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Although bacteria do not undergo true sexual reproduction as seen for eukaryotic organisms, there are mechanisms for gene transfer and genetic recombination in bacteria. The means by which genes are transferred between cells are divided into three general categories. You can read more about these phenomena in the chapter on Bacterial Genetics.

1. Transformation: Genes are absorbed by a cell from the environment where DNA from lysed cells is present.
2. Transduction: Bacterial genes are transferred by a bacteriophage.
3. Conjugation: Genes are transferred directly from one living cell to another with the aid of a protein bridge (pilus) allowing cell-to-cell contact.

In all three mechanisms, the DNA is transferred from a donor cell to a recipient cell. In most instances, only a small fraction of a chromosome is transferred. Once inside the recipient cell, the donor DNA has two pathways. If the DNA is capable of autonomous replication, it can exist indefinitely in the host cell. Any DNA entering the cell to position itself alongside the homologous portion of the recipient chromosome. Recombination may occur where, due to enzymatic action, a segment of the recipient DNA is excised and replaced by the integration of the homologous segment of donor DNA. In either case, the recombinant cell contains DNA derived from both the donor and recipient cells. Acquisition of donor genes may confer to the recipient an advantage such as being able to grow in a particular habitat or medium where growth was before impossible.

In Escherichia coli and presumably many other species, distinct mating types may be found. For E. coli, the F⁺ and F^- mating types are distinguished by the presence of a special plasmid, the F factor, in the former. This plasmid includes genes involved in the transfer by conjugation of DNA into compatible recipient cells; included are genes responsible for the synthesis of the F pilus. When the F plasmid is transferred to an F^- cell, the donor F⁺ cell retains a complete copy of the plasmid while the recipient F^- cell becomes an F⁺ donor cell. If the F plasmid becomes integrated into the chromosome of a F⁺ cell, an Hfr cell results. A cell of this mating type will be a donor, able to transfer part of its regular chromosome during conjugation (with part of the F factor on the leading end). However, an Hfr donor rarely transfers its mating ability to a recipient, since it would require complete transfer of the entire chromosome. Recombination involving any part of the transferred Hfr chromosome may subsequently occur in the recipient cell as discussed above.

In order for us to demonstrate conjugation and recombination effectively in this experiment, we must choose parent cells which are not only different mating types but are also different in certain phenotypic properties such that we may detect the result of recombination easily. We could not demonstrate the end result of these processes with phenotypically-identical mating types! For this experiment, we have chosen an Hfr strain which is a methionine auxotroph and an F^- strain which is a threonine auxotroph. Neither should grow on a minimal medium. However, after mixing the two strains and allowing for conjugation and subsequent recombination to occur, the appearance of any colony on the minimal medium can be attributed to an F^- cell which acquired the ability, from an Hfr cell (via conjugation and recombination), to synthesize threonine and thus all of its amino acids.

The real starting point in this experiment is the mixture of Hfr and F^- cells where we hope to have conjugation proceed. Knowing the concentration of each mating type in the mixture, we can calculate the percentage of F^- cells which acquire the ability to synthesize threonine and therefore produce colonies on the minimal medium. This percentage we call the recombination frequency.

The growth characteristics of relevant E. coli strains can be summarized as follows:

strain of E. coli	genotype designation	growth on Minimal Medium	growth on an all-purpose Medium
typical prototroph	Thr+ Met+	+	+
our Hfr strain (auxotrophic for methionine)	Thr+ Met-	-	+
our F- strain (auxotrophic for threonine)	Thr- Met+	-	+
possible recombinant 1	Thr+ Met+	+	+
possible recombinant 2	Thr- Met-	-	+

Period 1

Materials

Broth culture of E. coli: Hfr (donor) mating type; Thr⁺ Met^-; approximately 10⁸ cells/ml

Broth culture of E. coli: F^- (recipient) mating type; Thr^- Met⁺; approximately 10⁸ cells/ml

2 plates of Tryptone Yeast Extract Glucose Agar (TYEG)

(Alternatively, any all-purpose medium can be substituted; Minimal Medium plus threonine and methionine may also be used.)

