Conditionally Dimerizable Split Protein Systems for Genetic - - PowerPoint PPT Presentation

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Conditionally Dimerizable Split Protein Systems for Genetic - - PowerPoint PPT Presentation

Feb 22, 2023 218 likes •700 views

Conditionally Dimerizable Split Protein Systems for Genetic Logic and Genome Editing Applications Presented by: BostonU iGEM 2015 Background Motivation Design Achievements Results Engineering Synthetic

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Motivation Design Results Achievements Background

Presented by: BostonU iGEM 2015

Conditionally Dimerizable Split Protein Systems for Genetic Logic and Genome Editing Applications

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Motivation Design Results Achievements Background

Engineering Synthetic Control

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Synthetic biologists want to engineer precise control of biological systems

  • Original Phenotype

Desired Phenotype Protein DNA DNA

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Motivation Design Results Achievements Background

Control of Protein Activity

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Pre-Transcriptional Modification Post-Translational Modification

1. Faster response time 2. Potentially lower basal activity 3. Able to integrate post-translational modifications with previously characterized pre-transcriptional methods

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Motivation Design Results Achievements Background

Conditional Dimerization of Protein Systems

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Naturally proteins generally contain multiple domains that together coordinate protein function. By separating functional domains, we can regulate protein activity.

N-Terminal Domain + Dimerizable Domain C-Terminal Domain + Dimerizable Domain Inducer

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Achievements Results Design Background Design Motivation Background

Application #1: Conditionally Dimerizable Integrases and RDFs for Use in Controlled Gene Expression

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Motivation Design Results Achievements Background

Overview of Genetic Recombination

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Gene of Interest (GOI) Recombination Site 2 Recombination Site 1

Margaret Smith et. al JMB 2014

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Motivation Design Results Achievements Background

Catalyzing Reversible Inversion Reactions

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Site-Specific Recombinases Perform Directional Reactions: Inversion Deletion Insertion

GOI GOI GOI GOI GOI GOI

Margaret Smith et. al JMB 2014

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Motivation Design Results Achievements Background

Catalyzing Reversible Inversion Reactions

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“Off” State “On” State

Integrase Integrase Recombination Directionality Factor (RDF)

GOI GOI

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Motivation Design Results Achievements Background

Design Overview of a Conditionally Dimerizable System

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Our design answers these three questions:

Integrase, RDF Split Sites Dimerizable Domains Orientation

  • 2. Where should we split the proteins?
  • 3. What should we use to dimerize the proteins?
  • 1. Which integrases and RDFs do we split?
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Motivation Design Results Achievements Background

Which Integrases and RDF proteins do we split?

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GOI GOI

Integrase Integrase + RDF

Integrase, RDF Split Sites Dimerizable Domains Orientation

Integrases RDFs

TP901-1

+

  • rf7

PhiC31 gp3

+

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Motivation Design Results Achievements Background

Splitting Method: How Do We Choose Where to Split The Proteins?

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Primary Structure Secondary Structure Tertiary Structure Quaternary Structure

Integrase, RDF Split Sites Dimerizable Domains Orientation

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Motivation Design Results Achievements Background

Splitting Method: How Do We Choose Where to Split The Proteins?

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Catalytic Domain DNA Binding Domain

  • 1. Avoid interior regions
  • 2. Avoid secondary structures
  • 3. Avoid catalytic domain

Integrase, RDF Split Sites Dimerizable Domains Orientation

Alpha Helices Beta Sheets Catalytic Residues

Hydrophobic Hydrophilic

Courtesy of Billy Law and Wilson Wong

Billy Law

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Motivation Design Results Achievements Background

Identification of Conditionally Dimerizable Domains

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ABI PYL FKBP FRB CIBN CRY2

Integrase, RDF Split Sites Dimerizable Domains Orientation

Stuart Schreiber et al. Nature 1996 Gerald R. Crabtree et al. Science Signaling 2011 Chandra L Tucker et al. Nature 2010

Abscisic Acid

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Motivation Design Results Achievements Background N-terminal C-terminal C-terminal N-terminal

Integrase, RDF Split Sites Dimerizable Domains Orientation

ABI PYL FKBP FRB CIBN

Identification of Conditionally Dimerizable Domains

CRY2

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Stuart Schreiber et al. Nature 1996 Gerald R. Crabtree et al. Science Signaling 2011 Chandra L Tucker et al. Nature 2010

Abscisic Acid

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Motivation Design Results Achievements Background

Experimental Pipeline

  • 1. Cloning

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Motivation Design Results Achievements Background

  • 1. Cloning

Protein DNA Mammalian Expression Backbones

Experimental Pipeline

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Motivation Design Results Achievements Background

  • 1. Cloning

Experimental Pipeline

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Motivation Design Results Achievements Background

  • 1. Cloning
  • E. Coli

Experimental Pipeline

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Motivation Design Results Achievements Background

  • 1. Cloning
  • 2. Purification
  • 1. Cloning

Experimental Pipeline

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Motivation Design Results Achievements Background

  • 3. Transfection
  • 2. Purification
  • 1. Cloning

Experimental Pipeline

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Motivation Design Results Achievements Background

  • 4. Flow Cytometry
  • 1. Cloning
  • 3. Transfection
  • 2. Purification
  • 3. Transfection

Experimental Pipeline

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Motivation Design Results Achievements Background

Experimental Pipeline

mRuby

Integrase N-Terminal Domain Dimerizable Domain Dimerizable Domain Integrase C-Terminal Domain

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mRuby

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Motivation Design Results Achievements Background

Experimental Pipeline

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Dimerized Integrase

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Motivation Design Results Achievements Background

Experimental Pipeline

mRuby

RDF N-Terminal Domain Dimerizable Domain Dimerizable Domain RDF C-Terminal Domain

INDUCER

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mRuby

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Motivation Design Results Achievements Background

Experimental Pipeline

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mRuby mRuby

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Motivation Design Results Achievements Background

Characterization of Dimerizable Integrase and RDF Constructs

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137 78 18 6

Integrase Constructs Constructs Tested RDF Constructs Constructs Tested

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Motivation Design Results Achievements Background

Normalizing activity

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split protein mRuby fluoresence (a.u.) full protein mRuby fluoresence (a.u) 100 x % mRuby expression =

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Motivation Design Results Achievements Background

Functional split TP901-1 activity!

