IOD IOD BAND Rethinking modular design to harness the dynamics of - PowerPoint PPT Presentation

IOD IOD BAND Rethinking modular design to harness the dynamics of live cell processing power for affordable preventive cancer diagnostics

SECONDARY TUMOR 1.5M new cases in 2015 USA PRIMARY TUMOR 2

SECONDARY TUMOR 1.5M new cases in 2015 USA PRIMARY TUMOR 3

MOBILITY FOOTPRINTS UPREGULATED INVADE ADHERENS & INTEGRINS REPLENISH STEM CELL SECONDARY MARKERS RELEASE TUMOR DOWN REGULATED ADHERENS 1.5M new cases in 2015 USA PRIMARY TUMOR 4

MOBILITY FOOTPRINTS UPREGULATED INVADE ADHERENS & INTEGRINS REPLENISH STEM CELL SECONDARY MARKERS RELEASE TUMOR DOWN REGULATED ADHERENS 1.5M Dr. Pavel Pitule new cases in 2015 USA MEDICAL EXPERT PRIMARY TUMOR DIAGNOSTICS TODAY Ep Ep LATE CAM CAM LIMITED Ep BASIC COSTLY 5

Dr. Pavel Pitule MEDICAL EXPERT “…in research we use immunofluorescence assays to study CTCs under the microscope… 6

SCANNER FOR A TUMOR Dr. Pavel Pitule MARKER PROFILE ATLAS MEDICAL EXPERT CaLu-3 LN-229 RENAL CELL CARCINOMA PRIMARY cell line marker barcode 786-O CCD80 ASPN 786-O HCC4012 PRIMARY TUMOR SITES SECOND SECONDARY TUMOR SITES Liver Lungs Bladder Lymph “…in research we use immunofluorescence assays to study CTCs under the microscope… 7

SCANNER FOR A TUMOR Dr. Pavel Pitule MARKER PROFILE ATLAS MEDICAL EXPERT CaLu-3 LN-229 RENAL CELL CARCINOMA PRIMARY cell line marker barcode 786-O CCD80 ASPN 786-O HCC4012 PRIMARY TUMOR SITES SECOND SECONDARY TUMOR SITES Liver Lungs Bladder Lymph “…in research we use immunofluorescence assays to study CTCs under the microscope… … cool! … how can we help?” 8

FALSE new tests can + be tricksy Dr. Pavel Pitule MEDICAL EXPERT existing tests take too long $ insurance coverage 9

FALSE new tests can + be tricksy Dr. Pavel Pitule Prof. Milo š Schlegel MEDICAL EXPERT ENGINEERING PROF existing tests take too long $ insurance coverage need to be able to tune it…easily 10

MA ARKER 1 MARKE ER 2 THE IOD BAND 11

IOD1 IOD2 recognition of marker profiles MA ARKER 1 MARKE ER 2 THE IOD BAND 12

group logic enabled by localised signal transmission IOD1 IOD2 recognition of marker profiles MA ARKER 1 MARKE ER 2 THE IOD BAND 13

group logic enabled by localised signal transmission IOD1 IOD2 recognition of marker profiles MA ARKER 1 MARKE ER 2 agglutination $ signal visible to the naked eye THE IOD BAND 14

INPUTS OUTPUTS IODS: INPUT-OUTPUT DIPLOIDS intelligent units that work together to solve complex problems 15

Aberdeen STOP THE HAT iGEM 2014 WILL THERE BE FALSE POSITIVES? YES 16

WILL THERE BE FALSE POSITIVES? YES … IF DESIGNED POORLY robust group logic ensures FALSE IOD1 IOD2 + reliability of results MA ARKER 1 MARKE ER 2 17

transcription factor activation pheromone production location tag production IOD SYSTEM MODEL A SIMPLE SYSTEM OF 3 BIOCHEMICAL REACTIONS - SIGNAL TRANSDUCTION, PRODUCTION OF PHEROMONE, PRODUCTION OF LOCATION TAGS 18

CeCe: cell-cell simulator + biochemical interactions + hydrodynamic equations + diffusion in flow + cell-cell agglutination A priori multicellular simulator with integrated physical engine 19

a) b) c) d) Loc(1,2) Loc(1,2) IOD2 IOD2 Loc(1,2) Loc(1,2) Bind(1,2) IOD2 IOD2 IOD1 IOD1 Bind(1,2) Bind(2,2) Bind(2,2) Bind(1,1) Bind(1,1) 20

