Download How Can Transposons Accelerate Your Genomics

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?
Mobile genetic elements
Transposase
Recognition
Sequences
• Transposons are DNA sequences
that can move from one
genomic location to another
Transposase
Binding
Tn5
• Identified in all prokaryotic and
eukaryotic organisms
• Consists of two elements:
1. Transposase enzyme (Tn5),
catalyzes the transposition
reaction
2. Transposable DNA sequence
(Transposon) containing
transposase recognition
sequences
gDNA
Transposon
gDNA
Tn5
Tn5
Tn5
Cleavage
Tn5
Tn5
9bp Random
Insertion Site
Target DNA
Target
Capture
Tn5
Tn5
Transposon
9bp Repeat
Strand
Transfer
How are Transposons Used?
To insert DNA sequences into genomes or plasmids
A transposon contains a Desired DNA
Sequence:
• Resistance marker
• Origin of replication
• Promoter element
• Other DNA sequence
The Desired DNA sequence is flanked
by 19 bp Tn5 Transposase recognition
sequences (ME = Mosaic Ends).
The transposon is inserted into target
DNA:
• Genomic DNA
• Purified plasmid DNA
In a highly random, unbiased
manner.
The transposition reaction can be
accomplished in vitro or in vivo.
ME
Desired DNA Sequence
Tn5
ME
Transposon
Tn5
Tn5
Tn5
Genomic or
Plasmid
Target DNA
Target DNA
Target DNA
Transposome
Tn5
Tn5
Desired DNA Sequence
Analysis
Transposon Nomenclature
A “transposon” is the DNA Sequence
flanked by mosaic ends that will be
inserted into the target DNA.
ME
Desired DNA Sequence
Tn5
ME
Transposon
Tn5
A “transposome” consists of the
transposon complexed with the
transposase.
Tn5
Tn5
Transposome
Target DNA
“Transposition” is the reaction where
the transposon is inserted into the
target DNA.
“Transposed DNA” is target DNA with
a transposon insertion.
Target DNA
Tn5
Tn5
Transposition
Desired DNA Sequence
Transposed
DNA
Power of Transposomics
Transposons impart desired function to target DNA
Transposon
ME
ME
ME
ME
Replication
Origin
T7
Promoter
Selectable
Marker
Your
Sequence
Target DNA
Insertions
ME
Function
Replication
Plasmid
(in vitro)
ME
ME
ME
e.g. Reporter gene
Transcription
Genomic DNA
(in vivo)
Gene
Kan-r
Sequence
Disruption/
Inactivation
Your Desired
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 EZTn5™ 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
engineering
(CRISPR,
TALENS, Zinc
fingers)
Insertion/deletion of
desired gene or sequence
in known genomic
location
Specific, protocols are
becoming more
robust
Time-intensive, requires knowledge of exact
target insertion location, requires
optimization, possible off-target effects, may
require whole genome sequencing to
characterize, may not work well in all bacterial
strains
Spontaneous
mutagenesis
Expose the strain to
phenotypic screen for
desired attribute, identify
“positives”
“Positives”
automatically have
desired
function/phenotype
Low efficiency, may require multiple rounds of
screening and optimization, may require
whole genome sequencing to characterize
Chemical
mutagens
Expose the strain to
chemicals known to
mutagenize DNA
Easy, fast, low bias,
inexpensive
Low efficiency, may require optimization and
multiple chemicals/rounds of mutagenesis,
may require whole genome sequencing to
characterize
Random gene
disruption with
transposons
(knock-out
library)
Randomly disrupt a
genome by inserting a
selection marker or
replication origin
Easy to use, fast,
minimal optimization,
low-to-no-bias,
characterize insert by
rescue cloning
Requires characterization of insertion
location, non-specific
Random gene
insertion with
transposons
Randomly insert a desired
gene (which lends desired
phenotype) into the
bacterial genome
Easy to use, fast,
minimal optimization,
low-to-no bias, can
insert large
sequences
Requires characterization of insertion
location, non-specific
Bacterial Strain Engineering with Transposomes
Simple protocol for creating diverse mutant library
~3 days
EZ-Tn5
Transposome
Electroporate
Strain of
Interest
ME R6Kɣ ori / Kanr ME
Tn5
Tn5
Tn5
Tn5
Plate and
select
insertion
clones
(KanR)
Screen library for desired attribute(s)
A
B
C
D
E
F
G
H
Mutant
library
1
2
3
4 5
6 7
8 9 10 11 12
Identify positive
“hits”
Grow cells
and Extract
DNA
 Identify Insertion Point
 Characterize Mutant
Bacterial Strain Engineering with Transposomes
Identify integration site
~3 days
Extract
genomic
DNA
Rescue
Cloning
Digest
DNA (rare
cutter) End Repair &
Ligate DNA
Transform
into
EC100D*
Whole
Genome
Sequencing