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Motivation Design Results Achievements Background

Functional split PhiC31 activity!

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Motivation Design Results Achievements Background

Does split site location affect activity?

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Motivation Design Results Achievements Background

Does orientation of domain affect activity?

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Motivation Design Results Achievements Background

Integrase + RDF Part Characterization

Biobrick Prefix Biobrick Suffix Orf7 Biobrick Prefix Biobrick Suffix PYL Biobrick Prefix Biobrick Suffix ABI

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Achievements Results Design Background Design Motivation Background

Application #2: Conditionally Dimerizable SaCas9 for inducible in-vivo genome editing

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Motivation Design Results Achievements Background

Overview of Cas9

sgRNA PAM Sequence Target Sequence Mutation Deletion Insertion

Sander, Jeffry D., Joung, J. Keith, “CRISPR-Cas systems for editing, regulating, and targeting genomes”, Nature Biotechnology, 2013.

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Motivation Design Results Achievements Background

SpCas9 vs. SaCas9

We would like to control the activity of staphylococcus aureus Cas9 (SaCas9 SaCas9)

SpCas9 sgRNA SaCas9 sgRNA

Size ~4.3kb Size ~3.3kb

Adeno Associated Virus can hold ~4.7kb

SaCas9: ~3.3kb | NLS: 42 bp | FKBP: 327bp | FRB: 276bp = ~3.9kb

Daya, Shyam, Berns, Kenneth I., “Gene Therapy using Adeno-Associated Virus Vectors”, Clinical Microbiology Reviews, 2008. Feng Zhang et al. “In vivo genome editing using Staphylococcus aureus Cas9”, Nature, 2015

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Motivation Design Results Achievements Background PAM Sequence

Experimental Pipeline

Target Sequence

Scharenberg, Andrew M. et al., “Tracking genome engineering outcome at individual DNA breakpoints”, Nature Methods, 2011

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Motivation Design Results Achievements Background

Experimental Pipeline

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Motivation Design Results Achievements Background Non-homologous End Joining EGFP EGFP mCherry Homology Directed Repair

Scharenberg, Andrew M. et al., “Tracking genome engineering outcome at individual DNA breakpoints”, Nature Methods, 2011

renders this Gibberish 2-bp frameshift

Experimental Pipeline

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Motivation Design Results Achievements Background

Preliminary Experimental Validation of Our Model

Zhang, Feng et. al. Crystal Structure of Staphylococcus Aureus Cas9 in Complex with SgRNA and Target DNA (TTGAAT PAM). (2015).

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Motivation Design Results Achievements Background

iGEM Community:
 Beyond The Lab

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Motivation Design Results Achievements Background

Investigating iGEM Mammalian Research

  • 3. Limited awareness and incentive
  • 2. Difficulty submitting and using

BioBrick parts because of large size and internal complexity

  • 1. High cost of equipment,

reagents and upkeep of cells 41

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Motivation Design Results Achievements Background

Promoting iGEM Mammalian Research

P r

  • t
  • c
  • l

s P r

  • t
  • c
  • l

s

  • Increase

incentive and awareness Simplify submission rules Alleviate costs

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Motivation Design Results Achievements Background

Improving Public Awareness and Perception

“Building with Biology” at Museum of Science Wellesley collaboration

An effective way to educate children and adults alike: exploring learning and engaging in discussion about synthetic biology in a in a museum setting museum setting

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Motivation Design Results Achievements Background

Educational Outreach and Collaboration

Upward Bound Nanotechnology Lab NEGEM 44

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Motivation Design Results Achievements Background

Industry Relations

Monsanto Visit 45

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Motivation Design Results Achievements Background

Acknowledgements

Thank you to everyone who has helped us and provided us with support: Advisors: Mo Khalil and Wilson Wong Mentors: Divya Israni and Ben Weinberg

Others: : Traci Haddock – team organizer, recruiter Doug Densmore – – feedback on presentation Evan Appleton – ran interlab FACS Jangwhan Cho – lab assistance Deboki Charvarti – – feedback on presentation Nikit Patel – – feedback on presentation Chris Mancuso – – feedback on presentation Thomas Lozanoski – – lab assistance Hang Pham – lab assistance Teresa Wiese – – lab assistance Szilvia Kiriakov – – lab assistance, presentation Billy Law – creator of Matlab model Leidy Diana Carabllo – lab assistance Minhee Park – – feedback on presentation

  • Monica Brova – lab assistance

Maciej Walkosz – lab assistance, presentation Callen Bragden – lab assistance Swati Carr – lab assistance Yash Agarwal – help in lab Alan Pacheco – feedback on presentation Brandon Wong – – help in lab Prashanth Vishwanath – – organized Monsanto Visit Wellesley_The Tech iGEM Team Worcester Polytechnic Institute iGEM Team Northeastern University iGEM Team Massachusetts Institute of Technology iGEM Team Harvard University iGEM Team Tufts University iGEM Team UMass Dartmouth iGEM Team

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Motivation Design Results Achievements Background

  • Thank you for listening!