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Methods in Molecular Biology 1472 Randall A. Hughes Editor Synthetic DNA Methods and Protocols M M B ETHODS IN OLECULAR IOLOGY Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB , UK For further volumes: http://www.springer.com/series/7651 Synthetic DNA Methods and Protocols Edited by Randall A. Hughes Applied Research Laboratories, The University of Texas at Austin, Austin, TX, USA Editor Randall A. Hughes Applied Research Laboratories The University of Texas at Austin Austin, TX , USA ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-4939-6341-6 ISBN 978-1-4939-6343-0 (eBook) DOI 10.1007/978-1-4939-6343-0 Library of Congress Control Number: 2016948856 © Springer Science+Business Media New York 2 017 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifi cally the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfi lms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specifi c statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. Printed on acid-free paper This Humana Press imprint is published by Springer Nature The registered company is Springer Science+Business Media LLC New York Prefa ce The biological information revolution brought on by the advent of low-cost high- throughput sequencing has made available ever increasing troves of DNA sequences from all forms of life. This sequence information has provided researchers with databases of sequences which encode for all the functional (enzymes, proteins, stable RNAs) and regula- tory (promoters, enhancers, ribosomal binding sites, terminators) components which make cellular biochemistry possible. These DNA sequences have served as designer templates that have enabled the recent innovations and discoveries in the fi elds of synthetic biology, molecular biology, biochemistry, and biological engineering. However the availability of DNA sequence information is only partly responsible for the rise in the design, study, and use of programmable engineered biological systems. Signifi cantly, the concomitant development of tools and techniques to design, synthesize and assemble synthetic DNA into genes, synthetic circuits and even whole genomes continues to expand the applications of synthetic DNA in biology and biotechnology. This volume will present state-of-the art methods for the synthesis, design, assembly, post-synthesis processing, and application of synthetic DNA to modern biotechnology. This volume is divided into three general parts which incorporate protocols for the computational design of synthetic DNA sequences (Part I), the synthesis, assembly and cloning of synthetic DNA (Part II), and post-synthesis error reduction strategies (Part III). The historical origins of synthetic biology methods and the production and use of synthetic DNA itself can be traced to the development of chemistries for the abiotic synthesis of DNA. In the 1970s, synthetic organic chemistry methods were developed to synthesize DNA from synthetic nucleotides. Initially these chemistries were diffi cult and laborious to perform but they did result in the eventual synthesis of the fi rst functional synthetic DNA sequence which totaled 207 base pairs in length and encoded for a 75 bp tyrosine suppres- sor transfer RNA. Subsequent improvements to the synthetic chemistry methods in the 1980s led to the creation of the solid-phase phosphoramidite synthesis method which enabled the highly effi cient, robust, and scalable synthesis of synthetic DNA. In the 1980s–1990s, recombinant DNA technologies based around the discovery and use of bacterial restriction enzyme systems and the development of the polymerase chain reaction (PCR) further contributed to the development of techniques to produce and utilize synthetic DNA. In the last decade, the growth in the interest and application of engineered biological systems and materials has led to further advancements in the design, production, processing, and use of synthetic DNA. The protocols in this volume will aid researchers who wish to produce and utilize synthetic DNA in their work and undoubtedly contribute to continued innovation in synthetic biology. In Part I, protocols which outline the use of three computational tools for the design of synthetic oligonucleotide sequences for a variety of applications are outlined. In Chapter 1 , Damien O’Halloran provides a guide for the use of the online STITCHER algorithm for designing DNA sequences for overlap assembly. This program would be useful for those desiring to assemble longer DNA sequences from shorter starting materials by PCR v vi Preface assembly based methods for a variety of applications including gene synthesis and synthetic pathway construction. In Chapter 2 , Yu et al. present the online codon optimization tool called “COOL.” This protocol allows researchers to use the COOL program to recode and codon optimize synthetic DNA sequences to refl ect the codon preferences of a host organism or user defi ned codon tables. This tool would be especially useful for those that wish to optimize a synthetic gene for increased expression in a heterologous host organism. Codon shuffl ing of a DNA sequence could also be used to tweak the expression of genes in a biosynthetic pathway to optimize metabolic fl ux through a multigene system. In Chapter 3 , Milligan and Garry present the use of the Shuffl e Optimizer program for recoding homolo- gous genes for synthesis and subsequent molecular shuffl ing. This algorithm would be very useful for researchers who are interesting in the directed evolution of proteins via in vitro shuffl ing methods as it allows for maximizing the potential cross-overs during the shuffl ing protocol. Recoding homologs to more closely match host codon preferences and maximizing sequence homology between