Bacterial Conjugation 2v3e3e

Bacterial Conjugation Instructor: Dr. Amirul Mallick

PRINCIPLE: Transfer of genetic material from one bacteria strain (donor) to another strain (recipients) is common event that occurs in nature with the objective of mixing the gene pool , in otherwise asexually reproducing organisms.  Conjugation is the most widespread process of transferring genetic materials from one bacteria cell to another .  Unidirectional transfer DNA is mediated and require cell to cell . It was discovered by Lederberg and Tatum In 1946 .  Conjugation is best understood by considering properties of the “F” factor , which is a small circular plasmid DNA that can replicate autonomously in the cell or can integrate in the host chromosome  When individual cell with an integrated “F” are isolated an allowed to form pure colonies , the resulting strain can transfer chromosomal markers at very high frequency and are termed Hfr strain. Hfr stands for "high frequency of recombination".

Lederberg & Tatum (1946) Experiment demonstrating recombination in E. coli. Recombination of 2 complimentary auxotrophs gives rise to a strain that can synthesize all nutrients.

Bernard Davis experiment demonstrated that physical is required for bacterial recombination.

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E. coli has been the model organism in conjugation studies. Presence of an extrachromosomal genetic element (a plasmid called the F factor): mediates certain events such as the synthesis of a pilus (the F or sex pilus), and the rapid infection of all susceptible bacteria with this F factor. The pilus has been shown to facilitate attachment of the male (or F+ cells) to the female (or F- cells) which do not contain the F factor.

Conjugation-transfer of the sex factor F: 1.

William Hayes (1953) demonstrated that genetic exchange in E. coli occurs in only one direction.

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Genetic transfer is mediated by sex factor F.

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Donor is F+ and recipient is F-.

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F is a self-replicating, circular DNA plasmid (1/40 the size of the main chromosome).

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F plasmid contains an origin sequence (O), which initiates DNA transfer. It also contains genes for hairlike cell surface (F-pili or sex-pili), which aid in between cells. 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.

Transfer of the F factor

Inoculate 1ml of overnight donor culture into 25ml of LB broth( +tetracycline 30µg/ml.

Inoculate overnight recipient culture into 25ml of LB broth (+ streptomycin 100µg/ml Incubate at 37°C in a shaker

Grow recipient and donor culture upto O.D. reaches 0.8-0.9 at 600nm. Gently mix 0.2ml each recipient and donor cultures and incubate in an incubator at 37°C for 90min. Add 2.0ml of sterile LB into each tube Incubate the tubes at 37°C for another 90min Spread plate 0.1ml of each of the samples (donor, recipient, conjugated sample)