative abundances of Acidobacteria were higher, and with 10 or 50 mg/kg of DEHP, the relative abundances of Proteobacteria were higher.

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Microbial oils are regarded as promising alternatives to fossil fuels. For bio-oil production to be sustainable over the long term, utilizing low-cost substrates like volatile fatty acids (VFAs) is crucial. Increasing attention is being paid to one of the most common VFAs: propionate, a substrate that could be used to produce the odd-chain FAs of industrial interest. However, little is known about microbial responses to propionate-induced stress and the genes involved. Using genomic library screening, we identified two genes involved in propionate tolerance in Yarrowia lipolytica-MFS1 and RTS1. Strains containing each of the genes displayed enhanced tolerance to propionate even when the genes were expressed in truncated form via a replicative plasmid. Compared with the control strain, the strain overexpressing MFS1 under a constitutive promoter displayed greater tolerance to propionate: It had a shorter lag phase and higher growth rate in propionate medium (0.047 hr-1 versus 0.030 hr-1 for the control in 40 g/L propionate); it also accumulated more total lipids and more odd-chain lipids (10% and 3.3%, respectively) than the control. The strain overexpressing RTS1 showed less tolerance for propionate than the strains harboring the truncated form (0.057 hr-1 versus 0.065 hr-1 in 40 g/L propionate medium) but still had higher tolerance than the control strain. Furthermore, the overexpression of RTS1 seemed to confer tolerance to other weak acids such as lactate, formic acid, malic acid, and succinic acid. This work provides a basis for better understanding the response to propionate-induced stress in Y. lipolytica.

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Simple sequence repeat (SSR) markers are the major molecular tools for genetic and genomic researches that have been extensively developed and used in major crops. However, few are available for lentils (Lens culinaris M.), economically an important cool-season legume. The lack of informative simple sequence repeat (SSR) markers in lentil has been a major limitation for lentil molecular breeding studies. Therefore, in order to develop SSR markers for lentil, an enriched genomic libraries for AC and AG repeats were constructed from the Lens culinaris cv Kafkas. A total of 350 clones were inquired for the detection of SSRs. Of 350 clones, 68 had SSR motifs. In polymorphism analysis using 53 newly developed SSRs, a total of 144 alleles across 24 lentil cultivars were detected with an average of 4.64 per locus. The average heterozygosity was 0.588 and polymorphism information contents ranged from 0.194 to 0.895 with an average value of 0.520. These newly developed SSRs will constitute useful tools for molecular breeding, mapping, and assessments of genetic diversity and population structure of lentils.

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An efficient method to construct xenogeneic genomic libraries with low errors and bias by circumventing restriction-modification systems that restrict methylated DNA was developed. Un-methylated genomic DNA of Escherichia coli prepared by ϕ29 DNA polymerase was introduced to Corynebacterium glutamicum R after ligation with un-methylated vector plasmids.

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Alternative sigma factors control numerous aspects of bacterial life, including adaptation to physiological stresses, morphological development, persistence states and virulence. This is especially true for the physiologically complex actinobacteria. Here we report the development of a robust gene deletions system for Streptomyces lividans TK24 based on a BAC library combined with the λ-Red recombination technique. The developed system was validated by systematically deleting the most highly expressed genes encoding alternative sigma factors and several other regulatory genes within the chromosome of S. lividans TK24. To demonstrate the possibility of large scale genomic manipulations, the major part of the undecylprodigiosin gene cluster was deleted as well. The resulting mutant strains were characterized in terms of morphology, growth parameters, secondary metabolites production and response to thiol-oxidation and cell-wall stresses. Deletion of SLIV_12645 gene encoding S. coelicolor SigR1 ortholog has the most prominent phenotypic effect, resulted in overproduction of actinorhodin and coelichelin P1 and increased sensitivity to diamide. The secreted proteome analysis of SLIV_12645 mutant revealed SigR1 influence on trafficking of proteins involved in cell wall biogenesis and refactoring. The reported here gene deletion system will further facilitate work on S. lividans strain improvement as a host for either secondary metabolites or protein production and will contribute to basic research in streptomycetes physiology, morphological development, secondary metabolism. On the other hand, the systematic deletion of sigma factors encoding genes demonstrates the complexity and conservation of regulatory processes conducted by sigma factors in streptomycetes.

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Among the molecules of the immune system, antibodies, particularly monoclonal antibodies (mAbs), have been shown to be interesting for many biological applications. Due to their ability to recognize only a unique part of their target, mAbs are usually very specific. These targets can have many different compositions, but the most common ones are proteins or peptides that are usually from outside the host, although self-proteins can also be targeted in autoimmune diseases, or in some types of cancer. The parts of a mAb that interact with its target compose the paratope, while the recognized parts of the target compose the epitope. Knowing the epitope is valuable for the improvement of a biological product, e.g., a diagnostic assay, a therapeutic mAb, or a vaccine, as well as for the elucidation of immune responses. The current techniques for epitope mapping rely on the presentation of the target, or parts of it, in a way that it can interact with a certain mAb. Even though there are several techniques available, each has its pros and cons. Thus, the choice for one of them is usually dependent on the preference and availability of the researcher, opening possibility for improvement, or development of alternative techniques. Phage display, for example, is a versatile technology, which allows the presentation of many different oligopeptides that can be tested against different antibodies, fitting the need for an epitope mapping approach. In this chapter, a protocol for the construction of a single-target oligopeptide phage library, as well as for the panning procedure for epitope mapping using phage display is given.

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