Duke iGEM 2014 Methodology Scaling up Synthetic Biology Improving - - PowerPoint PPT Presentation

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Duke iGEM 2014 Methodology Scaling up Synthetic Biology Improving - - PowerPoint PPT Presentation

Sep 03, 2023 571 likes •1.07k views

Duke iGEM 2014 Methodology Scaling up Synthetic Biology Improving Improving Improving CRISPR Access Education Enabling digital logic with CRISPR/dCas9 CRISPR/Cas9 Background Figure adapted from : Nucleic Acids Res. 2013

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Duke iGEM 2014

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Scaling up Synthetic Biology

Improving Access Improving CRISPR Improving Education

Methodology

Enabling digital logic with CRISPR/dCas9

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Figure adapted from : Nucleic Acids Res. 2013 Aug;41(15):7429-37. doi: 10.1093/nar/gkt520. Epub 2013 Jun 12. Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system.Bikard D1, Jiang W, Samai P, Hochschild A, Zhang F, Marraffini LA.

CRISPR/Cas9 Background

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CRISPR/Cas9 Background

Figure adapted from : Nucleic Acids Res. 2013 Aug;41(15):7429-37. doi: 10.1093/nar/gkt520. Epub 2013 Jun 12. Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system.Bikard D1, Jiang W, Samai P, Hochschild A, Zhang F, Marraffini LA.

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CRISPR/Cas9 Background

Nucleic Acids Res. 2013 Aug;41(15):7429-37. doi: 10.1093/nar/gkt520. Epub 2013 Jun 12. Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system.Bikard D1, Jiang W, Samai P, Hochschild A, Zhang F, Marraffini LA.

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Gene Circuits

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Binding of a single dCas9 within the promoter region can almost fully repress expression of a gene. Additional dCas9 bound at the promoter act as redundancies - all must dissociate for expression to occur.

Multiple binding sites: Principles

Nucleic Acids Res. 2013 Aug;41(15):7429-37. doi: 10.1093/nar/gkt520. Epub 2013 Jun 12. Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system.Bikard D1, Jiang W, Samai P, Hochschild A, Zhang F, Marraffini LA.

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Multiple binding sites: Modeling

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Multiple binding sites: Design

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Molecular Titration: Principles

Molecular titration features pairs of neutralizing molecules. We attempt to recreate the characteristic sigmoidal titration curve

  • n a log-log plot

Two approaches to molecular titration:

  • 1. Decoy binding sites to neutralize fully-assembled dCas9
  • 2. Anti-tracrRNA to neutralize tracrRNA

Mol Syst Biol. 2009;5:272. doi: 10.1038/msb.2009.30. Epub 2009 May 19.Protein sequestration generates a flexible ultrasensitive response in a genetic

  • network. Buchler NE1, Cross FR.
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Decoy binding site: Design

Bikard et al.

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Decoy binding sites: Modeling

*dCas9 becomes the limiting factor for 10 uM decoy

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Decoy binding sites: Assembly

We have developed a modified PCR method for easily generating large arrays of repeat sequences for insertion into bacteria. Using this scheme, we produced 1X, 6X, and 12X decoy site arrays and inserted them into a high-copy plasmid.

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Decoy binding sites: Results

Restriction digest of pdCas9, GFP1, and decoy plasmids

Decoy arrays remain stable even as we increase the number of repeats. Repeat decoy site arrays are inserted into a high-copy plasmid (~200 copies), producing >2000 repeats per cell!

0x 1x 6x 12x

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Decoy binding sites: Results

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Decoy binding sites: Next Steps

We have a scheme for easily generating large arrays of repeat decoy binding sites (18X, 24X). As we increase the number, we get a sharper and sharper response. We can make decoy sites a better titrant by having dCas9 bind more readily to decoys than real targets. We do so by introducing mismatches into the target binding sites. Finally, we can combine decoy sites with a second titrant...

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Anti-tracrRNA titration: Design

Bikard et al.

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Anti-tracrRNA titration: Modeling

Estimated in vivo Kd for RNA hybridization is between 1 nM and 100 pM - well within the range for ultrasensitive response

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Policy and Practices

How can the Duke iGEM team increase access to synthetic biology?

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Scaling up Synthetic Biology

Improving Access Improving CRISPR Improving Education

Methodology

3D Printing of Lab Equipment

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Synthetic Biology: Improving Access 3D Printing Lab Equipment with PLA plastic

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Why PLA?

