Biotechniques: Biosafety Overview

Biotechniques (BMS 34010A)

• Course offered during Fall semester 2023-2024
• Instructor: Dr. Tania Tahtouh
• Email: [email protected]

Basic Principles: Biosafety in the Laboratory

• Laboratory settings present potential hazards.
• Safety procedures protect personnel and equipment.
• Disposal of contaminated materials/chemicals follows strict protocols.

Potential Laboratory Hazards

• Corrosive chemicals, sharp tools/glass, and open flames pose risks.
• Bunsen burners require extreme caution.
• Infectious organisms also pose hazards, classified into biosafety levels (BSL 1-4).
• Most undergrad labs use BSL 1 organisms.

Standard Practices in the Laboratory

• Lab coats are essential for protection against contamination.
• Lab coats should be long and not restrict movement.
• Lab coats are not chemical protection suits.
• Gloves should not touch surfaces outside the lab.
• Eating, drinking, chewing gum, or pipetting by mouth are forbidden in the lab.
• No makeup or contact lenses adjustments in the lab.
• Work area must be clean with only necessary materials.
• Cell phones, bags, and other items should be stored away from workbench.
• Avoid leaving active experiments unattended.
• Properly disinfect the workbench before and after each experiment.
• Maintain clean hands and use correct hand washing techniques.
• Unauthorized experiments are forbidden.
• Long hair must be tied up, and loose clothes must be secured when working with open flames.
• Safety glasses or face shields required when working with UV light or chemicals.
• Closed-toe shoes are mandatory to avoid chemical spills and sharp objects.
• Know location of safety equipment: eyewash, chemical shower, first-aid kit, and fire extinguisher.
• Follow chemical hygiene plans and use fume hoods/biosafety cabinets for chemical handling.
• Report spills, accidents, or injuries immediately.

Biohazard Bin

• Biohazardous waste goes in appropriately labeled bins/bags.
• Non-reusable items with contact to infectious material, body fluids, or animal tissue.
• Handled gloves must also be disposed of.
• Items can be autoclaved before disposal.

Sharps Containers & Trash Cans

• Sharps containers for broken glass, needles, used scalpels, glass pipettes, and other sharp objects.
• Non-contaminated items (paper, etc.) go in regular trash cans.

Reusable Materials

• Materials needing sterilization: autoclave after use (e.g., culture tubes and instruments).
• Materials not needing sterilization: wash in sink after use (e.g., dissection tools and glassware).

General Rules for Handling Chemicals

• Keep containers closed.
• Never handle wrong or unmarked reagents.
• Avoid carrying bottles by the neck or against the body.
• Labels should face up when pouring.
• Only place indicated chemical into bottle.
• Handle concentrated acids with caution (add acid to water and stir continuously).
• Disposal of some chemicals requires special procedures.

The Importance of Hand Washing

• Hand washing is crucial in labs, especially microbiology.
• Hands can come into contact with infectious agents, chemicals, stains, and other hazardous materials.
• Wash hands before, after, and whenever contact with potential hazards occurs.

Steps for Correct Hand Washing Technique

• Remove all jewelry.
• Wet hands and apply soap.
• Rub hands together to make a lather.
• Scrub for 20 seconds (e.g., "Happy Birthday" twice).
• Rinse and dry hands with a clean towel/air dry.

Metric Systems

• The SI system (International System of Units) is the standard used by scientists.
• The United States is the only industrialized nation to continue using the English system.
• SI system is based on the number 10.
• The SI system includes measurement units for length, mass, volume, temperature, and time.

Microscopy: Types and Principles

• Biological specimens often require microscopic visualization.

• Various types of microscopes exist for different applications.

• Types of microscopes include Light (compound, binocular, phase contrast, fluorescent, confocal) and Electron (transmission, scanning).

• Learning Outcomes: Recognize principles/uses of different microscopes, parts of a compound light microscope, total magnification, microscope rules of use, focus methods, field of view, longitudinal/cross sections, and resolution.

• Light Microscopes: Simplest consists of a single glass lens. -Specimen prep minimal. -Focusing by moving specimen relative to lens.

• Compound Light Microscopes: Multiple lenses (condenser, objective, eyepiece).

