Developing microbial systems capable of converting low cost lipids into value added products depends on the ability to acquire substrates from the growth media. Saccharomyces cerevisiae can acquire free fatty acids from the growth media and a portion of these lipids can be converted into new lipid products. However, they cannot acquire complex lipids from the growth media unless a nonspecific lipase is included. To circumvent lipase addition, we are genetically engineering S. cerevisiae to secrete a lipase into the growth media. We selected the LIP2 gene from Yarrowia lipolytica, which encodes a nonspecific lipase. Several modifications were made to the LIP2 gene to improve processing. Results identified strains secreting the most lipase. From these results, high producing strains were inserted into an oil inducible vector. Halo assays confirmed lipase secretion, while measuring the fatty acid composition confirmed triacylglycerol breakdown, and yeast uptake of the free fatty acids released.

Engineered cellular systems are a promising avenue for production of a wide range of useful products including renewable fuels, commodity and specialty chemicals, industrial enzymes, and pharmaceuticals. Achieving this breadth of biological products is facilitated by the diversity of organisms found in nature. Using biological and engineering principles, this diversity can be harnessed to make efficient and renewable bio-based products. Such advancements rely upon our ability to modify host genetics and metabolism. This work focuses on the development of new biotechnological tools which enable cellular engineering, and the implementation of these tools in eukaryotic systems. Mammalian cell engineering has important implications in protein therapeutics and gene therapy. One major limitation, however, is the ability to predictably control gene expression. We address this challenge by examining critical aspects of gene expression in human cells. First, we evaluate the impact of selection markers, a common mammalian expression element, on cell line development. In doing so, we determine that Zeocin is the best selection agent for human cells. Next, we identify loci across the genome that support high level expression of recombinant DNA and demonstrate their advantage for stable integration. Finally, we optimize a Cre recombinase based methodology that enables efficient retargeting of genomic loci. Collectively, this work augments the current genetic toolbox for human cell lines. Beyond basic gene expression, there is interest in understanding global interactions within the cell and how they relate to phenomena including gene regulation, expression and disease states. Although our tools are not yet sufficient to study these phenomena in many hosts, methods can be developed in lower eukaryotes and then adapted for more complex hosts later. We demonstrated two methods in S. cerevisiae that utilize a systems-level approach to understand complex phenotypes. First, we developed condition-specific codon optimization that utilizes systems biology information to optimize gene sequence in a condition-specific manner. Additionally, we developed a Graded Dominant Mutant Approach which can be used to dissect multifunctional proteins, understand epigenetic factors, and quantitatively determine protein-DNA interactions. Both can be implemented in many cellular hosts and expand our ability to engineer complex phenotypes in eukaryotic cell systems.

The genomes of diverse organisms are predicted to contain 20 – 30% membrane protein encoding genes and more than half of all therapeutics target membrane proteins. However, only 2% of crystal structures deposited in the protein data bank represent integral membrane proteins. This reflects the difficulties in studying them using standard biochemical and crystallographic methods. The first problem frequently encountered when investigating membrane proteins is their low natural abundance, which is insufficient for biochemical and structural studies. The aim of my thesis was to provide a simple method to improve the production of recombinant proteins. One of the most commonly used methods to increase protein yields is codon optimization of the entire coding sequence. However, our data show that subtle synonymous codon substitutions in the 5’ region can be more efficient. This is consistent with the view that protein yields under normal conditions are more dependent on translation initiation than elongation. mRNA secondary structures around the 5’ region are in large part responsible for this effect although rare codons, as well as other factors, also contribute. We developed a PCR based method to optimize the 5’ region for increased protein production in Escherichia coli. For those proteins produced in sufficient quantities several additional hurdles remain before high quality crystals can be obtained. A second aim of my thesis work was to provide a simple method for topology mapping membrane proteins. A topology map provides information about the orientation of transmembrane regions and the location of protein domains in relation to the membrane, which can give information on structure-function relationships. To this end we explored the split-GFP system in which GFP is split between the 10th and 11th β-strands. This results in one large and one small fragment, both of which are non-fluorescent but can re-anneal and regain fluorescence if localized to the same cellular compartment. Fusing the 11th β-strand to the termini of a protein of interest and expressing it, followed by expression of the detector fragment in the cytosol, allows determination of the topology of inner membrane proteins. Using this strategy the topology of three model proteins was correctly determined. We believe that this system could be used to predict the topology of a large number of additional proteins, especially single-spanning inner membrane proteins in E. coli. The methods for efficient protein production and topology mapping engineered during my thesis work are simple and cost-efficient and may be very valuable in future studies of membrane proteins. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.</p>

No description available.

