School of Botany - Theses
Permanent URI for this collection
Search Results
1 - 2 of 2
-
ItemDissecting antibiotic targeting in the malaria parasite Plasmodium falciparum(University of Melbourne, 2008)
-
ItemEstablishing and elucidating conditional genetic manipulation of the malaria parasite, Plasmodium falciparum( 2013)Methods to alter gene function in Plasmodium are barely out of infancy, with much growth occurring within the past five years via adaptation of protocols established in other species. In this thesis, a method where the parasite’s innate ability to control gene expression in response to environmental stimuli, and a synthetic small molecule regulated protein stability system were applied to change the phenotypic expression of introduced transgenes. In chapter 2, we aim to control activity of a temperature sensitive mutant FLP recombinase protein. Our goal was to induce FLP activity and thus change the open reading frame of a GFP expressing plasmid to mCherry using single site recombination. Dual transgenic parasites were placed in a variety of conditions to favour FLP activity and to prevent negative selection. No evidence of conditional genetic recombination or phenotypic change from green to cherry was observed. Direct expression of FLP during a short period of drug selection produced a mixed population of GFP and mCherry genotypes. The activity of FLP is inefficient and was deemed unsuccessful for our goal of raising transgenic parasites poised for conditional genetic recombination. A homogenic population of mCherry genotypes by successful recombination of the GFP ORF was achieved after drug-cycled expression of evolved FLP (FLPe). Chapter 3 focuses on turning the Plasmodium specific phenomenon of altering rRNA expression profiles in response to fluctuations of temperature and glucose concentration into a tool suitable for conditional gene expression. We copied the promoter region of the S2-Type rRNA gene and measured its ability to express luciferase in altered environmental conditions. A low level of basal activity was observed during the intra-erythrocytic developmental cycle, with expression peaking at levels similar to the weak chloroquine transporter (CRT) promoter during the trophozoite stage. The S2-Type rRNA promoter region did not produce a notable increase of luciferase transcription or activity in response to environmental change. Use of the S2-Type rRNA promoter to express FLP at the enzymatic favourable temperature of 26°C did not result in genetic recombination. The tools developed are capable of expressing transgenes, however their ability to conditionally alter the gene product is limited.