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Microbiology
Table of Contents
Unit 12 Overview: Modern Applications of Microbial Genetics
12.1 Microbes and the Tools of Genetic Engineering
12.2 Visualizing and Characterizing DNA, RNA, and Protein
12.3 Whole Genome Methods and Pharmaceutical Applications of Genetic Engineering
12.4 Gene Therapy

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12.3 Whole Genome Methods and Pharmaceutical Applications of Genetic Engineering

Citation:

Genomic methods revolutionize microbiology by unlocking the secrets of microbial genomes. From sequencing entire genomes to studying RNA transcripts and proteins, these techniques provide unprecedented insights into microbial life and function.

Genetic engineering transforms pharmaceutical development, enabling the production of life-saving drugs. By harnessing recombinant DNA technology and RNA interference, scientists create targeted therapies and combat viral infections with unprecedented precision.

Genomic Methods in Microbiology

Applications of omics in microbiology

  • Genomics involves sequencing and analyzing complete microbial genomes
    • Identifies genes and their functions in microorganisms (E. coli, Saccharomyces cerevisiae)
    • Enables comparative genomics to study evolutionary relationships and adaptations between different microbial species (Archaea vs Bacteria)
  • Transcriptomics studies RNA transcripts expressed by microorganisms under different conditions
    • Identifies genes that are actively transcribed in response to environmental stimuli (heat shock, nutrient limitation)
    • Elucidates regulatory networks and gene expression patterns controlling microbial physiology (quorum sensing, biofilm formation)
    • RNA-seq is a high-throughput sequencing method for quantifying gene expression levels and discovering novel transcripts (small RNAs, antisense RNAs)
  • Proteomics studies the complete set of proteins expressed by a microorganism
    • Identifies proteins and their functions in microbial cells (enzymes, transcription factors)
    • Elucidates protein-protein interactions and metabolic pathways (glycolysis, TCA cycle)
    • Mass spectrometry-based proteomics quantifies protein abundance and identifies post-translational modifications (phosphorylation, acetylation)
  • Metagenomics involves studying genetic material from environmental samples
    • Discovers novel microorganisms and genes from diverse habitats (soil, ocean, human gut)
    • Understands microbial communities and their interactions in complex ecosystems (symbiosis, competition)
    • Utilizes bioinformatics tools to analyze and interpret large-scale genomic data from complex microbial communities

Pharmaceutical Applications of Genetic Engineering

Recombinant DNA for pharmaceuticals

  • Recombinant DNA technology is used to produce pharmaceutical products by inserting genes encoding therapeutic proteins into host cells
    • Bacterial cells like E. coli are commonly used for their fast growth and easy genetic manipulation
    • Yeast cells such as S. cerevisiae are used for producing complex proteins with post-translational modifications
    • Mammalian cells like Chinese Hamster Ovary (CHO) cells are used for producing human-compatible proteins with proper folding and glycosylation
  • The therapeutic protein is expressed in the host cell by optimizing codon usage for efficient translation and using strong promoters for high-level expression
  • The expressed protein is purified from the host cell using various chromatography techniques
    • Affinity chromatography using tags like His-tag or GST-tag binds the protein of interest specifically
    • Size-exclusion chromatography separates proteins based on their molecular weight
    • Ion-exchange chromatography separates proteins based on their surface charge
  • Examples of recombinant pharmaceutical products include
    • Insulin produced in E. coli or yeast for treating diabetes
    • Erythropoietin (EPO) produced in CHO cells for treating anemia
    • Monoclonal antibodies produced in CHO cells for cancer therapy (Herceptin) and autoimmune disorders (Humira)
  • High-throughput screening methods are employed to identify and optimize potential drug candidates from large libraries of compounds

RNA interference vs viral infections

  • RNA interference (RNAi) mechanisms regulate gene expression by targeting complementary mRNA for degradation or translational repression
    • Small interfering RNAs (siRNAs) are 21-23 nucleotide double-stranded RNAs that bind to complementary mRNA and induce its degradation by the RISC complex
    • MicroRNAs (miRNAs) are endogenous small non-coding RNAs that regulate gene expression by binding to complementary sites in the 3' UTR of target mRNAs
  • RNAi has potential applications in treating viral infections by targeting viral genes essential for replication and pathogenesis
    • siRNAs can be designed to target conserved regions of viral genomes, inhibiting viral replication and reducing viral load in infected cells
    • siRNAs can be delivered to infected cells using lipid nanoparticles or viral vectors like adeno-associated virus (AAV)
    • Challenges and limitations of RNAi-based antiviral therapies include off-target effects, delivery efficiency, and the emergence of viral escape mutants
  • Examples of RNAi-based antiviral therapies under development include
    • siRNAs targeting influenza virus genes like PA and NP to inhibit viral replication
    • siRNAs targeting HIV genes like tat and rev to reduce viral load in infected cells
    • miRNA mimics or inhibitors targeting hepatitis C virus (HCV) genes to modulate viral replication and host immune responses

