Post on 16-Jul-2015
Engineering genetic machines with gBlocks® Gene
Fragments
gBlocks® Gene Fragments GEMs
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• Founded in 1987 by Dr Joseph Walder
• Largest custom oligonucleotide
manufacturer worldwide
• >840 employees in 6 locations
• >95% of ordered products are
manufactured and shipped in less
than 24 hours
Integrated DNA Technologies
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“The Sun Never Sets at IDT…”
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Coralville, IA, USA (Headquarters)• 14,300 m2
• Global infrastructure, production, and support offices
• ISO 9001:2008 certified• 310 m2, ISO 13485:2003 certified
and FDA-registered clean room
Coralville, IA (Crosspark Campus)• Software development and IT: 2200 m2
• Product R&D: 1028 m2
Skokie, IL, USA• Administrative and
financial office
Leuven, Belgium• 2660 m2
• ISO 9001:2008 certified• Europe, Africa, and Middle East
production and support
San Diego, CA, USA• 1654 m2
• ISO 9001:2008 certified• Next-day delivery for
western United States
Singapore• 560 m2
• Asia market productionand support
Redwood City, CA, USA• 600 m2
• NGS R&D
Key operating statistics:
• >82,000 active customers
• >44,000 oligos ordered per day
• >2500 orders shipped per day
• >91% of orders through the web
• >270,000 website visits per month
(108,000 unique visitors)
• >85,000 phone calls per year
• >23,000 web chats per year
The World’s Largest Supplier of Custom Nucleic Acids
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IDT Offers
More Than
Just Primers
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Creating Long and Accurate Synthetic
DNA Without Cloning
• High quality DNA fragments
• 125–2000 bp in length
• Sequence-verified
• Short delivery time and low price
• 200 ng provided, dry
• Mixed bases can be included!
gBlocks® Gene Fragments
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gBlocks® Gene Fragments—Formats and Pricing
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20 kb of DNA as gBlocks® Gene
Fragments free of charge to each
iGEM 2015 team!
• Register at www.IDTDNA.com/iGEM
2015 IDT iGEM Sponsorship
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The Many Uses of gBlocks® Gene Fragments
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• Customized BioBrick™ parts
What Can be Built With gBlocks® Gene Fragments?
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• Customized BioBrick™ parts
• Codon optimized components
What Can be Built With gBlocks® Gene Fragments?
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• Customized BioBrick™ parts
• Codon optimized components
• Complete circuits
What Can be Built With gBlocks® Gene Fragments?
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• Customized BioBrick™ parts
• Codon optimized components
• Complete circuits
• Anything else!
Important: The iGEM rules for parts construction
states that your parts sequences must not contain
the restriction sites of the BioBrick prefix and
suffix: EcoRI, XbaI, SpeI, PstI.
What Can be Built With gBlocks® Gene Fragments?
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• gBlocks Gene Fragments with
20–40 bp overlaps designed by the
researcher or by IDT specialists
• gBlocks Gene Fragments and vector
are assembled using the Gibson
Assembly® Method
• Construct is transformed and
screened for the correct sequence
Assembling Multiple gBlocks® With the Gibson Assembly®
Method
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How Isothermal Assembly of gBlocks® Gene
Fragments Works
Step 1: gBlocks Gene Fragments are designed with
30 bp overlaps on the 3′ strand.
Step 2: A mesophilic exonuclease cleaves bases from
the 5′ end of the double-stranded DNA fragments,
before being inactivated by the 50°C reaction
temperature.
Step 3: The newly generated, complementary, single-
stranded 3′ ends anneal.
Step 4: A high fidelity DNA polymerase fills in any
single-stranded gaps.
Step 5: Finally, a thermophilic DNA ligase covalently
joins the DNA segments.
The Gibson Assembly® Method
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The Many Uses of gBlocks® Gene Fragments
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gBlocks® Gene Fragments for CRISPR
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gBlocks® Gene Fragments for Homology Directed Repair
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gBlocks® Gene Fragments for CRISPR
CRISPR gBlocks® fragment, 364 bp, expression cassette includes a 265 bp U6
promoter driving transcription of a 99 bp sgRNA
AAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATA
ATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAG
TTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCT
TTATATATCTTGTGGAAAGGACGAAACACCGNNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAA
AATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTT
• www.idtdna.com/CRISPR
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Red = U6 promoterBlack = 20 bp guide sequenceGreen = sgRNA (tracrRNA fusion)
gBlocks® Gene Fragments as gRNAs—Two Methods
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1. Clone gBlocks Gene Fragments into
an expression plasmid
gBlocks® Gene Fragments as gRNAs—Two Methods
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1. Clone gBlocks Gene Fragments into
an expression plasmid
2. Directly transform gBlocks Gene
Fragments into cells without
cloning
• www.idtdna.com/CRISPR
• http://www.addgene.org/static/cms/files
/hCRISPR_gRNA_Synthesis.pdf
Current Protocols in Molecular Biology (2014) 31.1.1–31.1.17.
gBlocks® Gene Fragments Outperform IVT sgRNAs
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• IVT RNA was treated with phosphatase to remove 5′-ppp. Even so, these RNAs were very toxic using lipid transfection, triggering innate immune responses even in HEK293 cells.
