Lab Concepts

Streak Plating

Streak plating is a fundamental microbiology technique used to isolate pure colonies of microorganisms from a mixed sample. It involves spreading bacteria across the surface of an agar plate in a specific pattern.

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

• Common Errors: Not flaming the loop between streaks, using too much inoculum, or not streaking enough times can prevent isolation.

• Troubleshooting: Ensure proper sterilization, use a small amount of sample, and streak in multiple quadrants.

• Example: Isolating Escherichia coli from a mixed environmental sample.

DNA Extraction

DNA extraction is a process used to isolate DNA from cells for downstream applications such as PCR or sequencing. Safety and proper technique are essential, especially when using a centrifuge.

• Centrifuge Safety and Balancing: Always balance tubes by weight before centrifuging to prevent damage or accidents.

• Centrifuge Purpose: Separates components based on density; DNA is often found in the supernatant after cell debris is pelleted.

• Supernatant vs Pellet: Supernatant is the liquid above the pellet after centrifugation; Pellet is the solid material at the bottom.

Key Materials and Functions for Strawberry DNA Extraction

• Strawberries: Provide abundant nuclear DNA due to being octoploid.

• Dish Soap (Detergent): Dissolves cell and nuclear membranes, releasing DNA.

• Salt (NaCl): Neutralizes DNA charge, helps DNA strands clump, and separates proteins.

• Ice-Cold Ethanol/Isopropyl Alcohol: Precipitates DNA, making it visible as white fibers.

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

• Filter: Removes cell debris, allowing DNA-containing solution to pass through.

• Stirrer: Collects precipitated DNA.

Comparison to Other Methods: Bead bashing physically disrupts cells; DNA wash removes contaminants; column filtration binds DNA for purification.

Additional info: DNA extraction protocols may vary depending on the sample type and desired purity.

CH 5: Microbial Metabolism

Catabolism and Anabolism

Microbial metabolism consists of two main types of reactions: catabolic and anabolic. These reactions are essential for energy production and biosynthesis in cells.

• Catabolism: Breakdown of complex molecules into simpler ones; releases energy (exergonic, hydrolytic reactions).

• Anabolism: Synthesis of complex molecules from simpler ones; consumes energy (endergonic, dehydration synthesis).

• ATP: Catabolic reactions release ATP; anabolic reactions consume ATP.

Enzymes

Enzymes are biological catalysts that speed up chemical reactions without being consumed. They are crucial in metabolic pathways.

• Definition: Proteins (usually ending in -ase) that lower activation energy.

• Pathways: Enzymes facilitate sequential reactions in metabolic pathways.

• Substrate: The molecule upon which an enzyme acts.

• Active Site: Region of enzyme where substrate binds.

• Products: Common products include ATP, CO2, water, acids, alcohols.

Factors Affecting Enzyme Activity

• pH: Extreme pH can denature enzymes.

• Temperature: High temperatures can denature; low temperatures slow activity.

• Substrate Concentration: Higher concentration increases rate up to a point.

• Inhibitors: Competitive and noncompetitive inhibitors reduce enzyme activity.

Additional info: Enzyme activity is often regulated by feedback inhibition in metabolic pathways.

CH 8: Microbial Genetics

Central Dogma and -omics Analyses

The central dogma describes the flow of genetic information: DNA → RNA → Protein. Each step can be analyzed using specific '-omics' approaches.

• Genomics: Study of DNA (genome).

• Transcriptomics: Study of RNA transcripts.

• Proteomics: Study of proteins.

• Mutations: Changes at any step can impact subsequent steps and cellular function.

Transcription and Gene Expression

• Transcription: Process of copying DNA into RNA; essential for gene expression.

• Constitutively Expressed: Genes that are always transcribed, regardless of environmental conditions.

Operons and Regulation

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

• Inducible Operon: Usually 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 region where repressor binds.

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

Enzymes in Genetic Processes

• DNA Replication: DNA polymerase, helicase, ligase.

• Transcription: RNA polymerase.

• Translation: Ribosomes, tRNA, aminoacyl-tRNA synthetase.

RNA Differences: Prokaryotes vs Eukaryotes

• Prokaryotes: mRNA is often polycistronic; no introns; transcription and translation are coupled.

• Eukaryotes: mRNA is monocistronic; contains introns; transcription and translation are separated.

Ribosomes, Amino Acids, and Codons

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

• Amino Acids: Building blocks of proteins.

• Codon: Three-nucleotide sequence in mRNA specifying an amino acid; not all codons are the same (some code for the same amino acid, some are stop codons).

Gene Transfer and Mobile Genetic Elements

• Vertical Gene Transfer: Parent to offspring.

• Horizontal Gene Transfer: Between organisms (conjugation, transformation, transduction).

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

Plasmids

• Conjugative Plasmids: Carry genes for transfer (e.g., F factor).

• R Factors: Carry antibiotic resistance genes.

Transposons

• Function: Move within genome; can disrupt genes.

• Pros: Genetic diversity.

• Cons: Can cause mutations.

Phages

• Lytic Phase: Virus replicates and lyses host cell.

• Lysogenic Phase: Viral DNA integrates into host genome; can remain dormant.

Gene Transfer Mechanisms

• Conjugation: Direct transfer via pilus.

• Transformation: Uptake of naked DNA (Griffith's experiment demonstrated this).

• Transduction: Transfer via bacteriophage.

Additional info: Horizontal gene transfer is a major driver of microbial evolution and antibiotic resistance.

CH 9: Biotechnology and DNA Technology

Vector and Biotechnology

Biotechnology uses living organisms or their systems to develop products. Vectors are DNA molecules used to carry foreign genetic material into a host cell.

• Vector: Plasmid or virus used to transfer DNA.

• Biotechnology: Application of biological systems for industrial, medical, or agricultural purposes.

rDNA Technology and Genetic Modification

• rDNA Technology: Recombinant DNA methods to modify genetic material.

• Common Products: Insulin, growth hormones, genetically modified crops.

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

PCR (Polymerase Chain Reaction)

PCR is a technique to amplify DNA segments. It uses cycles of heating and cooling to denature DNA, anneal primers, and extend new strands.

• Heating: Denatures DNA (separates strands).

• Cooling: Allows primers to bind and DNA polymerase to synthesize new DNA.

Cloning

• Definition: Making identical copies of DNA, cells, or organisms.

• Applications: Gene analysis, protein production, therapeutic uses.

DNA Insertion Methods

• Transformation: Uptake of DNA by cells.

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

• Protoplast Fusion: Fusion of cells without cell walls.

• Microinjection: Direct injection of DNA into cells.

Sequencing Methods

• Amplicon Sequencing: Targets specific DNA regions; useful for microbial identification.

• Shotgun Sequencing: Randomly sequences DNA fragments; reconstructs whole genomes.

• Nanotechnology: Used to improve sequencing speed and accuracy.

Additional info: Modern biotechnology relies heavily on molecular tools for genetic engineering and analysis.