Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2014. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 123-130). / Experimenter control of target gene expression is a fundamental component of molecular biology research. In many systems, tools exist that allow generalizable control of gene expression at the transcriptional or post-transcriptional level. Plasmodium falciparum, the protozoan parasite responsible for the majority of death and sickness due to malaria, remains challenging to manipulate in the laboratory. No robust and generalizable tool for gene expression control has been developed in the parasite. To address this need, we engineered a new system for control of protein translation in eukarvotes, and applied it to P. falciparum. This system is based on the ligand-regulated interaction between an RNA aptamers and the TetR-repressor protein. Although such protein-RNA interactions are abundant in nature and are known to effectively mediate control of gene expression, our system is unique in its direct modulation by an exogenous chemical. By genetically encoding TetR-binding RNA aptamers in the 5' untranslated region (5'UTR) of an mRNA, translation of a downstream coding sequence is repressed by TetR in vivo and induced upon adding a non-toxic tetracycline analog. We first define the system's component molecular interactions in vitro, followed by optimization of the constituent parts for convenience and performance. We then further optimize the system and validate its performance in two model systems, the budding yeast Saccharomvces cerevisiae and cell-free rabbit reticulocyte extracts. We show the broad utility of the system in P. falciparum for controlling expression of reporter and endogenous proteins trafficked to a variety of subcellular compartments. Induction and repression are rapid and homogeneous across the cell population. Placing a drug resistance determinant tinder inducible control, we are able to modulate P. falciparum drug sensitivity, demonstrating the usefulness of the system for controlling relevant parasite biology. In the process of constructing and validating a novel tool for gene expression in P. falciparum. we built a new series of gene expression vectors for molecular biology work in the parasite. In addition to developing optimized protocols for plasmid construction, we built a standardized, sequence-defined family of plasmids for malaria research. In all, we present a generalizable, well-defined toolkit for genetic programming of P. falciparum. / by Stephen J. Goldfless. / Ph. D.
| Identifer | oai:union.ndltd.org:MIT/oai:dspace.mit.edu:1721.1/88903 |
| Date | January 2014 |
| Creators | Goldfless, Stephen J. (Stephen Jacob) |
| Contributors | Jacquin C. Niles., Massachusetts Institute of Technology. Department of Biological Engineering., Massachusetts Institute of Technology. Department of Biological Engineering. |
| Publisher | Massachusetts Institute of Technology |
| Source Sets | M.I.T. Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 130 pages, application/pdf |
| Rights | MIT theses are protected by copyright. They may be viewed, downloaded, or printed from this source but further reproduction or distribution in any format is prohibited without written permission., http://dspace.mit.edu/handle/1721.1/7582 |
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