| Advisor : | STEPHEN MAYFIELD | ||
|---|---|---|---|
| Abstract Title : | Expanding the Algal Genetic Engineering Toolkit | ||
| Abstract : | Genetic engineering of model organisms such as E. coli has greatly contributed to the increased production of recombinant therapeutic molecules. However, bacteria are limited in their ability to produce complex proteins. Engineered eukaryotic algae have a demonstrated ability to circumvent this problem due to the increased sophistication of algal protein-folding machinery, but a lack of well-characterized gene regulatory elements presents a major obstacle in making this a viable platform for protein production. An expanded genetic engineering toolkit must be developed within this system to overcome these limitations. We developed two new tools for algal engineering in the green alga Chlamydomonas reinhardtii - one for obtaining increased expression from transgenes in the chloroplast genome, and the other as an enhanced reporter for gene expression in the nuclear genome. We increased protein accumulation in the chloroplast by creating synthetic 5' untranslated regions (UTRs) that lead to higher transgene expression. We also improved upon C. reinhardtii's endogenous arylsulfatase gene ARS2 as an effective nuclear reporter, using a cDNA-derived construct that transforms more efficiently and expresses to higher levels than reporter constructs previously described in the literature.These new tools will advance algal genetic engineering for future applications in high-value protein production, metabolic engineering, or perhaps even bioenergy production. | ||
| Advisor : | MARK ESTELLE | ||
|---|---|---|---|
| Abstract Title : | Characterizing transcriptional outputs mediating circadian gene expression in Arabidopsis thaliana | ||
| Abstract : | The field of chronobiology studies the complex system driving biological rhythms. While there have been many advances in the effort to study the synchronization of endogenous circadian rhythms to information received from the environment, there is still much left to be understood. My project asks the question, what are the cellular processes in the model plant species Arabidopsis thaliana that lead to the translation of external inputs into functional clock phenotypes? The almost-ubiquitous extension of circadian expression to output pathways in plants points to an elaborate cascade mechanism involving transcription factors downstream of the core clock proteins LHY, CCA1, and TOC1. The goal of my project is to identify and characterize circadian output components that are a part of this cascade. I focused on a clock-controlled protein, WLIM1, that shows strong cycling but with previously unknown circadian regulation. WLIM1 is a broadly-expressed transcription factor crucial to actin bundle formation and higher-order cytoskeleton assembly. ChIP-sequencing of the WLIM1 promoter points to the possibility of a novel mechanism driving its output. Furthermore, I hope to identify its possible role in the circadian regulation of actin protein ACT7 | ||