Synchronized Chemotactic Oscillators S.M.U.G. Summer Synthetic Biology Competition Massachusetts Institute of Technology November 6, 2004
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1 Synchronized Chemotactic Oscillators S.M.U.G. Summer Synthetic Biology Competition Massachusetts Institute of Technology November 6, 2004
2 Motivation Our goal: an interesting, complex system something cool. But how to make it happen? We focused on implementing modularity Breaking biological systems into modular pieces At a low level, this is BioBricks Building a modular system allowed efficient division of labor key for a team this large
3 A Chemotactic Oscillator Chemoattractant Gradient Attractant Plug Bacterial Swimming Pool
4 A Chemotactic Oscillator Bacteria are added to the swimming pool
5 A Chemotactic Oscillator Chemotaxis is enabled, indicated by green bacteria Bacteria start swimming up the gradient towards the attractant plug
6 A Chemotactic Oscillator The bacteria congregate around the attractant plug
7 A Chemotactic Oscillator Each bacteria has an internal oscillator, driving switch between: chemotaxis enabled chemotaxis disabled
8 A Chemotactic Oscillator The bacteria communicate their internal oscillator phase with each other using cell-to-cell signaling
9 A Chemotactic Oscillator enabling the entire population to change state synchronously
10 A Chemotactic Oscillator In red bacteria, chemotaxis is disabled Bacteria start to randomly move away from the attractant plug
11 A Chemotactic Oscillator Eventually the bacteria are dispersed around the swimming pool
12 A Chemotactic Oscillator Again the bacteria communicate with each other using cell-to-cell signaling
13 A Chemotactic Oscillator The inter-cellular signaling molecule diffuses throughout out the swimming pool to the entire population
14 A Chemotactic Oscillator State changes: chemotaxis is enabled
15 A Chemotactic Oscillator... and onward they swim, doomed to a fate worse than that of Sisyphus
16 A Chemotactic Oscillator Futile, this wretched swimming!
17 Top-Level System Diagram Cell-to-Cell Signaling Module Cell Boundary Individual s Osc Phase Population s Osc Phase Oscillator Module Chemotaxis Enable Chemotaxis Module
18 Top-Level System Diagram Cell-to-Cell Signaling Module Cell Boundary Individual s Osc Phase Population s Osc Phase Oscillator Module Chemotaxis Enable Chemotaxis Module
19 Outline Overview Motivation System Description How we got there: Synopsis of Summer Activities Module Discussion Cell-to-cell Signaling Module Oscillator Module Chemotaxis Module Module Integration Final emarks Cell-to-Cell Synchronization Module Oscillator Module Cell Boundary Chemotaxis Module
20 Learning about SynthBio June July August Preliminary discussion and design work Previous Class Experiences at MIT Much design, little implementation Cool ideas, but we wondered: Can we do this? Had to hit the lab
21 Introduction to a Biology Lab June July August (Credit: GQ) Objective during this period was parts characterization Achieved useful work on BS characterization Attempted to build sets of linked inverters Began work on cell-to-cell signalling So...you're saying that was supposed to be refrigerated? Agarose gels, Agar gels...what's the difference?
22 Finalizing Design June July August Brainstormed several comprehensive and full-system designs Choosing one design gave us purpose and focus in lab
23 Thereafter: Making it Happen Cell-to-Cell Synchronization Module Cell Boundary Oscillator Module Chemotaxis Module Final element Integrating modules is surely easier said than done How to prepare the experimental setup for our work? Barry, Jason, Fred, and Vikki will now discuss these topics. Chris will offer final remarks.
