pGLO Report.docx - Kalli Rohloff Bhatia LSC 348 Genetics...

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Kalli Rohloff Bhatia LSC 348 Genetics Lab December 3, 2018 pGLO in E. coli Abstract Altering cells genetically by using a plasmid placed in a bacteria, is the process that was done in order to separate and purify the green fluorescent protein (GFP). This process is called genetic transformation. The GFP was being separated from E. coli . This was done to determine what was causing the bacteria to fluoresce. pGlo transformation, GFP SDS-PAGE, and HIC chromatography were all used to aid in the determination of the fluorescence. To breakdown the process again, the pGlo transformation of the E. coli was the introducing of the pGlo plasmid DNA to the bacteria. This transformation showed the +pGlo LB/AMP/ARA containing plate was the only one that fluoresced under the UV light. This was due to it having the DNA plasmid and arabinose. Arabinose is the sugar that permits the fluorescence. The second step involved carrying out the SDS-PAGE. This was done by using gel electrophoresis. This determined what the green fluorescent protein needed to be able to fluoresce. The results showed that the green fluorescent protein needed to be in a no heat environment to fluoresce. The final step was the HIC column chromatography. This was done to separate and purify the green fluorescent protein gene. This was done by running the supernatant containing the green fluorescent protein through several different salt buffers that had different concentrations of salt. To determine if the plasmid
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was inserted correctly for the transformation of the E. coli , the green fluorescent protein was viewed under UV light. It fluoresced under the UV light examination so it was done successfully. Introduction The process of an organism being modified due to the insertion of new DNA that alters the phenotype of the organism is what is called genetic transformation (Student Manual pGlo). In this lab experiment E. coli was used as the organism that was being modified. The E. coli was introduced to pGlo plasmid to be able to show the horizontal gene transfer of bacterial transformation of the green fluorescent protein (Student Manual pGlo). In bacteria plasmids can be found. Plasmids are “ small, circular, double-stranded DNA molecule that is distinct from a cell's chromosomal DNA” (“Plasmid”). The genes that are in the pGlo plasmid that is used in this experiment are GFP, bla , pBAD promoter, and araC operon (“ PGLO™ Plasmid Map and Resources” ). The green fluorescent protein is coded by the gene GFP. Bla is what encodes beta- lactamase, the enzyme that is an antibiotic resistor (” PGLO™ Plasmid Map and Resources”). This enzyme is what is used to breakdown the antibiotic ampicillin. The next gene, pBAD promoter, is the DNA sequence that is in charge of putting together araC -arabinose, RNA polymerase, and also the green fluorescence protein gene transcription (“PGLO™ Plasmid Map and Resources”). Arabinose will start to interact with araC after the binding of pBAD and the DNA is complete. If bacteria is in the environment it will absorb the arabinose and promote RNA
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