Browsing by Subject "Promoters"
Now showing 1 - 3 of 3
- Results Per Page
- Sort Options
Item Counterculture country : nation-building at 1960s rock music festivals(2017-05-08) Bendure, Matthew Thomas; Davis, Janet M.; Mickenberg, JuliaRock music festivals of the late 1960s were the sites of regular and violent contestation between promoters who organized them and the countercultural festival goers who attended. Throughout the final years of the 1960s, many countercultural youth began envisioning the rock music festival as the most practical space for realizing a revolutionary social order. Overwhelming local police and rural communities, these festival spaces were comprised of hundreds of thousands of countercultural youth who gathered and openly embodied countercultural values in plain sight and in solidarity. When promoters recognized the profitability of organizing rock music festivals, they began charging admission and excluding those who could not, or would not, pay admission. This tension between promoters and countercultural youth violently erupted at rock music festivals throughout the era.Item Design and synthesis of synthetic UP elements for modulation of gene expression in Escherichia coli(2017-01-05) Flexer Harrison, Madeleine Hannah; Alper, Hal S.To limit both cost and environmental impact, microorganisms are exploited by synthetic biologists for ecologically friendly ways to produce biochemicals essential to both industry and health. Synthetic biologists and metabolic engineers work to hijack the metabolism of accommodating hosts in order to produce useful chemicals. The alteration and optimization of endogenous and heterologous metabolic pathways require careful balancing and tuning of gene expression. While there are numerous ways to control gene expression in the cell, by far the most common way to regulate expression is transcriptionally through the promoter. In this work we explore a particular region of the bacterial promoter, the UP element, as an effective way modulate gene expression in Escherichia coli. We use both rational and library design of UP elements to search the upstream sequence space of the core promoter, rrnD. Using FACS and flow cytometry, we screen multiple libraries to identify a group of UP elements capable of modulating expression in E. coli. Additionally, we explore the effects of modifying sequences adjacent and upstream to the UP element site. We report that expression strength can be tuned both positively and negatively through the modifications in the UP element sequence and through modifications of sequences upstream and adjacent of the UP element. The strongest sequence identified, TP-24, amplifies our core promoter strength up to 30-fold based on GFP fluorescence. Our final selection of UP elements activate rrnD from 8 –– 26 fold, each stronger than the commonly used IPTG inducible promoter, Ptac1. This work provides a novel and minimal way to control promoter strength in E. coli expression without major alteration to the core promoter.Item Developing synthetic, minimal promoters in Saccharomyces cerevisiae(2017-05) Redden, Heidi Rosemary; Browning, Karen S.; Barrick, Jeffrey; Hoffman, David; Keatinge-clay, AdrianPromoters enable synthetic biologists to manipulate protein expression at the DNA level. For this reason, promoters are essential for almost all applications aiming to engineer an organism. Unfortunately, promoters available for eukaryotic organisms are derived directly from the genome. Such promoters are large and result in substantial and therefore, difficult DNA insertions to express a heterologous multi-gene pathway. Furthermore, their high sequence homology provides the organism the opportunity to perform homologous recombination resulting in undesirable gene deletions. For these reasons, there is a critical demand for short promoters with low sequence homology to the organism’s genome to continue synthetic biology advancements in eukaryotic hosts. This work addresses the need for yeast promoters by engineering the promoter’s two distinct DNA regions– the upstream activating sequence (UAS) and the downstream 3ˋ area comprised of the promoter’s core. The modularity of these regions is demonstrated in a non-conventional yeast, Yarrowia lipolytica by assembling multiple native UAS in tandem with a core. In doing so, the strongest promoters ever reported for Y. lipolytica were created. Drawing from these lessons, the length of promoters in the popular host strain Saccharomyces cerevisiae was minimized. The core region is first addressed by establishing promoter libraries with minimized de novo cores. Synthetic cores are isolated from a short promoter library and are evaluated in six DNA contexts to establish nine minimal cores with modularity, robustness, and context independence. Second, the UAS region was minimized. To do so, a randomized region of DNA was hybridized upstream of a synthetic minimal core to construct 18 de novo libraries of promoters. From these libraries, 26 short constitutive and inducible UAS elements were isolated. Collectively, this work highlights the utility of hybrid promoter engineering to increase the number of promoters available for host organisms, Y. lipolytica and S. cerevisiae. More importantly, it establishes a highly desirable set of 81 synthetic, minimal promoters of inducible and constitutive function that provides a 70-fold range of expression in S. cerevisiae. Furthermore, the workflow presented herein is generic enough for application in other eukaryotic host organisms to build their synthetic biology toolboxes.