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

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

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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

slide-1
SLIDE 1

IOD BAND

IOD

Rethinking modular design to harness the dynamics of live cell processing power for affordable preventive cancer diagnostics
slide-2
SLIDE 2 new cases in 2015 USA

1.5M

SECONDARY TUMOR PRIMARY TUMOR 2
slide-3
SLIDE 3 new cases in 2015 USA

1.5M

SECONDARY TUMOR PRIMARY TUMOR 3
slide-4
SLIDE 4 new cases in 2015 USA

1.5M

SECONDARY TUMOR PRIMARY TUMOR 4 INVADE UPREGULATED ADHERENS & INTEGRINS REPLENISH STEM CELL MARKERS RELEASE DOWN REGULATED ADHERENS MOBILITY FOOTPRINTS
slide-5
SLIDE 5 new cases in 2015 USA

1.5M

SECONDARY TUMOR PRIMARY TUMOR 5 INVADE UPREGULATED ADHERENS & INTEGRINS REPLENISH STEM CELL MARKERS RELEASE DOWN REGULATED ADHERENS MOBILITY FOOTPRINTS Ep CAM Ep DIAGNOSTICS TODAY LATE LIMITED BASIC COSTLY Ep CAM
  • Dr. Pavel Pitule
MEDICAL EXPERT
slide-6
SLIDE 6 6 “…in research we use immunofluorescence assays to study CTCs under the microscope…
  • Dr. Pavel Pitule
MEDICAL EXPERT
slide-7
SLIDE 7 7 “…in research we use immunofluorescence assays to study CTCs under the microscope… CaLu-3 786-O LN-229 PRIMARY TUMOR SITES SECONDARY TUMOR SITES HCC4012 Lungs Liver Lymph Bladder PRIMARY SECOND RENAL CELL CARCINOMA 786-O CCD80 ASPN cell line marker barcode SCANNER FOR A TUMOR MARKER PROFILE ATLAS
  • Dr. Pavel Pitule
MEDICAL EXPERT
slide-8
SLIDE 8 8 “…in research we use immunofluorescence assays to study CTCs under the microscope… … cool! … how can we help?” CaLu-3 786-O LN-229 PRIMARY TUMOR SITES SECONDARY TUMOR SITES HCC4012 Lungs Liver Lymph Bladder PRIMARY SECOND RENAL CELL CARCINOMA 786-O CCD80 ASPN cell line marker barcode SCANNER FOR A TUMOR MARKER PROFILE ATLAS
  • Dr. Pavel Pitule
MEDICAL EXPERT
slide-9
SLIDE 9 9

FALSE

+

new tests can be tricksy existing tests take too long

$

insurance coverage
  • Dr. Pavel Pitule
MEDICAL EXPERT
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SLIDE 10 10
  • Dr. Pavel Pitule
MEDICAL EXPERT
  • Prof. Miloš Schlegel
ENGINEERING PROF

FALSE

+

new tests can be tricksy existing tests take too long

$

insurance coverage need to be able to tune it…easily
slide-11
SLIDE 11

THE IOD BAND

11 MA ER 2 ARKER 1 MARKE
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SLIDE 12

THE IOD BAND

12 MA ER 2 ARKER 1 MARKE

IOD1 IOD2

recognition of marker profiles
slide-13
SLIDE 13

THE IOD BAND

13 MA ER 2 ARKER 1 MARKE

IOD1 IOD2

recognition of marker profiles group logic enabled by localised signal transmission
slide-14
SLIDE 14

THE IOD BAND

14 MA ER 2 ARKER 1 MARKE

IOD1 IOD2

recognition of marker profiles group logic enabled by localised signal transmission agglutination signal visible to the naked eye

$

slide-15
SLIDE 15

IODS: INPUT-OUTPUT DIPLOIDS

15

INPUTS OUTPUTS

intelligent units that work together to solve complex problems
slide-16
SLIDE 16 16

WILL THERE BE FALSE POSITIVES? YES

Aberdeen STOP THE HAT iGEM 2014
slide-17
SLIDE 17 17

WILL THERE BE FALSE POSITIVES? YES … IF DESIGNED POORLY

MA ER 2 ARKER 1 MARKE

IOD1 IOD2

robust group logic ensures reliability of results FALSE

+

slide-18
SLIDE 18 IOD SYSTEM MODEL A SIMPLE SYSTEM OF 3 BIOCHEMICAL REACTIONS - SIGNAL TRANSDUCTION, PRODUCTION OF PHEROMONE, PRODUCTION OF LOCATION TAGS 18 transcription factor activation pheromone production location tag production
slide-19
SLIDE 19 19