24 Cell-to-Cell Signaling Objectives Cell-to-Cell Synchronization Module Cell Boundary PoPS Sender Signal Individual s Osc Phase Population s Osc Phase Oscillator Module Chemotaxis Enable Chemotaxis Module Signal Signal Decompose signaling into elements Design, build and test elements Signal eceiver PoPS Explore how elements might function as part of the full system
25 The Lux System H L lux Lux box with left and right promoters luxi luxcdabeg
26 The Lux System Lux Dimerization H H H H L Luciferase lux Lux box with left and right promoters luxi luxcdabeg
27 The Lux System Lux Dimerization H H H H L Luciferase H H lux Lux box with left and right promoters luxi luxcdabeg
28 Utilizing Existing Components Lux Dimerization H H H H L Luciferase H H lux Lux box with left and right promoters luxi luxcdabeg PoPS Sender Signal
29 Utilizing Existing Components Lux Dimerization H H H H L Luciferase H H lux Lux box with left and right promoters luxi luxcdabeg Signal eceiver PoPS PoPS Sender Signal
30 Utilizing Existing Components Signal Signal Lux Dimerization H H H H L Luciferase H H lux Lux box with left and right promoters luxi luxcdabeg Signal eceiver PoPS PoPS Sender Signal
31 eceiver Design Signal eceiver PoPS H H H H BBa_I13270
32 eceiver Building Signal eceiver PoPS BBa_I13273 Varied Upstream Promoter - Ptet, luxp L High ( ) and Low Copy (10-20) Plasmid Used YFP Output Device as a PoPS eporter Built in DH5alpha using standardized assembly, Transformed into MC4100 and HCB1103
33 eceiver Testing Signal eceiver PoPS 1. Hi/Lo ratio 2. Switch Point 3. esponse Time
34 eceiver Testing Signal eceiver PoPS I psb1a2 Hi/Lo ratio 17 Switch Point 2nM
35 eceiver Testing Signal eceiver PoPS I psb1a2 - Growth Defects
36 eceiver Testing Signal eceiver PoPS I psb3k3 - Growth estored
37 eceiver Testing Signal eceiver PoPS I psb3k3 - esponse Time
38 Senders PoPS Sender Signal H L 10m 15m luxi 20m 25m 30m 40m
39 Transmission Signal Signal Diffusion rate Degradation due to dilution (e.g. in chemostat) Degradation due to raised ph Active enzymatic degradation - aiia
40 Transmission Signal Signal ph Dependent Degradation 60 minute incubation of HSL at various ph Use that HSL to activate receivers at neutral ph
41 Transmission Signal Signal aiia Enzymatic intracellular Degradation of HSL
42 Future Work Develop the ability to adjust receiver transfer function parameters at will Complete characterization of existing sender device using the receiver device Build and test the sender device used in the synchronized oscillator Continue to test the aiia degradation mechanism Quantify parameter robustness under different operating conditions chemostats, microscopes etc.
43 Oscillator Module Stand-alone Oscillator elaxation Oscillator ing Oscillator Synchronized Oscillator Synchronators Synchronized ing Oscillator Future Work Cell-to-Cell Synchronization Module Cell Boundary Individual s Osc Phase Population s Osc Phase Oscillator Module Chemotaxis Enable Chemotaxis Module
44 Input/Output Oscillator Device PoPS cell boundary
45 Lux/aiiA elaxation Oscillator L A Constitutive Promoter Lux P Lux LuxI aiia Lux is constitutively expressed, while LuxI and aiia are regulated by a Lux activated promoter
46 Lux/aiiA elaxation Oscillator Lux Dimerization H H L A Constitutive Promoter Lux P Lux LuxI aiia Lux forms a dimer while LuxI synthesizes HSL
47 Lux/aiiA elaxation Oscillator Lux Dimerization H H H H L A Constitutive Promoter H H Lux P Lux LuxI aiia Lux and HSL bind to form the transcriptional activator providing positive feedback
48 Lux/aiiA elaxation Oscillator Lux Dimerization H H H H L A Constitutive Promoter H H Lux P Lux LuxI aiia Lux and HSL bind to form the transcriptional activator providing positive feedback
49 Lux/aiiA elaxation Oscillator Lux Dimerization H Degradation of HSL by aiia H H L A Constitutive Promoter H H Lux P Lux LuxI aiia aiia degrades HSL providing negative feedback
50 Lux/aiiA elaxation Oscillator Lux Dimerization H Degradation of HSL by aiia H H L A Constitutive Promoter H H Lux P Lux LuxI aiia aiia degrades HSL providing negative feedback
