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A Synthetic Biological Engineering Approach to Secretion- Based Recovery of Polyhydroxyalkanoates and Other Cellular Products

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A Synthetic Biological Engineering Approach to Secretion- Based Recovery of Polyhydroxyalkanoates and Other Cellular Products Utah State University DigitalCommons@USU All Graduate Theses and Dissertations Graduate Studies 5-2010 A Synthetic Biological Engineering Approach to Secretion- Based Recovery of Polyhydroxyalkanoates and Other Cellular Products Elisabeth Linton Utah State University Follow this and additional works at: https://digitalcommons.usu.edu/etd Part of the Analytical Chemistry Commons, Biomedical Engineering and Bioengineering Commons, and the Molecular Biology Commons Recommended Citation Linton, Elisabeth, "A Synthetic Biological Engineering Approach to Secretion- Based Recovery of Polyhydroxyalkanoates and Other Cellular Products" (2010). All Graduate Theses and Dissertations. 678. https://digitalcommons.usu.edu/etd/678 This Thesis is brought to you for free and open access by the Graduate Studies at DigitalCommons@USU. It has been accepted for inclusion in All Graduate Theses and Dissertations by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. A SYNTHETIC BIOLOGICAL ENGINEERING APPROACH TO SECRETION- BASED RECOVERY OF POLYHYDROXYALKANOATES AND OTHER CELLULAR PRODUCTS by Elisabeth Linton A thesis submitted in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE in Biological Engineering Approved: Charles D. Miller Ronald C. Sims Co-Major Professor Co-Major Professor Daryll B. DeWald Byron R. Burnham Committee Member Dean of Graduate Studies UTAH STATE UNIVERSITY Logan, Utah 2010 ii Copyright © Elisabeth Linton 2010 All Rights Reserved iii ABSTRACT A Synthetic Biological Engineering Approach to Secretion- Based Recovery of Polyhydroxyalkanoates and Other Cellular Products by Elisabeth Linton, Master of Science Utah State University, 2010 Major Professors: Dr. Charles D. Miller and Dr. Ronald C. Sims Department: Biological Engineering The costs associated with cellular product recovery commonly account for as much as 80% of the total production expense. As a specific example, significant recovery costs limit commercial use of polyhydroxyalkanoates (PHA), which comprise a class of microbially-accumulated polyesters. PHAs are biodegradable compounds that are of interest as a sustainable alternative to petrochemically-derived plastics. Secretion- based recovery of PHAs was studied to decrease PHA production costs. Type I and II secretory pathways are commonly used for the translocation of recombinant proteins out of the cytoplasm of E. coli. Proteins were targeted for translocation using four signal peptides (HlyA, TorA, GeneIII, and PelB) that operate via type I and II secretory machinery. GFP translocation was investigated in parallel due to its relative ease of monitoring to gather information about the functionality of signal peptide sequences. The translocation of phasin was investigated because of its physical binding interaction iv with the PHA granule surface. Genetic fusion of phasin with targeting signal peptides creates a PHA-phasin-signal peptide complex that can then be potentially used for cellular export. An important design aspect of this investigation is that synthetic biological engineering principles and standardized technical formats BBF RFC 10 and BBF RFC 23 were applied for more efficient construction of genetic devices. As an additional part of this study, an 1H NMR-based PHA quantification method was developed to facilitate analysis of intracellular PHAs. Overall, this study demonstrated that the BioBrick model can be used to construct functional devices that promote secretion of cellular compounds. The information gathered from this work can be further optimized and applied to more complex cellular manufacturing systems. (160 pages) v ACKNOWLEDGMENTS This research would not have been possible without the help of a number of individuals. First, I am thankful to my husband, JD, for his continued support, patience, and encouragement. I am grateful to my parents and family for their support. I would like to sincerely thank Dr. Charles Miller for his enormous involvement throughout the course of this work. His dedication to his students has made this process very rewarding. I am also grateful to Dr. Ronald C. Sims not only for his research guidance, but also for the opportunities that he has afforded during my time as a Biological Engineering student at Utah State University. This research is also a result of contributions made by Dr. Sridhar Viamajala, Dr. Daryll B. DeWald, Dr. Marie K. Walsh, Dr. Jon Takemoto, Dean H. Scott Hinton, and the Synthetic Bio-Manufacturing Center. Lastly, I would like to thank my fellow students who kept me company during all of the many hours studying and in the lab. This entire process was made substantially more enjoyable because of their friendship. Elisabeth Linton vi CONTENTS Page ABSTRACT ..................................................................................................................... iii ACKNOWLEDGMENTS ................................................................................................ v LIST OF TABLES ........................................................................................................... ix LIST OF FIGURES .......................................................................................................... x LIST OF SYMBOLS, NOTATIONS, AND DEFINITIONS ........................................ xiii CHAPTER 1. INTRODUCTION .................................................................................... 1 Hypothesis........................................................................................... 3 Objectives ........................................................................................... 4 Thesis Outline ..................................................................................... 5 References ........................................................................................... 5 2. LITERATURE REVIEW ......................................................................... 8 PHA Biosynthesis ............................................................................... 8 Current Methods for PHA Recovery ................................................ 10 Recombinant Proteins ....................................................................... 12 Phasin .......................................................................................... 12 Green Fluorescent Protein........................................................... 14 Principles of Secretion in Gram-Negative Organisms ...................... 15 Secretion Pathways ..................................................................... 16 Cytoplasmic Membrane Translocation ....................................... 18 Signal Peptides ............................................................................ 21 Synthetic Biological Engineering ..................................................... 22 The BioBrick Standard ............................................................... 22 The BioFusion Standard ............................................................. 24 References ......................................................................................... 25 vii 3. TRANSLOCATION OF GREEN FLUORESCENT PROTEIN USING A SYNTHETIC BIOLOGY APPROACH WITH MULTIPLE SIGNAL PEPTIDES ............................................................................... 33 Abstract ............................................................................................. 33 Background ....................................................................................... 34 Materials and Methods ...................................................................... 38 BioBrick Construction, Primers, and Vectors............................. 38 Strains and Growth Conditions ................................................... 40 Cellular Fractionation ................................................................. 41 SDS-PAGE and Western Blotting .............................................. 42 Flourometry and Fluorescence Microscopy ................................ 42 Results ............................................................................................... 43 Construction of GFP BioFusion Devices .................................... 43 Analysis of GFP Translocation ................................................... 44 Fluorescence Analysis ................................................................ 48 Discussion ......................................................................................... 51 Conclusions ....................................................................................... 54 References ......................................................................................... 55 4. COMPARISON OF SINGLE-STEP EXTRACTION FOR 1H NMR ANALYSIS WITH GC AND FLUORESCENCE TECHNIQUES FOR POLYHYDROXYALKANOATE QUANTIFICATION .............. 62 Abstract ............................................................................................. 62 Introduction ....................................................................................... 63 Materials and Methods ...................................................................... 68 Chemicals .................................................................................... 68 Experimental Design ................................................................... 68 Microorganisms and Culture Methods for Pure Strains ............................................................................. 69 Environmental Samples .............................................................
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