Advanced Genetic Engineering Techniques in Pharmaceutical Development

  • CRISPR-Cas9 technology enables precise genome editing for developing new therapeutic approaches and improving drug production processes
  • Gene therapy utilizes genetic engineering to introduce therapeutic genes into patients' cells to treat or prevent diseases
  • Biosynthetic pathways in microorganisms can be engineered to produce complex pharmaceutical compounds or their precursors
  • Pharmacogenomics studies how genetic variations affect individual responses to drugs, enabling personalized medicine approaches

Key Terms to Review (57)

Antisense RNA: Antisense RNA is a single-stranded RNA molecule that is complementary to a messenger RNA (mRNA) strand in a cell. It functions by binding to the mRNA and preventing it from being translated into protein.
Bioinformatics: Bioinformatics is the interdisciplinary field that combines biology, computer science, and information technology to analyze and interpret biological data. It plays a crucial role in understanding genetic information and its applications in medicine and biotechnology.
Biomarkers: Biomarkers are measurable indicators of a biological state or condition, often used to detect or monitor diseases and the effects of treatments. They can be genes, proteins, or molecules that provide insights into microbial functions and genetic modifications.
DNA vaccines: DNA vaccines are a type of vaccine that uses genetically engineered DNA to induce an immune response. They work by introducing a plasmid containing the DNA sequence encoding the antigen(s) against which an immune response is sought.
Gene silencing: Gene silencing is a regulatory mechanism by which a gene's expression is inhibited. This can occur at the transcriptional or post-transcriptional level and is crucial in various genetic engineering applications.
Gene therapy: Gene therapy is a medical technique that modifies genes to treat or prevent disease. It often involves inserting, altering, or removing genes within an individual's cells using vectors like viruses.
Genomics: Genomics is the study of the entirety of an organism's genes, known as its genome. It involves sequencing, analyzing, and comparing genomes to understand genetic structure and function.
Green fluorescent protein: Green fluorescent protein (GFP) is a protein that exhibits bright green fluorescence when exposed to blue to ultraviolet light. It is widely used as a marker in molecular and cellular biology due to its ability to visualize gene expression and protein localization.
High-throughput screening methods: High-throughput screening methods are automated techniques used to quickly assess the biological activity of a large number of compounds against specific targets. These methods are crucial for discovering new antimicrobial agents.
Human growth hormone: Human growth hormone (HGH) is a peptide hormone that stimulates growth, cell reproduction, and regeneration in humans. It is produced by the pituitary gland and has significant applications in medicine and genetic engineering.
Insulin: Insulin is a hormone produced by the pancreas that regulates blood glucose levels by facilitating the uptake of glucose into cells. It plays a crucial role in energy metabolism and storage.
Interferons: Interferons are proteins produced by host cells in response to pathogens, such as viruses, bacteria, and parasites. They play a crucial role in the immune response by interfering with viral replication and modulating the immune system.
Metatranscriptomics: Metatranscriptomics is the study of RNA transcripts obtained from environmental samples, providing insight into gene expression and regulation in microbial communities. It helps to understand the functional activity of microorganisms in their natural habitats.
Metagenomics: Metagenomics is the study of genetic material recovered directly from environmental samples. It enables the analysis of microbial communities without the need for culturing.
Microarray: A microarray is a laboratory tool used to detect the expression of thousands of genes simultaneously. It consists of a small surface onto which DNA molecules are fixed in a grid-like pattern.
Mutational analysis: Mutational analysis is the process of identifying and studying mutations within an organism's genome to understand their effects on function and phenotype. It is widely used in genetic research and biotechnology for pinpointing gene functions and developing treatments.
Pharmacogenomics: Pharmacogenomics is the study of how an individual's genetic makeup affects their response to drugs. It combines pharmacology and genomics to develop effective, safe medications tailored to a person's genetic profile.
Post-treatment Lyme disease syndrome: Post-treatment Lyme disease syndrome (PTLDS) is a condition where patients experience symptoms such as fatigue, pain, and cognitive difficulties after completing antibiotic treatment for Lyme disease. The exact cause of PTLDS is not well understood and continues to be studied.
Proteomics: Proteomics is the large-scale study of proteins, particularly their structures and functions. It is a crucial field for understanding cellular processes and discovering new drug targets.
Proteome: The proteome is the entire set of proteins expressed by an organism, tissue, or cell under specific conditions. It reflects genetic information as well as environmental influences.
Recombinant DNA pharmaceuticals: Recombinant DNA pharmaceuticals are medications produced through the genetic engineering of organisms to express therapeutic proteins. These pharmaceuticals often include hormones, antibodies, and enzymes used in treating various diseases.
