Effects of Ampicillin on Transformation in Bacteria - Lab Report Example

Effects of Ampicillin on Transformation in Bacteria Yesenia Canciobello Panther ID: 3284180 Florida International Laboratory Partners: Andres Solano, Richard Christie and Nestor Camacho Group # 6 General Biology I Lab Section U-27 April 16, 2015 Abstract In the experiment, the bacterium that was able to take up the plasmid and successfully undertake the process of transformation was selected. The selection process was done by putting the bacteria in ampicillin medium that had all the factors necessary for the process of transformation. The bacterial cells that lived took the plasmid that contained the genes that are resistance to ampicillin. This was identified by the phenotype of transformed colonies in which some of the growth observed in the ampicillin rich media of LB/Ampc. The growth occurred as a result of the presence of resistance genes in the plasmid to ampicillin enabling the bacterial cells to undergo a transformation. In some plates, growth did not occur because of absent of the plasmids to offer the genes for resistance. Transformation efficiency was obtained from the growth observed in the media. Conclusively, some bacteria were transformed because they were able to survive in ampicillin rich medium due to the plasmid having genes that are resistant to ampicillin and the plasmid being incorporated into the bacteria’s DNA. The uptake of DNA is both beneficial and harmful to the organism. Introduction Bacteria are microorganisms that are prokaryotes and are single-celled but complex in their structure. The organism exists in different shapes and sizes and is found everywhere. They possess DNA and have organelle like cell wall that are either gram positive or negative and is important in the transformation process (Lorenz & Wackernagel, 1994). Ampicillin is an antibiotic, which is used in the treatment of many bacterial infections. The antibiotic is classified as beta-lactam that is from the family of penicillin and also equivalent to the amoxicillin antibiotic with regards to their activity. Ampicillin is very active against both gram-negative and gram-positive bacteria. It means therefore that the antibiotic is capable of killing bacterial cells (Albert, Pitzer & Calero, 2012). Its activity in the bacterial cells is enhanced by the presence of sulbactam that generally inhibits the beta lactamase enzyme that inactivates any antibiotic it is exposed to. Quorum sensing refers to the regulation of the gene expression with the response to the density of cell population in case of fluctuations (Miller, 1987). Plasmid is a DNA molecule that is double stranded and small in size. They exist in the cells of bacteria and also in other eukaryotes. The plasmid genes assist the bacteria in offering resistance to any infection or physical destruction on the outer cell surface. Plasmids can be inherited from parents to daughter cells in eukaryotic cells, and be conjugated in bacterial cells (Mello & Fire, 1995). Plasmids are used for cloning and manipulation of genes. The process makes them be referred to as vectors through the creation of a recombinant plasmid as they are introduced to another bacterium cell in the process called transformation. They can then copy large fragments of DNA since they have the ability to divide faster (Smith et al., 1993). LB broth is a medium that is nutritionally rich and used in the growth of bacteria species. An antibiotic when added to it to select cells containing genetic elements like the plasmids. The LB offers the growth surface in analyzing the colony morphology of bacteria. In the experiment, therefore, the medium was used to investigate the growth patterns of bacteria under different treatments of nutrient agar in carrying out transformation process. The scientific approach regarding the experiment is that bacteria that have successfully transformed should survive in ampicillin rich medium due to the plasmid having genes that are resistant to ampicillin and the plasmid being incorporated into the bacteria’s DNA. The null hypothesis is that there is no correlation between successfully transformed bacteria and its growth in ampicillin rich medium. The alternate hypothesis is that there is a correlation between successfully transformed bacteria and its growth in ampicillin rich medium. The report, therefore, explains the processes involved in transformation by plasmids and the advantages the uptake of DNA will have to the organism and environment and the disadvantages that may arise. Methods Three different types of tubes were prepared in which the first and second were labeled “C” and “lux” respectively and 70ml of complete cells together with 3µl plasmid added. The third tube was labeled NP and 35ml of complete cells added onto it. The tubes were then kept on ice baths to heat shock for 35 minutes then 275ml of LB added to tubes of C and lux. To the tube of NP, 150ml of the broth was added. Six plates were then prepared with different types of nutrient agar. Half were treated with antibiotic ampicillin. They were labeled LBc , LBlux , LB/Ampc , LB/Amplux, LBNPand LB/AmpNP and incubated for 370C for 45minutes for growth observations. A portion of controlled DNA was added to the plate of LB/Ampc so as to calculate the fraction of DNA that spread onto the plate through the ratio of total volume spread of the plate and total sample volume of DNA in control. Actual amounts of DNA were calculated to obtain the transformation efficiency of all the plates used. Results Table1 shows the expected results of growth from the different plates used in the experiment. Treatment Expected result (growth or no growth) Bioluminescence (yes or no) LBc Yes No LB/Ampc Yes No LBNP Yes No LB/AmpNP No No LB/Amplux Yes Yes LBlux Yes Yes Table2: shows the observed results after the experiment was carried out on the plates. Treatment Observed Growth Type Bioluminescence (yes/no) LBc Colonial growth None