Transformation
Purpose
Horizontal gene transfer refers to the transfer of genes from one organism to another through a method other than reproduction. Genetic transformation, a form of horizontal gene transfer, involves the altering of a cell through the uptake of naked DNA. Naked DNA refers to DNA which has been released from lysed or disrupted cells and is taken up by a recipient cell. If a cell is able to take up naked DNA, they are referred to as competent. This finding is accredited to Frederick Griffith, a bacteriologist who conducted an experiment in which a nonpathogenic strain of Streptococcus pneumoniae was exposed to a heat-killed pathogenic strain of the same bacteria. Griffith observed that although the virulent strain had been heat-killed; the DNA was able to survive the heating process and was taken up by the “resistant” strain of bacteria through gene transformation.
In this lab, an experiment was conducted to test the hypothesis that a susceptible strain of bacteria, known as the S-strain, will grow in the presence of an antibiotic if it is combined with the DNA from the lysed pathogenic cell. Methods To demonstrate Griffith’s findings, an experiment was conducted with a competent prokaryotic cell known as Acinetobacter. This bacterium is Gram-negative, rod-shaped, and typically found in soil and water. During this experiment, three sessions were completed in order to observe whether gene transformation had successfully taken place. In the first session, 1.0 mL of a resistant strain (R-strain) of Acinetobacter broth was transferred into a sterile tube, along with 0.1 mL of detergent sodium dodecyl sulfate (SDS) using a micropipette. The tube containing the solution was then incubated in a water bath for 30 minutes at 60o C. This was a crucial step in the experiment, as it caused the cells to lyse and release their DNA. If a cell did not lyse, it was heat-killed by the water during incubation. While the sample was being incubated, a TSY agar plate was divided into 5 sectors and labeled according to the solution each sector would contain. In the first sector, the lysed mixture of resistant cells (the source of DNA) was carefully transferred to the TSY agar plate using an inoculated loop. Using the same technique, both the R and S-strains of Acinetobacter were placed into their appropriate sectors. The preceding technique was used for the next sector, in which an S-strain of Acinetobacter was placed on the TSY agar plate and carefully topped with a loopful of DNA. In the fifth and final sector, an S-strain of Acinetobacter was placed on the plate followed by DNase, an enzyme that breaks down DNA, and finally DNA which was placed on top. For the next 48 hours, the plate was incubated at 37o C to allow time for growth to occur. In the following session, the TSY agar plate was observed and the results were noted after they had been properly incubated (Figure 1). In order to test if gene transformation occurred, a TSY agar plate containing streptomycin, an antibiotic, was divided into four sectors and labeled similar to the aforementioned TSY agar plate with the exception of the DNA sector, which was omitted because it showed no growth. After the samples were transferred to the TSY + streptomycin plate, it was incubated for 48 hours at room temperature. Figure 2 illustrates the appearance of the TSY + streptomycin plate prior to incubation. In the third and final session, the results from the incubated TSY + streptomycin plate were observed and recorded (Figure 3). If the sector containing S + DNA was able to grow on the plate, it would confirm that transformation had occurred because it was able to grow in the presence of an antibiotic. Results When the TSY agar plate was observed after 48 hours at room temperature, growth was found on all sectors apart from the DNA sector (Figure 1). When the TSY + streptomycin plate was observed, growth was found on all sectors apart from the sector containing the S-strain. Conclusion The experiment was successful in showing gene transformation. Visible growth of the S + DNA sector on the TSY + streptomycin plate confirmed the hypothesis that when mixed with DNA from lysed pathogenic Acinetobacter cells, susceptible or sensitive cells transformed to resistant cells which could withstand the presence of streptomycin. These findings verified that Acinetobacter was a competent cell that was able to take up naked DNA. In order to validate these results, it was very important that the control sectors be present. Although the sector containing the S-strain grew on the TSY plate, it showed no growth on the TSY + streptomycin plate, which proved that without the presence of DNA cells, the sensitive strand was not able to grow in the presence of streptmycin. The control sector containing S + DNase + DNA grew on the TSY plate because the S-strain of Acinetobacter had not been effected by the DNA, which was inhibited by DNase. However, our findings verified that when DNase on the TSY+ streptomycin plate broke down DNA, the DNA was no longer able to transfer genes to the sensitive strand, and no growth was seen. The R-strain showed growth on both the TSY agar plate and on the TSY agar + streptomycin plate. This experiment had several steps, which meant there was a potential for error. All measurements needed to be precise and accurate in order for the experiment to be valid. An experiment that could be performed in the future would be to change the antibiotic from streptomycin to another antibiotic and see if the same results are found. Another experiment includes adding DNA to the S + DNA sector before you add the S-strain and recording if a difference occurs in the results.

Works Cited Talaro, K. P., & Chess, B. (2012). Foundations in microbiology: basic principles (8th ed.). New York: McGraw-Hill.

1. What two components were mixed together to show transformation? a. Acinetobacter S-strain cells and DNA were mixed together to show transformation. An example of successful gene transformation was seen on the TSY + streptomycin agar plate when the S-strain did not grow on the plate and the S + DNA strain grew successfully. 2. If the S-strain cells had grown on the TSY + streptomycin plate, would you have been able to determine if transformation has taken place? b. No, because it would mean that the S-strain did not need the presence of DNA in order to grow on the plate, and therefore was not susceptible in the first place. 3. If you had used a DNA lysate containing viable cells, would it have been possible to determine whether transformation had taken place? c. No, if you had used a DNA lysate containing viable cells plate growth could have been attributed to the viable cells which were not sensitive to streptomycin.