51 Simplified elaxation Oscillator Initial modeling work used a system of continuous differential equations to examine a simplified oscillator Folds the positive feedback into a single Protein A ignoring the details of LuxI, HSL, and Lux Even with these simplifications, the model can give insight into what experimental constructs would be useful when building the actual Lux/aiiA oscillator Promoter Protein A Protein B
52 State Space Analysis Intersection of nullclines yields system equilibrium point Equlibrium point changes with Protein B degradation rate
53 Preliminary Modeling esults A vs B State Space Concentration of A vs Time
54 Experimental Work Modeling work suggested possible test constructs Experimental work on the Lux/aiiA relaxation oscillator was put on hold Initial results on aiia were discouraging Not enough degradation tags were available to effectively tune the aiia degradation rate
55 Input/Output Oscillator Device PoPS cell boundary
56 ing Oscillator
57 Input/Output PoPs Average oscillation phase of population receiver Oscillator Device sender Individual oscillation phase AHL cell boundary PoPs
58 Synchronized Oscillator Options epressilator & Synchronization Device Functional oscillator Need to design synchronization device Synchronator 4 designs available from the MIT 2003 Synthetic Biology course Designed to synchronize, completely built, but untested and uncharacterized See-ya-lator Modeled after Yankees playoff performance
59 Synchronator Designs Design 1 Design 2 gfp gfp Design 3 Design 4 gfp gfp
60 Synchronator Design 2 gfp
61 Synchronator Designs Design 1 Design 2 gfp gfp Design 3 Design 4 gfp gfp
62 Oscillator Lockdown Experiment Add IPTG = GFP high Add ATC = GFP high Add HSL = GFP low ATC gfp IPTG
63 Synchronator Lock-Down Fluorescence Units Control IPTG ATC HSL synch 1 synch 2 synch 3 synch 4
64 Synchronator 2 Movie
65 Synchronized ing Oscillator Add a synchronization element to the epressilator (Garcia-Ojalvo,Elowitz,Strogatz, PNAS 2004) receiver sender
66 Synchronized ing Oscillator Add a synchronization element to the epressilator (Garcia-Ojalvo,Elowitz,Strogatz, PNAS 2004) receiver sender
67 Construction of Synchronization Device I13905 I13974 I13975 S03167 I13973 S Q04121 I0461
68 Future Work Synchronized ing Oscillator Lock-down experiments Agarose Pad Time Lapse Movie Continuous Culture (chemostat) Plate eader Time Course elaxation Oscillator Explore aiia further to determine why it isn t functioning as expected Build test constructs and characterize
69 Chemotaxis Module Chemotaxis biology Chemotaxis devices estoring motility Deactivating motility Chemotaxis assay esults Future work Individual s Osc Phase Cell-to-Cell Synchronization Module Population s Osc Phase Cell Boundary Oscillator Module Chemotaxis Enable Chemotaxis Module
70 Chemotaxis in Escherichia Coli Four intracellular signaling proteins CheB, Che, CheY, and CheZ Maintained at specific levels and ratios
71 Motile Behavior Net movement toward or away from chemicals result from the combined effect of smooth runs and tumbles The expression and activity of signaling proteins (CheB, Che, CheY and CheZ) determines the frequency of tumbles and runs
72 Too Much or Too Little? Absence of any signaling protein affects motile behavior Genotype wt CheB Che CheY Motility Phenotype smooth runs and tumbles tumbles smooth runs none CheZ + smooth runs Overexpression of any signaling molecule affects motile behavior Genotype wt wt wt wt Overexpression CheB Che CheY CheZ Phenotype smooth runs tumbles tumbles smooth runs
73 Too Much or Too Little? Absence of any signaling protein affects motile behavior Genotype wt CheB Che CheY Motility Phenotype smooth runs and tumbles tumbles smooth runs none CheZ + smooth runs Overexpression of any signaling molecule affects motile behavior Genotype wt wt wt wt Overexpression CheB Che CheY CheZ Phenotype smooth runs tumbles tumbles smooth runs CheY concentration in P437: 8,200 ± 310 per cell in rich media and 6,300 ± 70 per cell in minimal media Li M, Hazelbauer G Cellular Stoichiometery of the Components of the Chemotaxis Signaling Complex Journal of Bacteriology, 2004, 186(12)
