Lab Report of the Experiment of Conjugation of E. Coli

Conjugation is a natural occurring process that involves the transfer of DNA from one cell into another through a physical connection between the cells. In the following experiment, two strains of Escherichia coli bacterial cells (donor F’lac+strs and recipient F-lac-strr) underwent conjugation to produce a transconjugant strain (F’lac+strr). MAC plates and streptomycin were utilized to determine if conjugation had occurred. When plated, the donor colonies appeared red and the recipient colonies appeared white. The transconjugant plates showed red and white colonies. Using alkaline lysis miniprep, a DNA plasmid was isolated from the donor and transconjugant strains and FIGE electrophoresis was used to determine the size of the plasmid. The conjugation efficiency was found to be 16.25% and the plasmid DNA was approximately 97 kilobases long. The results show that the F’ plasmid was effectively transferred from the donor cells into the recipient cells via conjugation.

Introduction:Bacterial conjugation is the unidirectional transfer of either genomic DNA or plasmid DNA from a donor bacterial cell to a recipient bacterial cell by cell-to-cell contact via a sex pilus (Snustad & Simmons, 2006). Conjugation was first discovered by Lederberg and Tatum in 1946. In their experiment, they grew two strains of bacteria in separate vessels with rich medium and then together in one vessel containing the same medium. Then, they spread the three vessel contents onto medium agar plates and incubated them overnight at 37˚C. The only plate that showed cell growth was the plate containing the mixture of the two bacterial strains. The other two plates showed no growth. This experiment proved that in order for recombination to occur, the two strains must come in contact with one another (Lederberg, Tatum, 1946).

In 1950, Bernard Davis discovered that cell-to-cell contact was required to obtain a transconjugant. Using a U tube containing a sintered filter between the two sides of the tube, he added two types of bacteria (donor and recipient) to each side of the tube. Because of the filter, Davis never observed conjugation. This further proved that in order for conjugation to occur, the cells must come into physical contact.

In order for cells to undergo conjugation, one cell must contain a fertility factor (F). William Hayes discovered this F factor in 1952. The F factor, which is a small circular molecule of DNA (plasmid), controls the synthesis of F pili that connect donor and recipient cells during conjugation. These F factors are approximately 105 basepairs in size.

In bacterial conjugation, a donor cell containing the F plasmid is referred to as an F+ cell while a recipient cell that lacks the plasmid is an F- cell. When an F+ cell mates with an F- cell (conjugation), the F+ plasmid is transferred. Both the donor and recipient cells become F+ cells and contain the F plasmid. While transferring the F+ plasmid, sometimes the plasmid is integrated into the recipient’s chromosome. These cells are referred to as Hfr cells. Sometimes chromosomal DNA is looped out of the F plasmid, and chromosomal genes are transferred into the recipient; the recipient cells are referred to as F’ strains. When donor F’ cells mate with recipient F- cells, genomic DNA is transferred from donor to recipient. This transfer is known as sexduction and the cell that receives the F’ plasmid from the donor is referred to as a transconjugant (Snustad & Simmons, 2006).

In the experiment performed, conjugation was studied in E. coli bacterial cells. The donor bacterial cells contained the F’ plasmid that had the lac+ gene integrated into it, making the cells F’lac+strs. The recipient bacterial cells were F-lac-strr. The donor and recipient cells were mixed and plated onto streptomycin indicator plates. Using FIGE electrophoresis, plasmid DNA was isolated and its size was determined. The plasmid was present in the donor and transconjugant cells; however, in the recipient cells the plasmid was absent.

Materials and Methods:One mL of each of donor (F’lac+strs) and recipient (F-lac-strr) the E.coli bacterial strains, from the American Type Culture Collection in Rockville, Md., was pipetted with a pipetman into a sterile culture tube and incubated, without shaking, at 37˚C for 90 minutes. Before plating the strains on agar plates, dilutions of the three strains of cells were prepared with LB broth.

100 μl of 10-5 and 10-6 dilutions of donor cells were each plated onto MacConkey (MAC) agar plates without streptomycin. 100 μl of 10-5 dilution of donor cells and 10-5 and 10-6 recipient were also plated onto MAC plates with streptomycin. 100 μl of 10-4 and 10-5 dilutions of the conjugation mixture cells were plated onto MAC agar with streptomycin. All seven plates were inverted and placed in a 37˚C incubator for about 24 hours. The bacterial colonies on each plate were counted the next day (colony counts seen in Table I).

