BIOL 3080 Lecture 5

Plasmids and Their Applications in Genetics

Genetic Transformation

  • Definition: The insertion of a gene into an organism.

  • Uses:

    • Agriculture: Genes coding for resistance against frost, pests, or spoilage can be introduced into plants.

    • Bioremediation: Bacteria can be genetically transformed with genes enabling them to digest oil spills.

    • Medicine: Diseases caused by defective genes can be treated by gene therapy.

  • Vectors: A manipulated plasmid that acts as a tool for transformation.

Characteristics of Plasmids

  • Bacterial Chromosome Plasmids:

    • Structure: Small, circular, double-stranded, extrachromosomal elements found in some strains of bacteria.

    • Size Range: 1 kb to >200 kb.

    • Copy Number: The number of plasmids per cell can range from 1 to >1000.

Plasmid Topology and Forms

  • Representation: Plasmids are usually drawn as a double circle to represent the two DNA strands.

  • Under Super-Helical Tension:

    • Plasmids are closed circular double-stranded DNA molecules that can exist in various forms:

    • Negative supercoiling

    • Linear form

    • Nicked circular form

    • Concatamers.

  • Migration: Different topological forms of plasmid DNA migrate at different rates on an agarose gel, although all forms have the same molecular weight.

Genes Encoded on Plasmid DNA

  • Essential Genes:

    • Genes required for its own replication and propagation, including:

    • Origin of Replication (ori)

    • Partitioning Gene: For segregating plasmids into daughter cells during division.

    • Advantageous Genes: Often include genes that provide benefits to the bacterial host, e.g., the ampicillin resistance gene (AmpR), which encodes the enzyme b-lactamase that inactivates ampicillin.

Development of Plasmids as Cloning Vectors

  • Key Features:

    • Selectable Marker: An antibiotic-resistant gene allowing only bacteria containing the plasmid to survive in the presence of the antibiotic.

    • Multiple Cloning Site (MCS): A region with various unique restriction enzyme sites for cloning DNA segments.

    • Regulatory Signals: Elements that regulate the expression of the cloned gene, including promoter sequences for gene transcription.

pGLO Plasmid

  • Key Components:

    • Green Fluorescent Protein gene (GFP): Isolated from the bioluminescent jellyfish Aequorea victoria.

    • Promoter Specificity: Induced by the sugar arabinose.

    • Ampicillin Resistance: Contains genes that confer resistance to ampicillin.

DNA Cloning Procedure

  • Process Overview:

    • Isolate the DNA fragment containing the gene of interest.

    • Insert the fragment into a plasmid to create a recombinant DNA molecule.

    • Introduce this recombinant DNA into a host cell, commonly E. coli.

    • Plate the transformed bacteria under selection conditions to allow only the intended cells to grow, forming colonies.

Bacterial Cloning Process

  • Integration Steps:

    • Cleavage with Restriction Nuclease: The DNA fragment to be cloned is cut with a restriction enzyme.

    • Covalent Linkage via DNA Ligase: Links the DNA fragments to form recombinant DNA.

Transformation Mechanisms

  • Methods of DNA Introduction into Bacteria:

    • Transformation: Nonpathogenic recipient cells take up free DNA.

    • Transduction: DNA incorporated by a DNA-containing virus (bacteriophage).

    • Conjugation: Natural plasmid transfer between bacterial cells.

Laboratory Techniques for Introducing Foreign DNA

  • Calcium Chloride (CaCl2) and Heat-Shock:

    • A commonly used method but with low efficiency (105 to 107 colonies/mg DNA). Bacterial cells are treated with cold CaCl2, DNA is added, heat-shocked, and then allowed to recover.

  • Electroporation:

    • High transformation efficiencies (108 to 109 colonies/mg DNA). Uses short pulses of current to create temporary pores in the cell membrane.

  • Lipofection:

    • DNA is encapsulated in liposomes for fusion with cell membranes; however, it is more expensive and seldom used with bacterial cells.

  • Microinjection, Ballistic/Gene Gun:

    • Techniques not typically used for bacterial transfections.

Restriction Enzymes (RE)

  • Functionality:

    • Restriction enzymes cut dsDNA at specific sequences, which helps prevent the invasion of DNA from foreign sources.

  • Naming Convention: Based on their origin and order of discovery, e.g.:

    • BamHI: Bacillus amyloliquefaciens H, first identified.

    • EcoRV: Escherichia coli RY13, fifth identified.

    • HaeII: Haemophilus aegypticus, second identified.

Restriction Enzyme Cut Sites

  • Characteristics:

    • Recognize and cut specific DNA usually 4 to 8 bases long, palindromic sequences.

  • Examples:

    • BamHI: Cuts at GGATCC.

    • EcoRV: Cuts at GATATC.

    • HaeII: Cuts at RGCGCY.

Alternative Restriction Enzymes

  • Isoschizomers: Recognize and cut the same recognition site.

  • Neoschizomers: Cleave at different positions despite recognizing the same site.

Probability of Restriction Enzyme Occurrence

  • The probability of a restriction enzyme occurrence is determined by:

    • The number of possible nucleotides (4) at any position.

    • Length of RE recognition site (n): rac{1}{4^n}. For example:

    • For a 6 bp site: rac{1}{4^6}.

    • For a 4 bp site: rac{1}{4^4}.

Restriction Enzyme Activity

  • Activity influenced by:

    • Buffer Composition: pH and ionic strength impact function.

    • Incubation Temperature: Most efficient around 37^{ ext{o}}C.

    • Star Activity: Conditions leading to non-canonical cleaving sequences due to excessive enzyme amounts, improper buffers, or prolonged reactions.

Restriction Mapping

  • Linear Mapping: Involves characterizing DNA by the location of restriction enzyme sites by analyzing fragments created from digestion.

  • Circular Mapping: Involves the same principle adapted for circular DNA structures, ensuring accurate representation of fragment sizes and positions.

Southern Blotting Technique

  • Steps Involved:

    1. Isolate genomic DNA.

    2. Cut DNA with restriction enzymes.

    3. Separate fragments via gel electrophoresis.

    4. Transfer denatured ssDNA to a nitrocellulose membrane.

    5. Hybridize with a labeled probe complementary to the target gene.

    6. Visualize results by exposure to X-ray film.

Northern Blotting Technique

  • Similar to Southern Blotting but involves:

    • Harvesting total RNA and using a labeled probe that hybridizes to the RNA on the membrane, with darker bands indicating higher abundance.

RNA Interference (RNAi)

  • Functionality: Targets mRNA for degradation or degradation of target RNA, employing processes such as "dicing" and "slicing."

  • Main Components: Argonaute proteins, dsRNA for specific target information.

CRISPR Technology

  • Components: Bacterial defense using crRNAs and Cas proteins to recognize and cleave invading viral DNA sequences.

  • Applications: DNA editing, creation of transgenic organisms by repairing or modifying specific genes.