74 Building Chemotaxis Output CheY quadparts: High Copy ( ) and Low Copy (15-20) laci+pl 0011 BOO34 chey C0020 B0015 Two methods for coupling to Chemotaxis estoration of normal chemotaxis in CheY mutant strain Deactivation of normal chemotaxis in wild type strain Characterizing quadpart expression Time frame of protein expression Observing the inactivation and deactivation of bacterial chemotaxis Swarm plate characterization Drop assay and bacterial clustering on glass slide Capillary Assay
75 Swarm Plate Assay Amino acids are bacterial chemoattractants Nutrient consumption produces gradient ing formation on agar corresponding to particular amino acid/chemoattractant consumed by motile bacteria Movement away from the center (point of inoculation) 10ul of bacterial suspension 30ºC for duration of swarming
76 Deactivation: Expected esults
77 Deactivation: High Copy esults High copy CheY expression in wild type
78 Deactivation: High Copy esults High copy CheY expression in LacI- strain (wild type background)
79 Deactivation: Low Copy esults 25µM IPTG induction for 2 hours, OD 660 = ul spot, 16 hrs LacI - Expected: Swarm Observed: Swarm wild type Expected: Swarm Observed: Swarm LacI - (transformed) Expected: Swarm/No rings Observed: Swarm/No rings wild type (transformed) Expected: Swarm Observed: No Swarm
80 Future Work More low copy construct (15-25 per cell) experiments Experimentation with chey mutants of E. coli P437 for the restoration of motility equires the tuned expression of CheY Drop assay or coverslip assay to observe bacterial aggregation Characterization of immediate chemotaxis response under varying levels of induction Coupling to population oscillator module and cell-cell signaling module
81 Module Integration Operating Conditions Strain ph Temperature Test setup Inter-Module Communication Signal Interpretation Timing Cell-to-Cell Synchronization Module Oscillator Module Cell Boundary Chemotaxis Module
82 Op Conditions: Strain Module Cell-Cell Signaling Oscillator Chemotaxis Known equirements LacI - Chemotactic Tested Conditions MC4100, DH5alpha MC4100 P437 (HCB33) Future Plans Testing Cell-Cell signaling module in P437 Create LacI- version of P437 Test combined construct in LacI- version of P437
83 Op Conditions: ph and Temp Component Cell Cell Signaling Oscillator Chemotaxis Known equirements HSL stability is ph dependent ph around 7, temperature around 30 o Celsius Tested Conditions 37 o Celsius, ph 7 37 o Celsius, ph 7 30 o Celsius, ph Future Plans Test combined system in ph7, 32 o Celsius
84 Op Conditions: Test Setup Component Cell Cell Signaling Oscillator Chemotaxis Known equirements Steady gradient of chemoattractants Tested conditions Culture tubes Chemostat Agarose pads Swarm plates Future Plans Plan I: Swimming Pool for continuous observation Plan II: Time Course Sampling with time course test for chemotaxis
85 Inter-Module: Signal Interpretation Signals given as a logical 1 or 0 output from one module must be interpreted as a logical 1 or 0 input by the other modules Module 1 Valid Output anges Low High Module 2 Valid Input anges Low High Increasing Signal Strength Future Plans Do signal strength characterization tests for modules If strengths don t match, fine tune by swapping promoter/bs
86 Inter-Module: Signal Interpretation Signals given as a logical 1 or 0 output from one module must be interpreted as a logical 1 or 0 input by the other modules Module 1 Valid Output anges Low High Module 2 Valid Input anges Low High Increasing Signal Strength Future Plans Do signal strength characterization tests for modules If strengths don t match, fine tune by swapping promoter/bs
87 Inter-Module: Signal Interpretation Signals given as a logical 1 or 0 output from one module must be interpreted as a logical 1 or 0 input by the other modules Module 1 Valid Output anges Low High Module 2 Valid Input anges Low High Increasing Signal Strength Future Plans Do signal strength characterization tests for modules If strengths don t match, fine tune by swapping promoter/bs
