Transformation of Bacterial Cells with Plasmid DNA

Transformation of Bacterial Cells with Plasmid DNA

Introduction: Transformation refers to the process in which the cell integrates foreign DNA to its genetic code, meaning it takes the genes and incorporates them into the cell’s current DNA. Cells that can do this naturally, most commonly bacteria and archea, are known as competent. The bacteria E. coli do not have high transformation competence under normal conditions, but can be manipulated to produce better results using a heat-shock system (Donahue and Bloom, 1998). Originally it was thought E. coli couldn’t be transformed until a study found that the pores of the bacteria’s cell wall could exhibit artificial competence with calcium chloride treatment (Mandel and Higa, 1970). Transformation of plasmid DNA would give the cell a nonessential gene, like resistance to antibiotics. Ampicillin is a toxin that causes bacterial cells to lyse by hindering cell wall synthesis. The selectable marker is the Ampicillin Resistance gene that codes beta-lactamase enzyme which breaks down the antibiotic. The transformed E. coli cells will be able to thrive in an ampicillin environment. The purpose of this experiment is to see how the resistant bacteria can grow into colonies with less than ten percent of the cells completing the transformation. We hypothesized that the bacteria will grow in petri dished 1, 2, and 4 (Figure 1). It is expected to see these three out of the four petri dishes contain growing bacteria; the fourth sample is predicted to die because they were untransformed cells in an ampicillin medium.
Results:
This experiment had four petri dishes containing the samples. They all had an LB medium. After being incubated overnight at 37°C, the first dish had the Ampicillin and transformed bacteria, the second had no Ampicillin with transformed bacteria. The third dish had the Ampicillin with untransformed bacteria, and the fourth had no Ampicillin with untransformed bacteria. The first dish grew small colonies E. coli, the second and fourth dishes had lawn growth colonies of bacteria, and the third dish had no growth (Figure 1). Plates three and four were the negative controls.

Figure 1. Photograph of the petri dish E. coli samples. Colonies grew in Dish 1(top left); there was also lawn growth in Dishes 2(top right) and 4(bottom right), and no growth in Dish 3(bottom left).
Discussion:
These results were exactly what were expected. Dish 1 with the Ampicillin and transformed bacteria grew in small colonies. The bacteria were resistant to Ampicillin and continued to reproduce and grow as long as a food source was available. There was a lot of lawn growth in Dishes 2 and 4 because there was no antibiotic to fight against. The bacteria died off in Dish 3 because it was untransformed and had no resistance to the Ampicillin. Dishes 3 and 4 were the negative controls. This validates the effectiveness of the experiment’s methods because they both had untransformed bacteria, Dish 3 with Ampicillin, Dish 4 without. This is to prove that in Dish 4 there is growth similar to normal conditions. Then in Dish 3 it is exposed to the antibiotic and all dies off. This ensures the components of the mediums are acting in the expected way in Dished 1 and 2. There was less growth of the transformed bacteria in Dish 1 because the approximately ten percent of cells that transform have to fight off the Ampicillin, while the other Dishes with growth have no resistance. This experiment was using the chemical method for bacterial transformation. Another way would be using the physical method, which is creating the pores by electroporation. It has better results, generally, as compared to the chemical method, and it could also present larger DNA fragments like chromosomal DNA into the bacteria. There are many applications for a specific bacterial gene, for example: vaccines, agriculture, and food like yogurt.
Literature Cited: Donahue RA, Bloom FR (1998). "Large-volume transformation with high-throughput efficiency chemically competent cells". Focus 20 (2): 54–56.
Mandel M., Higa A. 1970. Calcium-dependent bacteriophage DNA infection, Journal of Molecular Biology, 53(14), 159-162.

Cited: Donahue RA, Bloom FR (1998). "Large-volume transformation with high-throughput efficiency chemically competent cells". Focus 20 (2): 54–56. Mandel M., Higa A. 1970. Calcium-dependent bacteriophage DNA infection, Journal of Molecular Biology, 53(14), 159-162.