• Parts of Compound Light Microscope

  • Light source (lamp).
  • Condenser.
  • Objectives.
  • Eyepiece.
  • Stage & Clips.
  • Diaphragm
  • Coarse & Fine Focus Knobs.

-Lens types and Magnification.

• Field of view (at low and high power).

• Longitudinal & cross sections.

• Resolution.

• Scanning and Transmission Electronic Microscopes: Greater magnification to view smaller objects

• Stereomicroscope: Used to observe large specimens' surfaces in 3-D.

• Phase contrast microscopy: Improves contrast for transparent and colorless specimens.

• Fluorescent microscopy: High contrast for specific labeling of cellular components.

• Fluorescents and stains (RFP, GFP, and DAPI).

• Immunofluorescence: Using antibodies labeled with fluorescent dyes.

• Confocal microscopy: Produces optical sections of thick specimens.

• Microscope use rules outlined in slide deck

DNA Cloning Overview

• Inserting the target DNA into the plasmid vector.

Cut and Paste DNA: Plasmids

• Circular
• Self-replicating.
  • Host cell/chromosome separate.

Cut and Paste DNA: Euk expression vectors

-Eukaryotic DNA containing virus restriction sites and/or terminal repeats.

Cut and Paste DNA: Restriction

• Restriction Sites: Specific 4-8 bp.
• Type II Restriction Enzymes: Highly-specific, blunt, or sticky ends.

Cut and Paste DNA: Restriction (Inserting DNA sample into plasmid):

• Enzymes cut DNA at restriction sites, and ligase joins the fragments.

DNA Transfer into Cells (Transformation, Transduction, Transfection)

• Transformation (prokaryotic) - Non-viral method
• Transduction (prokaryotic/eukaryotic) - Viral method
• Transfection (eukaryotic) - Non-viral method

Chromosome Integration

• Integrase splices viral DNA into the cellular chromosome.

Cellular Screening

• Vectors derived from natural plasmids; genetically modified for convenient use, some more advanced than others in structure .

DNA Extraction

• Lysis
• Precipitation
• Purification
• Elution

Extraction of Plasmids

• Separation of plasmid/chromosomal DNA through differential denaturation and neutralization.

DNA Cloning Overview

• Gene amplification and digestion/ligation.
• Transformation & plasmid isolation/sequencing.

Crispr/Cas9

• A genome editing technique in bacteria.
• Creation of specific double-strand breaks at the target locus.
• Types: NHEJ and HR

DNA Synthesis

• Artificial gene synthesis not requiring DNA template.

DNA Array (Microarray)

• Tool for detecting gene expression.

Mutations and Causes

• Spontaneous Mutations: Errors in replication or chemical instability of DNA bases.
• Induced Mutations: Exposure to environmental factors.

Main Types of Mutations

• Germline: Inherited, in gametes.

• Somatic: In other body cells.  

• Point mutations: Substitutions, insertions, or deletions of single bases.

• Chromosomal mutations: Deletions. Duplications. Inversions, insertions, translocations.

• Copy Number Variation (CNV): Changes in the number of tandem copies of a locus.

Point Mutations

• Missense: Different amino acid.
• Nonsense: Premature stop codon.
• Synonymous: Same amino acid.
• Neutral: No effect.

Point Mutations: Categorization

• Loss-of-function: Decreased/absent gene product.
• Gain-of-function: Increased gene product or new activity

Chromosomal Mutations

• Deletion: Loss of chromosome region.
• Duplication: Repetition of chromosome region.
• Inversion: Flipping and reinserting a chromosome region.
• Insertion: Moving a chromosome region to another.
• Translocation: Rearrangement of chromosome regions.

Copy Number Variation (CNV)

• Gene amplification: Increasing tandem copies of a locus.
• Expanding trinucleotide repeat: Expanding repeated sequences.

Mutation Hotspots

• Regions with higher recombination rates or accessibility to mutagens.
• Repetitive sequences are often hotspots.

Mitochondrial DNA Mutations

• Inherited changes can cause growth/development/function problems.

• mtDNA mutations affect mitochondria's energy generation capacity.

• References: Provide links to relevant articles & resources.