Loc(1,2) SIMULATION CONDITIONS: IOD2 LAMINAR FLOW REGIME CeCe: IOD BAND IN SILICO Bind(1,2) VARIOUS VELOCITY PROFILES STATIONARY TARGETS IOD1 IRREVERSIBLE BONDS Bind(1,1) MARKER1+ MARKER1+ MARKER2+ MARKER2+ 21

Signal transmission network topology affects the robustness and efficiency of the agllutination signal. a) b) c) d) Loc(1,2) Loc(1,2) IOD2 IOD2 Loc(1,2) Loc(1,2) Bind(1,2) IOD2 IOD2 IOD1 IOD1 Bind(1,2) Bind(2,2) Bind(2,2) Bind(1,1) Bind(1,1) FALSE FALSE FALSE JUST RIGHT _ + + 22

WHAT WE LEARNED NO QUORUM SENSING NEED RELIABLE ALL OR NOTHING SIGNAL RESPONSE Aberdeen, STOP THE HAT iGEM 2014 AT LOW CONCENTRATIONS NEED A COLLECTION OF ORTHOGONAL SIGNALS AND RECEPTORS NEED STRONG BONDS BETWEEN LOCATION TAGS IOD2 IOD1 AND DIAGNOSED CELLS 23

IOD UNDER THE HOOD S. cerevisiae MODULE 2 C. albicans ORTHOGONAL C. parapsilosis SIGNALS & C. tropicalis L. elongisporus RECEPTORS Ste2syn α Fsyn ADH1 pGAL refactored MAT loci MODULE 1 mating genes & SYNTHETIC MATa-syn a specific genes Chr. III TetR Ste12 Ste12syn HAPLOIDS ADH1 asCYC1 ADH1 orthogonally expressed Chr. III α 1 Ste12 α 2 pTet pTet CYC1 MAPK MAT α -syn cascade α specific mating genes & a specific genes genes MODULE 3 Aga1 Aga2::LT ADH1 pGAL LOCATION TAGS tag2 EpCAM tag1 anti-EpCAM anti-c-Myc anti-HuA 24

PHERMONE RESPONSE PATHWAY LOCAL SIGNAL TRANSMISSION RECEPTOR PHEROMONE PAIR DICTATE PATHWAY SPECIFICITY GONCALVES (2010) COMMUNICATION AND CHOICE IN YEAST MATING. DISSERTATION. 25

THERE IS ONLY ONE USABLE NATIVE RECEPTOR AVAILABLE IN S. CEREVISIAE α -factor α -factor α -factor α -factor α -factor S. cerevisiae C. albicans C. parapsilosis C. tropicalis L. elongisporus STE2 STE2 STE2 STE2 STE2 S. cerevisiae C. albicans C. parapsilosis C. tropicalis L. elongisporus 26

LITERATURE INCLUDES ONLY PARTIAL RESULTS REGARDING THE ORTHOGONALITY OF RECEPTOR-PHEROMONE PAIRS FROM DIFFERENT YEAST STRAINS α -factor α -factor α -factor α -factor α -factor S. cerevisiae C. albicans C. parapsilosis C. tropicalis L. elongisporus STE2 STE2 STE2 STE2 STE2 S. cerevisiae C. albicans C. parapsilosis C. tropicalis L. elongisporus Janiak, A. et al. (2005). Fungal Genetics and Biology, 42. 27

HENCE WE DECIDED TO EXPLOIT PHEROMONE-RECEPTOR PAIRS FROM DIFFERENT YEAST STRAINS α -factor α -factor α -factor α -factor α -factor S. cerevisiae C. albicans C. parapsilosis C. tropicalis L. elongisporus STE2 STE2 STE2 STE2 STE2 S. cerevisiae C. albicans C. parapsilosis C. tropicalis L. elongisporus 28

DESIGN OF ORTHOGONAL RECEPTORS CUSTOMIZATION OF C-TERMINI 200 400 600 800 1,000 1,200 TM7 TM2 TM3 EL2 TM6 C-TERMINUS Kozak TM1 EL1 TM4 TM5 EL3 TU-Delft, Snifferymyces, iGEM 2012 29

