The Pennsylvania State University The Graduate School College of Engineering ENGINEERING A SYNTHETIC REPLICATION SYSTEM TO SECURELY STORE RECOMBINANT DNA A Thesis in Agricultural and Biological Engineering by Long Chen 2012 Long Chen Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science December 2012 The thesis of Long Chen was reviewed and approved* by the following: Howard Salis Assistant Professor of Agricultural and Biological Engineering Thesis Advisor Jeff Catchmark Professor of Agricultural and Biological Engineering Kenneth Keiler Associate Professor of Biochemistry and Molecular Biology Virendra Puri Distinguished Professor of Agricultural and Biological Engineering Graduate Program Coordinator *Signatures are on file in the Graduate School iii ABSTRACT The biotech industry spends millions to engineer genetic systems and manufacture products, and yet the DNA can be stolen and easily reverse-engineered for nefarious purposes. We are developing a synthetic DNA replication system to securely store high- value recombinant DNA and to prevent it from being manipulated by unauthorized third- parties. Our “LOCK and KEY” system only allows a high-value plasmid to replicate inside an authorized bacterial host, controlling a key route to reverse engineering a genetic system. Importantly, the LOCK and KEY system requires no special action by legitimate researchers and multiple orthogonal variants are available. iv TABLE OF CONTENTS List of Figures………………………………………………………………………………....v List of Tables………………………………………………………………………………….vi Acknowledgements…………………………………………………………………………....vii Chapter 1 Introduction ............................................................................................................. 1 Chapter 2 Methodology ........................................................................................................... 5 Strains, media ................................................................................................................... 5 Primary construct clone ................................................................................................... 5 LOCK and KEY variants clone ........................................................................................ 6 Variants characterization and transformants antibiotic susceptibility assay .................... 7 Chapter 3 Results ..................................................................................................................... 9 Construction of a dual-origin system ............................................................................... 9 Separation of RNAII primer and replication origin ......................................................... 10 Elimination of RNAII self-sufficiency ............................................................................. 15 Engineering synthetic “LOCK and KEY” v2.0 Variants ................................................. 16 Evaluating “LOCK and KEY” variants ........................................................................... 19 Translational optimization for LOCK and KEY v2.0 variants ........................................ 22 Chapter 4 Discussion ............................................................................................................... 24 Chapter 5 Conclusion ............................................................................................................... 26 Appendix .......................................................................................................................... 27 Reference ......................................................................................................................... 29 v LIST OF FIGURES Figure 1-1. Illustration of LOCK and KEY authorized replication system ............................. 2 Figure 1-3. Negative feedback loop formed by RNA I and RNA II. ....................................... 4 Figure 1-4. Schematic of synthetic ColE1 replication system ................................................. 4 Figure 3-1. Schematic map of P0 ............................................................................................. 9 Figure 3-2. qPCR data of P0 in DH10B and Pir-116................................................................ 10 Figure 3-3. Schematic map of construct “P1” .......................................................................... 10 Figure 3-4. Transformation results in DH10B and pir-116 of P1 under 50 µg/mL chloramphenicol selection ................................................................................................ 11 Figure 3-5. Schematic map of construct “P2” .......................................................................... 12 Figure 3-6. Transformation results in DH10B and pir-116 of P2 under 50µg/mL chloramphenicol selection. ............................................................................................... 12 Figure 3-7. Schematic map of “LK1.0” variants ..................................................................... 13 Figure 3-8. Schematic map of construct “C1” .......................................................................... 14 Figure 3-9. Transformation results in DH10B of “C1” and “C2” ............................................. 14 Figure 3-10. Schematic map of construct “C2” ........................................................................ 15 Figure 3-11. Schematic of minimizing cis-acting replication of RNAII ................................. 15 Figure 3-12. Antibiotic susceptibility assay outcome of poly “A” insertion ........................... 16 Figure 3-13. Schematic of LOCK and KEY v2.0 variants ...................................................... 17 Figure 3-14. 37 Alignment between biophysical parameter and length of 37 rescue sequence ........................................................................................................................... 19 Figure 3-15. Two types of constructs for characterizing LKv2.0 mutants .............................. 20 Figure 3-16. Antibiotic susceptibility data of 37 LKv2.0 variants .......................................... 21 Figure 3-17. Antibiotic susceptibility data of orthogonal test for 13 outstanding LOCK and KEY variants ............................................................................................................. 21 Figure 3-18. Comparison between previous CmR vector and optimized CmR vector ............ 23 vi LIST OF TABLES Table 3-1. Mutations of LOCK and KEY variants v1.0 at 3’ of both RNAII and origin of replication......................................................................................................................... 13 Table 3-2. Rescue sequence profile ......................................................................................... 17 Table 3-3. Synthetic RBS designed by RBS calculator ........................................................... 22 vii ACKNOWLEDGEMENTS I would like to thank my advisor Dr. Salis for guiding me perform my project and helping me figure out every trouble I met. I would like to thank all my committee professors: Dr. Catchmark and Dr. Keiler, I feel deeply appreciate them are interested in what I am doing and give me constructive suggestions. I would also like to give my acknowledgement to all of my labmates; we corporate with each other so well and I feel really good working with them. 1 Chapter 1 Introduction In the late 1970s, people engineered the first E.coli carried a synthetic gene to produce human insulin (Goeddel et al., 1979). At that time, most people didn’t believe microbes could be engineered to work for us. Today, thanks to the fast rising of metabolic engineering and synthetic biology, we are able to use cheap antibiotics and abundant vaccines. Besides, biofuel might be the next promising benefit we got from making good use of recombinant DNA. However, on the other hand the security of storing and utilization of recombinant DNA is becoming a serious issue. It is necessary to develop some technologies to secure the entire manufacture process. The project of “Gene Guard” is created by Dr. Salis in 2011. It has three different layers: “universal self-protection system”; “LOCK and KEY” authorized replication system and “obscurity system”. The goal of developing multiple genetic security systems is to create a comprehensive way to protect recombinant DNA information and prevent unauthorized utilization. This thesis focuses on the study of “LOCK and KEY” authorized replication system. The reason why we want to restrict plasmid’s replication is that plasmid could be easily transformed into a host, and replicate to numerous copies independently. If the plasmid contains particular information, people could transformed it to another hosts to maintain this plasmid and conduct further study on it. In metabolic engineering, recombinant DNA could always be unique and high-value, thus, it is risky if someone steals the plasmid and reverse engineers it. 2 Figure 1-1. Illustration of LOCK and KEY authorized replication system The goal of “LOCK and KEY” system is to engineer an authorized host, which carries valuable plasmid and allows its replication. However, if this valuable plasmid was transformed to an unauthorized host, it can’t replicate at all. In other words, only authorized hosts could maintain the DNA of the plasmid. Through this method, all the legitimate research could be conducted normally by using authorized hosts, and people don't need to be worried even if the plasmid was