Modular Synthetic Receptor System Interfaced with Nano Breadboard - - PowerPoint PPT Presentation

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Modular Synthetic Receptor System Interfaced with Nano Breadboard - - PowerPoint PPT Presentation

Nov 11, 2022 385 likes •644 views

Modular Synthetic Receptor System Interfaced with Nano Breadboard Synthetic receptor scheme Synthetic receptor model Active state Inactive state, protein split Principle of a construction kit FluA-Anticalin scFv-Anti-NIP Transmembraneregion

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Modular Synthetic Receptor System Interfaced with Nano Breadboard

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Synthetic receptor scheme

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Synthetic receptor model

Inactive state, protein split Active state

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Split β-Lactamase Split Cerulean CFP

Principle of a construction kit

Split Venus YFP FluA-Anticalin

Transmembraneregion B cell receptor

scFv-Anti-NIP

Transmembraneregion EGF-Receptor

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Antibody-anti-NIP Split-Cerulean-cCFP Split-β-Lactamase 1 Split-Cerulean-nCFP Split-Venus-nYFP Split-β-Lactamase 2

Cloning of our construction kit

Split-Luciferase 1 Split-Luciferase 2 Transmembrane- region BCR Lipocalin-FluA- Anticalin GGGS- Linker Transmembrane- region EGFR Signal- peptide Split- Linker Split-Venus-cYFP

Submitted: 13 basic and 28 composite parts in E. coli vectors. Further 16 parts were cloned in an eukaryotic transfection vector.

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EcoRI NotI XbaI NgoMIV AgeI SpeI NotI PstI | | | | | | | | GAATTCgcggccgctTCTAGAtgGCCGGCnnnnnnACCGGTtaatACTAGTagcggccgCTGCAG 1 ---------+---------+---------+---------+---------+---------+----- 65 CTTAAGcgccggcgaAGATCTacCGGCCGnnnnnnTGGCCAattaTGATCAtcgccggcGACGTC c I R G R F * M A G ? ? T G * Y * * R P L Q

  • NgoMIV

| CCGGC..SEQUENCE..

  • GGCCG..SEQUENCE..

G AgeI | . ..SEQUENCE..ACCGG

  • ..SEQUENCE..TGGCC

T ..SEQUENCE..ACCGGC..SEQUENCE..

  • ..SEQUENCE..TGGCCG..SEQUENCE..

T G

Using BioBrick 3.0

short flexible linker, no stop codons !!!

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Expression in mammalian cells

CMV- promotor

XbaI PstI

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Basic receptor dimerization: Free receptor and ligand Receptor dimerization Receptor activation Internalization Receptor ligand binding

Modeling: Receptor dimerization

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Receptor activity: Model characteristics:

5 ODEs, 12 parameters

Modeling: Receptor activity

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Dimerization of two distinct synthetic receptors: R1 and R1:

  • Dimerization, but no split protein activity

R2 and R2:

  • Dimerization, but no split protein activity

R1 and R2: Dimerization split protein activation

Modeling: Two distinct receptors

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Protein activity dependent on ligand amount: ‐ Higher activity for higher ligand concentrations until certain ligand level ‐ Decrease in activity for high amounts of ligands

Modeling: Ligand dependency

R1 R2 R1LL R2LL R1LLR1 R2LLR2 R1LLR2 A

LL LL R1 R2 R1 R2

Model characteristics: 9 ODEs 25 parameters

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Results: Programmable Input

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DNA Origami

DNA-Origami

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DNA Origami

T↓

  • P. Rothemund, 2006

Long (7526 nt) ssDNA Folded origami structures 216 staple

  • ligonucleotides

6 nm grid

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This origami yields a square width: 103.7 nm , 27 turns, (288 nt) height: approx. 60 nm, 24 helices

M13 ssDNA, length 7526 nt DNA Origami: Forcing a ssDNA in Shape by Staple Oligonucleotides

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DNA Origami

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5‘ linked nitro iodo phenol 3‘ linked fluorescein mid point linked Alexa 488

DNA Origami

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Results: Cellular Readout

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Membrane Integration in 293T Cells

YFP: cytosolic FluA-Anticalin – EGFR transmembranregion – β-Lactamase1 – YFP

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Transfected 293T cells without stimulation

Activation of Split-CFP

Transfected 293T cells with fluorescein-oligo stimulation

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β-Lactamase Activity Test

(CCF4-AM) Substrate Product Emission at 450 nm

+

ß-Lactamase

Substrate Product

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Negative Control:

Activation of Split Lactamase by Origami

Intensity 1.0 0.8 0.6 0.4 0.2 Intensity 1.0 0.8 0.6 0.4 0.2 420 440 460 480 500 520 540 Emission wavelength [nm] 420 440 460 480 500 520 540 Emission wavelength [nm]

Sample:

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Summary & Outlook

  • Devised modified DNA origami as input device
  • Verified DNA origami formation by AFM
  • Optimized buffers for origami stability and cell viability
  • Designed and cloned a modular synthetic receptor system
  • Demonstrated synthetic receptor membrane localization
  • Demonstrated “anticalin-split CFP receptor” activation
  • Demonstrated “anticalin-split lactamase receptor” activation
  • We showed that spatially arranged green fluorescent dyes trigger

a blue fluorescent output in a human cell line.

  • Technology provides the foundation for universal extracellular cell

programming.

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Acknowledgement

Instructors

  • Dr. Kristian Müller (Biology)
  • Dr. Katja Arndt (Biology, FRIAS, Bioss)

PD Wolfgang Schamel (MPI for Immunobiology) Support & Instrumentation Janina Speck Kilian Bartholomé

  • Dr. Christian Fleck (Physik)

PD Svetlana Santer(IMTEK)

  • Dr. Roland Nitschke (ZBSA)
  • Prof. Ralf Baumeister (Biology, ZBSA, FRIAS)
  • Prof. Michael Reth (Biology)
  • Prof. Ralf Reski (Biology)
  • Prof. Jan Korvink (IMTEK, FRIAS)

Collaboration ESBS Strasbourg iGEM team