The Effects of Ampicillin versus Tetracycline on the Plasmid Copy Numbers of pBR322

The pBR322 plasmid contains genes encoding for tetracycline and ampicillin resistance. Escherichia coli DH5α cells with pBR322 plasmid were grown and plated on selective plates containing 1) ampicillin only and 2) tetracycline only with varying antibiotic concentrations of half strength, three-fourth strength and full strength (15 µg/mL tetracycline and 50 µg/mL ampicillin for full strength). Colonies from the plates were grown overnight in Luria Broth (LB) and the cells were used for plasmid isolation. Gel electrophoresis was done on the isolated plasmids and pixel counts in the digital gel images were analyzed by Kodak 1D to determine the intensity of the bands on the gel. It was found that ampicillin selection led to a higher digital pixel counts and copy numbers of plasmids per cell than tetracycline selection. Furthermore, there seemed to be a correlation that there was an increase in plasmid copy numbers with an increasing antibiotic concentration. The pBR322 plasmid is a cloning vector containing both ampicillin and tetracycline resistance genes as selectable markers, and it is a widely used vector in microbial genetic experiments in research laboratories. Ampicillin resistance gene encodes for the enzyme β-lactamase. This secreted enzyme binds and inactivates the ampicillin molecule by catalyzing action of hydrolysis of the β-lactam ring of the ampicillin (13). Tetracycline resistance gene encodes a transmembrane efflux pump that pumps out tetracycline molecules that have entered the cell (15). E. coli DH5α cells containing pBR322 have a selective advantage in these antibiotic-containing environments. Previous experimental results found by Law et al. showed that the initial numbers of colonies formed on ampicillin plates were higher than tetracycline plates after E. coli DH5α cells were transformed with pBR322 plasmid (10). A number of factors were considered that could contribute to these results. One possible factor could be the level of resistance gene expression in E. coli DH5α cells, where there could be a higher expression of ampicillin resistance gene than the tetracycline resistance gene, thus resulting in higher number of cells grown on the ampicillin plates. This factor was considered because another experiment showed that increasing the copy numbers of the plasmid that carries chloramphenicol resistance gene enhanced bacterial efficiency at gaining drug resistance (8). This experiment investigated the pBR322 plasmid copy numbers in E. coli DH5α cells grown on ampicillin and tetracycline treated environments to see whether there are differences in resistance gene expression. A higher plasmid copy number is assumed to correlate with higher gene expression. Furthermore, these cells were treated with different strengths of antibiotics to observe whether the pBR322 plasmid copy numbers in E. coli DH5α cells was enhanced by the increasing antibiotic strengths. Three different approaches were used to test the hypothesis. The first method included isolation of the pBR322 plasmid from E. coli DH5α carrying the plasmid followed by transformation of the plasmid into E. coli DH5α with no plasmids. In the second approach, plasmid DNA from E. coli DH5α cells with pBR322 grown on ampicillin and tetracycline plates were isolated using a breakage buffer for rapid cell lysis. Finally a plasmid miniprep was used to isolate pBR322 from the E. coli DH5α with pBR322 grown in Luria broth containing ampicillin and tetracycline and the samples were electrophoresed on an agarose gel. Plasmid Isolation for Transformation by the Hanahan Protocol (Sambrook, 1989): The pBR322 plasmid was isolated from E. coli DH5α cells growing at mid-exponential phase. Cells were centrifuged at 8,500 x g using the IEC 818 rotor and then resuspended in lysozyme breakage buffer. Sodium hydroxide (0.4 M NaOH) and sodium dodecyl sulphate (2% SDS) was added to the cell suspension. The cell suspension was then treated with acidified salt buffer to precipitate the chromosomal DNA. The sample was then centrifuged at 7,700 x g (IEC 818 rotor) and was washed with phenol twice to remove residual proteins. The plasmid suspension was centrifuged at 4,400 x g (IEC 818 rotor) to separate it from the phenol layer. The plasmid was then precipitated with ethanol and then centrifuged at 6,200 x g (IEC 818 rotor). The plasmid pellet was resuspended in tris-ethylenediamine tetraacetic acid (Tris-EDTA) buffer and was treated with ribonuclease. This plasmid sample was then placed into a dialysis bag with the addition of chloroform and was dialyzed overnight in EDTA buffer. Concentration of DNA: The plasmid sample was diluted 1-in-25 and 1-in-50 at a final volume of 1 mL of Tris-EDTA buffer. The absorbance was read using the double beam spectrophotometer (Ultrospec 3000 UV/Visible Spectrophotometer, Biochrom) at wavelengths of 260, 270 and 280 nm. By using the conversion factor that one A260 unit is equal to 50 µg/mL of DNA, the DNA concentration in the undiluted sample was determined.