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Intro to Dna

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Introduction: The DNA molecule is the blueprint for every component of a cell. From the information in DNA, RNA is transcribed and then translated into proteins. GMOs or genetically modified organisms are results of biotechnological techniques such as regulations of gene expression which can be done through transformation, the uptake of foreign DNA.

Specifically, E. coli cells are readily modified to incorporate a pFG gene into their DNA. This modified piece of DNA contains information that is transcribed and then translated to express phosphorescence in the genetically modified cell. The presence of the pFG gene alone will not result in fluorescence within the cells. A lac promoter is needed to ensure the expression of the pFG gene. Also, a selection sequence, in this case AMP, is chosen to help identify which bacteria have been transformed. This is possible since the transformed E.coli will be ampicillin resistant and the plates used to grow E.coli contain ampicillum. Lastly, the specific biotechnological method used is the heat shock method which is used to transform competent E. coli.

Experimental: Calcium Chloride (0.25Ml) was added to two micro centrifuge tubes labeled +DNA and -DNA. E. coli cells from source plates were added to each test tube. Test Tubes were vortexed to ensure all cells are suspended in solution. To test tube +DNA, pFG (10 microliters) was added and vortexted again. Both test tubes, +DNA and -DNA, were incubated on ice for 15 minutes and then placed in a water bath at 42 degrees Celsius to make the competent E. coli cells transform. The test tubes were immediately returned to the ice for two minutes. Luria Broth (250 microliters) was then added to each tube and the test tubes were both incubated at 37 degrees celcius for thirty minutes as a recovery time. Next the cells were plated onto four plates. Two plates contain only a Luria broth media while the other two plates contain Luria broth media plus the AMP/IPTG media. From the -DNA test tube 0.25mL was added to both type of plates yielding a LB/-DNA and a LB/AMP/ITPG/-DNA plates. The same was done with the +DNA test tube resulting in four plates: LB/-DNA, LB/+DNA, LB/AMP/IPTG/-DNA, LB/AMP/IPTG/+DNA. These plates were then placed in a incubator with agar side up for 15-20 hours. The following week UV light was used to determine if the E. coli in the plate LB/AMP/IPTG/+DNA had transformed in turn fluorescence.

Results: Plate LB/-DNA and plate LB/+DNA both contained normal E. coli growth in the form of bacterial lawns. The plate with LB/AMP/IPTG/-DNA had no E. coli due to the ampicillum and no resistance. Lastly, the plate LB/AMP/IPTG/+DNA contained six colonies of E. coli that underwent transformation resulting in ampicillum resistance fluorerscing bacteria. Transformation efficiency was on the order of 1200.

Discussion: The results strongly support the initial hypothesis as stated in the introduction. As expected,



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