Sunday, August 18, 2019

Ligation of EGFP into pET41a(+) vector transformed into E. coli cells :: PCR amplification of extracted DNA plasmid

Ligation of EGFP into pET41a(+) vector transformed into E. coli cells followed by PCR amplification of extracted DNA plasmid for success evaluation along with gel electrophoresis at each step. Introduction Enhanced green fluorescent protein (EGFP) was originally isolated from a bioluminescent jellyfish called Aequorea victoria. As suggested by the name, this protein fluoresces green when exposed to light in the ultraviolet range. The ultimate goal of the following experiment was to successfully create a pET41a(+)/EGFP recombinant plasmid that was transformed into live E. coli cells. The success of this transformation could be evaluated based on whether EGFP’s fluorescence properties were displayed by the colony in question. The protein’s fluorescence properties â€Å"triggered the widespread and growing use of GFP as a reporter for gene expression and protein localization in a broad variety of organisms† (Ormo, et. al., 1996). Although EGFP and GFP differ for a few amino acids that make EGFP’s fluorescence mildly stronger, the basic principle that such a protein allows for the evaluation of transformation success remains intact. The first step of the experiment was ligation, and the objective was to insert EGFP cDNA into a restriction cut pET41a(+) vector to obtain a recombinant plasmid that would express green fluorescent gene. pET41a(+) was the choice of vector to ligate the EGFP into. Its structural design and genomic sequential properties render it especially well-suited for cloning and high-level expression of peptide sequences. This 5933 bp circular vector contains a built in sequence for Kanamayacin resistance gene. â€Å"Rooting of non-transgenic shoots was completely inhibited in all culture media containing kanamycin† (Montserrat, et. al., 2001). This allowed the growth of recombinant and non-recombinant colonies of E. coli, all of which contained the vector insert. Once the recombinant plasmid was obtained, it was then inserted into E. coli cells through transformation. From a successful transformation, we expected the bacterial cells to translate the inserted EGFP sequence into its protein form. The bacteria cultures were plated on petri dishes containing growth supplement, Luria Broth (LB), an antibiotic: Kanamycin, and IPTG which induced the fluorescence property within successfully transformed bacterial colonies. Different variants of the petri dishes were also included as control and unknown. The miniprep consisted of isolating the DNA plasmid from the bacterial cells. This was used to identify the success of EGFP ligation into pET41a(+) vector upon restriction digest and gel electrophoresis. Additionally, Polymerase Chain Reaction (PCR) was run on the isolated DNA plasmids with one of the primers specifically annealing to a part of pET41a(+) sequence and the other annealing to the EGFP gene.

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