The objective of this experiment was to test the transfer of antibiotic resistant genes using antibiotic resistant plasmids to inoculate competent E. coli cells. Each of the three cells produced (E.coliDH5α/pUC19, E.coliMM294/pKan, and E.coli/HB101) were resistant to either ampicillin, kanamycin, or neither. After exposure, these cells were spread onto various plates containing either the antibiotic ampicillin or kanamycin to determine if the antibiotic resistance genes had given them the ability to grow when antibiotics were present. The hypothesis was that each plasmid could grow in the presence of one antibiotic because antibiotic resistant genes are carried on plasmids. However, the results did not support this hypothesis.
Since their discovery, antibiotics have become a commonplace in the treatment of a range of illnesses. However, due to the sheer volume of prescriptions prescribed in the last century, many people have begun to develop a resistance to the antibiotics. Vast numbers of microorganisms have shown cases of being mostly if not completely immune to the effects of antibiotics. The majority of the time, they are able to accomplish this by resistance genes that are acquired from other bacterial species through means of conjugation, transformation, or transduction.
Each method of gene transfer is able to use plasmids as carriers. Plasmids are small, circular, double-stranded DNA found in bacteria that replicate independently of the bacterial genome. Bacteria traditionally replicate through asexual reproduction, however, they can also undergo “horizontal gene transfer” which is where genes can be acquired by a species through non-sexual means. There are three different mechanisms from which this can arise: conjugation, transformation, or transduction. Conjugation is where genetic information is exchanged between two cells through a sex pillus that temporarily forms between two cells. Transformation is where DNA is taken up by a bacterial cell from a medium on which it is growing. And, transduction is where new DNA is introduced into a cell through the use of a bacteriophage (Pierce, 2008).
For this experiment, transformation will be used to study the transfer of antibiotic resistant genes. First, we will be provided with plasmids that are hypothesized to be carriers of the antibiotic resistant genes. These will then separated from chromosomal DNA and isolated. A sample of plasmid DNA will be linearized to insure that the plasmid DNA will be isolated rather than chromosomal DNA. The size will then be measured via gel electrophoresis and compared to known lengths of DNA.
After, the isolated plasmids will be mixed with competent cells, or cells that are naturally able to take up exogenous DNA and undergo genetic transformation (Dubnau et al., 2004). The DNA will be mixed with the competent cells, then the cells with be heat shocked to induce the cells to engulf the plasmid DNA that will soon become part of its genome (Lindquist, 1986). After an hour of recovery, the cells should have undergone transcription and translation and the DNA will have integrated into the bacterial genome. The cells with the new DNA will then be spread onto three different plates. One will have the antibiotic kanamycin, one will have the antibiotic ampicillin, and the third- the control- will have no antibiotics. The results should prove or disprove the hypothesis that antibiotic resistance genes of E. Coli MM294/pKan and E.Coli DH5α/pUC19 are carried by plasmids.
Materials and Methods:
Prior to lab, two culture mediums (pUC19 and pKan) had been inoculated with a single bacterial colony, treated with the corresponding antibiotic, and incubated for 16-24 hours at 37 with vigorous shaking. Each lab group received a 1.5 mL sample of one of the two cell cultures on a 1.5 mL microcentrifuge tube.
Isolation of plasmid DNA:
The bacterial culture was microcentrifuged at maximum speed for 1 minute, and then