Monday, July 29, 2019
An Experiment Conducted in Attempt to Transform DNA off the Bacteria E. Coli
An Experiment Conducted in Attempt to Transform DNA off the Bacteria E. Coli Abstract: The main objective of this experiment was to transform DNA found in bacteria. A plasmid was used on E. coli and with the use of heat shock, was inherited by the bacteria which caused the E. coli to become resistant to ampicillin. Also in the plasmid was GFP which verified the hypothesis by glowing green under UV light. The predicted outcome held true and gives insight on what the future of medicine might hold. Introduction: Transforming DNA on bacteria will require a change in their genes. Using pGLO in this experiment allowed the DNA on Escherichia coli (E. coli) to transform. There are three genes in this plasmid: the araC gene, green fluorescent protein gene (GFP), and the bla gene. The ara C gene is a bifunctional regulator which transcripts araC mRNA which translates to produce araC proteins that act as a depressor or promoter to the GFP. The GFP also transcripts to produce GFP mRNA which translated to produce GFP that glows green under ultraviolet light (UV light). The last gene is the bla gene which gives the bacteria ampicillin resistance. E. Coli was used in this experiment because it is found inside our bodies so it is not very harmful to humans and grows rapidly. Bacteria transformation is the process by which foreign DNA is introduced into a cell (addgene.org). Using plasmids, A linear or circular double-stranded DNA that is capable of replicating independently of the chromosomal DNA(biolog y-online.org), transformation is able to occur. Using heat shock, most of the bacteria accepts the foreign DNA and incorporates it into its own DNA. In this experiment E. coli was transformed to become resistant to the antibiotic ampicillin and expressed the GFP. Using heat shock and pGLO will transform E. coli to be resistant to ampicillin while glowing green. These results could help transform human DNA genes and give resistance to life-threatening diseases such as AIDS. Methods and Materials: Two micro test tubes were labeled +pGLO and the other Ã¢â¬âpGLO. Using a sterile transfer pipette, 250Ã µL of transformation solution (CaCl2) were transferred to each test tube. The test tubes were placed in a bucket of ice for 3 minutes. A single colony of E. coli was placed in the transformation solution (CaCl2) of the test tube labeled +pGLO with a sterile loop and spun until the colony was all the way in the transformation solution. The same was done for the test tube labeled Ã¢â¬âpGLO. After, both test tubes were placed back in the bucket of ice for another 3 minutes. A new sterile loop was used to transfer pGLO Plasmid DNA to the test tube labeled +pGLO, not Ã¢â¬âpGLO. The test tubes were then back in the ice bucket for 10 minutes. While the test tubes were in the ice, the agar plates were gathered and labeled with the plate type and group name. After 10 minutes of the test tubes being in ice, they were both transferred to a water bath set at 42Ã °C for 50 seconds. After 50 seconds the test tubes were placed back in the ice bucket for 2 minutes. Taking both test tubes out, a sterile pipette was used to pipette 250Ã µL of LB nutrient broth into a test tube then mixed well. The same procedure was done to the other test tube using a new sterile pipette. Once both were mixed, they were incubated at room temperature for 20 minutes. After the 20 minutes are done and using a new sterile pipette for each tube, 100Ã µL of the transformation and control suspensions were transferred to the corresponding agar plates. Using a new sterile loop for each plate, the suspensions were evenly spread around the surface of the LB nutrient on each plate. The plates were stacked, labeled with a group name, and placed upside down in a fridge at 37Ã °C until next week. Independent Variable: Whether or not the plate contained pGLO Dependent Variable: Growth rate Controlled Variables: Amount of LB nutrient Amount of transformation solution Amount of suspensions Time in the ice bucket and room temperature Temperature of water bath and fridge Positive Control: -pGLO, LB Negative Control: -pGLO, LB, AMP These controls were selected because even without the plasmid, the plate labeled Ã¢â¬Ë-pGLO, LBÃ¢â¬â¢ should have growth on it because the only thing added to the plate was nutrients for the bacteria. The plate without the plasmid, LB nutrient, and ampicillin is a negative control because the ampicillin should kill off all the bacteria on the plate. Discussion: The hypothesis was supported in this experiment. In the plate labeled Ã¢â¬Ë+pGLO, LB, AMP, ARAÃ¢â¬â¢ there was growth when normally the ampicillin would kill of the bacteria, as shown in the plate labeled Ã¢â¬Ë-pGLO, LB, AMPÃ¢â¬â¢. Also looking at the UV Light figure, the same plate was able to glow as a cause of inheriting the GFP and being in the presence of arabinose sugar. In the plate labeled Ã¢â¬Ë-pGLO, LBÃ¢â¬â¢ there should be bacterial growth, but there is none present. This could be a possible error in not transferring a big enough colony of E. coli. The plate labeled Ã¢â¬Ë+pGLO, LB, ARAÃ¢â¬â¢ should have more distinct colonies glowing however on the UV Light figure only a film is seen glowing. This could be a cause of putting too much pressure on the colony when spreading the E. coli on the plate. Having the ability to genetically modify DNA could help researchers in the medicine field develop cells that would be resistant to any harmful thing, like the flu or HIV. In conclusion, altering bacteriaÃ¢â¬â¢s DNA is possible and so easy it can be performed by freshmans in college.