Conjugation and Transposition in Escherichia Coli Essay Example

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 and plate # 4(blue) only differ by approximately 100 comparing to the white that did not go conjugation is not too significant.The trend of increasing number of colonies on plates with a decreasing number of antibiotics is reflected by the counts of both the individual group and the class means.

Table 2 shows the percentage of plasmid transfers which took place between donor and the recipient cells, as well as the frequency of transposition. There is no great difference between the group and class results, both follow the same trend which is that there is a higher percentage on transfer F’lac and a very small on transposition, which correlated with the small percentage of transfer F’lac::Tn5. Table 3 shows the bacterial counts in the second part of the experiment. Plates #5,6 and 7 show similar numbers.

Both the class and group results have the same trend in which plate #5 has the largest bacterial counts and plate #6 the smaller. However, there is a great difference between plate # 1, where the group has 32 bacterial colonies and the class mean has 1114. 4.

Like Table 2, Table 4 summarizes the frequency of conjugation which took place between the donor and recipient cells. The % transfer of Kanamycin resistance which marks for the Tn5 transposon and the % transfer of Ampicillin which marks for Tn1 have similar frequency, being 107% and 111% respectively.

The group results follow the same logic but the group results differ widely from the class, since they are very large numbers. Table 1. Mean and group bacterial colonies counted for E. coli in the four plates containing antibiotics: streptomycin, kanamycin and ampicillin; and indicator X-gal. Standard errors are present.

Selects first recipient cell, contraselects donor Table 4: The frequncy of transfer of Kanamycin resistance (%), frequency of transfer of both ampicillin and kanamycin, and the frequency of ampicillin(%) of the group and class mean for bacteria E. coli. PlateGroupClass Mean . % Transfer Km Resistancea6 x 1030.

107 x 103 % Transfer Ap, Km Resistance4. 88 x 1030. 103 x 103 % Transfer Ap Resistanceb4. 81 x 1030.

111 x 103 a marks for Tn5 transposon b marks for Tn1 transposon Discussion: Experiment 1If we refer to the percentage of the transfer of the f’lac of the class, we could say that the majority of bacterial colonies went through conjugation due to the fact that once the recipient cell is converted into a donor cell, it can conjugate with other recipient cells, converting them into donor cells. If we refer to table 2 we will notice that the percentage of transfer of F’lac (along with tn1 since it was on the plasmid) is greater because conjugation is a common event and can easily happen while the case of transposition

is more difficult to occur since transposon tn5 has to be first shifted the bacterial chromosome to the plasmid.Then the plasmid must be transferred by conjugation to a recipient cell in order fo the recipient celll to ontain both transposons and therefore be resistant to both the kanamycin and the ampicillin antibiotics. The low frequency of conjugation of plasmids with both the Tn1 and the Tn5 transposons is therefore a result of the low frequency of transposition of the Tn5 transposon which took place between the F’lac plasmid and the bacterial chromosome. This conclusion is supported by both the individual group results and the class mean results, where the frequency of transposition was found to be 0. 29 x 10-3 and 0. 077 x 10-3 respectively. Questions 1 and 2: In order to determine whether an E. coli reconjugant cell was resistant to ampicillin, two different methods were used.

The first method involved plating some of the solution containing the donor and recipient cells onto a medium with streptomycin and ampicillin, where 33 bacterial colonies were counted according to the group results while in the class 254. 2 was the mean (plate # 3). The second method which was about measuring the plasmid transfer by using the IPTG/ X-gal colour indicator on a medium with streptomycin. In this case the excojungants with the lac genes on the F’lac turned blue, where only 4 bacterial colonies were counted according to the group results while in the class 144. 1 was the mean (plate # 4). We can conclude that the selection and contraselection with three antibiotics was more effective and accurate since plate # 3 had a

higher number of bacterial colonies. The number of exconjugants that turned blue was small because maybe the colour indicator inhibited them to conjugate, or interfered somehow with the process. Question 3The advantage of studying conjugation and the frequency of genetic material transfer in bacteria is that bacteria are convenient as a genetic model organism since they are fast-dividing, take up little space and only require the basic nutrients to survive. So in this way they can be culture in large amounts making it possible to study rare events that would be more complicated to see in eukaryotes.