CeCe: cell-cell simulator

+ biochemical interactions + hydrodynamic equations + diffusion in flow + cell-cell agglutination

A priori multicellular simulator with integrated physical engine
slide-20
SLIDE 20 20 IOD2 Loc(1,2) Bind(2,2) a) IOD2 Loc(1,2) Bind(2,2) Bind(1,2) b) IOD2 IOD1 Loc(1,2) Bind(1,1) c) d) IOD2 IOD1 Loc(1,2) Bind(1,1) Bind(1,2)
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SLIDE 21 21

CeCe: IOD BAND IN SILICO

IOD2 IOD1 Loc(1,2) Bind(1,1) Bind(1,2)

MARKER1+ MARKER2+ MARKER1+ MARKER2+

SIMULATION CONDITIONS: LAMINAR FLOW REGIME VARIOUS VELOCITY PROFILES STATIONARY TARGETS IRREVERSIBLE BONDS
slide-22
SLIDE 22 22 FALSE

+

FALSE

+

FALSE

_

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

JUST RIGHT

Signal transmission network topology affects the robustness and efficiency of the agllutination signal.

slide-23
SLIDE 23 23

WHAT WE LEARNED

NEED RELIABLE ALL OR NOTHING SIGNAL RESPONSE AT LOW CONCENTRATIONS NEED A COLLECTION OF ORTHOGONAL SIGNALS AND RECEPTORS NEED STRONG BONDS BETWEEN LOCATION TAGS AND DIAGNOSED CELLS IOD1 IOD2 NO QUORUM SENSING Aberdeen, STOP THE HAT iGEM 2014
slide-24
SLIDE 24

IOD UNDER THE HOOD

24
  • S. cerevisiae
  • C. albicans
  • C. parapsilosis
  • C. tropicalis
  • L. elongisporus
Ste2syn ADH1 αFsyn pGAL MODULE 2 ORTHOGONAL SIGNALS & RECEPTORS
  • rthogonally
expressed MAPK cascade MODULE 1 SYNTHETIC HAPLOIDS
  • Chr. III
  • Chr. III
refactored MAT loci MATa-syn MATα-syn α specific genes mating genes & a specific genes mating genes & a specific genes Ste12 pTet α2 CYC1 α1 pTet Ste12syn ADH1 Ste12 asCYC1 TetR ADH1 EpCAM anti-EpCAM anti-c-Myc anti-HuA Aga1 ADH1 Aga2::LT pGAL MODULE 3 LOCATION TAGS tag2 tag1
slide-25
SLIDE 25 PHERMONE RESPONSE PATHWAY

LOCAL SIGNAL TRANSMISSION RECEPTOR PHEROMONE PAIR DICTATE PATHWAY SPECIFICITY

GONCALVES (2010) COMMUNICATION AND CHOICE IN YEAST MATING. DISSERTATION. 25
slide-26
SLIDE 26 STE2
  • S. cerevisiae
α-factor
  • S. cerevisiae
α-factor
  • C. albicans
α-factor
  • C. parapsilosis
α-factor
  • C. tropicalis
α-factor
  • L. elongisporus
STE2
  • C. albicans
STE2
  • C. parapsilosis
STE2
  • C. tropicalis
STE2
  • L. elongisporus

THERE IS ONLY ONE USABLE NATIVE RECEPTOR AVAILABLE IN S. CEREVISIAE

26
slide-27
SLIDE 27 LITERATURE INCLUDES ONLY PARTIAL RESULTS REGARDING THE ORTHOGONALITY OF RECEPTOR-PHEROMONE PAIRS FROM DIFFERENT YEAST STRAINS 27 STE2
  • S. cerevisiae
α-factor
  • S. cerevisiae
α-factor
  • C. albicans
α-factor
  • C. parapsilosis
α-factor
  • C. tropicalis
α-factor
  • L. elongisporus
STE2
  • C. albicans
STE2
  • C. parapsilosis
STE2
  • C. tropicalis
STE2
  • L. elongisporus
Janiak, A. et al. (2005). Fungal Genetics and Biology, 42.
slide-28
SLIDE 28 STE2
  • S. cerevisiae
α-factor
  • S. cerevisiae
α-factor
  • C. albicans
α-factor
  • C. parapsilosis
α-factor
  • C. tropicalis
α-factor
  • L. elongisporus
STE2
  • C. albicans
STE2
  • C. parapsilosis
STE2
  • C. tropicalis
STE2
  • L. elongisporus