genes would enhance the creation of libraries for directed evolution and allow researchers to explore greater sequence diversity by directed evolution. Beyond the (re-)design of DNA sequences for assembly, expression, and evolution the assembly of DNA sequences from smaller pieces into larger genes, pathways, or constructs is important for the engineering of encoded biological function. This volume contains a series of protocols which allow the researcher to assemble and/or clone DNA of various sizes into larger constructs for subsequent testing and application. One popular application of synthetic DNA is for the directed evolution of biomolecular function. Directed evolution is a powerful tool that can be used to augment the substrate specifi city of enzymes and binding proteins as well as improve the stability and function of proteins and other biomol- ecules. In all directed evolution techniques a library of nucleic acid (DNA, RNA) variants is created and parsed by a functional screen or selection to select for a desired function. Effi cient library construction and cloning is essential to successfully perform direct evolution experiments. To this end, in Chapter 4 Zhong et al. present a protocol for overlap PCR-based cloning and preparation of random mutagenesis libraries for evolution in E. coli . This protocol provides an effi cient method for generating libraries of protein variants and cloning them using a straightforward PCR-based method. In Chapter 5 , Currin et al. present the “SpeedyGenes” method for assembling synthetic genes from single-stranded oligonucleotides. This method allows for the accurate assembly and cloning of genes and gene variant libraries directly from oligonucleotides. A similar method detailed in Chapter 1 0 entitled polymerase step reaction (PSR) developed by Brian DeDecker can be used to create genes or gene libraries from oligonucleotides without PCR in a simple and accurate protocol which has been successfully used to synthesize phage display libraries. These protocols add useful techniques to the molecular toolbox of experimenters using directed evolution techniques to augment biological function. In addition to directed evolution applications, several other protocols in this volume will aid researchers in the hierarchical assembly of DNA constructs for utilization in a number of other synthetic biology applications. In Chapter 6 , Storch et al. introduce a modular assembly method they call BASIC. This method uses DNA linkers to mediate the assembly of modular DNA assemblies into larger constructs guided by designed prefi x and suffi x sequences. This method allows for the fl exible and accurate assembly of modular DNA constructs into complete expression constructs, biosynthetic pathways, or combinatorial sequence libraries. The authors have demonstrated >90 % assembly accuracy for constructs containing up to seven component DNA parts. Similarly, the PaperClip assembly method Preface vii presented in Chapter 9 allows for the assembly of multiple DNA parts using short “clip” adapter sequences in a simple and fl exible protocol. Both of these methods are attractive alternatives to the BioBrick and similar assembly standards commonly used in synthetic biology applications and provide a level of accuracy and fl exibility which is superior to these traditional standards. Additional assembly methods in Chapters 8 and 1 2 present methods to allow researchers to assemble DNA sequences from single-stranded DNA oligonucleotides by the highly accurate ligase cycling reaction (Chapter 8 ) and to assemble DNA constructs using the robust and highly effi cient GoldenGATEway method (Chapter 12 ) . Assembly and manipulation of DNA in vivo by taking advantage of the native yeast homol- ogous recombination system has become a powerful tool for the assembly of DNA constructs especially those that are multi-kilobases or greater in length. To these ends, protocols in Chapters 13 and 1 4 allow researchers to use yeast to manipulate DNA sequences for genetic manipulation by gene deletion (Chapter 1 3 ) and the hierarchical assembly of DNA constructs from smaller pieces of DNA (Chapter 1 4 ) . The use of synthetic DNA for manipulating genomic DNA or mobile genetic elements such as expression vectors is a hallmark of many molecular biology experiments. Therefore methods which reduce the complexity, labor, time, and expense associated with these techniques would be of great utility to researchers in the molecular biosciences. In Chapter 1 1 , Cui and Sherwin present the Clonetegration method for assembling DNA and integrating it into bacterial genomes. This method uses bacteriophage integrases to facilitate the cloning, assembly, and integration of DNA into bacterial chromosomes. This method provides a simple and rapid strategy to manipulate bacterial genomic DNA and should prove useful to both academic and industrial scientists interested in bacterial genomic manipulation and strain creation. In Chapters 15 and 1 6 methods are presented which allow researchers to construct and manipulate plasmid DNA sequences for a variety of applications. Krishnamurthy and Zhang present a method for the rapid manipulation of plasmid DNA templates (Chapter 15 ) by simultaneous deletion and reassembly by PCR which will allow researchers to tailor existing vector sequences to their particular application. In Chapter 1 6 , Jajesniak and Wong introduce the Quickstep-Cloning method for construction of recombinant plasmids which provides a convenient method for assembling expression constructs and synthetic biological circuits. To round out this volume, methods which address sequence errors inherent in chemically synthesized DNA are presented. Sequence errors are introduced into synthetic DNA due to reaction ineffi ciencies