iGEM Success 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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“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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• Codon optimized components


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• Complete circuits


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• 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.


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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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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


• http://www.addgene.org/static/cms/files

/hCRISPR_gRNA_Synthesis.pdf

Current Protocols in Molecular Biology (2014) 31.1.1–31.1.17.

http://www.idtdna.com/CRISPR

http://www.addgene.org/static/cms/files/hCRISPR_gRNA_Synthesis.pdf

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.


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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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[email protected]

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



12.5 µM Probe B 250 nM



12.5 µM Probe C 250 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

[email protected]

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iGEM sponsorship

• www.IDTDNA.com/iGEM

CRISPR resources


Information for gBlocks® Gene Fragments

• www.idtdna.com/gBlocks

Help with design, experimental issues, and ordering

• [email protected]

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

http://www.idtdna.com/

Synthetic biology made simple

gBlocks® Gene Fragments

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iGEM Success Using High Quality Gene Fragments

Science

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