EZ-Tn5™ Transposon Tools: How Can Transposons Accelerate Your Genomics Research? Fred Hyde, PhD Staff Technical Applications Scientist Illumina, Inc. Madison, WI www.lucigen.com Agenda • What are transposons, and how are they used? • How do transposons insert into target DNA? • Applications for in vivo and in vitro transposomics • Transposon tools for your research • Designing a custom transposon • Tips for success with in vivo transposomics • Resources What are Transposons? Transposase Recognition Mobile genetic elements Sequences gDNA Transposon gDNA Transposase • Transposons are DNA sequences Binding that can move from one Tn5 Tn5 genomic location to another Tn5 • Identified in all prokaryotic and Tn5 eukaryotic organisms Cleavage • Consists of two elements: Tn5 1. Transposase enzyme (Tn5), Tn5 catalyzes the transposition 9bp Random Insertion Site reaction Target DNA 2. Transposable DNA sequence Target (Transposon) containing Capture transposase recognition Tn5 Tn5 sequences Strand Transfer Transposon 9bp Repeat How are Transposons Used? To insert DNA sequences into genomes or plasmids A transposon contains a Desired DNA ME Desired DNA Sequence ME Transposon Sequence: • Resistance marker • Origin of replication Tn5 Tn5 • Promoter element • Other DNA sequence Transposome Tn5 The Desired DNA sequence is flanked Tn5 by 19 bp Tn5 Transposase recognition Genomic or sequences (ME = Mosaic Ends). Plasmid Target DNA Target DNA The transposon is inserted into target DNA: Tn5 • Genomic DNA Tn5 • Purified plasmid DNA Target DNA In a highly random, unbiased manner. Desired DNA Sequence The transposition reaction can be accomplished in vitro or in vivo. Analysis Transposon Nomenclature ME Desired DNA Sequence ME A “transposon” is the DNA Sequence Transposon flanked by mosaic ends that will be inserted into the target DNA. Tn5 Tn5 A “transposome” consists of the Transposome Tn5 transposon complexed with the Tn5 transposase. Target DNA “Transposition” is the reaction where Tn5 the transposon is inserted into the Tn5 Transposition Target DNA target DNA. Desired DNA Sequence “Transposed DNA” is target DNA with a transposon insertion. Transposed DNA Power of Transposomics Transposons impart desired function to target DNA Transposon Target DNA Insertions Function Replication Origin ME ME Replication Plasmid T7 (in vitro) Promoter ME ME Transcription Selectable Marker Sequence ME ME Genomic DNA Disruption/ (in vivo) Gene Kan-r Inactivation Your Sequence ME ME Your Desired e.g. Reporter gene Function EZ-Tn5™ Transposon System Engineered for maximum transposase activity • First broad application/complete research system based on transposons • Developed in the 1990s (Reznikoff and Goryshin, 1995) based on a “hyperactive” modified Tn5 transposase • Transposase recognition sequences = 19-bp Mosaic Ends (ME): CTGTCTCTTATACACATCT • Combination of Hyperactive Transposase and ME sequences increases reaction kinetics by 1000-fold over native Tn5 • EZ-Tn5 Transposon Systems offer optimized reagents for both in vivo and in vitro applications EZ-Tn5 Transposon Insertions are Highly Random Little-to-no bias, stable insertion • Insertion events confirmed throughout the target DNA, regardless of target sequence content. • Small insertion bias toward GC-rich areas but high insertion efficiency compensates (no effect on most transposomics applications). • Insertion is stable – will not “hop” back out. Final EZ-Tn5™ Insertion Site 9 bp target gDNA repeats flank inserted transposon Transposon Structure Transposon Insertion EZ-Tn5™ Transposome Delivered to cells for in vivo transposomics • Transposomes are complexes of Tn5 Transposase + transposon DNA • High transposome stability allows direct electroporation into Gram negative and Gram positive bacteria • Easy, rapid method for generating library of gene knockouts in living bacteria and engineering novel bacterial strains EZ-Tn5 Transposome Graphic by Ivan Rayment and William Reznikoff, University of Wisconsin-Madison. What are the Most Common Applications of EZ- Tn5™ Transposon Tools? Bacterial Strain Engineering Many options for novel strain creation Goal: Create a bacterial strain with a desired phenotype or attribute. Requires a functional, phenotypic or genotypic screen to find “positives” Method Description Advantages Disadvantages Targeted Insertion/deletion of Specific, protocols are Time-intensive, requires knowledge of exact engineering desired gene or sequence becoming more target insertion location, requires (CRISPR, in known genomic robust optimization, possible off-target effects, may TALENS, Zinc location require whole genome sequencing to fingers) characterize, may not work well in all bacterial strains Spontaneous Expose the strain to “Positives” Low efficiency, may require