during the oligonucleotide synthesis process as well as by DNA polymerases during the assembly of dsDNA during the assembly process. To ensure sequence fi delity, enrichment of correct sequences from raw synthetic DNA populations which have sequences that both contain and lack errors is an important part of processing and using synthetic DNA. Applications which require ssDNA oligonucleotides with very high sequence fi delity may require expensive post-synthesis purifi cation or treatment of chemically synthesized ssDNA oligonucleotides before use. One alternative to chemically synthesized ssDNA oligonucleotides is to utilize DNA produced biologically which generally contain fewer errors. To this end, in Chapter 7 Ducani and Hogberg present a method for the enzymatic synthesis of ssDNA oligonucleotides for applications where high fi delity oligonucleotides are required. In the last few years the use of multiplex microarray synthesized oligonucleotide pools for gene synthesis has emerged as a low-cost alternative to traditional column synthesized oligonucleotides. However the synthesis fi delity of microarray synthesized oligonucleotides is generally lower than that of the traditional column synthesized oligonucleotides. In Chapter 17 Wan et al. present a MutS-mediated method to viii Preface enrich for oligonucleotides and assembled dsDNAs without errors from microarray synthesized oligonucleotide pools. This protocol provides researchers with a straightforward and inexpensive method to utilize oligonucleotide pools as starting materials for gene synthesis while eliminating the associated sequence errors. For DNA sequences assembled from oligonucleotides which contain a protein encoding sequence (or at least lack a termination codon), a simple and useful method for eliminating the majority of the errors is to fuse the synthetic coding sequence to a reporter gene and then screen for functional reporters by an in vivo phenotype screen. This type of screen works based on the necessity to maintain the correct reading frame of the fused reporter gene during translation to express a functional reporter protein. Since most synthesis- related errors are single or double nucleotide deletions, sequences which contain one of these deletion mutations will cause a shift in the translational reading frame which will result in the failed expression of the fused reporter protein. In Chapter 1 8 , Hoshida et al. outline the use of such a system in yeast for the selection of error-free synthetic genes from raw synthesis products. This method will be of use for any researcher synthesizing protein encoding genes and needs to enrich for correct sequences from synthetic products. In addition, since this reading frame selection is designed to work in yeast, researchers can take advantage of the power of the yeast recombination system to enable cloning and in vivo assembly of their synthetic DNA constructs prior to error selection. This book should be of use to researchers in the molecular biosciences who need to manipulate DNA sequences in the course of their work. The protocols and methods outlined herein contain innovative methods for the design, synthesis, assembly, cloning, and screening of synthetic DNA sequences which will help researchers advance the understand- ing of basic biology as well as the manipulation of biological systems for a variety of applications. I wish to thank all of the contributors to this volume for sharing their expertise and their methods with the research community. Finally, I want to thank my colleagues at the University of Texas-Applied Research Laboratories for their generous encouragement and support in the advancement of basic and applied science and engineering in the biological sciences and beyond. Austin, TX, USA R andall A . H ughes Contents Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x i PART I C OMPUTATIONAL TOOLS FOR DESIGN AND ASSEMBLY OF SYNTHETIC DNA 1 A Guide to Using S TITCHER for Overlapping Assembly PCR Applications . . 3 Damien M. O’Halloran 2 S ynthetic Gene Design using Codon Optimization On-Line (COOL). . . . . . . 1 3 Kai Yu , K ok Siong Ang , and Dong-Yup Lee 3 S huffle Optimizer: A Program to Optimize DNA Shuffling for Protein Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 5 John N. Milligan and Daniel J. Garry PART II DNA SYNTHESIS, ASSEMBLY, AND CLONING 4 S imple Cloning by Prolonged Overlap Extension-PCR with Application to the Preparation of Large-Size Random Gene Mutagenesis Library in Escherichia coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 9 Chao Zhong , C hun You , P ing Wei , and Yi-Heng Percival Zhang 5 S peedyGenes: Exploiting an Improved Gene Synthesis Method for the Efficient Production of Synthetic Protein Libraries for Directed Evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 Andrew Currin , N eil Swainston , P hilip J. Day , and Douglas B. Kell 6 BASIC: A Simple and Accurate Modular DNA Assembly Method . . . . . . . . . . 7 9 Marko Storch , A rturo Casini , B en Mackrow , T om Ellis , and Geoff S. Baldwin 7 E nzymatic Synthesis of Single-Stranded Clonal Pure Oligonucleotides . . . . . . 9 3 Cosimo Ducani and Björn Högberg 8 R apid Assembly of DNA via Ligase Cycling Reaction (LCR). . . . . . . . . . . . . . 1 05 Sunil Chandran 9 P aperClip: A Simple Method for Flexible Multi-Part DNA Assembly. . . . . . . . 1 11 Maryia Trubitsyna , C hao-Kuo Liu , A lejandro Salinas , A listair Elfick , and Christopher E. French 10 The Polymerase Step Reaction (PSR) Method for Gene and Library Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 29 Brian S. DeDecker 11 Clonetegration using OSIP Plasmids: One-Step DNA Assembly and Site-Specific Genomic Integration in Bacteria . . . . . . . . . . . . . . . . . . . . . . 139 Lun Cui and Keith E. Shearwin ix