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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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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!
280 registered teamsThis is about 5.6 Mbp of DNA!
2015 IDT iGEM Sponsorship
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Courtesy of iGEM Team Freiburg
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
Isothermal Assembly of Multiple gBlocks® Gene Fragments
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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 3ʹ′ strand.
Step 2: A mesophilic exonuclease cleaves bases from the 5ʹ′ end of the double-stranded DNA fragments, before being inactivated by the 50°C reaction temperature.
Step 3: The newly generated, complementary, single-stranded 3ʹ′ 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 bpU6 promoter driving transcription of a 99 bp sgRNA
AAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGNNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTT
• 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/fil
es/hCRISPR_gRNA_Synthesis.pdf
Current Protocols in Molecular Biology (2014) 31.1.1–31.1.17.
CRISPR gBlocks® gRNAs in the HPRT gene (HEK 293 cells)
38094S
38095S
38115S
38129S
38231S
38239S
38256S
38338S
38371S
38448S
38478S
38509S
38510S
38574S
38626S
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18% 27% 39% 19% 0% 22% 20% 47%0% 0%26% 5% 26% 19%0%
2% Agarose gel
Fragment Analyzer™
>400 sites tested in HPRT and EMX; more genes in progressNote: sequence analysis shows 30% cleavage in T7E1 assay = 60–75% real change
Predicted CRISPER Activity vs. Actual Activity
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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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 have assisted with:• 1900 bp sequence, no C bases on one strand, no G bases 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 [email protected]
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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• [email protected]
Other educational resources at www.IDTDNA.comDECODED 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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Questions?
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