NGS Analysis
Disrupted gene identified
*TransforMax EC100D cells allow replication of plasmids with R6Kɣ origins
Plate and
select clones
(KanR)
Isolate
R6Kɣ ori / KanR
“rescued”
plasmid DNA
Sanger
Sequencing
Disrupted gene identified
Metagenomics Applications
Characterize non-E. coli plasmids or circular genomes
Virus
Isolate
Circular
DNA
Environmental
samples
In vitro tranpsosition
R6Kɣ ori / KanR
ME R6Kɣ ori / KanR ME
Tn5
Tn5
Tn5
Tn5
Plasmid
Propagation
and “Rescue”
Transposon
Insertions
Bacteria with
plasmid
or DNA of
interest
Plasmid
Propagation
and “Rescue”
Transform
into EC100D
Plasmids
containing
R6Kɣ ori / KanR
transposon
Plate and select
clones (KanR)
Isolate
“rescued”
plasmid DNA
Analysis:
 Sequencing
 Genomics screening
 Functional analysis
Metagenomics Applications
Screen for essential genes using random gene KOs
Environmental Samples
Electroporate
Bacteria of
Interest
Bacteria of
interest
Whole
Genome
Sequencing
Insertions in essential
genes are lethal or
rapidly lost
Plate and
select
surviving
clones
EZ-Tn5
Transposome
Extract and
pool genomic
DNA
Whole
Genome
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
Mutant
library
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
mutants by
PCR/subcloning
PCR or RE-based
subcloning of
desired deletion
mutants into
expression vector
Specific deletion mutants can be
obtained
Time-intensive, low
throughput, requires
multiple steps, difficult
to accomplish on a
genome-scale level
Create library of
clones with randomly
inserted T7 promoters
with transposons
Randomly insert a
T7 promoter into
the cloned gene of
interest
Easy to use, fast, generates a large
library in one reaction, no subcloning
into expression vector, can be done with
single genes or libraries of cloned genes
Requires characterization
of insertion location via
Sanger sequencing or
NGS
ME
T7 / KanR
ME
T7 Promoter /
Kanamycin-r
Insertion generates
library of 1000’s of
mutants producing
novel transcripts
Tn5
Tn5
Tn5
Cloned Target(s)
T7
/ KanR
Gene
T7 / KanR
Etc.
T7 / KanR
Gene Expression Applications
Mutagenize cloned targets, analyze expression
~3 days
Transform the
reaction into E. coli
Combine and
Incubate:
T7 / KanR
Plate and select
clones (KanR)
Target
Clone
Transposon
R
ME T7 / Kan
Tn5
Tn5 Tn5
Transform
Expression Strain
[eg. BL21 (DE3)]
ME
Transposase
Tn5
Isolate
plasmid DNA
Sequence
In vitro
transcription
Induce
expression
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
into E. coli
Combine and
Incubate
Selection Marker &
Sequencing Primer
Target
Clone
or
Library
Plate and select
clones
Transposon
R
R
ME Kan , Tet , DHFR
Tn5
Tn5
Sequence using
transposon primers
Isolate
plasmid DNA
ME
Tn5
KanR
Gene
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,
insertional knock-outs
Kanamycin resistance
(KanR)
< KAN-2 >
EZ-Tn5 <KAN-2> Tnp
Transposome Kit
Strain engineering,
insertional knock-outs,
“rescue” genomic DNA or
plasmid
R6Kɣ origin of replication
and kanamycin resistance
(KanR)
< R6Kɣori/KAN-2 >
EZ-Tn5 <R6Kɣori/KAN-2>
Tnp Transposome Kit
Kits contain EZ-Tn5™ Tnp Transposome and Sequencing Primers
In vitro use in free, purified DNA:
Application
Transposon Function
Transposon Name
Product Name
Gene expression studies
T7 promoter and kanamycin
resistance (KanR)
< T7/KAN-2 >
EZ-Tn5 <T7/KAN-2>
Insertion Kit
Gene or plasmid rescue
R6Kɣ origin of replication
and kanamycin resistance
(KanR)
< R6Kɣori/KAN-2 >
EZ-Tn5 <R6Kɣori/KAN-2>
Insertion Kit
Sequencing, insertional
knock-outs, insertion of
selectable marker
Kanamycin, trimethoprim or
tetracycline resistance
< KAN-2 >
< DHFR-1 >
< TET-1 >
EZ-Tn5 <KAN-2>,
<DHFR-1>,
<TET-1> Insertion Kits
Kits contain EZ-Tn5 Transposon and EZ-Tn5 Transposase, Reaction Buffer, Stop Solution, Sequencing Primers, Control Target DNA
Customize Your Transposon
Insert your sequence of interest – up to 12 kb so far!
Desired Transposon/
Selection Marker/
Gene for Insertion
Gene-specific
primer with 5’-PO4
Mosaic End
Template DNA
Gene-specific
primer with
5’-PO4
Mosaic End
PCR amplify, purify
ME
in vitro
transposition
Mix transposon,
Tn5 Transposase,
target DNA
Target: purified DNA
ME
Transposon
in vivo
transposition
Mix transposon + Tn5
Transposase (no Mg2+),
form transposome
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
*
*
*
*
*
* 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
Thank You!
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