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Slide Set by Austin Davies, created more than 1 year ago

Lecture 9

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	Created by Austin Davies over 9 years ago

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Genetic mapping by conjugation

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An F plasmid is inserted into a bacterial chromosome by homologous recombination at an insertion sequence (of which there are plenty) - thus creating an HFR strain of bacteria.

Each of these HFR strains have their own unique OriT loci and direction of transfer - depending on the orientation of the IS (remember there are many to choose form).

Nature of HFR formation.

Once we have an HFR strain of bacteria, it will undergo conjugation (initiated by its OriT contained within the F factor). As seen in the diagram, based on the orientation of the OriT, whats adjacent to it will be transfered last and that following the OriT will be transferred first.

There will be different frequencies of transferred genes; this is due to the fragile nature of the mating pore through which conjugation is mediated. The longer conjugation is in effect, the more likely the mating pore is to break and bring gene transfer to a halt. Therefore we can map genes (from the OriT) based on their frequencies of transfer - this is named a gradient of transfer.

Complete HFR chromosome, showing directionality of transfer.

Experimentally, having an HRF strain with known selectable mutations, we can induce mating between the HFR and WT strain, only to interrupt this mating at a series of timer intervals. The selectable markers can be tested for at each interval and therefore can be plotted in frequency against time.
The most frequent and earliest presented markers are closest to the Ori. As frequencies decrease, we assume the genes become further displaced from the OriT.

Each HFR strain can be sequenced like this, and then a full map can be constructed based on the results:

Conjugation Map