The transformation of a cell is the process of a cell taking up and showing traits from a foreign genetic material, primarily DNA. First demonstrated by English bacteriologist Frederick Griffith, genetic transformation occurs in a large range of organisms. Several ways exist that allow cells to acquire this foreign DNA. The first is conjugation which is the process of mating between two bacterial cells, they share their own DNA and thus conjugate together. Another is transduction which involves viruses called bacteriophages (phages) that attach to and then inject their foreign DNA into the target cell. Lastly, there is transformation, simply the process of bacteria taking up the foreign DNA from their surrounding environment through their permeable cell walls. Overall, the process of being able to take up foreign DNA from the surrounding environment defines a cell as competent.
Genetic transformation of cells is a widely accepted and used practice among many areas of biology, genetics, medicine and even agriculture.
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In the area of medicine and treatment, one practice includes gene therapy, the inserting of genes into one?s cells and tissues to treat diseases. Despite that the technology is still experimental and somewhat controversial, it has lead to many scientific breakthroughs that serve to slowly incorporate it into mainstream medicine.
Practice of genetic transformation in agriculture is a growing study among scientists and farmers alike. CIAT, International Center for Tropical Agriculture, have worked to develop advances in the common bean. Current efforts have made improvements in the regeneration and transformation response in this bean stronger while preserving desirable traits.
However, it should be noted that competence of a cell is defined by two types: natural and artificial. It should also be noted that a large portion of use among industry and science leans towards the use of artificial competence in cells, intentionally forcing foreign DNA into a cell.
In this lab we will be observing the competence of E. coli cells. Using heat shock, we will transform E. coli cells with pGLO which has three parts. First a gene green fluorescent protein (GFP) that must be promoted by araC which is activated by arabinose in order to glow. There is also ampicillin resistance in pGLO which is a resistance to the antibiotic ampicillin that kills bacteria. This will allow us to observe which samples have effectively undergone transformation with pGLO, arabinose, or both based on the amount of bacteria that survives and whether or not it glows. We can hypothesize that if E. coli cells grow then they will have absorbed the ampicillin resistance. If cells also glow then they are considered competent because the pGLO was then transformed into the cell with heat shock and thus the glowing is a genetic trait that was taken up from the foreign DNA.
MATERIALS AND METHODS:
For this lab we first needed to gather the following: 4 LB nutrient agar plates, a micropipette, pipette tips, two microcentrifuge tubes, transformation solution, sterile loops, pGLO, and pGLO plasmid. After labeling two microcentrifuge tubes accordingly with one being + and the other ? pGLO, we proceeded to transfer 250ul of transformation solution into each tube. Both tubes were then placed on ice until further instruction. Picking up a single bacteria colony from the starter plate assigned and using a sterile loop we proceeded to immerse the used-loop into the +pGLO fluid tube. In order to disperse the entire colony throughout the fluid and avoid floating chunks in the mixture, we spun the loop using our fingers. Now, repeating the above process with a new sterile loop, we picked another bacterial colony and mixed it in with the ?pGLO tube. Using another new sterile loop, we then immersed it into the tube marked as ?pGLO plasmid DNA.? We inserted this loop, covered with a thin layer of substance, into the tube labeled +pGLO. Next, we incubated the ?pGLO and +pGLO tubes on ice for 10 minutes.
After returning the tubes to cooling, we labeled our 4 agar plates as follows: ?-pGLO LB/amp, +pGLO Lb/amp/ara, -pGLO LB/amp, and ?pGLO LB?. By then, it was time to take the tubes out from the ice and place them in hot water for 50 seconds; this was the process of heat shock on the cells, serving to make the membrane more permeable allowing for DNA to cross. After that step, we returned and placed both tubes on ice once again for 2 minutes. Now, with a fresh tip on our micropipetter and the tubes back on the rack, we added 250ul of LB nutrient broth in with the tube labeled +pGLO and then did the same for ?pGLO making sure to use another new tip. After adding broth to both, we closed the lids on both tubes and allowed them to sit at room temperature for 10 minutes.
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After the 10 minutes have passed, we mixed both tubes with a simple flick of the finger and proceeded to transfer 100ul of the +pGLO and ?pGLO onto the agar plates. We added +pGLO to the ?LB/amp? and ?LB/amp/ara? plates and the ?pGLO to the ?LB/amp? and ?LB? plates. Mixing contents on the surface of each plate, we used a new sterile loop to gently scratch the surface of each. Finally, we stacked and taped together all plates to store in 37 degrees C for 24 hours.
