College of Science and Health Theses and Dissertations

Date of Award

Fall 11-26-2019

Degree Type


Degree Name

Master of Science (MS)


Biological Science

First Advisor

Margaret Silliker, PhD

Second Advisor

Jingjing Kipp, PhD

Third Advisor

William Gilliland, PhD


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.

SLP Collection


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Biology Commons