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 37C. 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 of the 10-7 dilution of the donor. Two Luria plates were inoculated with 1 ml each of the 10-8 dilution of the donor. Two complete MM56 plates were inoculated with 1 ml each of the 10-7 dilution of the recipient. Two complete MM56 plates were inoculated with 1 ml each of the 10-8 dilution of the recipient. One complete MM56 plate was inoculated with 1 ml of the 10-2 dilution of the donor. Each of these were inoculated for 48 hours.
Conjugation: 30 sterile test tubes were filled with 9 ml each of sterile saline solution. A supply of sterile top agar was kept in a water bath to keep liquid until needed. One ml of the donor culture was added to 20 ml of the recipient culture in a flask. This mating mix was kept in the 37C water bath. Immediately (0 minutes), 1 ml of the mating mix was removed from the flask and added to 9 ml of sterile saline, making a 10-1 dilution. This was placed on the vortex for 60 seconds to interrupt any mating taking place in the mix. 1 ml of this mixture was added to another tube of 9 ml sterile saline, making a 10-2 dilution and this solution was vortexed for one minute. This was repeated twice more to produce a 10-3 and a 10-4 dilution. For each dilution, three tubes of top agar were inoculated with 1 ml of that dilution. One each of the agar-dilution mixtures were poured onto a threonine deficient plate, a proline deficient plate, and a histadine deficient plate. This procedure was repeated every ten minutes for sixty minutes. The top agar was allowed to harden and the plates were incubated at 37C for 48 hours.
RESULTS: After incubation, the plates were removed and colony counts were performed (figure 3). The time of entry of each gene was calculated, using this formula:
The calculated times of entry are found in figures 4 and 5. From this information, a map of E.coli K12 was constructed (figure 6).
DISCUSSION: The time of entry of the amino acid threonine was found to be consistent with that found in the lecture handout “Bacterial Gene Transfer – Conjugation”. The time of entry for proline (10 minutes) was longer that the handout value of about 5 minutes. The actual time for proline might have been 5 minutes, but the samples were taken at 10 minute intervals. The time of entry for histadine (10 minutes) differed drastically than the handout time of about 44 minutes. These discrepancies could be caused by a variety of errors. The cultures could have been contaminated. The media could have been prepared incorrectly. The wrong amino acids could have been added or the plates could have been labeled incorrectly. The amount of time spent vortexing the mixes (in order to separate the mating pairs) might have been insufficient. Also, the dilutions were placed on the vortex before making each dilution, instead of only after taking the sample directly from the flask in the water bath.
ReagentStock concentrationFinal concentrationCalculated
Streptomycin50 mg/ml200:g/ml2.4 ml
DL- Threonine1% in H2O10 ml/L6 ml
Vitamin B10.1% in H2O0.2 ml/L1.2 ml
Figure 4 – Time of entry table (percentages of recombinants)
Figure 5 – Time of entry graph (percentages of recombinants)