Transformation and Conjugation

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Conjugation and Transformation Presented By: Shruti Kashyap M.Tech Biotech

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Genetic recombination involves the physical exchange of genetic material between genetic elements. Homologous recombination results in genetic exchange between homologous DNA sequences from two different sources. This type of recombination is extremely important to all organisms. Genetic Recombination

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Mechanisms of Genetic Exchange Conjugation: Mechanism of transferring genetic information from one bacterium to another, followed by recombination with the recipient bacterium’s genetic material. Transformation: Uptake of DNA from the surrounding medium and recombination into the recipient’s bacterium’s genetic material. Transduction: Transfer of genetic material from one bacterium to another via bacteriophage.

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Discovered by Lederberg and Tatum (1946) Two auxotroph strains (one was met bio and the other thr leu thi ) Culture together on complete medium Subculture on minimal medium Recombinant prototrophic colonies appeared on the minimal medium. Bacterial Conjugation

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Lederberg & Tatum (1946) Experiment demonstrating recombination in E. coli .

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Bernard Davis (1950) experiment demonstrated that physical contact is required for bacterial recombination

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William Hayes (1953) demonstrated that genetic exchange in E. coli occurs in only one direction. Genetic transfer is mediated by sex factor F . Donor is F + and recipient is F - . F is a self-replicating, circular DNA plasmid (1/40 the size of the main chromosome). F plasmid contains an origin sequence ( O ), which initiates DNA transfer. It also contains genes for hair-like cell surface ( F - pili or sex- pili ), which aid in contact between cells. Conjugation-transfer of the sex factor F

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No conjugation can occur between cells of the same mating type. Conjugation begins when the F plasmid is nicked at the origin, and a single strand is transferred using the rolling circle mechanism. When transfer is complete, both cells are F + double-stranded.

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Transfer of F factor Two bacterial cells connected by long, tubular F- pilus

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No chromosomal DNA is transferred by standard sex factor F . Transfer of chromosome DNA is facilitated by special strains of F + integrated into the bacteria chromosome by crossing over. Hfr strains = high frequency recombination strains. Discovered by William Hayes and Luca Cavalli -Sforza. Hfr strains replicate F factor as part of their main chromosome. Conjugation of high-frequency recombinant strains

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Conjugation in Hfr strains begins when F + is nicked at the origin, and F + and bacteria chromosomal DNA are transferred using the rolling circle mechanism. Complete F + sequence (or complete chromosomal DNA) is rarely transferred (1/10,000) because bacteria separate randomly before DNA synthesis completes. Recombinants are produced by crossover of the recipient chromosome and donor DNA containing F + .

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Transfer of the Hfr F + factor

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Bacterial Transformation – Uptake of DNA This technique was used to first demonstrate that DNA was the genetic material and not protein. Griffith in 1928, demonstrated the ‘transformation principle’ Avery, Macleod & McCarty (1944) later demonstrated ‘transformation’ material was nucleic acid.

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Work was done with S. pneumoniae, 2 forms: normal or ‘S’ form (smooth shiny colonies), when injected into mice, it kills them mutant or ‘R’ form (rough colonies), it does NOT kill mice when injected into them.

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Transformation Begins with the uptake of DNA chromosomal fragments from the surrounding media into cells competent for uptake of DNA. The donor DNA then undergoes a physical exchange (recombination) and is incorporated into the host cell Most bacteria are proficient at recombination but very few are competent for uptake of DNA naturally. Even those that are naturally competent, only a small fraction are competent, but this can be enhanced by culturing under certain conditions .

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1. A donor bacterium dies and is degraded 2. A fragment of DNA from the dead donor bacterium binds to DNA binding proteins on the cell wall of a competent, living recipient bacterium

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3. The Rec A protein promotes genetic exchange between a fragment of the donor's DNA and the recipient's DNA 4. Exchange is complete