multiple rounds of mutagenesis phenotypic screen for automatically have screening and optimization, may require desired attribute, identify desired whole genome sequencing to characterize “positives” function/phenotype Chemical Expose the strain to Easy, fast, low bias, Low efficiency, may require optimization and mutagens chemicals known to inexpensive multiple chemicals/rounds of mutagenesis, mutagenize DNA may require whole genome sequencing to characterize Random gene Randomly disrupt a Easy to use, fast, Requires characterization of insertion disruption with genome by inserting a minimal optimization, location, non-specific transposons selection marker or low-to-no-bias, (knock-out replication origin characterize insert by library) rescue cloning Random gene Randomly insert a desired Easy to use, fast, Requires characterization of insertion insertion with gene (which lends desired minimal optimization, location, non-specific transposons phenotype) into the low-to-no bias, can bacterial genome insert large sequences Bacterial Strain Engineering with Transposomes Simple protocol for creating diverse mutant library ~3 days Plate and Electroporate Screen library for desired attribute(s) Strain of select Interest insertion EZ-Tn5 clones 1 2 3 4 5 6 7 8 9 10 11 12 Transposome (KanR) A B C D E F G H Mutant Identify positive library ME R6Kɣ ori / Kanr ME “hits” Grow cells and Extract DNA Tn5 Tn5 Tn5 Tn5 Identify Insertion Point Characterize Mutant Bacterial Strain Engineering with Transposomes Identify integration site ~3 days Extract Transform genomic Digest into EC100D* Isolate R6Kɣ ori / KanR DNA Rescue DNA (rare cutter) Plate and “rescued” Cloning End Repair & select clones plasmid DNA Ligate DNA (KanR) Whole Genome Sanger Sequencing Sequencing NGS Analysis Disrupted gene identified Disrupted gene identified *TransforMax EC100D cells allow replication of plasmids with R6Kɣ origins Metagenomics Applications Characterize non-E. coli plasmids or circular genomes Virus R Isolate R6Kɣ ori / Kan Environmental In vitro tranpsosition samples Circular DNA ME R6Kɣ ori / KanR ME Plasmid Propagation and “Rescue” Tn5 Tn5 Tn5 Tn5 Transposon Insertions Bacteria with plasmid or DNA of interest Plasmid Transform Propagation into EC100D Isolate and “Rescue” Plate and select Analysis: R “rescued” Plasmids clones (Kan ) plasmid DNA Sequencing containing Genomics screening R6Kɣ ori / KanR Functional analysis transposon Metagenomics Applications Screen for essential genes using random gene KOs Environmental Samples Insertions in essential Electroporate genes are lethal or Bacteria of rapidly lost Interest Plate and select Extract and surviving pool genomic clones DNA Bacteria of interest Mutant library Whole EZ-Tn5 Whole Genome Transposome Genome Sequencing Sequencing Compare NGS Analysis NGS Analysis Map insertion events Identify the genes not represented in the mutant library These genes are likely to be essential or advantageous Functional Analysis of Novel Genes Identify functional characteristics Goals: • Express novel gene, or cloned library of genes, in E. coli background • Identify regulatory or functional regions, study protein-protein interactions Method Description Advantages Disadvantages Create series of PCR or RE-based Specific deletion mutants can be Time-intensive, low mutants by subcloning of obtained throughput, requires PCR/subcloning desired deletion multiple steps, difficult mutants into to accomplish on a expression vector genome-scale level Create library of Randomly insert a Easy to use, fast, generates a large Requires characterization clones with randomly T7 promoter into library in one reaction, no subcloning of insertion location via inserted T7 promoters the cloned gene of into expression vector, can be done with Sanger sequencing or with transposons interest single genes or libraries of cloned genes NGS ME ME Tn5 T7 / KanR Tn5 T7 Promoter / Tn5 Kanamycin-r Cloned Target(s) Insertion generates T7Gene / KanR library of 1000’s of T7 / KanR mutants producing novel transcripts T7 / KanR Etc. Gene Expression Applications Mutagenize cloned targets, analyze expression ~3 days Transform the reaction into E. coli Combine and T7 / KanR Incubate: Plate and select Isolate Sequence clones (KanR) plasmid DNA Target Clone Transposon In vitro R Transform ME T7 / Kan ME Expression Strain transcription [eg. BL21 (DE3)] Transposase Induce Tn5 Tn5 expression Tn5 Tn5 in vivo Analysis: Functional analysis Protein:protein interactions Identify regulatory elements Insertional Inactivation and Sequencing DNA sequencing of large targets or cloned libraries ~3 days Transform Combine and into E. coli Incubate Selection Marker & Sequencing Primer Target Clone Plate and select Isolate Sequence using or transposon primers Library