LB/Ampc No growth None LBNP Lawn growth None LB/AmpNP None None LB/Amplux None None LBlux Colonial growth None Figure 1 Shows a picture of the LBNP, LBc, LB/Ampc. Figure 2 shows a picture of the LB/AmpNP, LB/Amplux, LBlux. Fraction of DNA spread on LB/Ampc is given by; Fraction = volume (µL) spread on LB/Ampc plate = (130 µL / 325 µL) Total sample volume (µL) in control DNA tube = 0.4 Total amount of DNA (µg) present in the LB/Ampc plate = µg of DNA X fraction of DNA spread (0.025 µg X 0.4) = 0.01µg. The transformed efficiency of LB/Ampc plate = total number of colonies on the plate/ total amount of DNA spread on the plate. (0 colonies/ 0.0336 µg) = 0 The total amount of plasmid DNA (µg) in the plate LB/Amp c used was calculated as concentration (µg/ µL) of DNA X volume of DNA (µL) (0.03 µg/ µL X 3 µL) = 0.009 µL. The total volume of cell suspension in the control DNA tube = amount (µL) of plasmid + amount of (µL) of LB + amount of (µL) of cell suspension. (3 µL + 275 µL +70 µL) = 348 µL. Discussion The results gotten did not meet the expectations because there was no growth in the case of plates of LB/Ampc and LB/Amp media in which growth were expected to occur. This could have been as a result of the factors like competency error in distribution, disinfection on agar plates that affected the efficiency of transformation (Albert, Pitzer & Calero, 2012). The plates of LBc and LBlux showed growth as a result of the absence of ampicillin that could have inhibited their growth. In the LBc plate, growth is expected with no bioluminescence. The reason to this is because there is no ampicillin to kill bacteria. The actual observation was that there was a colonial growth because there was no restriction from the ampicillin. In the LB/Ampc plate, growth was expected to occur because the ampicillin had the plasmid in it. However, the observed result produced no growth because the process of transformation did not occur. In the plate of LBNP, growth was to occur because of the absence of antibiotic ampicillin. The observed result produced a lawn growth because normal growth occurred in the absence of antibiotic to restrict growth. The plate of LB/AmpNP, growth was not expected as a result of the presence of ampicillin. Practically, growth was not observed because the bacterium was killed since plasmid was not there. Growth was expected in the plate of LB/Amplux since lux plasmids has resistance to ampicillin gene. Practically, growth was not observed in the actual experiment because the bacteria did not take in the plasmid. In the plate of LBlux, the expectation was growth occurrence because ampicillin is absent to restrict growth. From the experiment taken, the plate recorded colonial growth due to the absence of the antibiotic. The cells that are transformed with pUC18 and plasmid lux grow in the presence of ampicillin because they become immune to ampicillin as a result of the gene that produces the enzyme that can inactivate the antibiotic ampicillin. The enzyme that is produced during this process is the luciferase enzyme. The plate with the lux plasmid showed fewest numbers of colonies because lux has a greater molecular weight (Albert, Pitzer & Calero, 2012). Calcium chloride () was used to take away the charges from cell membrane to neutralize it. Plasmids were removed from the ice water to hot water to increase the competency and enhance the process of transformation. Conclusion The transformation efficiency is greatly affected by the size of the plasmid. The competency of transformation is enhanced through membrane neutralization. A chemical like CaCl2 is used in ice bath as it affects the lipids structure of the membrane by making a hole for the plasmids to go through. Heat shock provides a pathway in the cell wall of the organism so as to push the bacteria inside the cell through the holes (Albert, Pitzer & Calero, 2012). Uptakes of foreign DNA by different types of organism have benefits and demerits on the organism and the environment. Genetic transformation is advantageous to host in instances where the plasmid that contains resistance genes are well expressed in the organism. It becomes beneficial to the organism in terms of survival in any given environment to enhance growth (Davison, 1999).Moreover, the genetic transformation can be maladaptive to the host organism and, as a result, lead to some effects. The main effect could be less population growth and decrease because the plasmid it took was not resistant enough to suit it in the environment for interaction (Lorenz & Wackernagel, 1994). To sum it all, uptake of foreign DNA can be beneficial to an organism but disruptive to the ecosystem. It is beneficial when the DNA allows the cell to translate into a new protein but disruptive to the normal microbial flora in the ecosystem when the resistant genes destroy all of them (Davison, 1999). The experiment of transformation can be further improved by introducing a marker gene. The gene will help in identifying which specific bacteria have taken up the plasmid involved during the process and as a result, applied in areas of cloning (Mello & Fire, 1995). Reference Alberte J., Pitzer T., Calero K. (2012). General Biology Lab Manual / Second Edition. Florida International University: The McGraw Hill Companies. Davison, J. (1999). Genetic exchange between bacteria in the environment. Plasmid, 42(2), 73-91. Lorenz, M. G., & Wackernagel, W. (1994). Bacterial gene transfer by natural genetic transformation in the environment. Microbiological reviews, 58(3), 563. Mello, C., & Fire, A. (1995). DNA transformation. Methods Cell Biol, 48(48), 451-482. Miller, H. (1987). [13] Practical aspects of preparing phage and plasmid DNA: Growth, maintenance, and storage of bacteria and bacteriophage. Methods in enzymology, 152, 145-170. Smith, J. M., Smith, N. H., ORourke, M., & Spratt, B. G. (1993). How clonal are bacteria? Proceedings of the National Academy of Sciences, 90(10), 4384-4388. Read More