Reverse genetics: Reverse genetics is a method used to understand gene function by analyzing the phenotypic effects of specific gene sequences after they have been disrupted or altered. It is the opposite of forward genetics, which starts with a phenotype to find the gene responsible.
RNA interference (RNAi): RNA interference (RNAi) is a biological process where RNA molecules inhibit gene expression by neutralizing targeted mRNA molecules. It is a key tool in genetic research and biotechnology for silencing specific genes.
Streptomyces: Streptomyces is a genus of Gram-positive bacteria known for its complex life cycle and production of medically important antibiotics. It is characterized by its filamentous structure and soil-dwelling nature.
Subunit vaccines: Subunit vaccines are a type of vaccine that includes only the essential antigens, rather than whole pathogens, to stimulate an immune response. These vaccines focus on specific parts of the pathogen, such as proteins or polysaccharides.
Synovial fluid: Synovial fluid is a viscous, non-Newtonian fluid found in the cavities of synovial joints. It reduces friction between the articular cartilage of synovial joints during movement.
Transcriptomics: Transcriptomics is the study of the complete set of RNA transcripts produced by the genome under specific circumstances or in a specific cell using high-throughput methods. It helps understand gene expression and regulation.
Toxicogenomics: Toxicogenomics is the study of how genomes respond to environmental toxins. It combines toxicology with genomics to understand the genetic basis of adverse effects caused by toxic substances.
Vaccinia virus: Vaccinia virus is a member of the poxvirus family used as a vaccine to eradicate smallpox. It serves as a tool in genetic engineering due to its large genome and ability to accommodate foreign genes.
X-gal: X-gal is a substrate used in molecular biology to detect the presence of β-galactosidase enzyme activity. It turns blue when cleaved by this enzyme, providing a visual marker for gene expression.
CRISPR-Cas9: CRISPR-Cas9 is a gene-editing technology that uses a guide RNA and the Cas9 enzyme to precisely target and modify specific DNA sequences within a genome. It has revolutionized genetic engineering and has widespread applications in fields like microbiology, pharmaceuticals, and gene therapy.
Bioinformatics: Bioinformatics is the application of computational and analytical methods to the management and analysis of biological data, particularly DNA, RNA, and protein sequences. It combines biology, computer science, and information technology to extract meaningful insights from complex biological data.
Herceptin: Herceptin is a targeted cancer therapy drug used to treat certain types of breast cancer. It is a monoclonal antibody that specifically targets the HER2 protein, which is overexpressed in some breast cancer cells, and helps to slow or stop the growth of these cancer cells.
Humira: Humira is a biologic medication used to treat various autoimmune and inflammatory conditions. It is a monoclonal antibody that targets and neutralizes the inflammatory protein tumor necrosis factor (TNF), effectively reducing inflammation and symptoms associated with these diseases. Humira's development and applications are closely tied to the fields of whole genome methods and pharmaceutical applications of genetic engineering.
Gene Therapy: Gene therapy is a technique that uses genes to treat or prevent disease. It involves the introduction of genetic material into cells to compensate for abnormal genes or to create a beneficial genetic effect, such as fighting disease. Gene therapy has the potential to treat a wide range of genetic and acquired diseases by targeting the underlying genetic causes.
Transcriptomics: Transcriptomics is the comprehensive study of the complete set of RNA transcripts, known as the transcriptome, produced by the genome of a cell or a population of cells. It provides insights into gene expression patterns and their regulation, which is crucial for understanding cellular function, development, and disease processes.
Erythropoietin: Erythropoietin (EPO) is a glycoprotein hormone that regulates the production of red blood cells (erythrocytes) in the body. It is primarily produced by the kidneys and plays a crucial role in the process of erythropoiesis, the formation of new red blood cells.
Adeno-Associated Virus: Adeno-associated virus (AAV) is a small, non-enveloped virus that belongs to the Dependoparvovirus genus. It is a replication-deficient parvovirus that requires the presence of a helper virus, such as adenovirus or herpesvirus, to replicate and complete its life cycle. AAVs have gained significant attention in the field of genetic engineering and pharmaceutical applications due to their unique properties and potential for therapeutic gene delivery.
Chinese Hamster Ovary: Chinese Hamster Ovary (CHO) cells are a widely used mammalian cell line derived from the ovary of the Chinese hamster. These cells have become a prominent tool in the field of genetic engineering, particularly in the production of recombinant proteins and the study of whole genome methods.
TCA Cycle: The TCA (Tricarboxylic Acid) Cycle, also known as the Citric Acid Cycle or Krebs Cycle, is a series of chemical reactions that occur in the mitochondria of cells. It is a central metabolic pathway that plays a crucial role in the aerobic cellular respiration process, generating energy in the form of ATP for the cell.