6 plates of Minimal Medium (MM)

1 empty, sterile test tube

3 dilution blanks (9 ml)

Micropipettes and sterile tips

2 sterile swabs

Procedure I (quantitative)

1. For an experiment such as this, it helps to diagram the procedure and have the plates and tubes labeled appropriately for the upcoming steps in the procedure: Label one of each plating medium (i.e., TYEG and MM) for the Hfr culture, one of each plating medium for the F^- culture, the three dilution blanks for the dilutions to be made of the mixture of the two mating types (i.e., 10^-1, 10^-2 and 10^-3), and three plates of MM for the plated dilutions (i.e., 10^-2, 10^-3 and 10^-4).
2. Using separate pipette tips, aseptically transfer 1 ml of each strain to the empty sterile tube and mix gently. Note the time of the addition of the strains to each other. Set the tube aside where it will not be disturbed, and allow 30 minutes for conjugation to take place.
1. Considering the number of cells transferred in each 1 ml amount (see under Materials), what would be the number of cells per ml of the mixture you just made in the sterile tube?
2. Then, what would be the number of cells of either mating type per ml of the mixture?
3. If the cells were to pair up, what would be the number of pairs per ml of the mixture?
4. The last two values should be the same! Therefore, among these pairs of cells, we will be able to determine how many pairings resulted in F^- cells that received the Thr⁺ gene (after conjugation and recombination), as each of these cells will be able to grow (produce a countable colony) on MM.
3. Prepare control plates of the donor and recipient strains by streaking each culture onto a plate each of TYEG and MM. Any streaking method is acceptable as long as it is done the same for each plate. What do we expect to happen on each plate?
4. After the 30 minute period is up, prepare three serial, decimal (1/10) dilutions of the mixture and plate 0.1 ml from each dilution onto a plate of MM. Remember proper aseptic technique. Then, starting from the most dilute inoculum, spread the plates with a flame-sterilized hockey stick.
5. Incubate all plates (from steps 3 and 4) at 37°C for 2-3 days or at 30°C for 4-5 days.

Procedure II (qualitative)

6. Draw two perpendicular lines across the bottom of a plate of MM. Label one line for the donor and the other line for the recipient.
7. Dip a sterile cotton swab into the F^- broth culture and make a single streak across the medium in one direction, following the previously-marked line on the bottom of the plate. Discard the swab into the disinfectant.
8. Repeat the procedure for the Hfr culture, streaking along the other line. Notice how you are mixing Hfr and F^- cells when you cross the center of the plate. Therefore, where on the plate will you expect to see colonies of recombinant cells?
9. Incubate the plate at 37°C for 2-3 days or at 30°C for 4-5 days.

Period 2

Procedure I

1. Examine the streak plates on TYEG and MM of the donor and recipient cultures. On which medium was each strain able to grow?
2. Of the three MM dilution plates, count the one containing between 30 and 300 colonies. Multiplying this number by the appropriate dilution factor, determine the number of recombinant colony-forming units per ml of the mixture.
3. Compare the value obtained in step 2 to the total number of cells per ml of the mixture which had the potential to pick up DNA and undergo recombination - i.e., the recipient (F^-) cells. To determine the recombination frequency, use the following formula. (For convenience in this experiment we will treat colony-forming units and cells as equivalent.)



Figure 8.7. Calculating %recombinants. The percent of recombinants that successfully mating with the Hfr strain and recombined the thr region into the chromosome. The number of recipient cells per ml of the mixture was 5 x 10⁷.

Procedure II

4. Note any colonial or confluent growth on the plate. Do your results indicate that genetic recombination has taken place between the donor and recipient strains? Remember what genotype an organism needs to have in order to grow on this medium. Might it be possible to observe colonies resulting from revertants (back-mutations)? If so, where on the plate would they be evident?

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