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<meta http-equiv="Content-Type" content="text/html; charset=Windows-1252"></head><body style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space; color: rgb(0, 0, 0); font-size: 14px; font-family: Calibri, sans-serif; "><div><br></div><span id="OLK_SRC_BODY_SECTION"><div xmlns:v="urn:schemas-microsoft-com:vml" xmlns:o="urn:schemas-microsoft-com:office:office" xmlns:w="urn:schemas-microsoft-com:office:word" xmlns:m="http://schemas.microsoft.com/office/2004/12/omml" xmlns="http://www.w3.org/TR/REC-html40"><div lang="EN-US" link="blue" vlink="purple"><div class="WordSection1"><p class="MsoNormal" align="center" style="mso-margin-top-alt:auto;margin-bottom:6.0pt;text-align:center;line-height:18.0pt"><a name="_Toc434675189"><b><span style="font-size: 14pt; font-family: 'Times New Roman', serif; color: black; ">PhD DEFENSE STUDENT: </span></b></a><span style="font-size: 14pt; font-family: 'Times New Roman', serif; color: black; ">Christopher Frei</span><span style="font-size: 12pt; font-family: 'Times New Roman', serif; color: black; "><o:p></o:p></span></p><p class="MsoNormal" align="center" style="mso-margin-top-alt:auto;margin-bottom:6.0pt;text-align:center;line-height:18.0pt"><b><span style="font-size: 14pt; font-family: 'Times New Roman', serif; color: black; ">DATE: </span></b><span style="font-size: 14pt; font-family: 'Times New Roman', serif; color: black; ">November 20, 2015</span><span style="font-size: 12pt; font-family: 'Times New Roman', serif; color: black; "><o:p></o:p></span></p><p class="MsoNormal" align="center" style="mso-margin-top-alt:auto;margin-bottom:6.0pt;text-align:center;line-height:18.0pt"><b><span style="font-size: 14pt; font-family: 'Times New Roman', serif; color: black; ">TIME: </span></b><span style="font-size: 14pt; font-family: 'Times New Roman', serif; color: black; ">10:00 AM</span><span style="font-size: 12pt; font-family: 'Times New Roman', serif; color: black; "><o:p></o:p></span></p><p class="MsoNormal" align="center" style="mso-margin-top-alt:auto;margin-bottom:6.0pt;text-align:center;line-height:18.0pt"><b><span style="font-size: 14pt; font-family: 'Times New Roman', serif; color: black; ">PLACE: </span></b><span style="font-size: 14pt; font-family: 'Times New Roman', serif; color: black; ">Chemical Engineering Conference Room</span><span style="font-size: 12pt; font-family: 'Times New Roman', serif; color: black; "><o:p></o:p></span></p><p class="MsoNormal" align="center" style="mso-margin-top-alt:auto;margin-bottom:6.0pt;text-align:center;line-height:18.0pt"><b><span style="font-size: 14pt; font-family: 'Times New Roman', serif; color: black; ">DISSERTATION CHAIRS: </span></b><span style="font-size: 14pt; font-family: 'Times New Roman', serif; color: black; ">Dr. Patrick Cirino</span><span style="font-size: 12pt; font-family: 'Times New Roman', serif; color: black; "><o:p></o:p></span></p><div class="MsoNormal" align="center" style="margin-bottom:0in;margin-bottom:.0001pt;text-align:center;line-height:normal"><b><span style="font-size: 16pt; font-family: 'Times New Roman', serif; color: black; "><hr size="2" width="100%" align="center"></span></b></div><p class="MsoNormal" align="center" style="mso-margin-top-alt:auto;mso-margin-bottom-alt:auto;text-align:center;line-height:normal"><b><span style="font-size: 14pt; font-family: 'Times New Roman', serif; color: black; ">TITLE:<o:p></o:p></span></b></p><p class="MsoNormal" align="center" style="mso-margin-top-alt:auto;mso-margin-bottom-alt:auto;text-align:center;line-height:normal"><b><span style="font-size: 14pt; font-family: 'Times New Roman', serif; color: black; ">Engineered High-Throughput design of Whole-Cell Biosensors for Microbial Production of Value-Added Products<o:p></o:p></span></b></p><p class="MsoNormal" align="center" style="mso-margin-top-alt:auto;mso-margin-bottom-alt:auto;text-align:center;line-height:normal"><b><span style="font-size: 14pt; font-family: 'Times New Roman', serif; color: black; "><o:p> </o:p></span></b></p><p class="MsoNormal" style="margin-bottom:0in;margin-bottom:.0001pt;text-align:justify;text-indent:.25in;line-height:200%;text-autospace:none"><span style="font-family: 'Times New Roman', serif; color: black; ">Humans have developed an overwhelming dependence on synthetic materials, which are often costly and environmentally harmful. Over the past decade, there has been a boom in biotechnology, with one
emphasis on engaging easily manipulated microorganisms for relatively inexpensive industrial scale production of pharmaceuticals, polymers, and fuels. This challenging field of research offers invaluable benefits to society and the environment. Rapid and precise
screening of large libraries of genetically altered microorganisms for enhanced molecule production is a powerful approach to developing such “microbial factories.” Unfortunately, the lack of readily available high-throughput screening techniques inhibits
our ability to quickly engineer microorganisms. This limitation is addressed here using engineered whole-cell molecular biosensors based on a family of proteins known as transcriptional regulatory proteins (TRPs). The natural role of many TRPs is to link
molecule recognition with gene expression, making them ideal candidates for engineering endogenous molecule biosensors. Through powerful approaches such as directed evolution, TRPs can be altered to recognize targeted value–added molecules and their precursors.
Upon recognition of the target molecule, the TRP activates expression of genes with an easily measureable phenotype (e.g., luminescence, cell growth, fluorescence). In the present study, we sought to (i) improve the current screening strategies of TRP libraries,
(ii) investigate residue relationships governing the recognition and response of a TRP, and (iii) isolate novel whole-cell biosensors based on the TRP platform.<o:p></o:p></span></p><p class="MsoNormal" style="text-align:justify;text-indent:22.5pt;line-height:200%"><span style="font-family: 'Times New Roman', serif; ">High-throughput screening techniques for isolating functional clones from large genetic libraries ( > 10<sup>6</sup> mutants) is pivotal to the continued success of engineering microbial factories. Here, we
applied fluorescence-activated cell sorting (FACS) combined with antibiotic selections to dramatically improve the throughput of screening large libraries of AraC, an
<i>Escherichia coli </i>native TRP. After residue characterization and screening optimization, several functional AraC variants were isolated with desirable specificity and sensitivity toward target molecules, vanillin and salicylate. As we continue to characterize
new biosensors and optimize their design process, the limits of TRP molecule recognition are pushed further, thus allowing us to overcome the restraints imposed by natural TRPs and offering sustainable solutions to engineering microbial factories.</span><o:p></o:p></p></div></div></div></span><style><!--
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