Genome engineering /

Detalles Bibliográficos
Clasificación:Libro Electrónico
Otros Autores: Gurtler, Volder (Editor ), Calcutt, Michael (Editor )
Formato: Electrónico eBook
Idioma:Inglés
Publicado: London, United Kingdom : Academic Press, 2023.
Edición:First edition.
Colección:Methods in microbiology ; v. 52.
Temas:
Genetic engineering.
Genomes.
G�enomes.
Genetic engineering
Genomes
Acceso en línea:Texto completo
Tabla de Contenidos:
  • Intro
  • Genome Engineering
  • Copyright
  • Contents
  • Contributors
  • Preface
  • References
  • Section I: Genome transformation
  • Chapter 1: Genome transplantation in Mollicutes
  • 1. The historical scientific context associated with genome transplantation
  • 2. Mollicutes-The perfect model organisms for the establishment of GT
  • 2.1. General characteristics of the Mollicutes
  • 2.2. Transformation methods for Mollicutes
  • 2.3. Transformable replicative plasmids
  • 3. Baker's yeast-An engineering platform for microbial genomes
  • 4. The GT protocol
  • 4.1. Isolation of intact Mmc donor genomic DNA
  • 4.2. Preparation of recipient Mcap cells
  • 4.3. Transformation of Mcap with chromosomal DNA
  • 4.4. Transplanted Mollicutes chromosomes
  • 4.5. General comments about the GT protocol
  • 4.6. Limiting factors involved in the GT process
  • 4.6.1. Restriction-modification (R-M) systems
  • 4.6.2. Phylogenetic distance of donor and recipient species
  • 4.6.3. Selection of the recipient cell
  • 4.6.4. DNA recombination
  • 4.6.5. Nucleases
  • 4.6.6. Toxin/antitoxin systems
  • 4.6.7. Additional factors
  • 5. Future perspectives
  • 5.1. GT and Mollicutes
  • 5.2. Adaptation of GT to bacterial species other than Mollicutes
  • 6. Ethical considerations
  • Acknowledgements
  • Appendix
  • A.1. Entrapping of intact donor chromosomes in agarose plugs
  • A.1.1. Isolation of intact Mmc chromosomes from cultures
  • A.1.2. Preparation of agarose plugs from yeast cultures containing modified donor chromosomes
  • A.2. Genome transplantation using Mcap RE(-) as a recipient cell
  • A.2.1. Release of intact chromosomes from agarose plugs
  • A.2.2. Preparation of Mcap RE(-) recipient cells
  • A.2.3. Transplantation of donor chromosomes into Mcap RE(-) recipient cells
  • A.2.4. Screening of the transplants
  • References
  • Section II: Recombineering and engineering.
  • 5. Learn and general considerations
  • 5.1. Maximizing information from multi-factorial experiments: Sequential vs non-sequential optimisation
  • 5.2. Case studies
  • 6. Conclusions
  • References
  • Chapter 4: Recombineering
  • 1. Introduction
  • 2. History and development of recombineering
  • 3. Molecular tools for recombineering
  • 3.1. The RecBCD system
  • 3.2. The RecF pathway
  • 3.3. Lambda phage Red functions
  • 3.4. Rac prophage encoded RecE and RecT
  • 4. Steps involved in a typical recombineering experiment
  • 4.1. Substrate DNA and its meticulous design
  • 4.2. Provision for the Lambda Red recombination genes
  • 4.2.1. For bacterial chromosomal DNA
  • 4.2.2. For high and low copy number plasmids
  • 4.3. Inducing the Red genes
  • 4.4. Electroporation of the construct in the desired host
  • 4.5. Growing and maintaining the electroporated cells
  • 4.6. Selection and recombination of the clones
  • 5. Uses of recombineering
  • 5.1. Recombineering methods for inserting a selectable marker into the bacterial chromosome
  • 5.2. Recombineering can be used for inserting non-selectable DNA fragments (Sharan et al., 2009)
  • 5.2.1. Seamless method
  • 5.2.2. Scarred method
  • 6. Regulation and expression of recombineering gene
  • 6.1. Lac promoter
  • 6.2. Arabinose promoter
  • 6.3. Lambda phage's own promoter-repressor system
  • 7. Recombineering in various systems
  • 7.1. In BAC (bacterial artificial chromosome)
  • 7.1.1. Three step strategy
  • 7.1.2. Four step strategy
  • 7.1.3. ALFIRE (assisted large fragment insertion with red/ET recombination)
  • 7.2. Recombineering in E. coli phages
  • 7.3. Construction of Mycobacteriophage mutants by recombineering
  • 7.3.1. Bacteriophage Recombineering of Electroporated DNA (BRED)
  • 7.3.2. DADA-PCR: Deletion amplification assay PCR
  • 7.3.3. BRED for point mutation
  • 7.4. Recombineering in other strains.
