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

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 plate  Improved control device

Lab work Lab work

GECO GECO • Genetically encoded Ca 2+ indicators Genetically encoded Ca indicators • Detection of the intracellular Ca 2+ concentration concentration • R ‐ GECO • G ‐ GECO • B ‐ GECO (Campbell et al., Science 2011) ( p , )

CCH1 ‐ MID1 channel CCH1 MID1 channel • Over expression Over expression • Homologous to mammalian voltage ‐ gated calcium channels calcium channels • Depolarization plasma membrane  calcium i ions enter cytoplasm l (Iida et al., 2007) (Iida et al., 2007)

Ideal yeast cell Ideal yeast cell

Yeast Yeast • Stable Stable • Simple eukaryote • Native calcium channels i l i h l • Ideal homeostatic calcium concentration • Environmental safety • Shuttle vector

Approach Approach

GECO characterization GECO characterization R ‐ GECO 70 0 nM Ca 60 616 nM Ca 50 808 nM Ca 808 nM Ca 1000 nM Ca ensity (a.u.) 40 1200 nM Ca 1500 nM Ca 30 30 Inte 1700 nM Ca 2000 nM Ca 20 2400 nM Ca 2900 nM Ca 2900 M C 10 3600 nM Ca 3900 nM Ca 0 575 580 585 590 595 600 605 610 615 620 625 630 635 640 645 650 Wa elength (nm) Wavelength (nm)

Yeast characterization Yeast characterization Emission spectra of INVSc1 + GECO, CCH1, MID1 @ 565 nm excitation 3500 3000 tensity (a.u.) 2500 R ‐ GECO G ‐ GECO 2000 B ‐ GECO uorescence in 1500 R Ctrl. G Ctrl. 1000 B Ctrl. Flu 500 0 576 582 588 594 600 606 612 618 624 630 636 642 648 W Wavelength (nm) l h ( )

Initial model Initial model • Neuronal cell of bullfrog Neuronal cell of bullfrog • Parameter sensitivity analysis • Conclusion: M d l Model not fully applicable due to unknown t f ll li bl d t k yeast parameters (Koch and Segev, 1989)

Combined models Combined models • Calcium homeostasis in yeast Calcium homeostasis in yeast • Voltage ‐ dependent calcium channels • Kinetics of the GECO ‐ proteins i i f h G CO i (Cui et al 2009) (Cui et al., 2009) (Zhao et al., 2011)

Combined models Combined models

Calcium homeostasis Calcium homeostasis Conformational switch model

Calcium channels Calcium channels

GECO kinetics GECO kinetics

Modelling results Modelling results 80% decrease

Device Device

Device Device

Specifications Specifications • 16 pixels 16 pixels • Moving image using one signal • Refresh time: 1 ms per pixel R f h ti 1 i l • Maximum of 20 V • Software can easily be extended

In use In use

Accomplishments Accomplishments • Succeeded in an iGEM yeast ‐ project Succeeded in an iGEM yeast project • CCH1/MID1/X ‐ GECO in yeast • BioBricks of red, green and blue GECO i i k f d d bl G CO • Working computational model • Basis for future iGEM teams • Easy to extend • Working device and elaborate software

Human Interest Human Interest • Construct is suitable for calcium research Construct is suitable for calcium research • Insights in calcium fluxes and pathways • Insights in pathologies i h i h l i

Collaborations Collaborations • Two offers on ‘Matchmaker’ o 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 9 th and October 3 rd ) [teams: Dutch teams]

Teaching 14 year ‐ olds Teaching 14 year olds

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

Discovery Festival Discovery Festival

Discovery Festival Discovery Festival

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 • Our advisors: O d i • dr. ir. T.F.A. de Greef • prof. dr. ir. L. Brunsveld p • dr. P.Y.W. Dankers • S. Peters MSc • M van Rosmalen MSc • M. van Rosmalen MSc • M.J.J. Krom BSc