88 Inter-Module: Timing Timing of oscillator signals must match up with timing of cellcell signaling and chemotaxis modules Signal 1: (Oscillator) ise Time=0; Fall time=0; Period =2 Signal 2: (Follower) ise Time=1; Fall time=2 Signal 2 Signal 1 Increasing Time High Threshold Low Threshold High Threshold Low Threshold
89 Inter-Module: Timing Cell-to-Cell Signaling and Chemotaxis must be able to turn on and off in at most the amount of time it takes the Oscillator to turn on and off. Current Knowledge epressilator : Period ~2 hours Cell-to-Cell and Chemotaxis rising edge is fast Cell-to-Cell and Chemotaxis falling edge is slow Future Plans Try getting the HSL signals to degrade faster by operating at a higher ph (10) or in a chemostat Characterize off times for chemotaxis module
90 Integration Summary Integration is HAD! Operating Conditions Inter-module Communication Still a lot of work to be done
91 Final emarks Overview of summer accomplishments Advice for future summer competitions Key enablers for the field of synthetic biology Assembly process Device characterization Standard operating conditions
92 Summer Accomplishments Over 200 new BioBrick parts added to the registry Device characterization BS measurements Preliminary copy number measurements Basic chemostat constructed and tested Many inverter measurement constructs ready to be tested Cell-to-cell signaling module Working Lux sender/receiver constructed with BioBrick parts Characterizing receiver transfer curves Verified importance of low-copy constructs Characterization of cell-to-cell signaling channel
93 Summer Accomplishments Oscillator module Modeling work on Lux/aiiA relaxation oscillator efined techniques for creating time-lapse movies Verified repressilator ring oscillator Tested previously designed Synchronators New synchronized repressilator is built and ready for testing Chemotaxis module Working swarm plate chemotaxis assay esults on possibility of transcriptional control of chemotaxis Twelve synthetic biology students who are excited about the potential of this new field Five extremely frustrated advisors who are looking forward to a long winter vacation
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98 I13977 I13976 I13975 I13974 I13951 I13950 I13943 I13942 I13941 I13940 I13930 I13922 I13921 I13920 I13906 I13905 I13904 I13902 I13901 I13900 I13210 I13209 I13973 I13972 I13971 I13851 I13850 I13801 I13800 I13072 I13062 I13017 I6402 I6401 I13607 I13606 I13605 I13604 I13603 I13602 I13601 I13600 E0669 E0130 I13016 I13633 I13105 I0465 I13312 I13262 I0468 C0056 I13631 I13104 I0464 I13311 I13261 I0467 I13990 S04010 S04003 S04002 S04001 S04000 Q04740 Q02400 Q02121 Q00400 Q00121 P0474 I13006 I13013 I13012 I13011 I13010 I13009 I13008 I13007 I13667 I13665 I13664 I13657 I13654 I13653 I13651 I13647 I13645 I13644 I13637 I13634 I13627 I13103 I0463 I13310 I13220 I0466 I13626 I13102 C0261 I13309 I13213 I13005 I13625 I13101 C0260 I13308 I13212 I13004 I13624 I13100 C0163 I13307 I13211 I13002 I13623 I13033 C0063 I13306 I13208 I13002 I13621 I13032 I13207 I13305 I13206 I13001 I13617 I13031 I I13304 I13205 G00701 I13614 I13030 I I13303 I13203 G00700 I13613 I13029 P34160 I13283 I13202 S10000 I13611 I13028 P34060 I13280 I13201 I13730 I13115 I13027 I1468 I13279 I13200 I13721 I13114 I13026 I1466 I13277 I13053 I13712 I13113 I13025 I13915 I13274 I13038 I13711 I13112 I13024 I13914 I13273 I13037 I13710 I13111 I13023 I13913 I13271 I13036 I13702 I13110 I13022 I13912 I13270 I13035 I13701 I13109 I13021 I13911 I13266 I13034 I13700 I13108 I13020 I13910 I13265 I13018 C0028 I13107 I13019 I13314 I13264 I13015 C0024 I13106 I13014 I13313 I13263 I13000 C0020 BioBrick Parts 29 Parts Completely Working