HENCE WE DECIDED TO EXPLOIT PHEROMONE-RECEPTOR PAIRS FROM DIFFERENT YEAST STRAINS

28
slide-29
SLIDE 29

DESIGN OF ORTHOGONAL RECEPTORS

CUSTOMIZATION OF C-TERMINI 29 Kozak TM1 TM2 TM3 TM4 TM5 EL2 EL1 TM6 EL3 TM7 C-TERMINUS 200 400 600 800 1,000 1,200 TU-Delft, Snifferymyces, iGEM 2012
slide-30
SLIDE 30

DESIGN OF ORTHOGONAL RECEPTORS

CUSTOMIZATION OF C-TERMINI 30 Kozak TM1 TM2 TM3 TM4 TM5 EL2 EL1 TM6 EL3 TM7 C-TERMINUS 200 400 600 800 1,000 1,200 Kozak TM1 TM2 TM3 TM4 TM5 EL2 EL1 TM6 EL3 TM7

C-TERMINUS

200 400 600 800 1,000 1,200

CANDIDA ALBICANS | SACCHAROMYCES CEREVISIAE

slide-31
SLIDE 31

DESIGN OF ORTHOGONAL RECEPTORS

CUSTOMIZATION OF C-TERMINI 31 Kozak TM1 TM2 TM3 TM4 TM5 EL2 EL1 TM6 EL3 TM7 C-TERMINUS 200 400 600 800 1,000 1,200

CANDIDA ALBICANS | SACCHAROMYCES CEREVISIAE

DESIGN OF ORTHOGONAL SIGNALS

Kozak TM1 TM2 TM3 TM4 TM5 EL2 EL1 TM6 EL3 TM7 C-TERMINUS 200 400 600 800 1,000 1,200 50 100 150 200 250 300 350 400 450 Alpha factor 1 Alpha factor 2 Alpha factor 3 Alpha factor 4 Signal peptide Pro region Alpha factor secretion signal
slide-32
SLIDE 32 50 100 150 200 250 300 350 400 450 Alpha factor 1 Alpha factor 2 Alpha factor 3 Alpha factor 4 Signal peptide Pro region Alpha factor secretion signal

DESIGN OF ORTHOGONAL RECEPTORS

CUSTOMIZATION OF C-TERMINI 32 Kozak TM1 TM2 TM3 TM4 TM5 EL2 EL1 TM6 EL3 TM7 C-TERMINUS 200 400 600 800 1,000 1,200

CANDIDA ALBICANS | SACCHAROMYCES CEREVISIAE

DESIGN OF ORTHOGONAL SIGNALS

Kozak TM1 TM2 TM3 TM4 TM5 EL2 EL1 TM6 EL3 TM7 C-TERMINUS 200 400 600 800 1,000 1,200
slide-33
SLIDE 33

DESIGN OF ORTHOGONAL RECEPTORS

CUSTOMIZATION OF C-TERMINI 33 Kozak TM1 TM2 TM3 TM4 TM5 EL2 EL1 TM6 EL3 TM7 C-TERMINUS 200 400 600 800 1,000 1,200

CANDIDA ALBICANS | SACCHAROMYCES CEREVISIAE

DESIGN OF ORTHOGONAL SIGNALS

Kozak TM1 TM2 TM3 TM4 TM5 EL2 EL1 TM6 EL3 TM7 C-TERMINUS 200 400 600 800 1,000 1,200 50 100 150 200 250 300 350 400 450 Alpha factor 1 Alpha factor 2 Alpha factor 3 Alpha factor 4 Signal peptide Pro region Alpha factor secretion signal Kozak
slide-34
SLIDE 34

