E. Coli Lab

Introduction:

Genetic transformation is a change caused by genes, involving the insertion of a gene into an organism to change a particular trait of tht organism. In the lab, bacteria, specifically E. coli, will be transformed with a gene that codes for GFP. Those that are exposed to that gene will glow a green color under ultraviolet light. Plasmid DNA contain genes for traits that may be helpful for survival. I his lab, pGLO is resistant to the antibiotic ampicillin. The gene for GFP gets switched on in transformed cells when sugar, in this case arabinose, is added to the cells.

Purpose: In the pGLO E. coli lab, the main goal of our experiment was to find the condition under which the bacteria would grow in its antibiotic resistant form and glow. Two of the plates did not contain the plasmid (-pGLO), and therefore would not appear to glow underneath the black light. The purpose of these two plates was using them as a source of comparison. In order for the lab to be successful, we needed to complete the lab by making one of the plates antibiotic resistant as well as able to glow under the black light. We were trying to prove that in order for E. coli to grow and glow, the presence of a sugar was necessary.

Methods: First we labeled 2 micro test tubes (+pGlo and -pGlo) and placed them in the tube rack. We then transferred 250 micro liters of the transformation solution into each tube. After that we placed the tubes in a cup full of ice. Using a sterile loop, we took some bacteria from the starter plate and placed it into the +pGlo tube and made sure the colony was mixed inside. We repeated the process for the -pGlo tube and placed both tubes back in the ice. A new loop was used to extract plasmid DNa from the stock tube. We then mixed added it to the +pGlo tube ONLY. We let the tubes stay in the ice cup for 10 min. While waiting, we labeled our agar plates respective to the lab instructions. After time had passed, we trasnfered the tubes into a water bath (at 42 degrees Celsius) for 50 seconds, then took them out to be placed in ice again for 2 minutes. After removing the rack from the ice, we added about 250 micro liters of broth to each tube (pGlo+ and pGlo-). The tubes were mixed properly and then 100 micro liters were extracted from each and placed respectively in 4 agar plates. We then had to spread the suspensions around the plates surface.

Discussion: The procedure of the pGLO resulted in the expected outcome. Each plate came back from the incubator with the correct and predicted growth: the -pGLO LB plate had innumerable bacterial growth, the -pGLO LB/AMP had no growth (therefore the antibiotic was successful), the +pGLO LB/AMP had growth that did not glow beneath a black light, and the +pGLO LB/AMP/ARA had glowing bacterial growth. A pattern became evident that unless the plasmid DNA was present, there would be no growth in a plate that contained the antibiotic. We were able to determine that glowing E. coli needs sugar to grow, while non-glowing bacteria with or without the newly inserted plasmid DNA. There were no inconsistencies with our data: we obtained the results that we should have in our attempt at transforming the bacteria. 

Conclusion: We started this lab to ultimately answer the question, will the presence of sugar be  necessary to make the E. Coli grow/glow. Looking back at our results, we concluded that our data supported to hypothesis that the sugar should make the bacteria experience growth and glow. The +pGLO LB/AMP/ARA plate showed the resulted that were expecting further proving that we were successful in our steps when trying to transform the plasmid.