Also, because bacteria can be cultured in liquid or solid medium, geneticists can observe different interaction between the strains using genetic markers and selective mediums. The last advantage would be that because they have simpler genomes they are easier for mapping. Question 4 The reason why fewer colonies were grown on the kanamycin plates than on the plates containing ampicillin is because kanamycin marks the transposon Tn5, which must be transposed onto the plasmid before it can be transferred through conjugation onto a recipient cell. Since the frequency of transposition is low, the number of recipient cells containing Tn5, being resistant to kanamycin is very low as a consequence. Experiment 2In Table 4, Tn5 and Tn1 have similar rates of transfer being 107% and 111%, because both transposons are located on the plasmid and not on the bacterial chromosome, this explains why both transposons can be transferred at the same time. The percentage of transfer in experiment one was about 75% while in the second part was greater 107% this could have been because the donor had already the

plasmid with both transposons tn5 and tn1 making the bacteria resistant to all the antibiotics.

Question 1 The number of colonies in plates #5, 6 and 7 were similar because the frequency of transfer across the different antibiotics. This similarity is because the donor already contained transposons tn1 and tn5 on the F’lac plasmid, so after conjugation the recipient cell got resistance to kanamycin and ampicillin. Question 2 Transpositions on conjugative plasmids have great reaching medical implications since it can give rise to bacteria resistant to a number of different antibiotics, which could make a disease difficult to treat because if the bacteria are immune to drugs then they are not able to stop the spread of the virus, making the process longer.Also, the fact that resistance is transferred as a single genetic package means that such a resistance could spread rapidly throughout a population.

Transpositions onto conjugative plasmids also has evolutionary implications. Antibiotics can become powerful selective agents if they are not controlled and kept to a minimum, because certain bacteria who are resistant will flourish through conjugation and the ones that are not will die. In most cases the group bacterial counts showed the same trends as of the class mean and section data in both parts of the experiment. However, for the first part of the experiment the bacterial group counts were much smaller than of the class mean. In the second part of the experiment the bacterial counts were close except for plate # 1 in which the group bacterial count was smaller than plates #5, # 6 and # 7, comparing with the section and class data, plate # 1 always had

the largest count by a significant difference, which is contradictory.

The percentage of transfer on experiment 1 from the group bacterial colonies was not too far away from the class and section data, but it followed the same trend, meaning that the percent of transfer of transposition was lower than of conjugation. For the second part of the experiment the percentage transfer of the group was much higher than of the section and data results, this has correlation with the probable mistake on plate # 1, but in both cases the percent of transfer for tn1 and tn5 are similar. There can be different sources of errors, it is important to consider that there must have been mistakes on the bacterial counting, since it is not accurate enough to count by the naked eye, there must have been overcounting and undercounting of bacterial colonies. Another big error could have occurred during the transfers of bacteria from culture flask to test tube, test tube to test tube containing the solution mixture or transferring the right amount of mixture because there were 2 different size pipettes (1ml vs. 2 ml). And finally there could have been an error when entering the data by confusing the plates or by not using appropriately the sterile technique.

To improve the experiment, highlight the different size of pipettes and find a more accurate method to count the bacterial colonies, also trying to reduce the number of transfers will reduce mistakes. The results of the group agreed with the hypothesis stated in the introduction since the frequency of transfer of the F’lac plasmid were high, whereas the frequency of transfer of the Tn5 transposon

was very low. The second part of the experiment also agreed with the hypothesis which stated that the frequency of transfer for the different selection agents would be quite close to each other. According to the literature, the F’ plasmid transfers rapidly, and can be transmitted in an infectious manner, as was seen in part 1. This experiment was successful, as it allowed students to study and understand the transfer of genetic material between bacteria cells through plasmids using conjugation and transposition.It made clear the concepts of plasmid and showed how the F’ plasmid can be used to transfer not only the fertility factor but also antibiotic resistance from one cell to another.

References

1. Becker, Kleinsmith and Hardin. 2005. Cell biology. 627-628. Benjamin Cummings, San Francisco.
2. Anthony J. F. Griffiths, Susan R. Wessler, Richard C. Lewontin, and Sean B. Carrol.
3. Introduction to Genetic Analysis. Pp. 185-189. Sara Tenney, New York.
4. 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.
5. Heinermann, P. and Johnson, D. 2008. Bio 2133 Genetics Lab Manual. University of Ottawa. Experiment 1 and Experiment 2. Appendix Sample Calculations: Experiment 1(using group results).