99 I13977 I13976 I13975 I13974 I13951 I13950 I13943 I13942 I13941 I13940 I13930 I13922 I13921 I13920 I13906 I13905 I13904 I13902 I13901 I13900 I13210 I13209 I13973 I13972 I13971 I13851 I13850 I13801 I13800 I13072 I13062 I13017 I6402 I6401 I13607 I13606 I13605 I13604 I13603 I13602 I13601 I13600 E0669 E0130 I13016 I13633 I13105 I0465 I13312 I13262 I0468 C0056 I13631 I13104 I0464 I13311 I13261 I0467 I13990 S04010 S04003 S04002 S04001 S04000 Q04740 Q02400 Q02121 Q00400 Q00121 P0474 I13006 I13013 I13012 I13011 I13010 I13009 I13008 I13007 I13667 I13665 I13664 I13657 I13654 I13653 I13651 I13647 I13645 I13644 I13637 I13634 I13627 I13103 I0463 I13310 I13220 I0466 I13626 I13102 C0261 I13309 I13213 I13005 I13625 I13101 C0260 I13308 I13212 I13004 I13624 I13100 C0163 I13307 I13211 I13002 I13623 I13033 C0063 I13306 I13208 I13002 I13621 I13032 I13207 I13305 I13206 I13001 I13617 I13031 I I13304 I13205 G00701 I13614 I13030 I I13303 I13203 G00700 I13613 I13029 P34160 I13283 I13202 S10000 I13611 I13028 P34060 I13280 I13201 I13730 I13115 I13027 I1468 I13279 I13200 I13721 I13114 I13026 I1466 I13277 I13053 I13712 I13113 I13025 I13915 I13274 I13038 I13711 I13112 I13024 I13914 I13273 I13037 I13710 I13111 I13023 I13913 I13271 I13036 I13702 I13110 I13022 I13912 I13270 I13035 I13701 I13109 I13021 I13911 I13266 I13034 I13700 I13108 I13020 I13910 I13265 I13018 C0028 I13107 I13019 I13314 I13264 I13015 C0024 I13106 I13014 I13313 I13263 I13000 C0020 BioBrick Parts 13 Parts Canceled
100 I13977 I13976 I13975 I13974 I13951 I13950 I13943 I13942 I13941 I13940 I13930 I13922 I13921 I13920 I13906 I13905 I13904 I13902 I13901 I13900 I13210 I13209 I13973 I13972 I13971 I13851 I13850 I13801 I13800 I13072 I13062 I13017 I6402 I6401 I13607 I13606 I13605 I13604 I13603 I13602 I13601 I13600 E0669 E0130 I13016 I13633 I13105 I0465 I13312 I13262 I0468 C0056 I13631 I13104 I0464 I13311 I13261 I0467 I13990 S04010 S04003 S04002 S04001 S04000 Q04740 Q02400 Q02121 Q00400 Q00121 P0474 I13006 I13013 I13012 I13011 I13010 I13009 I13008 I13007 I13667 I13665 I13664 I13657 I13654 I13653 I13651 I13647 I13645 I13644 I13637 I13634 I13627 I13103 I0463 I13310 I13220 I0466 I13626 I13102 C0261 I13309 I13213 I13005 I13625 I13101 C0260 I13308 I13212 I13004 I13624 I13100 C0163 I13307 I13211 I13002 I13623 I13033 C0063 I13306 I13208 I13002 I13621 I13032 I13207 I13305 I13206 I13001 I13617 I13031 I I13304 I13205 G00701 I13614 I13030 I I13303 I13203 G00700 I13613 I13029 P34160 I13283 I13202 S10000 I13611 I13028 P34060 I13280 I13201 I13730 I13115 I13027 I1468 I13279 I13200 I13721 I13114 I13026 I1466 I13277 I13053 I13712 I13113 I13025 I13915 I13274 I13038 I13711 I13112 I13024 I13914 I13273 I13037 I13710 I13111 I13023 I13913 I13271 I13036 I13702 I13110 I13022 I13912 I13270 I13035 I13701 I13109 I13021 I13911 I13266 I13034 I13700 I13108 I13020 I13910 I13265 I13018 C0028 I13107 I13019 I13314 I13264 I13015 C0024 I13106 I13014 I13313 I13263 I13000 C0020 BioBrick Parts 69 Parts for Cell-to-Cell Module
101 I13977 I13976 I13975 I13974 I13951 I13950 I13943 I13942 I13941 I13940 I13930 I13922 I13921 I13920 I13906 I13905 I13904 I13902 I13901 I13900 I13210 I13209 I13973 I13972 I13971 I13851 I13850 I13801 I13800 I13072 I13062 I13017 I6402 I6401 I13607 I13606 I13605 I13604 I13603 I13602 I13601 I13600 E0669 E0130 I13016 I13633 I13105 I0465 I13312 I13262 I0468 C0056 I13631 I13104 I0464 I13311 I13261 I0467 I13990 S04010 S04003 S04002 S04001 S04000 Q04740 Q02400 Q02121 Q00400 Q00121 P0474 I13006 I13013 I13012 I13011 I13010 I13009 I13008 I13007 I13667 I13665 I13664 I13657 I13654 I13653 I13651 I13647 I13645 I13644 I13637 I13634 I13627 I13103 I0463 I13310 I13220 I0466 I13626 I13102 C0261 I13309 I13213 I13005 I13625 I13101 C0260 I13308 I13212 I13004 I13624 I13100 C0163 I13307 I13211 I13002 I13623 I13033 C0063 I13306 I13208 I13002 I13621 I13032 I13207 I13305 I13206 I13001 I13617 I13031 I I13304 I13205 G00701 I13614 I13030 I I13303 I13203 G00700 I13613 I13029 P34160 I13283 I13202 S10000 I13611 I13028 P34060 I13280 I13201 I13730 I13115 I13027 I1468 I13279 I13200 I13721 I13114 I13026 I1466 I13277 I13053 I13712 I13113 I13025 I13915 I13274 I13038 I13711 I13112 I13024 I13914 I13273 I13037 I13710 I13111 I13023 I13913 I13271 I13036 I13702 I13110 I13022 I13912 I13270 I13035 I13701 I13109 I13021 I13911 I13266 I13034 I13700 I13108 I13020 I13910 I13265 I13018 C0028 I13107 I13019 I13314 I13264 I13015 C0024 I13106 I13014 I13313 I13263 I13000 C0020 BioBrick Parts 22 Parts for Oscillator Module
