Sunday, September 29, 2019
Bacterial Transformation Using pGLO Involving X and Y Genes
Genetic transformation is due to a direct cause in the change by genes, due to the cell in taking and expressing traits from a separate piece of DNA. Naturally proficient bacteria are able to absorb exogenous DNA and go through genetic transformation. (Chen & Dubnau, 2004) The purpose of this experiment was to discover how a gene could be moved from one organism to a different organism with the help of plasmid. The cells that are capable of acquiring these traits from the other organism are known as being competent.Weedman, 2013). In this particular experiment we will genetically transform the bacteria E. coli by inserting a gene through heat shock, this gene codes for Green Fluorescent Protein, also known at GFP. The GFP gene originally comes from a Jellyfish and under an ultraviolet light the bacteria that acquired the gene with glow a brilliant fluorescent green color. (Portman et al. 2013). If the cells' nutrient medium has the sugar arabinose added to it then GFP can be turned o n. (Weedman, 2013). To determine if our hypothesis was correct, we used four differently prepared plates.The four plates each contained a different combination of the following; arabinose, ampicillin, LB nutrient broth, and pGLO plasmid. The combinations were; +pGLO LB/amp, +pGLO LB/amp/ara, -pGLO LB/amp, and -pGLO LB. Our hypothesis was: the plates with pGLO will have growth because they are resistant to the antibiotics involved, the plate with ampicillin and without pGLO will show no growth due to the fact that the antibiotic compromises the bacteria, and the plates that will grow will be the ones containing pGLO since they obtain the trait for glowing.Materials and Methods: All methods were obtained from (Weedman, 2013) Before beginning the experiment obtain latex gloves, two microcentrifuge tubes, a beaker filled with ice, a micropipetter, micropipetter tips, transformation solution containing calcium chloride, sterile loops, pGLO, E. coli, and four plates containing different s ubstances. To begin label the two microcentrifuge tubes +pGLO and Ã¢â¬â pGLO. Then proceed to obtain 250ul of transformation solution and put it in each one of the tubes using a different miropipetter tip each time, this solution will help enhance the permeability of the cell membranes.Then use a sterile loop to acquire single colony of E. coli to add to the tube labeled +pGLO; add this by twisting the sterile loop until the pGLO is off. Then repeat the last step for the -pGLO tube using a new sterile loop. Next add pGLO to the tube labeled +pGLO, to do this take a new sterile loop and inserted it into a vile containing the plasmid pGLO. Then twist the loop into the tube labeled +pGLO, then place both tubes into the beaker filled with ice for approximately 10 minutes. While the tubes are on ice grab the four LB (Luria Bertani broth) nutrient agar plates.Each plate should be labeled either +pGLO or Ã¢â¬â GLO; you should nave 1 LB/amp/ara plate (+pGLO), 1 LB plate (-pGLO 2 LB/am p plates (+pGLO)(-pGLO). After 10 minutes in the ice bath place the tubes in a floating rack and put them in a 420C water bath for exactly 50 seconds, giving them a heat shock. Immediately place both tubes back in the ice after the water bath for approximately 2 minutes. Once 2 minutes is up remove the tubes from the ice and put them in the rack at room temperature. Using a new tip each time, add 250ul of nutrient broth to both tubes. Then close the tubes and let them sit at room temperature for 10 minutes.After 10 minutes flick both tubes with your fingers to ix the contents, then using a fresh tip each time add 100ul of the transformation solution (+pGLO) and the control (-pGLO) to their appropriately labeled plates. Using a new sterile loop each time spread the contents around in each dish. Then tape the plates together and placed them upside-down in an incubator set at 370 C for 24 hours. Results: This experiment shows how a gene can be transferred from one organism to a differe nt organism through the help of plasmid. Traits are exchanged from one DNA stand toa different one in the bacteria E. coli.Two of the plates were a control group, hich meant there was no growth after the plates were taken out of the incubator. These two control plates were the ones containing -pGLO LB/amp and -pGLO LB. The transformation plates were the two plates containing +pGLO LB/amp and +pGLO LB/ amp/ara. These two plates showed a substantial growth in bacteria after being taken out of the incubator, one plate showing a considerably larger growth than the other and they both glowed under UV light due to the pGLO. The plate that obtained the arabinose had the largest amount of growth over the 24-hour period. http://mol-bi014masters. masters. grkraJ. g/html/Genetic_Engineering4A- Transformation-Bacterial Cells. htm http://faculty. clintoncc. suny. edu/faculty/michael. gregory/files/bio%20101 [bio %20101 %201aboratory/bacterial%20transformation/results. htm Discussion: Our hypothe sis was: the plates with pGLO will have growth because they are glowing. Our results supported our hypothesis, the plates that showed growth were the plates containing +pGLO LB/amp and +pGLO LB/amp/ara. Where as the other two plates showed no growth at all, which matched our hypothesis. Michael Gregory did a previous experiment; he came to the same conclusion that our experiments' results oncluded.His experiment was identical to ours, involving the same materials and procedure. The same plates showed growth in his experiment as ours, as well as the plates that didn't show growth were the same. (Gregory, 2004). The only weakness that I could think of that would have a major effect on the results would be not using sterile equipment and causing cross contamination. Our experiments did not have any problems arise that would affect the results we obtained.