Being that we now had applied to all the proper agar plates and allowed the plates to culture for a full 24 hours at 37 degrees C, it was time to observe the results. The goal of this experiment was to decide the competence of E. coli and thus if these cells were transformed with the pGLO or not.
Of the four total plates used in this lab, two of these were controls to monitor the changes in the other two plates. The two controls plates were the ones that were labeled ?-pGLO LB/amp? and ?-pGLO LB?. ?Amp? or ampicillin is the antibiotic that kills bacteria, while ?LB? or Luria Broth is nutrients for bacteria that promote growth and colony development. The first of the control plates, -pGLO with LB/amp, showed no signs of bacteria and therefore demonstrates that Luria Broth and ampicillin combined yields no growth of bacteria. It is also visible that this dish did not receive any pGLO, thus it is named -pGLO. This control was successful in showing that without possessing the ampicillin resistance, bacteria cannot form on the plate even with LB present and is thus killed off completely.
The second of the control plates, that of ?pGLO with LB, showed large colonies all over the plate (roughly 95% covered). These colonies show a white-yellow color and serve to demonstrate that with Luria Broth alone and without pGLO, growth of bacteria is rampant. This plate clearly demonstrates the effects of LB in that it promotes bacteria growth, especially without the presence of any ampicillin.
Of the four total plates, the two remaining plates were considered transformation plates and contained pGLO, unlike the control plates. The first of these two transformation plates, +pGLO with LB/amp, given that with the presence of pGLO comes ampicillin resistance, it produced bacterial colonies. These colonies show that some cells were successfully transformed, with help from both LB and amp-r, roughly 20% of the plate was covered with bacterial colonies.
The last of the two transformation plates, +pGLO with LB/amp/ara, having contained the same elements of the previous transformation plate it however also had the addition of ?ara? which is arabinose. Arabinose serves to activate araC in its presence, the activated araC acts as a promoter for the green fluorescence gene. This plate showed plate growth equal to roughly 15% of the total plate and also showed fluorescence of a blue-green color when put under a black light.
Now that the experiment has been performed and the results collected and analyzed, it can be concluded that the null hypothesis of this experiment was disproven in that when E. coli was subjected to pGLO plasmids, with heatshock, it did take in foreign DNA that permitted growth despite the presence of ampicillin. Of the experimental dishes, the one that contained arabinose(ara), showed that ara served to activate the green fluorescence protein (present as an allele in some jellyfish) which showed a green-blue glow under a U.V. light and also proving that transformation has occurred in these cells. For this to have occurred, the dish must have LB/amp/ara to successfully show a fluorescent color. In a more broad application, the ability for an organism to turn on or off a particular gene, in this case fluorescence, in response to environmental conditions serves many a purpose. Being able to control genes in a particular setting can serve as a defense mechanism to scare away predators but also allows for camouflage potential when turned off. While the two experimental plates showed the effects of the substances on them, the two control plates were used to prove that our resources were effective and the methods used were correct. Both control plates, with varying combinations of Luria Broth and ampicillin, proved both the effectiveness of Luria Broth in promoting bacteria growth and the ampicillin in neutralizing bacteria altogether. Not only, once again, did the controls prove the effectiveness of our variables but it also showed that the methods used were also effective and accurate in procedure. There were few problems or errors in the experiment and observation of the results, except that it was rather time consuming and with frequent intervals of waiting potentially allowed for more error. However, the bacterial growth of our experimental plates seemed to be less than that of many other groups, a difference that would most likely be contributed to some form of minor contamination. The data generally seemed to be on track with that of the general experiment and paired well with the initial expectations of the experiment. While human error can be a killing-factor in many experiments, this was generally avoided by closely following the procedure and taking the time needed for each step.
- CIAT, Joe Tohme and Zaida Lintini. ?Using Agrobiodiversity through Biotechnology: Genetic Transformation?
- Wikipedia. ?Transformation (genetics)? - From Wikipedia, the free encyclopedia
- Cohen. ?Nonchromosomal antibiotic resistance in bacteria: genetic transformation of
Escherichia coli by R-factor DNA?
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