102 I13977 I13976 I13975 I13974 I13951 I13950 I13943 I13942 I13941 I13940 I13930 I13922 I13921 I13920 I13906 I13905 I13904 I13902 I13901 I13900 I13210 I13209 I13973 I13972 I13971 I13851 I13850 I13801 I13800 I13072 I13062 I13017 I6402 I6401 I13607 I13606 I13605 I13604 I13603 I13602 I13601 I13600 E0669 E0130 I13016 I13633 I13105 I0465 I13312 I13262 I0468 C0056 I13631 I13104 I0464 I13311 I13261 I0467 I13990 S04010 S04003 S04002 S04001 S04000 Q04740 Q02400 Q02121 Q00400 Q00121 P0474 I13006 I13013 I13012 I13011 I13010 I13009 I13008 I13007 I13667 I13665 I13664 I13657 I13654 I13653 I13651 I13647 I13645 I13644 I13637 I13634 I13627 I13103 I0463 I13310 I13220 I0466 I13626 I13102 C0261 I13309 I13213 I13005 I13625 I13101 C0260 I13308 I13212 I13004 I13624 I13100 C0163 I13307 I13211 I13002 I13623 I13033 C0063 I13306 I13208 I13002 I13621 I13032 I13207 I13305 I13206 I13001 I13617 I13031 I I13304 I13205 G00701 I13614 I13030 I I13303 I13203 G00700 I13613 I13029 P34160 I13283 I13202 S10000 I13611 I13028 P34060 I13280 I13201 I13730 I13115 I13027 I1468 I13279 I13200 I13721 I13114 I13026 I1466 I13277 I13053 I13712 I13113 I13025 I13915 I13274 I13038 I13711 I13112 I13024 I13914 I13273 I13037 I13710 I13111 I13023 I13913 I13271 I13036 I13702 I13110 I13022 I13912 I13270 I13035 I13701 I13109 I13021 I13911 I13266 I13034 I13700 I13108 I13020 I13910 I13265 I13018 C0028 I13107 I13019 I13314 I13264 I13015 C0024 I13106 I13014 I13313 I13263 I13000 C0020 BioBrick Parts 11 Parts for Chemotaxis Module
103 I13977 I13976 I13975 I13974 I13951 I13950 I13943 I13942 I13941 I13940 I13930 I13922 I13921 I13920 I13906 I13905 I13904 I13902 I13901 I13900 I13210 I13209 I13973 I13972 I13971 I13851 I13850 I13801 I13800 I13072 I13062 I13017 I6402 I6401 I13607 I13606 I13605 I13604 I13603 I13602 I13601 I13600 E0669 E0130 I13016 I13633 I13105 I0465 I13312 I13262 I0468 C0056 I13631 I13104 I0464 I13311 I13261 I0467 I13990 S04010 S04003 S04002 S04001 S04000 Q04740 Q02400 Q02121 Q00400 Q00121 P0474 I13006 I13013 I13012 I13011 I13010 I13009 I13008 I13007 I13667 I13665 I13664 I13657 I13654 I13653 I13651 I13647 I13645 I13644 I13637 I13634 I13627 I13103 I0463 I13310 I13220 I0466 I13626 I13102 C0261 I13309 I13213 I13005 I13625 I13101 C0260 I13308 I13212 I13004 I13624 I13100 C0163 I13307 I13211 I13002 I13623 I13033 C0063 I13306 I13208 I13002 I13621 I13032 I13207 I13305 I13206 I13001 I13617 I13031 I I13304 I13205 G00701 I13614 I13030 I I13303 I13203 G00700 I13613 I13029 P34160 I13283 I13202 S10000 I13611 I13028 P34060 I13280 I13201 I13730 I13115 I13027 I1468 I13279 I13200 I13721 I13114 I13026 I1466 I13277 I13053 I13712 I13113 I13025 I13915 I13274 I13038 I13711 I13112 I13024 I13914 I13273 I13037 I13710 I13111 I13023 I13913 I13271 I13036 I13702 I13110 I13022 I13912 I13270 I13035 I13701 I13109 I13021 I13911 I13266 I13034 I13700 I13108 I13020 I13910 I13265 I13018 C0028 I13107 I13019 I13314 I13264 I13015 C0024 I13106 I13014 I13313 I13263 I13000 C0020 BioBrick Parts 63 Parts for Device Characterization
104 I13977 I13976 I13975 I13974 I13951 I13950 I13943 I13942 I13941 I13940 I13930 I13922 I13921 I13920 I13906 I13905 I13904 I13902 I13901 I13900 I13210 I13209 I13973 I13972 I13971 I13851 I13850 I13801 I13800 I13072 I13062 I13017 I6402 I6401 I13607 I13606 I13605 I13604 I13603 I13602 I13601 I13600 E0669 E0130 I13016 I13633 I13105 I0465 I13312 I13262 I0468 C0056 I13631 I13104 I0464 I13311 I13261 I0467 I13990 S04010 S04003 S04002 S04001 S04000 Q04740 Q02400 Q02121 Q00400 Q00121 P0474 I13006 I13013 I13012 I13011 I13010 I13009 I13008 I13007 I13667 I13665 I13664 I13657 I13654 I13653 I13651 I13647 I13645 I13644 I13637 I13634 I13627 I13103 I0463 I13310 I13220 I0466 I13626 I13102 C0261 I13309 I13213 I13005 I13625 I13101 C0260 I13308 I13212 I13004 I13624 I13100 C0163 I13307 I13211 I13002 I13623 I13033 C0063 I13306 I13208 I13002 I13621 I13032 I13207 I13305 I13206 I13001 I13617 I13031 I I13304 I13205 G00701 I13614 I13030 I I13303 I13203 G00700 I13613 I13029 P34160 I13283 I13202 S10000 I13611 I13028 P34060 I13280 I13201 I13730 I13115 I13027 I1468 I13279 I13200 I13721 I13114 I13026 I1466 I13277 I13053 I13712 I13113 I13025 I13915 I13274 I13038 I13711 I13112 I13024 I13914 I13273 I13037 I13710 I13111 I13023 I13913 I13271 I13036 I13702 I13110 I13022 I13912 I13270 I13035 I13701 I13109 I13021 I13911 I13266 I13034 I13700 I13108 I13020 I13910 I13265 I13018 C0028 I13107 I13019 I13314 I13264 I13015 C0024 I13106 I13014 I13313 I13263 I13000 C0020 BioBrick Parts 12 New eporter Parts
