� � Conditionally Dimerizable Split Protein Systems for Genetic Logic and Genome Editing Applications � Presented by: BostonU iGEM 2015 � Background Motivation � Design � Achievements � Results �
� Engineering Synthetic Control � Synthetic biologists want to engineer precise control of biological systems � Protein � DNA � DNA � Original Phenotype � Desired Phenotype � 2 � Background Motivation � Design � Achievements � Results �
Control of Protein Activity � 1. Faster response time � 2. Potentially lower basal activity � 3. Able to integrate post-translational modifications with previously characterized pre-transcriptional methods � Pre-Transcriptional Modification Post-Translational Modification 3 � Background Motivation � Design � Achievements � Results �
Conditional Dimerization of Protein Systems 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 � Inducer � C-Terminal Domain + Dimerizable Domain � 4 � Background Motivation � Design � Achievements � Results �
Application #1: � Conditionally Dimerizable Integrases and RDFs for Use in Controlled Gene Expression Background Motivation Design � Design Results Achievements Background �
Overview of Genetic Recombination � Recombination Gene of Interest Recombination Site 1 (GOI) Site 2 Margaret Smith et. al JMB 2014 6 � Motivation Design � Achievements � Background � Results �
Catalyzing Reversible Inversion Reactions � Site-Specific Recombinases Perform Directional Reactions: � Inversion � Deletion � Insertion � GOI GOI GOI GOI GOI GOI Margaret Smith et. al JMB 2014 7 � Motivation Design � Achievements � Background � Results �
Catalyzing Reversible Inversion Reactions � “Off” State � GOI Integrase � Integrase � Recombination Directionality Factor (RDF) � GOI “On” State � 8 � Motivation Design � Achievements � Background � Results �
Design Overview of a Conditionally Dimerizable System � Our design answers these three questions: � 1. Which integrases and RDFs do we split? � 2. Where should we split the proteins? � 3. What should we use to dimerize the proteins? � Integrase, RDF � Split Sites � Dimerizable Domains � Orientation � 9 � Motivation � Design Achievements � Background � Results �
Which Integrases and RDF proteins do we split? � Integrases RDFs GOI Integrase � Integrase + RDF � + � TP901-1 � orf7 � + � PhiC31 � gp3 � GOI Integrase, RDF � Split Sites � Dimerizable Domains � Orientation � 10 � Motivation � Design Achievements � Background � Results �
Splitting Method: How Do We Choose Where to Split The Proteins? � Primary Structure � Tertiary Structure � Quaternary Structure � Secondary Structure � Integrase, RDF � Split Sites � Dimerizable Domains � Orientation � 11 � Motivation � Design Achievements � Background � Results �
Splitting Method: How Do We Choose Where to Split The Proteins? � 1. Avoid interior regions � 2. Avoid secondary structures � 3. Avoid catalytic domain � Hydrophobic � Hydrophilic � Alpha Helices � Beta Sheets � Courtesy of Billy Law and Wilson Wong � Catalytic Residues � Catalytic Domain DNA Binding Domain Billy Law Integrase, RDF � Split Sites � Dimerizable Domains � Orientation � 12 � Motivation � Design Achievements � Background � Results �
Identification of Conditionally Dimerizable Domains � CRY2 � FKBP � PYL � Abscisic Acid FRB � ABI � CIBN � Stuart Schreiber et al. Nature 1996 Gerald R. Crabtree et al. Science Signaling 2011 Chandra L Tucker et al. Nature 2010 Integrase, RDF � Split Sites � Dimerizable Domains � Orientation � 13 � Motivation � Design Achievements � Background � Results �
Identification of Conditionally Dimerizable Domains � CRY2 � FKBP � PYL � Abscisic Acid FRB � ABI � CIBN � N-terminal � C-terminal � Stuart Schreiber et al. Nature 1996 N-terminal � C-terminal � Gerald R. Crabtree et al. Science Signaling 2011 Chandra L Tucker et al. Nature 2010 Integrase, RDF � Split Sites � Dimerizable Domains � Orientation � 14 � Motivation � Design Achievements � Background � Results �
Experimental Pipeline � 1. Cloning � 15 � Motivation � Design Achievements � Background � Results �
