Utah State University DigitalCommons@USU All Graduate Theses and Dissertations Graduate Studies 5-2011 Design of a BioBrickTM Compatible Gene Expression System for Rhodobacter sphaeroides Junling Huo Utah State University Follow this and additional works at: https://digitalcommons.usu.edu/etd Part of the Biology Commons Recommended Citation Huo, Junling, "Design of a BioBrickTM Compatible Gene Expression System for Rhodobacter sphaeroides" (2011). All Graduate Theses and Dissertations. 877. https://digitalcommons.usu.edu/etd/877 This Dissertation 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]. DESIGN OF A BIOBRICKTM COMPATIBLE GENE EXPRESSION SYSTEM FOR RHODOBACTER SPHAEROIDES by Junling Huo A dissertation submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Biological Engineering Approved: Professor H. Scott Hinton Dr. Daryll B DeWald Major Professor Committee Member Dr. Charles Miller Dr. Ronald C. Sims Committee Member Committee Member Dr. Jon Y Takemoto Dr. Byron R. Burnham Committee Member Dean of Graduate Studies UTAH STATE UNIVERSITY Logan, Utah 2011 ii Copyright © Junling Huo 2011 All Rights Reserved iii ABSTRACT Design of a BioBrickTM Compatible Gene Expression System for Rhodobacter sphaeroides by Junling Huo, Doctor of Philosophy Utah State University, 2011 Major Professor: Professor H. Scott Hinton Department: Biological Engineering The concept of introducing engineering principles of abstraction and standardization into synthetic biology has received increasing attention in the past several years and continues to be in the forefront of synthetic biology. One direction being pursued by synthetic biologists is creation of modular biological parts (BioBrickTM) that can be readily synthesized and mixed together in different combinations. However, most standard BioBrickTM parts in the Registry were designed for E. coli, although synthesis of specific BioBrickTM parts for other bacteria, such as for yeast and cyanobacteria, have begun. Besides, at the present time, there are only three chassis, which include E. coli, Bacillus subtilis, and a cell-free chassis, available in the Registry. Thus, the choices of BioBrickTM chassis are very limited. In addition, most BioBrickTM parts in the Registry have not been characterized. In the present study, the BioBrickTM concept was extended to the photosynthetic bacteria, Rhodobacter sphaeroides. In order to do that, a BioBrickTM compatible gene iv expression system was designed to convert R. sphaeroides to potential solar powered bio-factories or bio-refineries. This gene expression system was composed of BioBrickTM promoters, Ribosome Binding Sites (RBSs), and terminators in a BioBrickTM compatible cloning vector and its function has been validated through the expression of fluorescent proteins. In addition, a bioluminescence-based BioBrickTM characterization method was developed in this study. This method was based on a cloning vector that includes two adjacent operons, with each expressing a different luciferase reporter gene. The measured optical signals from the two expressed bioluminescent reporters were then used to predict the performance of promoters, RBSs, and terminators. Based on this bioluminescence-based BioBrickTM characterization method, two BioBrickTM characterizations kits, one for E. coli and one for R. sphaeroides, were developed. BioBrickTM parts that include seven promoters, six RBSs, and six terminators were characterized using the E. coli characterization kit. R. sphaeroides BioBrickTM parts were characterized when R. sphaeroides containing the BioBrickTM measurement constructs were cultured by both anaerobic photosynthesis and by aerobic respiration respectively. The experimental results showed that the activities of these R. sphaeroides BioBrickTM parts were very similar for the cells growing under two different conditions. (182 pages) v ACKNOWLEDGMENTS I am deeply grateful and indebted to my major advisor, Professor H. Scott Hinton, and Dr. Ronald C. Sims, for their consistent guidance, support, and encouragement during my graduate studies at Utah State University. Without their help and patience, this work would not have been possible to reach its final stage. In addition to introducing me to the subject of synthetic biology, the most important skill that I have learned from them was how to be an effective researcher and a good mentor. I would like to thank all my committee members, Dr. Daryll B DeWald, Dr. Charles Miller, and Dr. Jon Y Takemoto, for their guidance, criticisms, and insightful comments on my dissertation. I would like to convey my appreciation to Dr. Michelle Grilley (Dr. Takemoto‘s