DESIGN OF ORTHOGONAL RECEPTORS

CUSTOMIZATION OF C-TERMINI 34 Kozak TM1 TM2 TM3 TM4 TM5 EL2 EL1 TM6 EL3 TM7 C-TERMINUS 200 400 600 800 1,000 1,200

CANDIDA ALBICANS | SACCHAROMYCES CEREVISIAE

DESIGN OF ORTHOGONAL SIGNALS

Kozak TM1 TM2 TM3 TM4 TM5 EL2 EL1 TM6 EL3 TM7 C-TERMINUS 200 400 600 800 1,000 1,200 50 100 150 200 250 300 350 400 450 Alpha factor 1 Signal peptide Pro region Alpha factor secretion signal Kozak Caplan, S., et al. (1991). Journal of Bacteriology, 173.
slide-35
SLIDE 35

SIGNAL TRANSMISSION WAS TESTED AND CHARACTERISED USING STANDARD METHODS AND MICROFLUDICS

35

MATa cell producing synthetic pheromone to its natural STE2 receptor was captured exhibiting schmooing

slide-36
SLIDE 36 [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 36 Previously known receptor-pheromone interactions in S. cerevisiae
slide-37
SLIDE 37 [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

5 RECEPTORS 5 PHEROMONES MINIMAL CROSS-TALK

37
slide-38
SLIDE 38 38

IOD UNDER THE HOOD

  • S. cerevisiae
  • C. albicans
  • C. parapsilosis
  • C. tropicalis
  • L. elongisporus
Ste2syn ADH1 αFsyn pGAL MODULE 2 ORTHOGONAL SIGNALS & RECEPTORS
  • rthogonally
expressed MAPK cascade MODULE 1 SYNTHETIC HAPLOIDS
  • Chr. III
  • Chr. III
refactored MAT loci MATa-syn MATα-syn α specific genes mating genes & a specific genes mating genes & a specific genes Ste12 pTet α2 CYC1 α1 pTet Ste12syn ADH1 Ste12 asCYC1 TetR ADH1 EpCAM anti-EpCAM anti-c-Myc anti-HuA Aga1 ADH1 Aga2::LT pGAL MODULE 3 LOCATION TAGS tag2 tag1
slide-39
SLIDE 39

LOCATION TAGS

39

RECOGNISE COMMON TUMOR SURFACE MARKERS AND AGGLUTINATE CELL POPULATIONS

POSSIBLE HUMAN SURFACE MARKERS anti- EpCAM anti- c-Myc EpCAM c-Myc
slide-40
SLIDE 40 POSSIBLE HUMAN SURFACE MARKERS RED BLOOD CELLS anti- EpCAM anti- c-Myc EpCAM/ c-Myc anti- HuA EpCAM c-Myc HuA YEAST FACILITATED AGGLUTINATION WITH COMPLIMENTARY LOCATION TAGS RBC FACILITATED AGGLUTINATION WITH anti-HUMAN ANTIGEN A DISPLAY

LOCATION TAGS

40

LOCATION TAGS AGGLUTINATE CELL POPULATIONS

RECOGNISE COMMON TUMOR SURFACE MARKERS AND AGGLUTINATE CELL POPULATIONS

slide-41
SLIDE 41

DISPLAY OF LOCATION TAGS VALIDATED BY IMMUNOFLUORESCENCE 41

EpCAM anti-EpCAM anti-HuA c-MYC

slide-42
SLIDE 42 EpCam + anti-EpCAM AGGLUTINATION UNDER THE MICROSCOPE 42
slide-43
SLIDE 43

STAINED AND MOUNTED CELLS ON GLASS SLIDES

43

RESULTS VISIBLE TO NAKED EYE

slide-44
SLIDE 44

YEAST-INDUCED BLOOD AGGLUTINATION ON CHIP

44
slide-45
SLIDE 45 45

IOD UNDER THE HOOD

  • S. cerevisiae
  • C. albicans
  • C. parapsilosis
  • C. tropicalis
  • L. elongisporus
Ste2syn ADH1 αFsyn pGAL MODULE 2 ORTHOGONAL SIGNALS & RECEPTORS
  • rthogonally
expressed MAPK cascade MODULE 1 SYNTHETIC HAPLOIDS
  • Chr. III
  • Chr. III
refactored MAT loci MATa-syn MATα-syn α specific genes mating genes & a specific genes mating genes & a specific genes Ste12 pTet α2 CYC1 α1 pTet Ste12syn ADH1 Ste12 asCYC1 TetR ADH1 EpCAM anti-EpCAM anti-c-Myc anti-HuA Aga1 ADH1 Aga2::LT pGAL MODULE 3 LOCATION TAGS tag2 tag1
slide-46
SLIDE 46 46

