GENERATING MUTANT LIBRARIES USING ERROR-PRONE PCR

By L. KUSIN

PART I POINT MUTAGENESIS 1 ERROR PRONE PCR AND EFFECTIVE GENERATION OF GENE VARIANT LIBRARIES FOR DIRECTED EVOLUTION 3 2 ERROR PRONE ROLLING CIRCLE AMPLIFICATION GREATLY SIMPLIFIES RANDOM MUTAGENESIS 23 3 RANDOM MUTAGENESIS BY ERROR PRONE POL PLASMID REPLICATION IN ESCHERICHIA COLI 31 4 THE SEQUENCE SATURATION MUTAGENESIS SESAM METHOD 45 5 GENERATION OF EFFECTIVE LIBRARIES BY NEUTRAL DRIFT 69 6 SITE SATURATION MUTAGENESIS BY OVERLAP EXTENSION PCR 83 7 ITERATIVE SATURATION MUTAGENESIS A POWERFUL APPROACH TO ENGINEER PROTEINS BY SYSTEMATICALLY SIMULATING DARWINIAN EVOLUTION 103 8 GENERATING TARGETED LIBRARIES BY THE COMBINATORIAL INCORPORATION OF SYNTHETIC OLIGONUCLEOTIDES DURING GENE SHUFFLING ISOR 129 9 OMNICHANGE SIMULTANEOUS SITE SATURATION OF UP TO FIVE CODONS 139 10 RANDOM INSERTIONAL DELETIONAL STRAND EXCHANGE MUTAGENESIS RAISE A SIMPLE METHOD FOR GENERATING RANDOM INSERTION AND DELETION MUTATIONS 151 CONTENTS 1 11 TRANSPOSON BASED APPROACHES FOR GENERATING NOVEL MOLECULAR DIVERSITY DURING DIRECTED EVOLUTION 159 PART II RECOMBINATORIAL METHODS 12 RESTRICTION ENZYME MEDIATED DNA FAMILY SHUFFLING 175 13 ASSEMBLY OF DESIGNED OLIGONUCLEOTIDES A USEFUL TOOL IN SYNTHETIC BIOLOGY FOR CREATING HIGH QUALITY COMBINATORIAL DNA LIBRARIES 189 14 ONE POT SIMPLE METHODOLOGY FOR CASSETTE RANDOMIZATION AND RECOMBINATION FOR FOCUSED DIRECTED EVOLUTION OSCARR 207 15 USER FRIENDLY DNA RECOMBINATION USEREC GENE LIBRARY CONSTRUCTION REQUIRING MINIMAL SEQUENCE HOMOLOGY 213 16 ITCHY INCREMENTAL TRUNCATION FOR THE CREATION OF HYBRID ENZYMES 225 17 GENERATING RANDOM CIRCULAR PERMUTATION LIBRARIES 245 PART III STRUCTURE GUIDED METHODS AND COMPUTATIONAL TOOLS FOR THE DESIGN AND ANALYSIS OF MUTANT LIBRARIES 18 PROBABILISTIC METHODS IN DIRECTED EVOLUTION LIBRARY SIZE MUTATION RATE AND DIVERSITY 261 19 THE MUTAGENESIS ASSISTANT PROGRAM 279 20 COMPUTATIONAL TOOLS FOR DESIGNING SMART LIBRARIES 291 21 COMPUTATIONAL TOOLS FOR DIRECTED EVOLUTION A COMPARISON OF PROSPECTIVE AND RETROSPECTIVE STRATEGIES 315 22 DESIGNING LIBRARIES OF CHIMERIC PROTEINS USING SCHEMA RECOMBINATION AND RASPP 335 23 NONCONTIGUOUS SCHEMA PROTEIN RECOMBINATION 345 24 ENGINEERING PROTEINS BY RECONSTRUCTING EVOLUTIONARY ADAPTIVE PATHS 353 PART I POINT MUTAGENESIS 1 CHAPTER 1 ERROR PRONE PCR AND EFFECTIVE GENERATION OF GENE VARIANT LIBRARIES FOR DIRECTED EVOLUTION ABSTRACT ANY SINGLE ENZYME DIRECTED EVOLUTION STRATEGY HAS TWO FUNDAMENTAL REQUIREMENTS THE NEED TO EFFICIENTLY INTRODUCE VARIATION INTO A GENE OF INTEREST AND THE NEED TO CREATE AN EFFECTIVE LIBRARY FROM THOSE VARIANTS GENERATION OF A MAXIMALLY DIVERSE GENE LIBRARY IS PARTICULARLY IMPORTANT WHEN EMPLOYING NONTARGETED MUTAGENESIS STRATEGIES SUCH AS ERROR PRONE PCR EPPCR WHICH SEEK TO EXPLORE VERY LARGE AREAS OF SEQUENCE SPACE HERE WE PRESENT COMPREHENSIVE PROTOCOLS AND TIPS FOR USING EPPCR TO GENERATE GENE VARI ANTS THAT EXHIBIT A RELATIVELY BALANCED SPECTRUM OF MUTATIONS AND FOR CAPTURING AS MUCH DIVERSITY AS POSSIBLE THROUGH EFFECTIVE CLONING OF THOSE VARIANTS THE DETAILED LIBRARY PREPARATION METHODS THAT WE DESCRIBE ARE GENERALLY APPLICABLE TO ANY DIRECTED EVOLUTION STRATEGY THAT USES RESTRICTION ENZYMES TO CLONE GENE VARIANTS INTO AN EXPRESSION PLASMID KEY WORDS DIRECTED EVOLUTION RANDOM MUTAGENESIS ERROR PRONE PCR GENEMORPH II MUTAZYME II DNA POLYMERASE LIBRARY GENERATION LIBRARY ANALYSIS 1? INTRODUCTION DIRECTED EVOLUTION AT A SINGLE GENE LEVEL IS A POWERFUL TOOL FOR EFFECTING RAPID IMPROVEMENT IN A DESIRED PROTEIN FUNCTION RECENT YEARS HAVE SEEN AN INCREASING TREND TOWARD THE DESIGN OF TARGETED SMART LIBRARIES WHICH EXPLOIT AVAILABLE STRUCTURAL FUNCTIONAL AND OR SEQUENCE DERIVED INFORMATION TO PRESELECT PROMISING RESI DUES FOR MUTAGENESIS THESE APPROACHES SEEK TO MINIMIZE THE NUMBER OF GENE VARIANTS THAT MUST BE SCREENED TO ACHIEVE A SUC CESSFUL OUTCOME 1 3 AND ARE PARTICULARLY VALUABLE WHEN ONLY A LOW TO MODERATE THROUGHPUT SCREEN IS AVAILABLE E G 102 104 VARI ANTS PER DAY IN CONTRAST APPROACHES LIKE ERROR PRONE PCR EPPCR PROVIDE ACCESS TO AN ALMOST UNLIMITED NUMBER OF VARIANTS BUT GENERALLY HAVE A LOWER HIT RATE AND ARE THEREFORE BETTER SUITED TO HIGH THROUGHPUT SCREENING OR SELECTION STRATEGIES NEVERTHELESS IT IS WORTH NOTING THAT EPPCR CAN STILL YIELD RAPID GAINS IN FITNESS 3