DESIGN OF ORTHOGONAL RECEPTORS CUSTOMIZATION OF C-TERMINI 200 200 400 400 600 600 800 800 1,000 1,000 1,200 1,200 TM7 TM7 TM2 TM2 TM3 TM3 EL2 EL2 TM6 TM6 C-TERMINUS C-TERMINUS Kozak Kozak TM1 TM1 EL1 EL1 TM4 TM4 TM5 TM5 EL3 EL3 CANDIDA ALBICANS | SACCHAROMYCES CEREVISIAE 30

DESIGN OF ORTHOGONAL RECEPTORS CUSTOMIZATION OF C-TERMINI 200 200 400 400 600 600 800 800 1,000 1,000 1,200 1,200 TM7 TM7 TM2 TM2 TM3 TM3 EL2 EL2 TM6 TM6 C-TERMINUS C-TERMINUS Kozak Kozak TM1 TM1 EL1 EL1 TM4 TM4 TM5 TM5 EL3 EL3 CANDIDA ALBICANS | SACCHAROMYCES CEREVISIAE DESIGN OF ORTHOGONAL SIGNALS 50 100 150 200 250 300 350 400 450 Alpha factor 3 Pro region Alpha factor 2 Signal peptide Alpha factor 1 Alpha factor 4 Alpha factor secretion signal 31

DESIGN OF ORTHOGONAL RECEPTORS CUSTOMIZATION OF C-TERMINI 200 200 400 400 600 600 800 800 1,000 1,000 1,200 1,200 TM7 TM7 TM2 TM2 TM3 TM3 EL2 EL2 TM6 TM6 C-TERMINUS C-TERMINUS Kozak Kozak TM1 TM1 EL1 EL1 TM4 TM4 TM5 TM5 EL3 EL3 CANDIDA ALBICANS | SACCHAROMYCES CEREVISIAE DESIGN OF ORTHOGONAL SIGNALS 50 100 150 200 250 300 350 400 450 Alpha factor 3 Pro region Alpha factor 2 Alpha factor 1 Alpha factor 4 Signal peptide Alpha factor secretion signal 32

DESIGN OF ORTHOGONAL RECEPTORS CUSTOMIZATION OF C-TERMINI 200 200 400 400 600 600 800 800 1,000 1,000 1,200 1,200 TM7 TM7 TM2 TM2 TM3 TM3 EL2 EL2 TM6 TM6 C-TERMINUS C-TERMINUS Kozak Kozak TM1 TM1 EL1 EL1 TM4 TM4 TM5 TM5 EL3 EL3 CANDIDA ALBICANS | SACCHAROMYCES CEREVISIAE DESIGN OF ORTHOGONAL SIGNALS 50 100 150 200 250 300 350 400 450 Alpha factor 3 Pro region Alpha factor 2 Alpha factor 1 Alpha factor 4 Signal peptide Alpha factor secretion signal Kozak 33

DESIGN OF ORTHOGONAL RECEPTORS CUSTOMIZATION OF C-TERMINI 200 200 400 400 600 600 800 800 1,000 1,000 1,200 1,200 TM7 TM7 TM2 TM2 TM3 TM3 EL2 EL2 TM6 TM6 C-TERMINUS C-TERMINUS Kozak Kozak TM1 TM1 EL1 EL1 TM4 TM4 TM5 TM5 EL3 EL3 CANDIDA ALBICANS | SACCHAROMYCES CEREVISIAE DESIGN OF ORTHOGONAL SIGNALS 50 100 150 200 250 300 350 400 450 Pro region Alpha factor 1 Signal peptide Alpha factor secretion signal Kozak Caplan, S., et al. (1991). Journal of Bacteriology, 173. 34

MATa cell producing synthetic pheromone to its natural STE2 receptor was captured exhibiting schmooing SIGNAL TRANSMISSION WAS TESTED AND CHARACTERISED USING STANDARD METHODS AND MICROFLUDICS 35

[RFU] SC alpha CA alpha CP alpha CT alpha LE alpha SC R 1.00 CA R 0.22 1.00 CP R CT R LE R Previously known receptor-pheromone interactions in S. cerevisiae 36

5 RECEPTORS 5 PHEROMONES MINIMAL CROSS-TALK [RFU] SC alpha CA alpha CP alpha CT alpha LE alpha SC R 1.00 0.61 0.57 0.65 0.59 CA R 0.22 1.00 0.31 0.11 0.38 CP R 0.54 0.36 1.00 0.31 0.23 CT R 0.31 0.36 0.36 1.00 0.30 LE R 0.15 0.30 0.28 0.26 1.00 37