Bacterial Genetics: Competency, Plasmids, PCR, and Electrophoresis

Posted on Mar 9, 2025 in Biotechnology

Bacterial Genetics

  • Explain Competency

    • The physical state of a bacterial cell which allows nucleotide bases in primers to anneal to the complementary base present in a DNA template.

  • Describe the Transformation Experiment

    • The uptake of free-floating DNA from the environment by bacterial cells.

  • Characteristics of a Plasmid: pGLO Example

    • Plasmids carry full-length genes and, therefore, can introduce new genotypes into bacterial cells, which can cause new phenotypes.

  • Function of Buffers in Plasmid DNA Isolation

    • An electrically conductive solution.

  • Components Needed for a PCR Reaction

    • 1. DNA template
    • 2. Primers (two per sequence of interest)
    • 3. Nucleotides—dNTPs (Guanine, Adenine, Thymine, Cytosine)
    • 4. DNA polymerase
    • 5. Thermal cycler machine

  • Three Steps in a PCR Cycle

    • Denaturation occurs at 94 °C when the hydrogen bonds in the DNA double helix come apart, resulting in single-stranded DNA molecules. Annealing occurs at lower temperatures (45-65 °C) when the primers base pair with complementary sequences on the DNA molecules. Extension occurs at 72 °C when the thermostable DNA polymerase binds to the 3’ ends of the primers and copies the DNA. Every cycle results in a doubling of the DNA sequence of interest.

  • How PCR Cycles Create Copies of Target DNA

  • Theory of Agarose DNA Gel Electrophoresis

    • DNA gel electrophoresis separates DNA fragments by size and charge. The gel is made of a gelatin-like substance, either agarose (a highly purified form of agar).
    • DNA is negatively charged, so the fragments move towards positive electrodes.

  • Purpose of SYBR® Green in Agarose Gel Electrophoresis

  • Competency and DNA Insertion into Bacteria

    • Competence can also be induced in a laboratory setting by cooling bacterial cells on ice and treating them with calcium chloride followed by a short heat shock. This method is widely used to introduce plasmids into bacterial cells.

  • Why Put DNA into Bacteria?

    • Genetic engineering can be accomplished by transforming bacteria with plasmids carrying genes of interest. For example, interleukin-2, a protein used in the treatment of metastatic melanoma, is produced by transforming bacterial cells with a plasmid carrying the human interleukin-2 gene.

  • How to Tell if DNA is in the Bacteria

    • A selectable marker like an antibiotic resistance gene must be present in order to directly select for transformed cells.

  • What is Ampicillin and Why Was it Used?

    • pGLO contains an ampicillin resistance gene, so ampicillin was added to the media in the Petri plates in order to select for ampicillin resistant cells. Any colonies that grew on the media containing ampicillin began with a single cell that was transformed with the pGLO plasmid.

  • What is Arabinose and Why Was it Used?

    • In addition, the plasmid pGLO contains the GFP gene under control of the arabinose operon promoter. When the sugar, arabinose, is present in the media, the arabinose repressor is inactivated, which allows expression of arabinose operon genes. Therefore, in the presence of arabinose, the GFP gene is expressed, which results in green fluorescent colonies when UV light is shown on the plate.