IOD UNDER THE HOOD

  • S. cerevisiae
  • C. albicans
  • C. parapsilosis
  • C. tropicalis
  • L. elongisporus
Ste2syn ADH1 αFsyn pGAL MODULE 2 ORTHOGONAL SIGNALS & RECEPTORS
  • rthogonally
expressed MAPK cascade MODULE 1 SYNTHETIC HAPLOIDS
  • Chr. III
  • Chr. III
refactored MAT loci MATa-syn MATα-syn α specific genes mating genes & a specific genes mating genes & a specific genes Ste12 pTet α2 CYC1 α1 pTet Ste12syn ADH1 Ste12 asCYC1 TetR ADH1 EpCAM anti-EpCAM anti-c-Myc anti-HuA Aga1 ADH1 Aga2::LT pGAL MODULE 3 LOCATION TAGS tag2 tag1

AND NO … WE DID NOT FORGET ABOUT THE ENGINEERS

slide-47
SLIDE 47 47

THIS IS HOW IODS ARE MADE

a unique clone-free assembly feature brings limitless design flexibility to the non-synthetic biologist

SYN BIO BREADBOARD

assembly of a unique signal transmission network in a day on a Petri dish
slide-48
SLIDE 48 48

CHALLENGE: WANT TO USE PHEROMONE PATHWAY FOR TWO THINGS

HAPLOID CONJUGATION SIGNAL TRANSMISSION ONE PATHWAY - TWO APPLICATIONS

slide-49
SLIDE 49 SIGNALLING PATHWAY GENES

MATING TYPE SPECIFIC HAPLOID SPECIFIC*

49
slide-50
SLIDE 50 50 SIGNALLING PATHWAY GENES

MATING TYPE SPECIFIC NON-SPECIFIC (WT) INDUCIBLE (SYN)

slide-51
SLIDE 51 51 SIGNALLING PATHWAY GENES

MATING TYPE SPECIFIC NON-SPECIFIC (WT) INDUCIBLE (SYN)

STE12-SYN DESIGN

Pi H, Chien CT (1997) Molecular and Cellular Biology.
slide-52
SLIDE 52
  • S. cerevisiae
  • C. albicans
  • C. parapsilosis
  • C. tropicalis
  • L. elongisporus
Ste2syn ADH1 αFsyn pGAL MODULE 2 ORTHOGONAL SIGNALS & RECEPTORS MODULE 1 SYNTHETIC HAPLOIDS
  • Chr. III
  • Chr. III
MATa-syn MATα-syn α specific genes haploid secific genes α1 α2 MATα MATα a2 MATa a1 MATa EpCAM anti-EpCAM anti-c-Myc anti-HuA Aga1 ADH1 Aga2::LT pGAL MODULE 3 LOCATION TAGS tag2 tag1 52

IOD UNDER THE HOOD

slide-53
SLIDE 53
  • S. cerevisiae
  • C. albicans
  • C. parapsilosis
  • C. tropicalis
  • L. elongisporus
Ste2syn ADH1 αFsyn pGAL MODULE 2 ORTHOGONAL SIGNALS & RECEPTORS
  • rthogonally
expressed MAPK cascade MODULE 1 SYNTHETIC HAPLOIDS
  • Chr. III
  • Chr. III
refactored MAT loci MATa-syn MATα-syn α specific genes mating genes & a specific genes mating genes & a specific genes Ste12 pTet α2 CYC1 α1 pTet Ste12syn ADH1 Ste12 asCYC1 TetR ADH1 EpCAM anti-EpCAM anti-c-Myc anti-HuA Aga1 ADH1 Aga2::LT pGAL MODULE 3 LOCATION TAGS tag2 tag1 53

IOD UNDER THE HOOD

Cyc1v3: Curran, K. A., et al. (2014). Nat Commun, 5. pTet: Ellis, T., et al. (2009). Nature Biotechnology, 27(5). asCyc1: Zhong, H., et al. (1999). Genome Research, 9(11).
slide-54
SLIDE 54 54

