Bacterial conjugation experiment
Bacterial conjugation experimentBACTERIAL CONJUGATION EXPERIMENT
INTRODUCTION: Bacteria, in general, reproduce asexually, but in order to increase diversity, they have developed a mechanism for transfer of genetic material from one bacterium to another. The ability to perform this transfer is conferred by a set of genes which are called F for 'fertility.' These genes exist on a small, circular piece of deoxyribonucleic acid (DNA) that replicates independently from the bacterial chromosome, or they can be integrated into the chromosome. The bacterium containing this gene (sometimes referred to as 'male' or F+) extends its pilus to a neighboring bacterium (sometimes referred to as 'female' or F-), and the two cells are attached. This process is called conjugation. The third manifestation of the F factor is Hfr, which is the term for the F element becoming integrated into the genome. When conjugation occurs, the F genes start travelling across the pilus, bringing the remainder of the genome behind it. Most often, the entire genome isn't transferred. The bacterial genome that is delivered can be measured in minutes from the origin of transfer. That is, the amount of time it takes for a particular gene to be transferred from one bacterium to another indicated how far it is from the origin of replication.
METHODS AND MATERIALS: Media Preparation: The starting material was Medium 56-glucose agar (MM560). The components of the MM56 are found in figure 1. From this, we made two types of media, complete and selective. The reagents used, along with their stock and final concentrations are found in figure 2. The formula:
Stock volume = [(Final concentration)(final volume)] / (stock concentration)
was used to calculate the amounts of each reagent added to the complete and selective media. (The final volume was 600 ml.) These values are also found in figure 2. To make the complete media, appropriate amounts of each amino acid, glucose, vitamin B1, and streptomycin were aseptically added to the MM56 media and poured into sterile petri plates. The selective media was prepared so that 84 plates were made. These plates were made much in the same way as the complete plates, except that 28 of the plates contained all of the reagents except proline, another 28 were without histadine, and another 28 were without threonine. These were labeled with "pro-", "his-", and "thr-".
Viable Counts: The strain used was Escherichia coli K12. The donor and recipient cultures were kept as much as possible in a water bath at 37�C. Viable counts of the donor (Hfr) and recipient were done on complete MM56 and Luria agar. Serial dilutions were performed to obtain a 10-7 and 10-8 dilutions of the recipient, and 10-2, 10-7, and 10-8 dilutions of the donor. Two Luria plates were inoculated with 1 ml each of the 10-7 dilution of the recipient. Two Luria plates were inoculated with 1 ml each of the 10-8 dilution of the recipient. Two Luria plates were inoculated with 1 ml each...
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