Donor colonies were picked with a sterile loop and placed into a sterile test tube containing LB broth. Recipient and transconjugant colonies were also isolated and placed into sterile test tubes containing LB broth and streptomycin. The tubes were then placed in a 37˚C shaking incubator at 250 rpm overnight.

After the incubation, 1.5 mL of each of the three cultures were added to eppendorf tubes and centrifuged at 13,200 rpm for 1 minute. An alkaline lysis procedure like that of Birnboim and Doly was then performed to extract the plasmid DNA with 200 μl of alkaline SDS detergent solution (Birnboim & Doly, 1979). After the alkaline lysis procedure was complete, the pellets were washed with a 100% ethanol and stored in a -20°C freezer.

A 1% agarose gel in 0.5 X TBE buffer was prepared for gel electrophoresis in a gel tray. The gel tray was placed into the BIO-RAD FIGE Mapper apparatus. Loading dye was added and each sample (aprox. 25 μl) was then loaded into a well. DNA markers were loaded into the first and last wells. The gel was run under program 4 for 16 hours, 180 volts forward and 120 volts reverse. When the program was finished, the gel was placed into an ethidium bromide solution to stain. After staining, the gel was gently rocked in distilled water. Using a Kodak EDAS 290 imaging system, a picture of the gel was taken (which can be seen in Figure 1.0).

Results:During the experiment, donor (F+lac+strs) and recipient (F-lac-strr) cells were mixed and plated onto streptomycin indicator plates. Plasmid DNA was extracted from the donor and transconjugant cells and FIGE electrophoresis was used to determine the plasmid’s size. After plating and incubating the bacterial dilutions, the cell colonies were counted. It was observed that all of the donor cells were red, all of the recipient cells were white, and the conjugation culture cells were a mix of red and white. There were too many (>300) red colonies to count on the donor 10-5 MAC agar plate and 60 red colonies on the donor 10-6 MAC agar plate. No colonies were seen on the donor 10-5 MAC agar + strep plate. There were 126 white colonies on the recipient 10-5 MAC + strep plate and 32 white colonies seen on the recipient 10-6 MAC + strep agar plate. The transconjugant 10-4 MAC + strep agar plate had 206 red and too many white colonies to count, while the transconjugant 10-5 MAC + strep agar plate had 26 red colonies and 86 white colonies (seen in Table I).

Using the cell counts and their dilutions, the culture concentration was calculated. The concentration of donor cells in the 10-6 dilution was 6×108 cells/mL. The concentration of recipient cells in the 10-6 dilution was 3.2×108 cells/mL. The concentration of transconjugant cells in the 10-5 dilution was 2.6×107 cells/mL (Table II). The conjugation efficiency was calculated to be 16.25% (Table III).

Upon completion of a FIGE electrophoresis, marker standards were used to determine the plasmid size and the distance travelled. The size and mobility of the bands in Marker II (Figure 1.0) were measured and a standard curve was generated (Figure 2.0). This curve was then used to determine the plasmid size present in the donor and transconjugant cells. (The plasmid was not present in the recipient cells.) The plasmid travelled 14.5 mm and was approximately 101 kilobases long.

Discussion:After plating the donor cells onto MAC plates that did not contain the streptomycin antibiotic, red colonies grew. This result is plausible because the donor cells contained the lac operon, which codes for enzymes that can utilize lactose as food. Cells containing this operon can grow on MAC plates because the plates contain lactose sugar. These two plates were then compared to the donor plate that contained the streptomycin antibiotic. No colonies grew on the streptomycin plate. This is because the donor cells did not contain the gene for streptomycin resistance. After plating the recipient cells onto MAC+strep plates, white colonies grew. This result is seen because the recipient cells lack the lac operon.

These cells cannot utilize lactose as a food source. Also, the recipient cells were able to grow in the presence of streptomycin because they contained a gene for resistance to the antibiotic. On the plates containing MAC+strep and 10-5 transconjugant cells, there were 26 red cells present. Ideally, because the cells were too dilute for conjugation to be seen, there should have been no red cells present. On the plates containing MAC+strep and 10-4 transconjugant cells, both red and white colonies were observed. The white colonies were recipient cells and the red were transconjugants. It can be determined that the red cells were the transconjugants because previously, red cells (which indicate donor cells) were not able to grow on plates containing streptomycin. Because they were present on streptomycin plate, the cells must have undergone conjugation.