CHALLENGE: WANT TO USE PHEROMONE PATHWAY FOR TWO THINGS

HAPLOID CONJUGATION SIGNAL TRANSMISSION

ONE PATHWAY - TWO APPLICATIONS

slide-55
SLIDE 55

MATx-syn & MATa-syn: INTEGRATION MAPS

55
slide-56
SLIDE 56 56 CROSSING TEST RESULTS STRAIN CROSSING PATTERN MATa-wt leu2 trp1 MATa-wt ura3 leu2 trp1 MATa-syn leu2 trp1 MATa-syn ura3 trp1 MATα-wt ura3 leu2 his3 MATα-syn leu2 his3 MATα-syn ura3 his3 MATa-syn trp1 MATα-syn leu2 his3 MATα-syn his3 EXPECTED MATING POSITIVE CONTROL

PREDICTED RESULTS TEST RESULTS

SYNTHETIC HAPLOID MATING TEST

slide-57
SLIDE 57 57 CROSSING TEST RESULTS STRAIN CROSSING PATTERN MATa-wt leu2 trp1 MATa-wt ura3 leu2 trp1 MATa-syn leu2 trp1 MATa-syn ura3 trp1 MATα-wt ura3 leu2 his3 MATα-syn leu2 his3 MATα-syn ura3 his3 MATa-syn trp1 MATα-syn leu2 his3 MATα-syn his3 EXPECTED MATING POSITIVE CONTROL

PREDICTED RESULTS TEST RESULTS

HAPLOID CONJUGATION

SYNTHETIC HAPLOID MATING TEST

slide-58
SLIDE 58 58 Fluorescence [RFU] 102 103 104 105 106 Counts 1000 2000 3000 4000 5000 6000 7000 MATα-wt Counts 2000 4000 6000 8000 10000 12000 MATα-syn NONE MATa SPECIFIC - SYN MATa SPECIFIC - STE2 HAPLOID SPECIFIC - STE5 REPORTERS COLLECTION

SYNTHETIC HAPLOID MATING EXPRESSION TEST

slide-59
SLIDE 59 59 Fluorescence [RFU] 102 103 104 105 106 Counts 1000 2000 3000 4000 5000 6000 7000 MATα-wt Counts 2000 4000 6000 8000 10000 12000 MATα-syn NONE MATa SPECIFIC - SYN MATa SPECIFIC - STE2 HAPLOID SPECIFIC - STE5 REPORTERS COLLECTION Fluorescence [RFU] 102 103 104 105 106 1000 2000 3000 4000 5000 6000 MATa-wt 1000 2000 3000 4000 5000 6000 7000 MATa-syn

SYNTHETIC HAPLOID MATING EXPRESSION TEST

slide-60
SLIDE 60 60 Fluorescence [RFU] 102 103 104 105 106 Counts 1000 2000 3000 4000 5000 6000 7000 MATα-wt Counts 2000 4000 6000 8000 10000 12000 MATα-syn NONE MATa SPECIFIC - SYN MATa SPECIFIC - STE2 HAPLOID SPECIFIC - STE5 REPORTERS COLLECTION Fluorescence [RFU] 102 103 104 105 106 1000 2000 3000 4000 5000 6000 MATa-wt 1000 2000 3000 4000 5000 6000 7000 MATa-syn Fluorescence [RFU] 102 103 104 105 106 1000 2000 3000 4000 5000 6000 7000 MATa-wt x MATα-wt 1000 2000 3000 4000 5000 6000 7000 MATa-syn x MATα-syn

SYNTHETIC HAPLOID MATING EXPRESSION TEST

slide-61
SLIDE 61 61

SYNTHETIC HAPLOID MATING EXPRESSION TEST

Fluorescence [RFU] 102 103 104 105 106 Counts 1000 2000 3000 4000 5000 6000 7000 MATα-wt Fluorescence [RFU] 102 103 104 105 106 1000 2000 3000 4000 5000 6000 MATa-wt Fluorescence [RFU] 102 103 104 105 106 1000 2000 3000 4000 5000 6000 7000 MATa-wt x MATα-wt 1000 2000 3000 4000 5000 6000 7000 MATa-syn x MATα-syn 1000 2000 3000 4000 5000 6000 7000 MATa-syn Counts 2000 4000 6000 8000 10000 12000 MATα-syn NONE MATa SPECIFIC - SYN MATa SPECIFIC - STE2 HAPLOID SPECIFIC - STE5 REPORTERS COLLECTION

