An Introduction to Molecular Biotechnology

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Completely updated in line with the rapid progress made in the field, this new edition of the highly-praised textbook addresses powerful new methods and concepts in biotechnology, such as genome editing, reprogrammed stem cells, and personalized medicine.<br> An introduction to the fundamentals in molecular and cell biology is followed by a description of standard techniques, including purification and analysis of biomolecules, cloning techniques, gene expression systems, genome editing methods, labeling of proteins and in situ-techniques, standard and high resolution microscopy. The third part focuses on key areas in research and application, ranging from functional genomics, proteomics and bioinformatics to drug targeting, recombinant antibodies and systems biology. The final part looks at the biotechnology industry, explaining intellectual property issues, legal frameworks for pharmaceutical products and the interplay between start-up and larger companies. The contents are beautifully illustrated throughout, with hundreds of full color diagrams and photographs.<br> Provides students and professionals in life sciences, pharmacy and biochemistry with everything they need to know about molecular biotechnology.<br>

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Table 1.2 Compartments of animal and plant cells and their main functions.

Compartment Occurrence Functions
Nucleus A P Harbors chromosomes, site of replication, transcription, and assembly of ribosomal subunits
Endoplasmic reticulum (ER)
Rough ER A P Posttranslational modification of proteins
Smooth ER A P Synthesis of lipids and lipophilic substances
Golgi apparatus A P Posttranslational modification of proteins, modification of sugar chains
Lysosome A Harbors hydrolytic enzymes, degrades organelles and macromolecules, macrophages eat invading microbes
Vacuole P Sequestration of storage proteins, defense and signal molecules, contains hydrolytic enzymes, degrades organelles and macromolecules
Mitochondrium A P Organelle derived from endosymbiotic bacteria; contains circular DNA, own ribosomes; enzymes of citric acid cycle, β‐oxidation, and respiratory chain (ATP generation)
Chloroplast P Organelle derived from endosymbiotic bacteria; contains circular DNA, own ribosomes; chlorophyll and proteins of photosynthesis, enzymes of CO 2fixation and glucose formation (Calvin cycle)
Peroxisome A P Contains enzymes that generate and degrade H 2O 2
Cytoplasm A P Harbors all compartments, organelles, and the cytoskeleton of a cell; many enzymatic pathways (e.g. glycolysis) occur in the cytoplasm

A, animal; P, plant.

A highly resolved tree of life is based on completely sequenced genomes (Ciccarelli 2006). The image was generated using Interactive Tree Of Life (iTOL) (Letunic 2007), an online phylogenetic tree viewer and Tree of Life resource. Eukaryotes are colored red, archaea green, and bacteria blue.

The most important biochemical and cell biological charactersof Archaea, Bacteria, and Eukarya are summarized in Table 1.1.

As virusesand bacteriophages( Figure 1.3) do not have their own metabolism, they therefore do not count as organisms in the true sense of the word. They share several macromolecules and structures with cells. Viruses and bacteriophages are dependent on the host cells for reproduction, and therefore their physiology and structures are closely linked to that of the host cell.

Figure 13 Schematic structure of bacteriophages and viruses a Bacteriophage - фото 5

Figure 1.3 Schematic structure of bacteriophages and viruses. (a) Bacteriophage T4 and (b) structure of a retrovirus (human immunodeficiency virus causing AIDS).

Eukaryotic cells are characterized by compartmentsthat are enclosed by biomembranes (Table 1.2). As a result of these compartments, the multitude of metabolic reactions can run in a cell at the same time.

In the following discussion on the shared characteristics of all cells, the diverse differences that appear in multicellular organismsshould not be forgotten. The human body has more than 200 different cell types, which show diverse structures and compositions. These differences must be understood in detail if cell‐specific disorders, such as cancer, are to be understood and consequently treated. Modern technology with Next‐Generation Sequencing (NGS) allows a study of single cells at a genomic and transcriptomic level.

Before a detailed discussion of cellular structures and their functions (see Chapters 3– 5), a short summary of the biochemical basics of cellular and molecular biology is given in Chapter 2.

Progress in cell biology and biotechnology largely depends on innovative methods, as new methods often open windows to look deeper into biology and to solve old questions. Table 1.3summarizes some of the important tools, which are important for cell and molecular biology today.

Table 1.3 Important methodological tools of modern biology.

Problem Technique/instrument Remarks
Structure elucidation of proteins Protein isolation, column chromatography (gel filtration, ion exchange, affinity) Chapter 7
Gel electrophoresis Chapter 7
Protein–protein interactions (FRET, two hybrid systems, FRAP) Chapters 19and 23
Crystallization
X‐ray diffraction
NMR
Cryoelectron microscopy
Mass spectrometry Chapter 8
Protein sequencing
DNA PCR and quantitative PCR (qPCR) Chapter 13
DNA/RNA isolation Chapter 9
DNA hybridization Chapter 11
Sanger sequencing Chapter 14
Restriction enzymes Chapter 12
Gel and capillary electrophoresis Chapter 10
Next generation sequencing Chapter 14
Microsatellite analysis Chapter 11
SNP analysis Chapters 14and 21
FISH Chapter 11
In situ hybridization Chapter 11
RNA (transcriptomics) RNA‐seq (NGS) Chapters 14and 21
DNA microarrays Chapter 11
In situ hybridization Chapter 11
Cell and tissue culture Cells with reporter genes
Cell sorting
Organoid cultures
Stem cells
Cancer cells
Hybridoma cells for production of monoclonal antibodies
Cell cycle analysis Chapter 18
Patch clamp recording Chapter 17
Microscopy Light microscope (bright field, dark field, phase contrast, differential interference contrast) Chapter 19
Fluorescence microscope (confocal) Chapters 19and 20
Immunofluorescence and GFP fusion proteins Chapter 19
Super‐resolution microscopy (STED, SIM, PALM, STORM) Chapter 19
Atomic force microscopy Chapter 19
Electron microscope Chapter 19
Scanning electron microscope (SEM)
Cryoelectron microscopy Chapter 19
Image processing
Cloning and expression Plasmid and viral vectors Chapter 15
Expression vectors Chapters 15and 16
Fermenters
Genomic and cDNA libraries Chapter 21
Reverse genetics
Genetic engineering Transformation Chapter 15
Transfection Chapter 15
RNAi
CRISPR–Cas gene editing
Transgenic organism
New active agents Recombinant antibodies Chapter 16
Recombinant vaccines Chapter 16
Recombinant enzymes Chapter 16
Information DNA sequences Chapter 24
Genomes Chapter 24
Proteins Chapter 23
System biology Chapter 23

Abbreviations: SNP, single nucleotide polymorphism; GFP, green fluoresecnt protein; NGS, next generation sequencing.

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