Synthetic Bio- Communication 1 S YNTHETIC B IO -C OMMUNICATION 1. A - PowerPoint PPT Presentation

O RIGIN OF L IFE : C YS E R ESULTS 0.23 0.18 0.18 OD OD 0.13 0.13 0.08 0.08 0 10000 20000 30000 40000 50000 60000 70000 0 10000 20000 30000 40000 50000 60000 70000 Time (secs) Time (secs) Vmax Points = 21 Vmax Points = 21 Well H1 H2 H3 H4 H5 H6 Well E1 E2 E3 E4 E5 E6 Vmax 0.085 0.078 0.059 0.067 0.049 0.073 Vmax 0.159 0.104 0.085 0.078 0.082 0.090 R^2 0.770 0.850 0.905 0.883 0.891 0.906 R^2 0.557 0.769 0.727 0.629 0.659 0.776 Positive Control (Trento 2012) Ancestral Gene

O RIGIN OF L IFE : H IS C R ESULTS 0.28 0.23 0.23 0.18 OD OD 0.18 0.13 0.13 0.08 0.08 0 10000 20000 30000 40000 50000 60000 70000 0 10000 20000 30000 40000 50000 60000 70000 Time (secs) Time (secs) Vmax Points = 21 Vmax Points = 21 Well F1 F2 F3 F4 F5 F6 Well C1 C2 C3 C4 C5 C6 Vmax 0.142 0.164 0.107 0.105 0.115 0.117 Vmax 0.138 0.117 0.102 0.132 0.107 0.130 R^2 0.762 0.949 0.965 0.744 0.933 0.937 R^2 0.811 0.862 0.828 0.847 0.871 0.904 Positive Control Ancestral Gene

A PPLICATION : C RISPR Target: CasA • CASCADE Complex • Mechanism • Protein Modeling Testing • Test functionality - purified proteins • Compare to modern sequences

D OES THE M ODEL W ORK ? A-1 A- E. coli Thanks to Rich Lenski (MSU) we can validate the model A+ A+6

D OES THE M ODEL W ORK ? L ENSKI D ATA The Experiment • E coli evolution • 25 years • 12 strains • 40,000 generations Our Goals • Ancestral Reconstruction • Whole Genome • Proof of Concept • Modeling Evolution

P ROOF OF C ONCEPT : L ENSKI D ATA Results • Lazarus ~90% • Consensus ~99% Implications • Timeline too short? • Problems with model? • Not enough strains?

F UTURE A IMS Proof of Concept • Alternative Models: ML, Bayes, MP • Selective Pressure Application • Biochemical Assay • Functional Assay

D E -E XTINCTION : B IO B RICKS 7 BioBricks: Ancestral HisC Modern HisC 3 characterized AroE Ancestral CysE 4 currently being tested Ancestral CasA Modern CasA CasBCDE

D E -E XTINCTION : E THICS

D E -E XTINCTION : A CCOMPLISHMENTS ✓ Predicted Ancestral Genes ✓ Successfully Tested Ancestral Gene Function ✓ Modeled Ancestral Proteins ✓ Collaborated with Dr. Rich Lenski to Model and Tested Reconstruction Methods ✓ Submitted 7 BioBricks!

S YNTHETIC B IO -C OMMUNICATION B IO W IRES 1. A TOMIC C RISPR -C AS 2. I NTERCELLULAR 3. T IME D E -E XTINCTION E U :CROPIS 4. S PACE

E U :CROPIS Euglena Combined Regenerative Organic Food Production A German satellite In mission scheduled to Space launch in 2016

E U :CROPIS Brown-Stanford iGEM team pioneers PowerCell for in situ resource utilization on Mars 2011 2016

P OWER C ELL Goal Develop a universal, sustainable energy source to power biological tools Application Feed the biological tools that will transform raw materials into fuel, food, drugs, and other products useful to settlers Brown-Stanford 2011

E U :CROPIS We create a chromogenic biosensor for PowerCell in B. subtilis 2011 2016 2013

E U :CROPIS Load microfluidics chip into satellite 2011 2015

E U :CROPIS Launch! Activate first gravity level and ground controls. 2011 2016

Eu:CROPIS Microfluidics Chip

Anabaena Bacillus subtilis (Brown-Stanford 2011) (Stanford-Brown 2013)

P ROGRESS

P ROGRESS We identified genes associated with regulation of sucrose metabolism (sacY) sporulation (spo0A)

P ROGRESS We identified genes associated with regulation of sucrose metabolism (sacY) sporulation (spo0A) Used oligos to construct gene promoters

P ROGRESS We identified genes associated with regulation of sucrose metabolism (sacY) sporulation (spo0A) Used oligos to construct gene promoters Assembled 2 plasmids containing: sucrose metabolism promoter, a ribosome binding site, and a red chromoprotein/RFP

P ROGRESS We identified genes associated with regulation of sucrose metabolism (sacY) sporulation (spo0A) Used oligos to construct gene promoters Assembled 2 plasmids containing: sucrose metabolism promoter, a ribosome binding site, and a red chromoprotein/RFP Synthesized the equivalent construct for sporulation

P ROGRESS We identified genes associated with regulation of sucrose metabolism (sacY) sporulation (spo0A) Used oligos to construct gene promoters Assembled 2 plasmids containing: sucrose metabolism promoter, a ribosome binding site, and a red chromoprotein/RFP Synthesized the equivalent construct for sporulation Transformed into B. subtilis using an integration vector

R ESULTS Bacillus with sucrose metabolism promoter + RFP (Phase Contrast)

R ESULTS B. subtilis with sucrose inducer Bacillus with sucrose inducer promoter + fluorescent promoter + RFP chromoprotein eForRed (Fluorescence) (Fluorescence)

F UTURE A IMS

F UTURE A IMS Fall 2013: Isolate colonies containing our construct and characterize promoters

F UTURE A IMS Fall 2013: Isolate colonies containing our construct and characterize promoters Winter 2013: Helium balloon test

F UTURE A IMS Fall 2013: Isolate colonies containing our construct and characterize promoters Winter 2013: Helium balloon test 2014: Biocompatibility tests with microfluidics chip

F UTURE A IMS Fall 2013: Isolate colonies containing our construct and characterize promoters Winter 2013: Helium balloon test 2014: Biocompatibility tests with microfluidics chip 2015: Prepare for mission

F UTURE A IMS Fall 2013: Isolate colonies containing our construct and characterize promoters Winter 2013: Helium balloon test 2014: Biocompatibility tests with microfluidics chip 2015: Prepare for mission 2016: EuCROPIS Satellite Mission

E U :CROPIS - A CCOMPLISHMENTS Constructed and synthesized prototype prototype chromogenic biosensors to detect sucrose induction and sporulation 5 BioBricks: BBa_K1218020 BBa_K1218021 BBa_K1218001 BBa_K1218025 BBa_K1218023

E U :CROPIS - A CCOMPLISHMENTS Constructed and synthesized prototype prototype chromogenic biosensors to detect sucrose induction and sporulation 5 BioBricks: BBa_K1218020 Contributed on project to validate PowerCell on the EuCROPIS BBa_K1218021 BBa_K1218001 satellite mission and send synthetic biology into outer space BBa_K1218025 BBa_K1218023

O UTREACH

C ALIFORNIA A CADEMY OF S CIENCES

Synthetic Bio- Communication 1 S YNTHETIC B IO -C OMMUNICATION 1. A - PowerPoint PPT Presentation

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