iGEM Progress Using High Quality Gene Fragments

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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Questions?genes@idtdna.com

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 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.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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