Date of Award
Fall 11-26-2019
Degree Type
Thesis
Degree Name
Master of Science (MS)
Department
Biological Science
First Advisor
Margaret Silliker, PhD
Second Advisor
Jingjing Kipp, PhD
Third Advisor
William Gilliland, PhD
Abstract
The plasmodial slime mold and member of Mycetozoa, Didymium iridis, has been studied in a variety of contexts such as RNA editing (Traphagen et al., 2010; Hendrickson and Silliker, 2010a; Hendrickson and Silliker, 2010b), mitochondrial inheritance (Silliker et al., 2002), biological speciation (Betterley and Collins, 1983; Clark et al., 1991) and mating competency (Shipley and Holt, 1982). Further studies are hindered by the lack of a transfection protocol, which would allow for gene manipulation in D. iridis (hereafter Didymium). Transfection methods developed in the related slime molds, Physarum polycephalum (Burland et al., 1993) and Dictyostelium discoideum (Fey et al., 1995; Pang et al., 1999), have only been successful when native regulatory sequences were used. This study tested whether Didymium could recognize vectors with regulatory elements (a promoter and terminator) from related slime molds, and whether vectors with regulatory elements native to Didymium would be recognized and expressed when introduced by standard transfection methods. We constructed vectors using overlap extension PCR and the Gibson Assembly. These vectors were comprised of a cloning vector backbone, regulatory elements from close relatives of Didymium or Didymium, and a green fluorescent - reporter gene gfp. Four plasmids were introduced into Pan 2-16 amoebae; pDicty, pPhys, pDidy 1.0 and pDidy 2.0, by means of electroporation, lipofection, and XFECT transfection. Expression of the reporter gene, gfp, from these constructs was observed by fluorescence microscopy. Though gfp expression was observed with Didymium and Physarum based vectors, the number of transformants by any transfection method employed was extremely low. However, for each method of transfection, there was consistency in the parameters that worked, even with different constructs.
Recommended Citation
Baray, Uriel Adolfo, "Developing a transfection method for Didymium iridis" (2019). College of Science and Health Theses and Dissertations. 351.
https://via.library.depaul.edu/csh_etd/351
SLP Collection
no