Lab Report of the Experiment of Conjugation of E. Coli Essays

Lab Report of the Experiment of Conjugation of E. Coli Essays Lab Report of the Experiment of Conjugation of E. Coli Paper Lab Report of the Experiment of Conjugation of E. Coli Paper Using alkaline lysine nipper, a DNA lassie was isolated from the donor and transcontinental strains and FIG 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 kilobytes 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 pills (Sonatas Simmons, 2006). Conjugation was first discovered by Elderberry and Datum 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 ETC. 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 (Elderberry, Datum, 1946). In 1950, Bernard Davis discovered that cell-to-cell contact was required to obtain a transcontinental. 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 auricular molecule of DNA (plasmid), controls the synthesis of F pill that connect donor and recipient cells during conjugation. These F factors are approximately 105 bagpipers 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 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 recipients chromosome. These cells are referred to as Hoff 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 seduction and the cell that receives the F plasmid from the donor is referred to as a transcontinental (Sonatas 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 lack+ gene integrated into it, making the cells Flag+stars. The recipient bacterial cells were F- lack-stir. The donor and recipient cells were mixed and plated onto streptomycin indicator plates. Using AGE electrophoresis, plasmid DNA was isolated and its size was determined. The plasmid was present in the donor and transcontinental cells; however, in the recipient cells the plasmid was absent. Materials and Methods:One ml of each of donor (Flag+stars) and recipient (F- lack-stir) the E. Oil bacterial strains, from the American Type Culture Collection in Rockville, Md. , was pipettes with a pitman into a sterile culture tube and incubated, without shaking, at 370 C for 90 minutes. Before plating the strains on agar plates, dilutions of the three strains of cells were prepared with LB broth. 100 Pl of 10-5 and 10-6 dilutions of donor cells were each plated onto McCracken (MAC) agar plates without streptomycin. 100 Pl of 1 0-5 dilution of donor cells and 10-5 and 10-6 recipient were also plated onto MAC plates with streptomycin. 00 Pl 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 ETC incubator for about 24 hours. The bacterial colonies on each plate were counted the next day (colony counts seen in Table l). Donor colonies were picked with a sterile loop and placed into a sterile test tube containing LB broth. Recipient and transcontinental 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 offender tubes and centrifuged at 13,200 RPM for 1 minute. An alkaline lysine procedure like that of Bromine and Doll was then performed to extract the lassie DNA with 200 Pl of alkaline SD detergent solution (Bromine Daly, 1979). After the alkaline lysine procedure was complete, the pellets were washed with a 100% ethanol and stored in a -ICC freezer. A 1% agrees gel in 0. 5 X TUBE buffer was prepared for gel electrophoresis in a gel tray. The gel tray was placed into the BIO-RADAR FIG Mapped apparatus. Loading dye was added and each sample (apron. 25 VI) 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 knishes, the gel was placed into an tedium bromide solution to stain. After staining, the gel was gently rocked in distilled water. Using a Kodak IDEAS 290 imaging system, a picture of the gel was taken (which can be seen in Figure 1. 0). Results:During the experiment, donor (F+lack+stars) and recipient (F-lack-stir) cells were mixed and plated onto streptomycin indicator plates. Plasmid DNA was extracted from the donor and transcontinental cells and FIG electrophoresis was used to determine the plasmids size. After plating and incubating the bacterial dilutions, the cell colonies were counted. It was observed that all of the donor ells 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 transcontinental 10-4 MAC + strep agar plate had 206 red and too many white colonies to count, while the transcontinental 10-5 MAC + strep agar plate had 26 De colonies and 86 white colonies (seen in Table l). Using the cell counts and their dilutions, the culture concentration was calculated. The concentration of donor cells in the 10-6 dilution was xx cells/ ml_. The concentration of recipient cells in the 10-6 dilution was 3. Axis cells/ml. The concentration of transcontinental cells in the 10-5 dilution was 2. Xx cells/ ml (Table II). The conjugation efficiency was calculated to be 16. 25% (Table Ill). Upon completion of a FIG electrophoresis, marker standards were used to determine the plasmid size and the distance traveled. The size and mobility f 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 transcontinental cells. The plasmid was not present in the recipient cells. ) The plasmid traveled 14. 5 mm and was approximately 101 kilobytes 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 lack Oberon, which codes for enzymes that can utilize lactose as food. Cells containing this Oberon 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 lack Oberon. 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 gene for resistance to the antibiotic. On the plates containing MAC+strep and 10-5 transcontinental 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 transcontinental cells, both red and white colonies were observed. The white colonies were recipient cells and the red were transcontinental. It can be determined that the red cells were the transcontinental because previously, red cells (which indicate donor cells) were not able to grow on plates containing streptomycin. Because they ere present on streptomycin plate, the cells must have undergone conjugation. After isolating the plasmids and running them on a FIG electrophoresis, it was observed that the plasmid was only present in the donor and transcontinental 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 traveled in the gel, and comparing it to a known marker (Marker II). The size of the F plasmid as determined to be approximately 97 kilobytes long. This was compared to the literature value, of approximately 100 kilobytes (Sealing, Paulson, 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 transcontinental cells. Genomic DNA was not transferred and no Hoff strains were formed. The conjugation efficiency was calculated and found to be 16. 25% on the transcontinental plates, meaning for every 100 cells on the plate, 16. 5 were transcontinental. 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 (Kiang 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.