  • 7.5. Gram negative bacteria
  • 7.5.1. Recombineering in Shewanella
  • 7.5.2. Recombineering in Vibrio natriegens
  • 7.5.3. Recombineering in Vibrio cholerae
  • 7.5.4. Recombineering in Photorhabdus luminescens
  • 7.5.5. Recombineering in Pseudomonas
  • 7.5.6. Recombineering in Salmonella enterica
  • 7.5.7. Recombineering in Klebsiella pneumoniae
  • 7.5.8. Recombineering in Yersinia pestis
  • 7.5.9. Recombineering in Zymomonas mobilis
  • 7.6. Gram positive strains
  • 7.6.1. Recombineering in mycobacteria
  • 8. Future prospects of recombineering
  • References
  • Further reading
  • Section III: CRISPR
  • Chapter 5: Applications of CRISPR/Cas9 in the field of microbiology
  • 1. Overview of CRISPR/Cas9 biology
  • 2. Applications of CRISPR/Cas9
  • 3. Recent uses of CRISPR/Cas9-based technologies in microbiology
  • 3.1. Bacterial gene expression and CRISPR/Cas9
  • 3.2. Bacterial resistance and CRISPR/Cas9
  • 3.3. Delivery strategies via CRISPR/Cas9
  • 3.4. Bacterial infections and CRISPR/Cas9
  • 4. Techniques utilizing CRISPR/Cas9
  • 4.1. Mouse model techniques
  • 4.2. Techniques based on organoid models
  • 4.3. Techniques based on cell lines
  • 4.4. Techniques based on targeting miRNA
  • 4.5. CRISPR/Cas9 in clinical trails
  • 5. Challenges in the field of CRISPR/Cas9 system
  • 6. Conclusion
  • References
  • Chapter 6: Genome engineering in Aspergillus niger
  • 1. Introduction
  • 2. Materials
  • 2.1. Nucleotide preparation or construction
  • 2.2. Manipulation of strains
  • 2.3. Measurement of enzyme activity
  • 2.4. Detection of secondary metabolism
  • 3. Methods
  • 3.1. Choice of appropriate Cas9 protein expression plasmids
  • 3.2. Construction of sgRNAs
  • 3.2.1. sgRNAs expression in vivo through plasmids
  • 3.2.2. sgRNAs synthesis in vitro
  • 3.3. Preparation of donor DNAs
  • 3.3.1. Construction of donor DNAs with short homologous arms (39bp).
  • 3.3.2. Construction of donor DNAs with long homologous arms (500-2000bp)
  • 3.4. Transformation of host strains and verification of positive transformants
  • 3.5. Detecting the yield of target products and evaluation of the genome edit effect or efficiency
  • 3.5.1. Detecting the activity of glucose oxidase
  • 3.5.2. Secondary metabolism detection
  • 4. Notes
  • References
  • Section IV: Transformation
  • Chapter 7: Natural transformation as a tool in Acinetobacter baylyi: Evolution by amplification of gene copy number
  • 1. Introduction
  • 2. General considerations
  • 2.1. Design of the amplicon and synthetic bridging fragment
  • 2.2. Chromosomal integration by natural transformation
  • 2.3. Selection of amplification mutants
  • 2.4. Interpreting gene copy number estimations and obtaining single-copy mutants
  • 3. Material and equipment
  • 3.1. Strains and culture media
  • 3.2. Reagents for DNA manipulation
  • 3.3. Equipment
  • 4. Experimental procedures
  • 4.1. Construction of the amplicon and chromosomal integration
  • 4.2. Construction of the SBF and amplification of gene copy number
  • 4.3. Adaptive laboratory evolution and monitoring of gene copy number over time
  • 4.3.1. Adaptive laboratory evolution by serial transfer
  • 4.3.2. Gene copy number analysis by quantitative PCR
  • 4.4. Obtaining single-copy mutants from EASy
  • 4.4.1. Isolation and screening by colony PCR
  • 4.4.2. Allelic replacement in evolved populations
  • 5. Summary and concluding remarks
  • Acknowledgements
  • References
  • Chapter 8: Natural transformation as a tool in Acinetobacter baylyi: Streamlined engineering and mutational analysis
  • 1. Introduction
  • 2. General considerations
  • 2.1. Convenience and optimization
  • 2.2. Preparation of recipient cells and donor DNA
  • 2.3. Introduction of DNA into cells, and growth conditions following transformation.

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