e 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:[(Recipient/ml)/(donor/ml)] * 100.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. Figure 1 - MM56 ComponentsChemicalAmount in 1L of MM56Na2HPO4 (0.1M)611 mlKH2PO4 (0.1M)384 mlMgSO4o7...
