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Spring 2013

Carlson, Haley J

of work had gone into developing and improving GFP-based Ca2+ biosensors1,2, but there were no reports of a red FP-based biosensor. The work in this thesis describes the engineering of an RFP-based Ca2+ biosensor using a circular permutated RFP, mCherry. The first step in this process was to

The discovery of green fluorescent protein (GFP) from the Aequorea victoria jellyfish revolutionized many fields in the scientific community, including molecular biology, protein engineering, and neuroscience. The ability to genetically link a fluorescent protein to a protein of interest has

allowed scientists to probe the exact structural localization of proteins. Another important application of FPs is their design for use in biosensors, whereby the fluorescence of the protein is intrinsically dependent on a small molecule of interest, such as calcium ion (Ca2+) or a physiological process

Fall 2016

Abdelfattah, Ahmed

). Using protein engineering and directed evolution, we engineered FlicR1 to have sufficient speed and sensitivity to report single action potentials in single-trial recordings. Because it is excitable with yellow light, FlicR1 is the first FP-based voltage indicator that can be used in conjunction with

Fluorescence microscopy is currently the most powerful imaging technique for interrogation of neural circuits. Accordingly, tremendous efforts have been invested in engineering fluorescent proteins (FPs) to act as indicators for various biochemical processes in neural circuits, resulting in a

plethora of genetically encoded FP-based indicators for neural imaging. In this thesis, we report the development of various FP-based indicators for neuronal imaging. Specifically, we describe our efforts to expand the current repertoire of neuronal activity indicators to include a voltage indicator with

Fall 2015

Lai, Yan Tiffany

ubiquitin modifier 1 (SUMO-1) in pancreatic islet cells. The creation of these plasmids is a proof of the versatility and modularity of the ddFP and its related FP exchange (FPX) biosensor design strategy. We also report the engineering of a dark “A” copy (DA) for ddFP applications. By a simple rational

The expansion of the fluorescent protein (FP) colour palette through discovery of more species possessing FPs and by subjecting them to intensive protein engineering efforts has greatly aided the effectiveness of FP-based biosensor designs. Currently, the main FP-based methods for detecting protein

may facilitate multicolour imaging of biosensors in live cells due to the fact that they have a narrower spectral profile than a FRET pair. In this thesis, we describe our efforts to expand the range of applications of ddFP in live cells. We report the creation of systems for the sensing of global O

Fall 2014

Zhao, Yongxin

-based indicators remains a challenging engineering problem, mainly due to lack of structural information for rational design and effective methodologies of protein engineering. The goal of this thesis work is to tackle the long-standing challenge of engineering FP-based indicators for improved

engineering of single FP-based Ca2+ indicator and led to several variants with improved performance and various new colors. This palette of new Ca2+ indicators enables simultaneous monitoring of Ca2+ transients in different cellular compartments or different types of cells, which opens up a new era of

Fall 2017

Zhang, Wei

proteins have required some degree of protein engineering for improved optical and functional properties. Encouragingly, an expanded range of optogenetic tools with novel functions has been developed by coupling the natural photosensory mechanisms with certain proteins. Through such efforts, the

optogenetic toolkit is rapidly growing, though it is still relatively limited. In this thesis, we describe our efforts to expand the scope of photosensory mechanisms by engineering a photocleavable protein (PhoCl) that splits into two fragments upon violet light (~400 nm) illumination. PhoCl is a

-forming glycoprotein, Pannexin-1. We also report the development, optimization, and characterization of the first genetically encoded near infrared (NIR) fluorescent pH indicator, pH-mIFP. The pH dependent fluorescence was introduced to the NIR FP by rational design, which was followed by

Fall 2014

Wu, Jiahui

than ever. Despite the tremendous developments and efforts invested in the field of FP-based indicators, there remain numerous opportunities in engineering indicators with improved or novel properties for studying biological processes in vivo. In this thesis we describe our efforts in developing a

series of FP-based calcium ion (Ca2+) and glutamate indicators with various colors and useful spectral properties as versatile tools for interrogating cell signaling in cell biology. In this thesis, we first describe our efforts in employing protein engineering to expand the color palette of genetically

protein (GFP)-based reporter. We also report in this thesis the development, optimization and characterization of the first red fluorescent protein (RFP)-based glutamate indicator, GltR1. We demonstrate GltR1 can detect glutamate changes on the surface of cultured human cells, as well as the glutamate