105 Advice for Future Competitions June July August Structured introductory two-week curriculum Daily lectures in the mornings and specific lab tutorials in the afternoons Students would model, assemble, and characterize a simple synthetic system such as a single O gate Teaches synthetic biology basics and experimental lab technique as well as providing a solid foundation for initial design work
106 Advice for Future Competitions June July August More milestones and incremental deliverables eport on simple synthetic system
107 Advice for Future Competitions June July August More milestones and incremental deliverables eport on simple synthetic system Preliminary design specification
108 Advice for Future Competitions June July August More milestones and incremental deliverables eport on simple synthetic system Preliminary design specification Interim progress report
109 Advice for Future Competitions June July August More milestones and incremental deliverables eport on simple synthetic system Preliminary design specification Interim progress report Periodic logs kept by each student and lab-group
110 Advice for Future Competitions Inter-team collaboration Periodic conference calls Distribute design specifications and interim reports to all teams Logs managed in online forum accessible by all teams
111 Key Enablers for SynthBio Through our summer experiences we identified three key enablers which will greatly help future work in the field of synthetic biology Assembly Process Device Characterization Standard Operating Conditions
112 Assembly Process emarkable that a group of students with very little biology background was able to build working biological parts relatively quickly Even so, current assembly process placed significant constraints on what was possible Took on average one week per stage When assembly failed very difficult to determine why Assembly is an important research topic Optimize each stage Characterize and model error rates Develop more assembly tools
113 Device Characterization Modeling work is significantly hampered by lack of useful device characterization Device characterization is challenging What do we actually measure? How do we measure it? How do we make measurements repeatable? Accurate device characterization will enable Effective parameterized models More rational design Easier reuse of previously developed parts
114 Operating Conditions Currently no standard operating conditions Strain, media, growth phase are usually documented but they still vary with each experiment Standard conditions enable easier result comparisons Standard operation conditions also make it easier to predict how future systems will behave Standard operating conditions is challenging Difficult to choose a single set of conditions since different experiments have different requirements Continuous culture using chemostat is an attractive possibility but needs more work
115 Acknowledgements Strains + Plasmids Howard Berg (Harvard) Karen Fahrner (Harvard) Michael Elowitz (CalTech) Cell-to-Cell Signaling Advice on Weiss (Princeton) Assembly Caitlin Conboy Jen Braff Advisors Drew Endy andy egistry anger ettberg Gerry Sussman Pam Silver Tom Knight
116 Conclusions We have designed and made strong progress towards building a synchronized chemotactic oscillator The assembly process, device characterization, and standard operating conditions are key enablers which will greatly benefit the field of synthetic biology Cell-to-Cell Synchronization Module Cell Boundary Oscillator Module Chemotaxis Module
117 Conclusions We have designed and made strong progress towards building a synchronized chemotactic oscillator The assembly process, device characterization, and standard operating conditions are key enablers which will greatly benefit the field of synthetic biology Cell-to-Cell Synchronization Module Questions? Cell Boundary Oscillator Module Chemotaxis Module
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