Brinjal shoot and fruit borer (Leucinodes orbonalis Guenee) is a major limiting factor in commercial cultivation of eggplant in southeast Asia. Extensive use of pesticides as well as the conventional breeding methods have been ineffective in controlling the borer so there is a need for Integrated Pest Management (IPM) strategies for its control. Bacillus thuringiensis (Bt) is known to produce a variety of insecticidal crystal proteins toxic to lepidopteran, dipteran and coleopteran pests. The Cry2Aa protein has been found to be more toxic to brinjal shoot and fruit borer than Cry1Ab. My objective was to develop eggplant cultivars that express a codon-optimized cry2Aa gene, the sequence of which is based on that of an Indian isolate of Bt, with the eventual goal of producing fully resistant cultivars. The cry2Aa gene was modified for optimal expression in eggplant using the codon usage frequencies based on solanaceous sequences (eggplant, tomato and pepper). The GC content was increased from 34.3% in the native gene to 41.3% in the optimized gene, thus removing the AT-rich regions that are typical for Bt cry genes. Also, other mRNA destabilizing and hairpin forming structure sequences were removed. The gene was synthesized in four different parts with complementary restriction sites. A total of 152 oligonucleotides (oligos) was used to assemble the 1.9 kb gene using dual asymmetric (DA) and overlap extension (OE) PCR techniques. The individual parts were subsequently ligated using the complementary restriction sites and inserted into vector pCAMBIA 1302. Also, the transformation efficiency of 12 different eggplant cultivars was tested using plasmid pHB2892 to predict utility for transformation with the synthetic cry2Aa. / Master of Science

The development of shape in multicellular organisms has intrigued human minds for millenia. Empowered by modern genetic techniques, molecular biologists are now striving to not only dissect developmental processes, but to exploit their modularity for the design of custom living systems used in bioproduction, remediation, and regenerative medicine. Currently, our capacity to harness this potential is fundamentally limited by a lack of spatiotemporal control over gene expression in multicellular systems. While several synthetic genetic circuits for control of multicellular patterning have been reported, hierarchical induction of gene expression domains has received little attention from synthetic biologists, despite its fundamental role in biological self-organization. In this thesis, I introduce the first synthetic genetic system implementing population-based AND logic for programmed hierarchical patterning of bacterial populations of Escherichia coli, and address fundamental prerequisites for implementation of an analogous genetic circuit into the emergent multicellular plant model Marchantia polymorpha. In both model systems, I explore the use of bacteriophage T7 RNA polymerase as a gene expression engine to control synthetic patterning across populations of cells. In E. coli, I developed a ratiometric assay of bacteriophage T7 RNA polymerase activity, which I used to systematically characterize different intact and split enzyme variants. I utilized the best-performing variant to build a three-color patterning system responsive to two different homoserine lactones. I validated the AND gate-like behavior of this system both in cell suspension and in surface culture. Then, I used the synthetic circuit in a membrane-based spatial assay to demonstrate programmed hierarchical patterning of gene expression across bacterial populations. To prepare the adaption of bacteriophage T7 RNA polymerase-driven synthetic patterning from the prokaryote E. coli to the eukaryote M. polymorpha, I developed a toolbox of genetic elements for spatial gene expression control in the liverwort: I analyzed codon usage across the transcriptome of M. polymorpha, and used insights gained to design codon-optimized fluorescent reporters successfully expressed from its nuclear and chloroplast genomes. For targeting of bacteriophage T7 RNA polymerase to these cellular compartments, I functionally validated nuclear localization signals and chloroplast transit peptides. For spatiotemporal control of bacteriophage T7 RNA polymerase in M. polymorpha, I characterized spatially restricted and inducible promoters. For facilitated posttranscriptional processing of target transcripts, I functionally validated viral enhancer sequences in M. polymorpha. Taking advantage of this genetic toolbox, I introduced inducible nuclear-targeted bacteriophage T7 RNA polymerase into M. polymorpha. I showed implementation of the bacteriophage T7 RNA polymerase/PT7 expression system accompanied by hypermethylation of its target nuclear transgene. My observations suggest operation of efficient epigenetic gene silencing in M. polymorpha, and guide future efforts in chassis engineering of this multicellular plant model. Furthermore, my results encourage utilization of spatiotemporally controlled bacteriophage T7 RNA polymerase as a targeted silencing system for functional genomic studies and morphogenetic engineering in the liverwort. Taken together, the work presented enhances our capacity for spatiotemporal gene expression control in bacterial populations and plants, facilitating future efforts in synthetic morphogenesis for applications in synthetic biology and metabolic engineering.