  • Made from lactic acid

⇒ Biodegradable

  • Yale iGEM 2013

– Synthesize PLA from

  • E. coli

– Perform experiments on E. coli using materials produced by E. coli?!

  • Used by many 3D printers, including

the MakerBot

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Tube Rack

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96-well plate Vortex adaptor

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Roller Drum!

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Price Comparison

Full Product Purchased Online 3D Printed Equivalent Decrease in Price (%) Tube Rack $10-20 $1.62 83.8-91.9% decrease 96-well plate accessory $100 $1.67 98.3% decrease Roller drum $2162 $85.50 96% decrease

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Buying a 3D printer along with all the materials needed for our final product is cheaper than buying a roller drum!

Consequently…

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  • Provide access of these designs to

the high schools and universities

Future Directions: 3D Printing

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  • Provide access of these designs to

the high schools and universities

  • Develop new designs for lab

equipment

Future Directions: 3D Printing

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  • Provide access of these designs to

the high schools and universities

  • Develop new designs for lab

equipment

  • Ultimately, lower the cost of entry

into synthetic biology research

Future Directions: 3D Printing

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Interested?

Contact me at matthew.faw@duke.edu for access to .stl files

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Scaling up Synthetic Biology

Improving Access Improving CRISPR Improving Education

Methodology

Duke House Course NCSSM iGEM

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Synthetic Biology: Improving Education: University Level

Duke House Course

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House Course?

  • Student-led, ½ credit course
  • pen to all Duke students
  • Title: Creating Life: the

emergence of synthetic biology

  • Spring 2015
  • Taught by Duke iGEM with help

and lectures by Duke faculty

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Objectives:

1) Provide students with a foundation of understanding upon which they can critically evaluate developments in synthetic biology and society 2) Introduce students to the synthetic biology movement, as well as catalyze a dialogue about the potential applications of the young field and its implications for society

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Future Directions: House Course

  • To transform the House Course into a

course that is collaboratively designed and taught by iGEM teams in universities and high schools across the world

  • Creating MOOCs and a standardized

curriculum

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Synthetic Biology: Improving Education: High School Level

Assisting the NCSSM Team

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Objectives

1) Educate high school students on synthetic biology and the iGEM competition 2) Increase public awareness of synthetic biology as bioethical implications of the subject concern everyone

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Approach 1: Symposium

  • Summer Research Symposium
  • Place for NCSSM students to present

research, ideas, and projects to each other and staff of NCSSM

  • Duke iGEM presented their project

and an overview of synthetic biology to feel for interest

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Approach 2: NCSSM Seminar

  • Developed a curriculum to teach

synthetic biology and molecular genetics basics to high school students

  • introducing the idea of an NCSSM

iGEM team

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Approach 3: Startup of NCSSM iGEM

  • Creating a project
  • Teaching wet lab skills
  • Assisting with beginning stages of

project

  • Registering for HS division
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Approach 4: TEDx

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Future Ideas: NCSSM iGEM

  • Spread the curriculum to other iGEM

teams at the collegiate level to share with local high schools

  • Increase interest in high school iGEM
  • Start public seminars to teach about

synthetic biology

  • similar to the goal of TEDx
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Molecular Titration: Decoy Binding Sites Molecular Titration: anti-tracrRNA

Scaling up Synthetic Biology

Improving Access Improving CRISPR Improving Education

University Level: Duke House Course High School Level: NCSSM iGEM 3D Printing Equipment

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Acknowledgements

Anthony Ciesla1, Matthew Farnitano1, Matthew Faw1, Delta Ghoshal1, Garima Tomar1, Janan Zhu1, Mike Zhu1, Charles Cooper2, Dr. Nicholas Buchler346, Dr. Charles Gersbach356

1iGEM team member, 2iGEM team mentor, 3Faculty Advisor, 4Department of Biology, Duke

University, Durham, NC, 5Department of Biomedical Engineering, Duke University, Durham, NC,

6Duke Center for Genomic and Computational Biology, Duke University, Durham, NC

We thank the Lord-Alstadt Foundation for funding the Duke iGEM

  • team. We would also like to thank Dr. Mike Lynch, Chip Bobbert,

and faculty at the NC School of Science and Mathematics for their assistance in this project.

The Pratt School of Engineering, Duke Department of Biology, and the Duke Center for Genomics and Computational Biology (GCB) support us, Dr. Buchler, and Dr. Gersbach.

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WE WELCOME QUESTIONS!

Thank you very much for your time.