SIGNAL TRANSMISSION

slide-62
SLIDE 62 62

IOD UNDER THE HOOD

  • S. cerevisiae
  • C. albicans
  • C. parapsilosis
  • C. tropicalis
  • L. elongisporus
Ste2syn ADH1 αFsyn pGAL MODULE 2 ORTHOGONAL SIGNALS & RECEPTORS
  • rthogonally
expressed MAPK cascade MODULE 1 SYNTHETIC HAPLOIDS
  • Chr. III
  • Chr. III
refactored MAT loci MATa-syn MATα-syn α specific genes mating genes & a specific genes mating genes & a specific genes Ste12 pTet α2 CYC1 α1 pTet Ste12syn ADH1 Ste12 asCYC1 TetR ADH1 EpCAM anti-EpCAM anti-c-Myc anti-HuA Aga1 ADH1 Aga2::LT pGAL MODULE 3 LOCATION TAGS tag2 tag1
slide-63
SLIDE 63 63

WE DISCUSSED OUR PROJECT WITH ENGINEERS AND MEDICAL EXPERTS

Most engineers are eager to see the immediate reaction of the system. So we designed IODs with the clone- free assembly feature. Use of GFP was suggested at the

  • utset. Agglutination verification on

a slide was also recommended. Both suggestions were implemented.

slide-64
SLIDE 64 64 Czech Republic teamed up with Chalmers Gothenburg to conduct a multinational survey on the public perception of Synthetic Biology. In return we helped Chalmers to model their "Study in Scarlet" and simulate it in our CeCe simulator.
slide-65
SLIDE 65 65 IOD INPUT HAPLOID OUTPUT HAPLOID

ASSEMBLY FOR THE NON- SYNTHETIC BIOLOGIST SIGNAL LOCALISATION USING THE LOCATION TAG CONCEPT

IOD1 IOD2 EpCAM anti-EpCAM STE2
  • S. cerevisiae
α-factor
  • S. cerevisiae
α-factor
  • C. albicans
α-factor
  • C. parapsilosis
α-factor
  • C. tropicalis
α-factor
  • L. elongisporus
STE2
  • C. albicans
STE2
  • C. parapsilosis
STE2
  • C. tropicalis
STE2
  • L. elongisporus

ORTHOGONAL SIGNALLING AT THE LOCAL LEVEL

MA ER 2 ARKER 1 MARKE IOD1 IOD2

THE IOD BAND DIAGNOSTIC TEST

ACCOMPLISHMENTS

slide-66
SLIDE 66 MA ER 2 ARKER 1 MARKE IOD1 IOD2

THE IOD BAND DIAGNOSTIC TEST

66 IOD INPUT HAPLOID OUTPUT HAPLOID

ASSEMBLY FOR THE NON- SYNTHETIC BIOLOGIST SIGNAL LOCALISATION USING THE LOCATION TAG CONCEPT

IOD1 IOD2 EpCAM anti-EpCAM STE2
  • S. cerevisiae
α-factor
  • S. cerevisiae
α-factor
  • C. albicans
α-factor
  • C. parapsilosis
α-factor
  • C. tropicalis
α-factor
  • L. elongisporus
STE2
  • C. albicans
STE2
  • C. parapsilosis
STE2
  • C. tropicalis
STE2
  • L. elongisporus

ORTHOGONAL SIGNALLING AT THE LOCAL LEVEL

ACCOMPLISHMENTS

slide-67
SLIDE 67 STE2
  • S. cerevisiae
α-factor
  • S. cerevisiae
α-factor
  • C. albicans
α-factor
  • C. parapsilosis
α-factor
  • C. tropicalis
α-factor
  • L. elongisporus
STE2
  • C. albicans
STE2
  • C. parapsilosis
STE2
  • C. tropicalis
STE2
  • L. elongisporus

ORTHOGONAL SIGNALLING AT THE LOCAL LEVEL

MA ER 2 ARKER 1 MARKE IOD1 IOD2

THE IOD BAND DIAGNOSTIC TEST

67 IOD INPUT HAPLOID OUTPUT HAPLOID

ASSEMBLY FOR THE NON- SYNTHETIC BIOLOGIST SIGNAL LOCALISATION USING THE LOCATION TAG CONCEPT

IOD1 IOD2 EpCAM anti-EpCAM

ACCOMPLISHMENTS

slide-68
SLIDE 68

SIGNAL LOCALISATION USING THE LOCATION TAG CONCEPT

IOD1 IOD2 EpCAM anti-EpCAM STE2
  • S. cerevisiae
α-factor
  • S. cerevisiae
α-factor
  • C. albicans
α-factor
  • C. parapsilosis
α-factor
  • C. tropicalis
α-factor
  • L. elongisporus
STE2
  • C. albicans
STE2
  • C. parapsilosis
STE2
  • C. tropicalis
STE2
  • L. elongisporus

