The Lipid Sensing Eukaryotic Toolkit Debrecen Team 2010 University - - PowerPoint PPT Presentation

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The Lipid Sensing Eukaryotic Toolkit Debrecen Team 2010 University - - PowerPoint PPT Presentation

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The Lipid Sensing Eukaryotic Toolkit Debrecen Team 2010 University of Debrecen, Medical and Health Science Centre, Department of Biochemistry and Molecular Biology City of Debrecen (Hungary) The University Of Debrecen Objective I

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Debrecen Team 2010

University of Debrecen, Medical and Health Science Centre, Department of Biochemistry and Molecular Biology

The Lipid Sensing Eukaryotic Toolkit

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City of Debrecen (Hungary)

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The University Of Debrecen

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Objective I

  • Addition of lipid sensing tools to the Parts Registry,

which may be used for the rational design of lipid responsive transcription factors

  • Expanding the possibilities of the eukaryote chassis in

synthetic biology Debrecen Team 2010

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Objective II

  • Creating a toolkit containing
  • Lipid sensors which may be activated by extracellular

lipids and comes equipped with a PoPs (Polymerase Per Second) output

  • DNA binding domains, chimeric receptors, expression

vectors and more

  • Side projects
  • Creating a library of video tutorials for elaboration
  • f basic lab techniques
  • Collaboration with team Edinburgh 2006 (arsenic

biosensor) assessment of arsenic contamination in drinking water

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Function and structural

  • rganization of nuclear receptors
  • a. Function
  • Metabolism
  • Development
  • Homeostasis
  • b. Structure

Nat Struct Mol Biol. 2008 Sep;15(9):924-31. Itoh et al.

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Nuclear receptors in C. elegans

Robinson et al. J Mol Evol (2005) 60:577–586

  • Drosophila 21
  • Human 48
  • C. Elegans 284
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Making single part

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Results I - Cloning

Basic Parts

DNA Binding Domains

  • Estrogen Receptor
  • Gal4
  • Retinoic X Receptor

Ligand Binding Domains

  • Constitutive Androstane

Receptor

  • Ecdysone Receptor
  • Estrogen Receptor
  • Pregnane X receptor
  • DAF-12
  • NHR-8, NHR-23, NHR-25
  • NHR-31, NHR-64, NHR-80

Others

  • TRE-CMV
  • PolyA tail
  • VDR hinge region
  • BAX
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Results II - Cloning

Chimeric Nuclear Receptors

Gal4-EcR LBD Gal4-C.elegans LBDs Others

  • Gal4 - ER LBD
  • ER DBD – VDR

Hinge – ER LBD Gal4-PXR LBD

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Results III - Cloning

Eukaryotic expression vectors

TRE-Gal4-PXR TRE-Gal4-EcR TRE-Gal4-EcR-PolyA TRE-Gal4-PXR

  • PolyA
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Results IV – Western Blot

anti-Gal4 anti-Actin Confirmation of protein expression proved by WB in transfected 293T cells

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Mammalian Two-Hybrid System

  • b. Ligand testing

Detected luciferase activity correlates with ligand binding or strength of interaction

  • a. Interaction experiment

LUCIFERASE ENZYME

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Lipid Extraction

  • Diethyl-ether
  • DMSO-ETOH
  • MQ water

Over night incubation at 65c After centrifugation and Filtration

Oil sands for testing C. elegans nuclear receptor

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Testing receptors in action

  • 1. Activity
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Dose-dependent activity (pcDNA-Gal-EcR-LBD)

no ligand 1 uM 5 uM 25 uM 5 10 15

Ponasterone A

Testing receptors in action

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Testing receptors in action

  • 2. Dimer formation
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Testing receptors in action

  • 2. Dimer formation
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Dimer formation of receptors

Dimer formation of PXR (with RXR) Positive control

Gal: in CMX-vector

VP-RXR Gal-PXR Gal-PXR/VP-RXR

500 1000 1500 2000 2500 3000 3500

VP-RXR Gal-PXR Gal-PXR/VP-RXR

100 200 300 400 500

Gal: in pcDNA-vector

Gal: in pSB1A3-vector

VP-RXR Gal-PXR Gal-PXR/VP-RXR

10 20

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Arsenic side project

Importance: 10 µg per liter is the WHO threshold for arsenic in drinking water Background: Team Edinburgh, 2006 made an arsenic biosensor, which can be a useful tool for on site arsenic detection Our aim: We tested the system on real samples from South-East Hungary, and the results are the followings

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Video Project: “Film in Lab”

Debrecen Team 2010

  • Recording optimized protocols
  • Uploading the video tutorials to an online source

http://www.youtube.com/user/debrecenigem2010

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In a Nutshell

  • a. Eleven different lipid sensors were designed cloned,

tested and added to the parts registry

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In a Nutshell

  • a. Eleven different lipid sensors were designed cloned,

tested and added to the parts registry

  • b. Our Team has worked to expand synthetic biological

possibilities in the eukaryotic chasis by adding a standard (BBF RFC 64) and an expression vector as a composite part

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Importance

  • a. Addition of Lipid sensing parts to the parts registry
  • b. Our parts allow the design of complex biological

Systems which require defined PoPs input or PoPs titration

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  • a. Remote control of gene therapy or smart cells
  • b. Synthetic organisms with an environmental “sixth sense”

for pollutants

Possible application

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Future directions

  • a. Addition of DNA binding domains to the kit
  • b. Creation of devices and modules which use our parts

as input and deliver an apoptosis output synthetic

  • rganisms with an environmental “sixth sense” for pollutants
  • c. Develop devices that can be used for screening

enviromental lipid contaminanted samples