The purpose of this experiment was to study the transfer of genetic information on plasmid F’lac by using Escherichia coli. Plasmid transfer was measured by using two different methods. The first one was by using selection and contraselection with three antibiotics: streptomycin(which was replaced by naladixic acid for the second part of the experiment),ampicillin and kanamycin and the second one by using a colour indicator ( X-gal). As significant results, the percentage of transfer for F’lac was higher than the percentage for transposition. Also, the experiment demonstrated that E.coli can quickly acquire resistance to several different antibiotics through the transfer of the F’lac plasmid. It was concluded that significant changes on the genetic makeup can be achieved through transposition and conjugative plasmids in a short amount of time, which can have severe implication on the effectiveness of antibiotics for bacterial diseases.
The purpose of this experiment is to study the transfer of genetic information on plasmid F’lac by using the bacteria Escherichia coli. The genome of the bacteria consists of a single circular DNA molecule. Many bacteria also contain plasmids which assist on the transfer of genetic material, in E.coli it is called the fertility factor (F). The donor bacteria containing the F factor are designated F+, whereas the recipient, which usually lacks the F factor are designated F- (Becker et al). Bacterial conjugation enables the horizontal transfer of plasmids through appendages formed by the F+ called sex pili, which creates the bridges necessary between the donor and the recipient, in order for the process to occur (Bates et al, 1998). Sometimes transposition can occur with conjugation, this happens when insertion sequences shift from the bacterial chromosome to the plasmid so they can be transferred from the donor to the recipient. (Griffiths et al, 2008) In the first part of the experiment, the donor contains the lac operon , and Tn1 transposon while its bacterial chromosome contains the Tn5 transposon. The lac operon controls lactose metabolism in E.coli and is marked by the IPTG/X-gal colour indicator. According to this, the hypothesis is that the percentage of transfer of f’lac is going to be greater than the percentage of transposition. The Tn1 transposon is marked by the antibiotic ampicilin (Ap), while the Tn5 transposon is marked by kanamycin (Km). The recipient is resistant to streptomycin (Sm). For the second part, the donor will be a former exconjugant, resistant to streptomycin that will the contain the lac operon and both transposons Tn1 and Tn5.The recipient will not contain F’ plasmid and will be resistant to naladixic acid (Na). The hypotheses for this second part will be that the 2 transposons will give resistance to two antibiotics so the percentage of transfer will be higher than on the first part of the experiment. Materials and Methods
The following sterile transfer techniques were used (please refer to the listed page numbers in the lab manual for more information) : Culture flask to test tube (pg 5)
Test tube to test tube (pg. 5)
Test tube to petri dish (pg 6)
The protocol used in the first part of the experiment is found on pages 7 - 8 of the Bacterial Genetics 1 lab in the lab manual. The protocol was followed verbatim. The protocol used in the second part of the experiment is found on pages 5 – 6 of the Bacterial Genetics 2 lab in the lab manual. The protocol was followed verbatim. Results
Tables 1 and 2 show the results of the first part of the experiment while Tables 3 and 4 summarize the results of the second part of the experiment. Table 1 summarizes the bacterial counts from the first part of the experiment. As we can notice, plate # 2 contains the least amount of bacterial colonies as it is selected for the three antibiotics streptomycin, kanamycin, and ampicillin. The bacterial colonies in plate # 3...
References: 3. Bates, S; Cashmore, AM and Wilkins, BM. 1998. IncP Plasmids Are Unusually effective in mediating Conjugation of Escherichia coli and Saccharomyces cerevisiae: Involvement of the Tra2 Mating System. Journal of Bacteriology. 180:6538-6540.
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