Microbiology Lab Concepts and Core Topics: Metabolism, Genetics, and Biotechnology

Lab Concepts

Streak Plating

Streak plating is a fundamental microbiological technique used to isolate pure colonies from a mixed culture. It involves spreading bacteria across the surface of an agar plate in a pattern that thins out the sample and separates individual cells.

• Purpose: To obtain isolated colonies for further study or identification.

• Common Errors: Not flaming the loop between quadrants, using too much inoculum, or not streaking enough can lead to poor isolation.

• Troubleshooting: Ensure proper sterilization of the loop, use gentle pressure, and streak in a systematic pattern.

DNA Extractions

DNA extraction is the process of isolating DNA from cells for analysis. It is essential for genetic studies, cloning, and biotechnology applications.

• Centrifuge Safety and Balancing: Always balance tubes with equal volumes opposite each other to prevent damage or accidents.

• Centrifuge Purpose: To separate components based on density, such as pelleting cell debris while leaving DNA in the supernatant.

• Supernatant vs Pellet: The supernatant is the liquid above the solid residue (pellet) after centrifugation. The pellet contains heavier particles.

Key Materials and Functions for Strawberry DNA Extraction

• Strawberries: High DNA yield due to being octoploid (eight sets of chromosomes).

• Dish Soap (Detergent): Disrupts cell and nuclear membranes by dissolving phospholipids, releasing DNA.

• Salt (NaCl): Neutralizes DNA's negative charge, allowing it to aggregate and separate from proteins.

• Ice-Cold Ethanol/Isopropyl Alcohol (70-90%): Precipitates DNA, making it visible as white fibers.

• Plastic Bag/Mash: Mechanically breaks down cell walls.

• Filter (Coffee filter/cheesecloth): Removes cell debris, leaving DNA in the filtrate.

• Stirrer (Wooden skewer/hook): Collects precipitated DNA.

Comparison to Other Methods: Bead bashing uses mechanical force to lyse cells; DNA wash removes contaminants; column filtration binds DNA to a matrix for purification.

Additional info: DNA extraction is foundational for PCR, cloning, and sequencing.

CH 5: Microbial Metabolism

Catabolism and Anabolism

Microbial metabolism encompasses all chemical reactions in a cell, divided into catabolism (breakdown) and anabolism (synthesis).

• Catabolism: Degradative, exergonic reactions that release energy (often as ATP). Example: Glycolysis.

• Anabolism: Biosynthetic, endergonic reactions that consume energy (ATP). Example: Protein synthesis.

• Exergonic (Hydrolytic) Reactions: Release energy by breaking bonds using water.

• Endergonic (Dehydration Synthesis) Reactions: Require energy input to form new bonds, releasing water.

ATP Release and Consumption:

• Catabolic reactions release ATP.

• Anabolic reactions consume ATP.

Enzymes

Enzymes are biological catalysts, usually proteins ending in -ase, that speed up chemical reactions without being consumed.

• Role in Pathways: Enzymes lower activation energy, allowing metabolic pathways to proceed efficiently.

• Substrate: The molecule upon which an enzyme acts.

• Active Site: The region on the enzyme where the substrate binds.

• Products: Common products include ATP, acids, gases, and alcohols.

Factors Affecting Enzyme Activity

• pH: Extreme pH can denature enzymes.

• Temperature: High temperatures can denature enzymes; low temperatures slow reactions.

• Substrate Concentration: Higher concentrations increase reaction rate up to a saturation point.

• Inhibitors: Competitive inhibitors block the active site; noncompetitive inhibitors bind elsewhere, altering enzyme shape.

Equation for Enzyme-Catalyzed Reaction:

$\mathrm{E + S \rightleftharpoons ES \rightarrow E + P}$

Where E = enzyme, S = substrate, ES = enzyme-substrate complex, P = product.

CH 8: Microbial Genetics

Central Dogma and -Omics Analyses

The central dogma describes the flow of genetic information: DNA → RNA → Protein.

• Genomics: Study of the entire genome (DNA).

• Transcriptomics: Study of RNA transcripts.

• Proteomics: Study of the protein complement.

• Mutations: Changes at any step can affect downstream processes and phenotypes.

Gene Expression and Operons

• Transcription: Synthesis of RNA from DNA; gene expression.

• Constitutively Expressed: Genes that are always active.