ORTHOGONAL SIGNALLING AT THE LOCAL LEVEL

MA ER 2 ARKER 1 MARKE IOD1 IOD2

THE IOD BAND DIAGNOSTIC TEST

68 IOD INPUT HAPLOID OUTPUT HAPLOID

ASSEMBLY FOR THE NON- SYNTHETIC BIOLOGIST

ACCOMPLISHMENTS

slide-69
SLIDE 69

SIGNAL LOCALISATION USING THE LOCATION TAG CONCEPT

IOD1 IOD2 EpCAM anti-EpCAM STE2
  • S. cerevisiae
α-factor
  • S. cerevisiae
α-factor
  • C. albicans
α-factor
  • C. parapsilosis
α-factor
  • C. tropicalis
α-factor
  • L. elongisporus
STE2
  • C. albicans
STE2
  • C. parapsilosis
STE2
  • C. tropicalis
STE2
  • L. elongisporus

ORTHOGONAL SIGNALLING AT THE LOCAL LEVEL

MA ER 2 ARKER 1 MARKE IOD1 IOD2

THE IOD BAND DIAGNOSTIC TEST

69 IOD INPUT HAPLOID OUTPUT HAPLOID

ASSEMBLY FOR THE NON- SYNTHETIC BIOLOGIST

ACCOMPLISHMENTS

REPORTER COLLECTION COLLECTION RECEPTOR- PHEROMONE COLLECTION COLLECTION
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SLIDE 70 70 We are engineers first. Biologists and students of medicine complete us.
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SLIDE 71 71

ATTRIBUTIONS

MODULE 1

SYNTHETIC HAPLOIDS

MODULE 2

ORTHOGONAL SIGNALS

MODULE 3

LOCATION TAGS

MODULE 4

MODELLING & SIMULATION

MODULE 5

MICROFLUIDIC EXPERIMENTS TEAM MEMBERS ADVISORS

GENOME DATA RECEPTOR & PHEROMONE LITERATURE / ORGANISATION PHYSICAL ENGINE CELL SIMULATOR FOR CeCe OVERSIGHT ON ALL MICROFLUIDIC ACTIVITIES
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SLIDE 72 72

ATTRIBUTIONS

INSTRUCTORS

DIRECTION & PRESENTATION

ADVISOR

INSTITUTIONAL SUPPORT LAB MANAGEMENT
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SLIDE 73
  • Mgr. Lucie Vistejnova, PhD
  • Mgr. Pavel Pitule, PhD
  • Mgr. Pavel Ostasov, PhD
  • Prof. Ing. Milos Schlegel, CSc

MSc Jana Aletta, PhD

73

ACKNOWLEDGEMENTS

Team Chalmers Gothenburg

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SLIDE 74 SPONSORS 74
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SLIDE 75

pFUS1::GFP MA M ARKER 1 EpCAM IOD1 IOD2 anti-MARKER 1 anti-EpCAM

MODULE X

75
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SLIDE 76

pFUS1::GFP IOD1 IOD2 EpCAM anti-EpCAM

THE MASTER EXPERIMENT SETUP

76

IOD1 represents the IOD bound to the CTC MARKER1 The remainder of the system remains the same

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SLIDE 77

Microplate assays suggests modest activation of Strain A in mixed cultures when compared to background activation in pure cultures and controls

77

The icing on the cake

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SLIDE 78

SYNTHETIC TRANSCRIPTION FACTOR

78
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SLIDE 79

PROPOSED PROTOCOL

79 PROCEDURE* STEP 1: combine two part IOD powder with a prepared sample
  • f peripheral blood.
STEP 2: incubate for 60 minutes at room temp. STEP 3: analyse the sample for visible solid clumps. The number of round clumps of at least 5mm in diameter indicates the number of circulating tumor cells of type matching the package label.