After isolating the plasmids and running them on a FIGE electrophoresis, it was observed that the plasmid was only present in the donor and transconjugant cells. This occurred because only the donor cells contained the plasmid. Because donor cells were not present in the recipient cells, no conjugation could occur; therefore, no plasmid would be seen in the recipient lane on the gel. The size of the F plasmid was determined by measuring the distance the plasmid travelled in the gel, and comparing it to a known marker (Marker II). The size of the F plasmid was determined to be approximately 97 kilobases long. This was compared to the literature value, of approximately 100 kilobases (Keasling, Palsson, and Cooper, 1991). Because the plasmid size is very close to the literature value, it can be concluded that the DNA plasmid was successfully isolated from the donor to the transconjugant cells. Genomic DNA was not transferred and no Hfr strains were formed.

The conjugation efficiency was calculated and found to be 16.25% on the transconjugant plates, meaning for every 100 cells on the plate, 16.25 were transconjugants. A 16.25% conjugation efficiency is a reasonable value. The value seen could be due to the fact that even though a donor cell contains the F’ plasmid, the plasmid is not always transferred into every single recipient cell. If this were the case, a conjugation efficiency of 100% would be seen every time. Comparing this value to an efficiency value seen in the literature of 94%, the value is a bit low (Kuang et al., 2000). To increase the conjugation efficiency, the mixed donor and recipient cells could be left to sit for a longer period of time. This would encourage more conjugation.

In this experiment, the size and mobility of E. coli plasmid DNA was observed. To further investigate plasmids, more experiments could be used to determine the size and mobility of other plasmids, such as genomic DNA and Hfr cell strains.

Table I. Colony Counts and ColorPlate# of colonies and colorMAC Agar PlatesDonor 10-5Too many red colonies to countDonor 10-660 RedMAC Agar + Strep PlatesDonor 10-50Recipient 10-5126 WhiteRecipient 10-632 WhiteTransconjugant 10-4206 RedToo many white colonies to countTransconjugant 10-526 Red86 WhiteTable II. Culture ConcentrationsPlateCalculationCulture Concentration (cells/mL)Donor10-660 cells X 106/0.1 mL6x108Recipient10-632 cells X 106/0.1 mL3.2x108Transconjugant10-526 cells X 105/0.1 mL2.6x107Table III. Conjugation EfficiencyTransconjugantCalculationConjugation Efficiency10-5(2.6×107)x100(3.2×108)/216.25%Table IV. Marker II Size and MobilitySize of Marker II (kb)Mobility (mm)153033.52748.52463.52082179714112.510130.58145.54Figure 1.0 Kodak EDAS 290 picture of FIGE electrophoresis. The first and last lanes on the gel represent standard Marker I and Marker II DNA. The thick bands seen in the middle of the lanes represent coiled genomic DNA. The thinner, fainter bands seen farther down the lanes represent plasmid DNA. The fifth lane represents donor cells, the sixth lane represents recipient, and the seventh represents transconjugant. Each group of three lanes represents a donor, recipient, and transconjugant for each lab group.

Figure 2.0 See attached graph. Graph of the standard curve of the DNA Marker II, seen as the far right lane in gel picture. The distance of each band travelled was measured in millimeters then matched with the known size of the bands in kilo bases. The standard curve was then used to determine the size of the plasmid DNA present in the donor and transconjugant cells from the experiment.

References:

Birnboim & Doly. 1979. Nucleic Acid Research 7:1513.

Keasling, J.D., B.O. Palsson, and S Cooper. (1991, April). Cell-cycle-specific F plasmidreplication: regulation by cell size control of initiation, 173, 2673-2680. Retrieved on November 26, 2008.

Kuang, Hao, D. Haring, D. Qi, A. Mazhary, and M.D. Distefano. (2000, April). Synthesis of acationic pyridoxamine conjugation reagent and application to the mechanistic analysis of an artificial transaminase. American Psychologist, 10(18), 2091-2095. Retrieved on December 2nd, 2008.

Lederberg, J., & Tatum, E.L. (1946). Gene recombination in Escherichia coli. Nature. 158, 558.

Snustad , D, & Simmons, M (2006). Principles of Genetics. John Wiley & Sons. Pgs. 178-182.