The Many Uses of gBlocks® Gene Fragments
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Adding mixed bases to gBlocks Gene Fragments:
• Basic designs can be ordered directly from the web, if they meet the criteria described at www.idtdna.com/gblocks
Uses of gBlocks Gene Fragment libraries:
• Binding site engineering
• Catalytic site analysis
• Antibody engineering
• Vaccine development
• DNA binding analysis
gBlocks® Gene Fragments Libraries
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Questions?genes@idtdna.com
Synthetic Templates for qPCR
ACVR2B-LIMK1-ACVR1B-CDK7 wtTCATACCTGCATGAGGATGTGCCCTGGTGCCGTGGCGAGGGCCACAAGCCGTCTATTGCCCACAGGGACTTTAA
AAGTAAGAATGTATTGCTGAAGAGCGACCTCACAGCCGTGCTGGCTGACTTTGGCTTGGtttttGAACATCATC
CACCGAGACCTCAACTCCCACAACTGCCTGGTCCGCGAGAACAAGAATGTGGTGGTGGCTGACTTCGGGCTGGC
GCGTCTCATGGTGGACGAGAAGACTtttttGTATGTGATCAGAAGCTGCGTCCCAACATCCCCAACTGGTGGCA
GAGTTATGAGGCACTGCGGGTGATGGGGAAGATGATGCGAGAGTGTTGGTATGtttttgGATGTATGGTGTAGG
TGTGGACATGTGGGCTGTTGGCTGTATATTAGCAGAGTTACTTCTAAGGGTTCCTTTTTTGCCAGGAGATTCAG
ACCTTGATCAGCTAACAgcggccgc
• Separate sequences by a series of T bases
• If the template will be cloned, add a restriction site(s) for linearization of
plasmid
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Fourplex Reaction Conditions
Reagent Final Conc.
10X buffer 1X
100 mM dNTPs 800 nM
50 mM MgCl2 3 mM
25 µM Forward Primer 1 500 nM
25 µM Reverse Primer 1 500 nM
12.5 µM Probe A 250 nM
25 µM Forward Primer 2 500 nM
25 µM Reverse Primer 2 500 nM
12.5 µM Probe B 250 nM
25 µM Forward Primer 3 500 nM
25 µM Reverse Primer 3 500 nM
12.5 µM Probe C 250 nM
25 µM Forward Primer 4 500 nM
25 µM Reverse Primer 4 500 nM
12.5 µM Probe D 250 nM
Immolase polymerase 0.8 U
H2O ----
Template
gBlocks® Gene Fragments as Quadruplex qPCR Standards
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0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
2.00E+06 2.00E+04 2.00E+02
Cq
Va
lue
s
Copies
Hs LIMK1
Hs CDK7
Hs ACVR1B
Hs ACVR2B
Project: CRISPR as a biological sensor
Detection of tuberculosis drug
resistance genes using a phage-
delivered cassette containing a:
• Cas9/gRNA targeting a drug
resistance gene
• LacZ gene driven by an SOS
dependent promoter
Past iGEM Success—2013 Overgrad Winner Paris-Bettencourt
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A quick and inexpensive field diagnostic test!
Past iGEM Success—2013 Overgrad Winner Paris-Bettencourt
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Project:
Constructed a set of modular
components for TALEN assembly using
gBlocks® Gene Fragments and Golden
Gate assembly
Past iGEM Success—2012 Team Freiberg
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www.idtdna.com/CRISPR
• Webinars
• CRISPR articles
• Peer-reviewed publications
• FAQs
• Much more!
Find More CRISPR Information
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gBlocks® Gene Fragments Custom Design Requests
Examples of the types of requests we’ve assisted with:
• 1900 bp sequence, no C’s on one strand, no G’s on the other
• Generate codon usage tables given unique sequencing data
• Minimize CpG dinucleotides, but at same time maximize G/C in the 3rd
codon position
What other custom requests do you have?
For any questions regarding IDT gene synthesis products, please email
genes@idtdna.com
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iGEM sponsorship
• www.IDTDNA.com/iGEM
CRISPR resources
• www.idtdna.com/CRISPR
Information for gBlocks® Gene Fragments
• www.idtdna.com/gBlocks
Help with design, experimental issues, and ordering
• Genes@idtdna.com
Other educational resources at www.IDTDNA.com:
• DECODED newsletter (www.idtdna.com/DECODED)
• Video library
• Frequently asked questions
• Much More!
Additional Resources
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4 Manufacturing Locations:
• Coralville, IA
• San Diego, CA
• Leuven, Belgium
• Singapore
Synthetic biology made simple
gBlocks® Gene Fragments
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