Lab 5 Bacterial Transformation Study Notes

Lab 5: Bacterial Transformation

Introduction

  • Overview of living organisms:
    • Living organisms organize themselves uniquely.
    • Blueprint of this organization is transferred to offspring.
    • Cells are the smallest functional units capable of independent reproduction.
    • Bacteria can survive as single cells.
    • Inside each cell, molecules work in concert to perform various functions.

Cell Culture

  • Definition: The process of harvesting cells from natural locations and growing them under controlled laboratory conditions.
  • Requirements for cell growth:
    • Appropriate nutrients and environmental conditions must be provided.
  • Ease of cultivation:
    • Bacteria and yeast are easy to culture.
    • Cells from plants, insects, and animals are more challenging to maintain.
  • Post-culture, cells can be harvested and analyzed.

Cellular Molecules

  • Types of molecules:
    • Each type of molecule performs a specific function.
    • DNA: Stores information similar to a computer's hard drive.
    • Proteins: Act as the workhorses of the cell.
  • Example Method: Cloning a population from a specific cell type, breaking open cells, and sorting contents.
  • Purification techniques:
    • Distinguishing between proteins and DNA easily.
    • Each protein has specific physical and chemical properties allowing for separation based on size, charge, or hydrophobicity.

Special Molecules in Cells

  • DNA:
    • Functions as the universal template for biological information.
    • Composition:
    • Long chains made up of repeating units—nucleotides.
    • Each nucleotide contains one of four bases: Adenine (A), Thymine (T), Cytosine (C), Guanine (G).
    • Orientation of strands:
    • Joined head-to-tail, with a chemical backbone and protruding bases along the side, read from the 5' end to the 3' end.
  • Complementary base pairing:
    • A pairs with T and C pairs with G.
    • Details about double-stranded DNA structures:
    • Must run in opposite directions to pair.
    • Form a double helix structure, often coiled like a spring.

DNA Replication

  • Process of unwinding and “unzipping” the double helix.
  • Enzyme: DNA polymerase synthesizes new complementary strands, resulting in two identical daughter molecules.

Proteins and RNA: The Workhorses of the Cell

  • RNA (Ribonucleic Acid):

    • Composed of four bases: A, G, C, U (Uracil); differs from DNA where A pairs with U instead of T.
    • Structural differences:
    • Generally single-stranded, with ribose sugar in the backbone.
    • Shorter than DNA, as each RNA is a transcript of a short DNA segment.
    • Process of transcription performed by RNA polymerase.

  • Proteins (Polypeptides):

    • Diverse structures due to 20 variations of amino acids used as building blocks.
    • The structure determines functionality:
    • Functions of proteins include:
      • Acting as enzymes (catalysts in chemical reactions).
      • Carrying signals within cells (e.g., hormones).
      • Serving as antibodies against foreign intruders.
      • Acting as structural components within cells.
      • Regulating cellular activities.

Linear Code and Three-Dimensional Outcomes

  • DNA contains linear information used to code traits:
    • The primary transfer of information: DNA > RNA > PROTEIN > TRAIT.
  • Gene Definition: A segment of DNA singled out for copying into RNA.
    • Gene variation in traits and expressions:
    • Genes can govern multiple traits and vary in length.
    • Expression levels of genes may differ based on cell type; cells express only the genes necessary for their functions (e.g., liver vs. skin cells).

Genetic Transformation

  • Definition: Inserting new DNA into organisms (e.g., E. coli).
    • Bacteria often contain one large chromosome and additional small circular DNA pieces called plasmids.
    • Use of plasmids in genetic engineering for trait insertion (e.g., pGLO plasmid contains GFP gene for green fluorescent protein and a gene for antibiotic resistance).

Transformation Process Steps

  1. Competency of Cells: Use E. coli with calcium chloride to facilitate transformation.
  2. Heat Shock: Rapidly increased temperature to enable plasmid entry into cells.
  3. Nutrient Provision: After transformation, incubate to express acquired genes.

Molecular Tools Used in Transformation

  • Restriction Enzymes: Proteins cutting DNA at specific sequences (e.g., BamHI).
  • DNA Ligase: Joins cut DNA pieces together to create recombinant DNA.
  • Plasmids: Circular DNA that supports gene cloning and can carry antibiotic resistance genes for selection purposes.

Overview of Genetic Engineering Techniques

  • DNA Libraries: Cloned DNA fragments from cells for screening desired genes.
  • Gene Regulation: Control expression of genes based on environmental conditions (e.g., digesting arabinose in E. coli only when present).
Arabinose Operon Example
  • The operon cluster includes three genes (araA, araB, araD) under a single promoter and regulated by araC protein, facilitating transcription in arabinose presence.
  • Outcome of arabinose involvement in gene transcription leads to enzymes for arabinose breakdown.

Practical Exercises in the Lab

  1. Labeling Tubes and Pipetting Competent Cells.
  2. Procedure for Using Plasmids in Transformations - outlined with specific steps for experimental design.
  3. Data Collection and Analysis: Observation of transformation effects under visible and UV light and interpretations of experimental results.

Measuring Transformation Efficiency

  • Formula for calculating efficiency: Transformation Efficiency=Total number of colonies growing on the agar plateAmount of DNA spread on the agar plate (in ug)Transformation\,Efficiency = \frac{Total\,number\,of\,colonies\,growing\,on\,the\,agar\,plate}{Amount\,of\,DNA\,spread\,on\,the\,agar\,plate\,(in\,ug)}
    • Key to calculating total amount of plasmid DNA and determining transformation success rates.

Historical Context of Biotechnology and Transformation

  • Highlighting pivotal moments in the timeline of genetic transformation:
    • 1928: Frederick Griffith's transformation experiments.
    • 1944: Oswald Avery proved DNA as the transforming material.
    • 1973: Paul Berg created the first recombinant DNA molecule.
    • 1980 onwards: Major advancements in gene therapy, cloning, and the understanding of genetic regulation.

Glossary of Terms

  • Agar: Gelatinous medium for bacterial growth.
  • Antibiotic Selection: Using antibiotics to select for genetically modified organisms.
  • Beta-Lactamase: Protein providing antibiotic resistance, produced by modified organisms.
  • Recombinant DNA Technology: Process to manipulate genetic material by joining fragments of DNA.