The team The team Inspiration Valencia 2009 Inspiration Valencia - - PowerPoint PPT Presentation

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The team The team Inspiration Valencia 2009 Inspiration Valencia - - PowerPoint PPT Presentation

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The team The team Inspiration Valencia 2009 Inspiration Valencia 2009 Our idea Our idea Goals Goals Multiple colors Multiple colors Blue Blue GECO Aequorin Milliseconds Minutes i illi d 96 well

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The team The team

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Inspiration – Valencia 2009 Inspiration Valencia 2009

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Our idea Our idea

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Goals Goals

  • Blue

 Multiple colors Blue  Multiple colors

  • Aequorin

 GECO i  illi d

  • Minutes

 Milliseconds

  • 96‐well plate  Improved control device
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Lab work Lab work

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GECO GECO

  • Genetically encoded Ca2+ indicators

Genetically encoded Ca indicators

  • Detection of the intracellular Ca2+

concentration concentration

  • R‐GECO
  • G‐GECO
  • B‐GECO

(Campbell et al., Science 2011) ( p , )

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CCH1‐MID1 channel CCH1 MID1 channel

  • Over expression

Over expression

  • Homologous to mammalian voltage‐gated

calcium channels calcium channels

  • Depolarization plasma membrane  calcium

i l ions enter cytoplasm

(Iida et al., 2007) (Iida et al., 2007)

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Ideal yeast cell Ideal yeast cell

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Yeast Yeast

  • Stable

Stable

  • Simple eukaryote

i l i h l

  • Native calcium channels
  • Ideal homeostatic calcium concentration
  • Environmental safety
  • Shuttle vector
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Approach Approach

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GECO characterization GECO characterization

70

R‐GECO

50 60

0 nM Ca 616 nM Ca 808 nM Ca

30 40

ensity (a.u.) 808 nM Ca 1000 nM Ca 1200 nM Ca 1500 nM Ca

20 30

Inte 1700 nM Ca 2000 nM Ca 2400 nM Ca 2900 M C

10 575 580 585 590 595 600 605 610 615 620 625 630 635 640 645 650

Wa elength (nm) 2900 nM Ca 3600 nM Ca 3900 nM Ca Wavelength (nm)

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Yeast characterization Yeast characterization

Emission spectra of INVSc1 + GECO, CCH1, MID1 @ 565 nm excitation

3000 3500 2000 2500

tensity (a.u.) R‐GECO G‐GECO B‐GECO

1000 1500

uorescence in R Ctrl. G Ctrl. B Ctrl.

500 576 582 588 594 600 606 612 618 624 630 636 642 648

Flu W l h ( ) Wavelength (nm)

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Initial model Initial model

  • Neuronal cell of bullfrog

Neuronal cell of bullfrog

  • Parameter sensitivity analysis
  • Conclusion:

M d l t f ll li bl d t k Model not fully applicable due to unknown yeast parameters

(Koch and Segev, 1989)

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Combined models Combined models

  • Calcium homeostasis in yeast

Calcium homeostasis in yeast

  • Voltage‐dependent calcium channels

i i f h G CO i

  • Kinetics of the GECO‐proteins

(Cui et al 2009) (Cui et al., 2009) (Zhao et al., 2011)

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Combined models Combined models

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Calcium homeostasis Calcium homeostasis

Conformational switch model

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Calcium channels Calcium channels

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GECO kinetics GECO kinetics

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Modelling results Modelling results

80% decrease

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Device Device

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Device Device

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Specifications Specifications

  • 16 pixels

16 pixels

  • Moving image using one signal

R f h ti 1 i l

  • Refresh time: 1 ms per pixel
  • Maximum of 20 V
  • Software can easily be extended
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In use In use

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Accomplishments Accomplishments

  • Succeeded in an iGEM yeast‐project

Succeeded in an iGEM yeast project

  • CCH1/MID1/X‐GECO in yeast

i i k f d d bl G CO

  • BioBricks of red, green and blue GECO
  • Working computational model
  • Basis for future iGEM teams
  • Easy to extend
  • Working device and elaborate software
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Human Interest Human Interest

  • Construct is suitable for calcium research

Construct is suitable for calcium research

  • Insights in calcium fluxes and pathways

i h i h l i

  • Insights in pathologies
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Collaborations Collaborations

  • Two offers on ‘Matchmaker’
  • o e s o

atc a e [team: NTNU‐Trondheim]

  • Modelling help

g p [team: Queens‐Canada] [team: Trieste]

  • Responded to request on ‘Matchmaker’
  • Input ‘tips and tricks’ new teams

[team: Cinvestav‐IPN‐UNAM]

  • Two symposia (July 9th and October 3rd)

[teams: Dutch teams]

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Teaching 14 year‐olds Teaching 14 year olds

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Discovery Festival Discovery Festival

  • Art – Music – Science

t us c Sc e ce

  • Synthetic biology workshop
  • National effort:

National effort:

  • Three locations
  • Four iGEM teams: Amsterdam, Groningen,

TU‐Eindhoven and Wageningen_UR

  • Day: 300 high‐school students
  • Night: 5000+ visitors
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Discovery Festival Discovery Festival

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Discovery Festival Discovery Festival

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Thanks to Thanks to

  • Institute for Complex Molecular Systems
  • ICMS Animation Studio
  • Our supervisors:

p

  • dr. M. Merkx
  • dr. ir. N.A.W. van Riel

O d i

  • Our advisors:
  • dr. ir. T.F.A. de Greef
  • prof. dr. ir. L. Brunsveld

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  • dr. P.Y.W. Dankers
  • S. Peters MSc
  • M van Rosmalen MSc
  • M. van Rosmalen MSc
  • M.J.J. Krom BSc
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