• Repressible Operon: Normally on; can be turned off by a repressor (e.g., trp operon).

• Inducible Operon: Normally off; can be turned on by an inducer (e.g., lac operon).

• Enzymes and Substrates: Induction requires substrate presence; repression occurs when product accumulates.

• Operon: Cluster of genes under control of a single promoter.

• Operator: DNA segment where repressor binds.

• Promoter: DNA sequence where RNA polymerase binds to initiate transcription.

Enzymes in Genetic Processes

• Enzymes are essential for DNA replication (DNA polymerase), transcription (RNA polymerase), and translation (ribosomes, tRNA synthetases).

RNA Differences: Prokaryotes vs Eukaryotes

• Prokaryotes: mRNA is often polycistronic (encodes multiple proteins), no introns, transcription and translation are coupled.

• Eukaryotes: mRNA is monocistronic, contains introns (spliced out), transcription in nucleus, translation in cytoplasm.

Ribosomes, Amino Acids, and Codons

• Ribosomes: Sites of protein synthesis; 70S in prokaryotes, 80S in eukaryotes.

• Amino Acids: Building blocks of proteins; 20 standard types.

• Codon: Sequence of three nucleotides in mRNA that specifies an amino acid. Not all codons are the same; some are stop codons.

Gene Transfer and Mobile Genetic Elements

• Vertical Gene Transfer: Parent to offspring.

• Horizontal Gene Transfer: Between organisms, not by descent.

• Mobile Genetic Elements (MGEs): DNA segments that can move within or between genomes (e.g., plasmids, transposons).

Plasmids and Transposons

• Plasmids: Small, circular DNA molecules; can carry antibiotic resistance (R factors) or conjugative genes.

• Transposons: "Jumping genes" that move within the genome; can disrupt genes (cons: mutations) or spread beneficial traits (pros: adaptation).

Phages and Gene Transfer Mechanisms

• Phages: Viruses that infect bacteria; can undergo lytic (destroy host) or lysogenic (integrate into host genome) cycles.

• Conjugation: Direct transfer of DNA via pilus between bacteria.

• Transformation: Uptake of naked DNA from environment (Griffith's experiment demonstrated this in Streptococcus pneumoniae).

• Transduction: Transfer of DNA by bacteriophages.

CH 9: Biotechnology and DNA Technology

Key Concepts

• Vector: DNA molecule (e.g., plasmid, virus) used to carry foreign DNA into a host cell.

• Biotechnology: Use of living organisms or their products for practical applications.

• rDNA Technology: Recombinant DNA technology; combining DNA from different sources to create genetically modified organisms (GMOs).

• Common Products: Insulin, growth hormones, pest-resistant crops.

• Recombination: Exchange of genetic material; results in new gene combinations.

PCR (Polymerase Chain Reaction)

PCR is a technique to amplify specific DNA sequences using cycles of heating and cooling.

• Heating: Denatures DNA (separates strands).

• Cooling: Allows primers to anneal and DNA polymerase to extend new strands.

Equation for PCR Cycle:

$\text{DNA}_{\text{template}} + \text{Primers} + \text{dNTPs} + \text{Polymerase} \xrightarrow{\text{PCR cycles}} \text{Amplified DNA}$

Cloning and DNA Insertion Methods

• Cloning: Producing identical copies of DNA or organisms; used for gene studies, protein production.

• DNA Insertion Methods:

  • Transformation: Uptake of naked DNA by cells.

  • Electroporation: Electric shock creates pores in cell membrane for DNA entry.

  • Protoplast Fusion: Fusion of cells without cell walls to combine genetic material.

  • Microinjection: Direct injection of DNA into cells.

Sequencing Methods

• Amplicon Sequencing: Targets specific DNA regions (e.g., 16S rRNA gene) for microbial identification.

• Shotgun Sequencing: Randomly sequences all DNA in a sample; useful for metagenomics.

• Nanotechnology: Used to enhance sequencing accuracy and throughput.

Table: Comparison of DNA Insertion Methods

Method	Principle	Application
Transformation	Uptake of naked DNA	Bacterial genetic engineering
Electroporation	Electric field induces membrane pores	Broad host range, including eukaryotes
Protoplast Fusion	Fusion of cell membranes after wall removal	Hybrid cell creation
Microinjection	Direct injection of DNA	Animal and plant cells

Additional info: These methods are essential for creating genetically modified organisms and for research in functional genomics.