Bachelor of Science
Major or Concentration
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Due to its natural fluorescence, Green Fluorescent Protein (GFP) is a preferred reporter protein for cellular localization studies. For use in nuclear localization studies, a fusion protein consisting of multiple copies of GFP must be used to eliminate simple diffusion as a mechanism through which the protein can enter the nucleus. The long-term goal of this research project is to create a plasmid expression vector containing three copies of the GFP gene linked to a single monopartite nuclear localization signal (3GFP-1NLS) and containing the commonly used pCMV eukaryotic promoter. The specific objective of the research study reported here was to create an intermediate plasmid, containing three copies of the GFP gene, from which the 3GFP-1NLS plasmid could be eventually produced. Using site-directed mutagenesis techniques, the parent plasmid used in this study, the pCMV/myc/nuc/GFP pShooter plasmid (Life Technologies, Inc.), was modified to create 2 intermediate plasmids, one serving as a vector and the other serving as a source for a single copy GFP insert. The restriction enzyme PstI was then used to successfully open the vector and isolate the insert. After multiple attempts, GFP insert has been successfully ligated into the vector creating the intermediate plasmid, 2GFP-3NLS. Again, using site-directed mutagenesis and restriction endonuclease digestion techniques, the 2GFP_3NLS plasmid was modified to create 2 intermediate plasmids, one serving as a vector and the other serving as a source for a single copy GFP insert. The restriction enzyme PacI was then used to successfully open the vector and isolate the insert. After multiple attempts, the GFP insert has been successfully ligated into the vector creating the 3GFP-3NLS. This plasmid will lead to the eventual production of the final 3GFP-1NLS plasmid. Once produced, the final plasmid could be used to study various aspects of NLS-dependent nuclear import.
Krumpos, Kristina Chantel, "Genetically Engineering Plasmid Expression Vectors for Nuclear Localization Studies" (2016). Student Research Submissions. 90.