Experimental Pipeline � Protein DNA � Mammalian � Expression � Backbones � 1. Cloning � 16 � Motivation � Design Achievements � Background � Results �
Experimental Pipeline � 1. Cloning � 17 � Motivation � Design Achievements � Background � Results �
Experimental Pipeline � 1. Cloning � E. Coli � 18 � Motivation � Design Achievements � Background � Results �
� Experimental Pipeline � 1. Cloning � 1. Cloning � 2. Purification � 19 � Motivation � Design Achievements � Background � Results �
� � � Experimental Pipeline � 1. Cloning � 2. Purification � 3. Transfection � 20 � Motivation � Design Achievements � Background � Results �
� � � � � � � � Experimental Pipeline � 1. Cloning � 2. Purification � 3. Transfection � 3. Transfection � 4. Flow Cytometry � 21 � Motivation � Design Achievements � Background � Results �
Experimental Pipeline � mRuby Integrase N-Terminal Domain Dimerizable Domain � Dimerizable Domain Integrase C-Terminal Domain � mRuby 22 � Motivation � Design Achievements � Background � Results �
Experimental Pipeline � Dimerized Integrase � 23 � Motivation � Design Achievements � Background � Results �
Experimental Pipeline � mRuby INDUCER � RDF N-Terminal Domain Dimerizable Domain � Dimerizable Domain RDF C-Terminal Domain � mRuby 24 � Motivation � Design Achievements � Background � Results �
Experimental Pipeline � mRuby mRuby 25 � Motivation � Design Achievements � Background � Results �
Characterization of Dimerizable Integrase and RDF Constructs � 137 78 Integrase Constructs Constructs Tested 18 6 RDF Constructs Constructs Tested 26 � Motivation � Design Achievements � Background � Results �
Normalizing activity � split protein mRuby fluoresence (a.u.) % mRuby expression = 100 x full protein mRuby fluoresence (a.u) 27 � Motivation � Design � Results Achievements � Background �
Functional split TP901-1 activity! � 28 � Motivation � Design � Results Achievements � Background �
Functional split PhiC31 activity! � 29 � Motivation � Design � Results Achievements � Background �
Does split site location affect activity? � 30 � Motivation � Design � Results Achievements � Background �
Does orientation of domain affect activity? � 31 � Motivation � Design � Results Achievements � Background �
Integrase + RDF Part Characterization � ABI Biobrick Prefix Biobrick Suffix PYL Biobrick Prefix Biobrick Suffix Orf7 Biobrick Prefix Biobrick Suffix 32 � Motivation � Design � Results � Achievements Background �
Application #2: Conditionally Dimerizable SaCas9 for inducible in-vivo genome editing � Background Motivation Design � Design Results Achievements Background �
Overview of Cas9 � sgRNA � Target Sequence � PAM Sequence � Insertion Deletion Mutation Sander, Jeffry D., Joung, J. Keith, “CRISPR-Cas systems for editing, regulating, and targeting genomes”, Nature Biotechnology, 2013. 34 � Motivation Design � Achievements � Background � Results �
SpCas9 vs. SaCas9 � We would like to control the activity of staphylococcus aureus Cas9 (SaCas9 SaCas9) � Adeno Associated Virus can hold ~4.7kb � SaCas9: ~3.3kb | NLS: 42 bp | FKBP: 327bp | FRB: 276bp Size ~4.3kb = ~3.9kb � sgRNA SpCas9 Size ~3.3kb SaCas9 sgRNA 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 35 � Motivation Design � Achievements � Background � Results �
Experimental Pipeline � PAM Sequence � Target Sequence � Scharenberg, Andrew M. et al., “Tracking genome engineering outcome at individual DNA breakpoints”, Nature Methods, 2011 36 � Motivation � Design Achievements � Background � Results �
Experimental Pipeline � 37 � Motivation � Design Achievements � Background � Results �
Experimental Pipeline � EGFP mCherry 2-bp frameshift � renders this Gibberish � EGFP Non-homologous End Joining � Homology Directed Repair � Scharenberg, Andrew M. et al., “Tracking genome engineering outcome at individual DNA breakpoints”, Nature Methods, 2011 38 � Motivation � Design Achievements � Background � Results �
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