Cloning a Gene

Overview of Bacterial DNA Types

  • Bacteria possess two primary forms of DNA:

    • Chromosomal DNA

    • A single chromosome is composed of a large DNA molecule.

    • Contains all essential information for the organism's survival and reproduction.

    • Plasmids

    • Small circular DNA molecules ranging from 1,000 to 200,000 base pairs.

    • Present in multiple copies, separate from the chromosomal DNA (up to 500 plasmids per cell).

    • Depicts a bacterial cell with flagella and two forms of DNA:

      • Chromosomal DNA: Occupies the left half in a tangled mass.

      • Plasmid DNA: Shown as small rings on the right.

Characteristics of Plasmids as Vectors

  • Four critical characteristics make plasmids suitable vectors:

    1. Replication

    • Plasmids can autonomously replicate independently of the bacterial chromosome.

    • Contain a specific sequence for DNA synthesis enzyme binding.

    • This sequence is known as the origin of replication (ori) site.

    1. Transcription Initiation

    • Plasmids can initiate transcription, transferring DNA-encoded information to messenger RNA (mRNA).

    • mRNA, an RNA molecule, serves as the template for protein synthesis using the host cell RNA polymerase.

    • RNA: A single-stranded biomolecule consisting of:

      • A nitrogenous base.

      • A ribose sugar.

      • A phosphate group.

    • Promoter: A specific DNA sequence crucial for binding RNA polymerase, initiating transcription. Located next to every gene.

    • For human therapeutic protein genes to be expressed in bacteria, they must be inserted adjacent to the plasmid's promoter.

    1. Antibiotic Resistance Genes

    • Plasmids often carry genes that confer resistance to antibiotics, allowing bacteria to grow despite the presence of antibiotics.

    • These genes produce proteins that inhibit antibiotic actions.

    • This can lead to a selective advantage, enhancing survival in competitive environments.

    1. Horizontal Gene Transfer through Conjugation

    • Plasmids can be transferred between bacterial strains via a process called bacterial conjugation.

    • Conjugation allows for genetic exchange. Bacteria that gain plasmids with resistance genes become resistant.

    • The efficiency of natural conjugation is low, with only a small percentage of bacteria capable of receiving plasmid DNA at any one time.

      • Shows plasmid structure, including:

      • ori: Black segment of the ring.

      • Promoter: Small purple segment (arrow-shaped) adjacent to the ori.

      • Antibiotic-resistant gene: Blue segment also shaped like an arrow.

Application of Plasmids in Gene Cloning

  • Plasmids serve as vectors in gene cloning, enabling:

    • Uptake by bacteria.

    • Replication to produce multiple copies of a gene.

    • Transcription of inserted genes.

    • Containing antibiotic resistance genes for selection purposes.

  • Common genes included in plasmids used in laboratories for cloning:

    • Resistance to ampicillin.

    • Resistance to kanamycin.

  • Mechanisms through which these resistance genes work involve chemical modification of antibiotics, effectively inactivating them.

Restriction Enzymes: Discovery and Function

  • Discovery in the early 1950s: Certain E. coli strains resistant to bacteriophage infections led to the identification of restriction enzymes.

  • Function of Restriction Enzymes:

    • Proteins that restrict bacteriophage growth by recognizing and destroying phage DNA while preserving host DNA.

    • Different bacterial strains have unique restriction enzymes that cut DNA at specific locations known as restriction sites.

  • Understanding restriction enzymes facilitates genetic engineering and cloning practices, providing precise methods for cutting DNA to incorporate foreign genes.

Types of Bacterial DNA

Bacteria contain two types of DNA:

  1. Chromosomal DNA

    • A single, large molecule containing essential survival and reproductive information.

  2. Plasmids

    • Small circular DNA (1,0001,000 to 200,000200,000 base pairs).

    • Replicate independently of the chromosome, with up to 500500 copies per cell.

Key Characteristics of Plasmids

  • Origin of Replication (ori): A DNA sequence that allows plasmids to replicate autonomously.

  • Promoters: DNA sequences where RNA polymerase binds to initiate transcription (copying DNA into mRNA). For a human gene to work in bacteria, it must be placed next to a bacterial promoter.

  • Antibiotic Resistance: Plasmids often carry genes that protect bacteria from antibiotics (e.g., ampicillin or kanamycin), providing a selective survival advantage.

  • Bacterial Conjugation: The process of transferring plasmids between bacteria, allowing for genetic exchange.

Genetic Engineering Applications

  • Vectors: Plasmids are used as vehicles (vectors) to carry foreign genes into bacteria for cloning and protein production.

  • Restriction Enzymes:

    • Discovered in the 1950s1950s as a defense against bacteriophages (viruses).

    • These proteins cut DNA at specific restriction sites.

    • Scientists use them like "scissors" to precisely insert genes into plasmids.