Date of this Version
Brueggeman, A.J. (2013). Transcriptomic Analyses of the CO2-Concentrating Mechanisms and Development of Molecular Tools for Chlamydomonas reinhardtii. PhD Dissertation, University of Nebraska.
Microalgae, such as Chlamydomonas reinhardtii, account for a large percentage of photosynthesis that occurs on the planet. Many algae possess a Carbon-Concentrating Mechanism, or CCM, that actively transports inorganic carbon (Ci) into the cell to create artificially high internal levels of CO2, enhancing their rate of carbon fixation. The production of biofuels from algal sources can serve as both a renewable and carbon-neutral energy source. This thesis details research in Chlamydomonas, in the effort to both better understand the CCM in algae and improve laboratory and industrial manipulations with algae.
In the first chapter of this thesis, changes in the Chlamydomonas transcriptome in response to changes in environmental CO2 levels are investigated. In addition to witnessing changes in the transcription of 38% of Chlamydomonas genes, numerous patterns of genetic response are detailed, including a transient reduction in overall transcription that is later alleviated as cells became acclimated to the new environment. Our data also reveal a vast bidirectional promoter system in Chlamydomonas, detail markedly different responses of gene families, and elucidated several putative binding motifs for transcriptional regulators involved in responses to low CO2.
The remaining two chapters detail efforts to develop tools for use in Chlamydomonas. In the second chapter, drawing upon prior knowledge of herbicide resistance in higher plants, we developed several herbicide-resistant genes for optimal function in Chlamydomonas. As a result, we now have resistance markers for three classes of herbicides. These genes can be used either as selectable markers in transgenic experiments or as tools for maintaining axenic cultures in large-scale biofuel production facilities. The final chapter details efforts to develop an in-house method of synthesizing Transcription Activator-Like Effector (TALE) proteins to target specific DNA sequences in Chlamydomonas. Final TALE constructs were fused with FokI nuclease domains to create TAL Effector Nucleases (TALENs) that can create DNA double-strand breaks at specific sites. With an efficient method of synthesizing TALEN genes de novo, efforts can proceed to utilize this technology to create specific modifications within the Chlamydomonas genome.
Advisor: Donald P. Weeks