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 clones plasmid DNA
Transposon KanR, Tet R, DHFR ME ME
Tn5 Tn5 GeneKanR Tn5 Tn5
Produce 1000’s of insertional mutants ...... EZ-Tn5 Transposomes for in vivo Use Simple insertion of a transposon into a genome
1. Mix EZ-Tn5 Transposome with electrocompetent cells of choice 2. Place in electroporator cuvette and electroporate 3. Transfer to SOC/LB medium, incubate with shaking for 1 hour 4. Plate on antibiotic-containing selective media EZ-Tn5 Insertion Kits for in vitro Use Easy insertion into purified DNA
1. Mix purified Target DNA with Tranposon and EZ-Tn5 Transposase
2. Incubate 37oC for 2 hours
3. Transform into E. coli, plate on selective media
4. Prepare template DNA for downstream analysis EZ-Tn5™ Transposon Toolbox Tools to generate the mutants you need In vivo use in bacteria:
Application Transposon Function Transposon Name Product Name Strain engineering, Kanamycin resistance < KAN-2 > EZ-Tn5
In vitro use in free, purified DNA:
Application Transposon Function Transposon Name Product Name Gene expression studies T7 promoter and kanamycin < T7/KAN-2 > EZ-Tn5
Gene or plasmid rescue R6Kɣ origin of replication < R6Kɣori/KAN-2 > EZ-Tn5
Gene-specific primer with 5’-PO PCR amplify, purify 4 Mosaic End
ME ME Transposon in vitro in vivo transposition transposition Mix transposon, Mix transposon + Tn5 Tn5 Transposase, Transposase (no Mg2+), target DNA form transposome
Target: purified DNA Target: bacteria
Largest known insert = 12kb, large enough for an operon or biosynthetic pathway! TypeOne™ Restriction Inhibitor for Metagenomics Increase transposition efficiency in non-E. coli bacteria
• Bacterial Type I restriction and modification (R-M) systems can attack and degrade transposomes, decreasing transposition frequency • Widespread in Eubacteria and Archaebacteria • TypeOne Restriction Inhibitor blocks Type I R-M systems – ocr gene product from T7 bacteriophage, a DNA structural mimic – Prevents transposon DNA binding and degradation by endogenous host restriction enzymes – Also inactivates Type III nucleases, but does NOT inhibit Type II “normal” restriction endonucleases used for cloning applications (BamHI, EcoR1, HindIII, etc) • Increases transposome resistance to Type I and III R-M systems = increases transposition efficiency • Can also increase plasmid transformation efficiency in non-E. coli strains! Improve Transposome Insertion Efficiency Use TypeOne™ during bacterial electroporation
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* TypeOne Restriction Inhibitor improves transformation efficiency of plasmid DNA and transposomes in strains with active Type I R-M systems * Agrobacterium does not contain an active Type I system Tips for Success: EZ-Tn5 in vivo Transposomics Improve transposome delivery
• Success depends on bacterial type, electroporation conditions, antibiotic resistance characteristics and endogenous restriction systems. • Bacterial strain of interest: – Ensure antibiotic resistance markers and promoters in your transposon are functional in your organism of interest. – Identify appropriate antibiotic concentration with a Minimum Inhibitory Concentration test. – If your bacteria is resistant to our available selection markers, design your own transposon containing an alternate marker! Contact [email protected] for design help. – Include TypeOne Restriction Inhibitor to inhibit transposon degradation. – Optimize electroporation conditions using plasmid DNA (as a starting point, try 50 µL cells, 1 µL Transposome, 2 mm cuvette, 2500V, 5 msec time constant). – Recover cells immediately after electroporation. Tips for Success: EZ-Tn5 in vivo Transposomics Efficient mutagenesis of non-E. coli strains
• Best practices: – Use a high efficiency electrocompetent cell preparation, at least 1 x 106 cfu/µg. – Do not use chemically competent cells! Transformation efficiency is too low to generate a sufficient population of transposition clones with good mutation coverage. – Screen multiple colonies, especially if mutagenesis of a particular target gene is your goal. – Note: if your desired gene is not represented in the final transposed library, successful insertion may have created a lethal mutation. – Run a control transposition reaction in high-efficiency TransforMax™ EC100 Electrocompetent E. coli (>109 cfu/µg). Conclusions EZ-Tn5 Systems are Powerful and Easy-to Use
• EZ-Tn5 system is an easy-to-use, powerful system optimized for maximum transposase activity
• Use transposomics for almost any application requiring insertion or inactivation of DNA!
• Transposomes can be used in vivo for mutagenesis, bacterial strain development, and gene silencing/knockouts
• Transposons can be used in vitro for sequencing, plasmid or gene rescue, and functional analysis
• Proven success in a wide variety of bacteria including Gram positive strains
• Large payload (up to 12 kb) enables custom transposon generation and engineering of entire operons or pathways
• Only limited by your imagination! Explore References for 100’s of Applications Contact [email protected] for help Questions? www.lucigen.com
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