lab) and Dr. Sitaram Harihar (Dr. DeWald‘s Lab) for their technical help on my lab design. I would like to thank Ninglin Yin and Sarah Bastian from USU Center for Integrated Biosystems for their hard work on DNA sequencing support. I am extremely grateful to all members of SBC. I owe special thanks to Dr. Dong Chen and Dr. Jixun Zhan for their technical support and suggestions. I would like to thank all of my lab colleagues, Jason Brown, Mathew Kurian, Sanjib Shrestha, for useful discussion and cooperation. I would like to thank Melanie Ivans (executive assistant to the Dean), Jaclyn Evans (former staff assistant at SBC), Shelly Wardell (staff assistant at SBC), and Anne Martin (recruiter in Biological Engineering) for their assistance and enthusiasm during my research. vi I would also like to thank my good friends Gary Chiu, Frankie Chiu, and Kuanchiang Chen for their support in my daily life during this research. This work is supported by the USTAR synthetic Bio-manufacturing center (SBC). This support is gratefully acknowledged. Special thanks go to Dr. Samuel Kaplan and Dr. Jesus M. Eraso from University of Texas-Houston Medical School for providing technical support, Rhodobacter sphaeroides 2.4.1, E. coli S17-1, E coli DH5αPhe, and E. coli HB101/pRK2013; Dr. Masayuki Inui from Molecular Microbiology and Biotechnology group, Kyoto, Japan, for kindly providing cloning vectors pMG170/pMG171; Dr. Judith P. Armitage from Oxford center for integrative systems biology, University of Oxford, United Kingdom; and Elaine Byles (Dr. Armitage‘s lab), and Dr. Steven Porter from School of Biosciences, University of Exeter, United Kingdom for kindly providing cloning vectors pIND4 and technical discussions including the use of lactose inducible promoter in R. sphaeroides. Finally, I would like to express my deepest appreciation to my wife, Dr. Qin Li, my sons, Andy and Randy, my parents-in-law, and my sisters for their endless love, support, and encouragement. Junling Huo vii CONTENTS Page ABSTRACT……………………...…….…………………..………………….iii ACKNOWLEDGMENTS…………………………………..……………….…v LIST OF TABLES………………………...…..…..…………………………..xi LIST OF FIGURES.………………...………..…………………………….…xii CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW……………....…1 1.1 Background……………………………………….......….…..1 1.1.1 Synthetic Biology……………………………….....…1 1.1.2 Standardization of Biological Parts: BioBrickTM….....2 1.2 Current status of BioBrickTM research.....................................7 1.2.1 BioBrickTM parts characterization ...............................7 1.2.2 BioBrickTM chassis.......................................................8 1.3 Rhodobacter sphaeroides……………………………….....…9 1.3.1 Cloning vectors for R. sphaeroides.…..….................11 1.3.2 Restriction endonucleases in R. sphaeroides……….15 1.3.3 Operonic structure of R. sphaeroides…...………......16 1.4 Motivation and Research objective………………………....19 1.5 Outline of the Dissertation…………………………........….19 References………………………………………....….………….....21 2 DESIGN OF A BIOBRICKTM COMPATIBLE GENE EXPRESSION SYSTEM FOR RHODOBACTER SPHAEROIDES …………...…...27 2.1 Background………………………………….…………...…28 2.1.1 Cloning vectors for R. sphaeroides….….....………..30 2.1.2 Promoters for R. sphaeroides……........………….…31 2.1.3 RBSs for R. sphaeroides…........................................32 viii 2.1.4 Terminators for R. sphaeroides……..…....…………32 2.2 Results……………………………………….………...……33 2.2.1 BioBrickTM vectors for R. sphaeroides………...…...33 2.2.2 BioBrickTM promoters for R. sphaeroides…….....….34 2.2.3 BioBrickTM RBS for R. sphaeroides…...…….……..35 2.2.4 BioBrickTM terminator for R. sphaeroides……....….38 2.2.5 Expression of cyan fluorescent protein (CFP) in R. sphaeroides.................................................................40 2.2.6 Gene expression system tested with fluorescent proteins.......................................................................41 2.3 Discussion and conclusion.......…………….……………….42 2.4 Methods……...………………….…………..………………43 2.4.1 Bacterial strains and plasmids...….………………....43 2.4.2 Culture conditions…….......……….………………..43 2.4.3 DNA techniques…………………........………….…44 References……………...………………………..……….…………47 3 A NEW BIOLUMINESCNECE -BASED METHOD FOR CHARACTERIZING BIOBRICKTM PROMOTERS, RIBOSOME BINDING SITES, AND TERMINATORS.......................................51 3.1 Background………….……………….…………………......52 3.2 Results.......................………………….…...……………….55 3.2.1 Definition and model of BioBrickTM activity.............55 3.2.2 Principle of promoter measurement....................…...56 3.2.3 Principle of RBS measurement..................................60 3.2.4 Principle