MiRNAs: miRNAs, or microRNAs, are small, non-coding RNA molecules that play a crucial role in gene expression regulation. These molecules are involved in a variety of cellular processes and have important implications in the context of whole genome methods and pharmaceutical applications of genetic engineering.
GST-tag: A GST-tag is a fusion protein that combines a protein of interest with the glutathione S-transferase (GST) protein. This tag is commonly used in molecular biology and biochemistry to facilitate the purification and detection of recombinant proteins.
SiRNAs: Small interfering RNAs (siRNAs) are double-stranded RNA molecules that play a crucial role in gene silencing and regulation, with applications in both whole genome methods and pharmaceutical genetic engineering.
Biosynthetic Pathways: Biosynthetic pathways refer to the series of enzyme-catalyzed chemical reactions that living organisms use to synthesize complex molecules from simpler precursors. These pathways are crucial for the production of essential biomolecules, such as proteins, lipids, and carbohydrates, which are necessary for cellular structure, function, and energy production.
RNA-seq: RNA-seq, or RNA sequencing, is a powerful technique used to analyze the transcriptome, which is the complete set of RNA molecules expressed in a cell or organism. It allows for the identification and quantification of gene expression levels by sequencing the RNA molecules present in a sample.
Proteomics: Proteomics is the large-scale study of proteins, including their structure, function, and interactions within an organism. It is a crucial field that complements genomics, the study of genes, and provides valuable insights into the complex biological processes that sustain life.
Genomics: Genomics is the comprehensive study of the entire genome, which encompasses the complete set of genetic information within an organism. It involves the analysis of the structure, function, and evolution of genomes, providing insights into the fundamental mechanisms of life and enabling advancements in various fields, including medicine, biotechnology, and evolutionary biology.
His-tag: A His-tag, or Histidine-tag, is a protein purification and detection tool that involves adding a short peptide sequence, typically consisting of six to ten consecutive histidine residues, to a recombinant protein. This tag allows for the efficient purification and identification of the target protein.
High-Throughput Screening: High-throughput screening (HTS) is a method used in drug discovery and genetic engineering to rapidly test a large number of chemical compounds or genetic samples to identify those with a desired biological activity. It is a powerful tool that enables the efficient exploration of vast chemical libraries and genomic data to uncover potential therapeutic agents or identify gene functions.
Metagenomics: Metagenomics is the study of genetic material recovered directly from environmental samples, bypassing the need for culturing individual microorganisms. It provides a powerful approach for understanding the collective genetic makeup and metabolic potential of complex microbial communities in their natural habitats, with applications in fields such as whole genome methods and pharmaceutical discovery.
Biofilm Formation: Biofilm formation is the process by which microorganisms, such as bacteria and fungi, adhere to surfaces and develop into complex, structured communities encased in a self-produced extracellular matrix. This phenomenon is closely linked to the pathogenesis and persistence of various microbial infections, as well as the effectiveness of antimicrobial agents and disinfectants.
RISC Complex: The RISC (RNA-Induced Silencing Complex) is a key component in the RNA interference (RNAi) pathway, which is a gene silencing mechanism that uses small RNA molecules to regulate gene expression and defend cells against viruses. The RISC complex is responsible for recognizing, binding, and cleaving target mRNA molecules, thereby preventing their translation into proteins.
Pharmacogenomics: Pharmacogenomics is the study of how an individual's genetic makeup influences their response to drugs. It combines the fields of pharmacology, which is the study of drugs, and genomics, the study of the human genome, to develop more effective and safer medications tailored to a person's genetic profile.
Archaea: Archaea are a domain of single-celled microorganisms that are distinct from bacteria and eukaryotes. They are prokaryotic in nature, but possess unique characteristics that set them apart from other prokaryotes, making them a separate domain of life. Archaea are found in a wide range of habitats, from extreme environments to the human microbiome, and have important applications in biotechnology and genetic engineering.
Quorum Sensing: Quorum sensing is a communication mechanism used by bacteria to coordinate gene expression and behavior in response to changes in cell population density. It allows bacteria to sense and respond to the presence of other bacteria, enabling them to act in a coordinated manner as a community rather than as individual cells.
Glycolysis: Glycolysis is the metabolic pathway that converts glucose, a six-carbon sugar, into two molecules of pyruvate, a three-carbon compound. This process is the first stage of cellular respiration and occurs in the cytoplasm of cells, providing a crucial source of energy for the cell.
RNA Interference: RNA interference (RNAi) is a biological process in which RNA molecules inhibit gene expression or translation, by neutralizing targeted mRNA molecules. This mechanism is used by cells to regulate gene expression and defend against viral infections.