FlashBacter UPO-Sevilla Team APPLICATIONS Biosensors Killer - PowerPoint PPT Presentation

FlashBacter UPO-Sevilla Team

APPLICATIONS Biosensors Killer proteins production Multiple alleles expression And more…

OVERVIEW BASIC FLIP FLOP IMPROVED FLIP FLOP EPIGENETIC FLIP FLOP MINI TN7 BIOBRICK CREATOR HUMAN PRACTICES

BASIC IMPROVED EPIGENETIC MINITN7 BB.CREATOR H.PRACTICES BASIC FLIP-FLOP

BASIC IMPROVED EPIGENETIC MINITN7 BB.CREATOR H.PRACTICES Basic flip-flop E. coli chassis GFP cI ( ts ) Plac Prm LacI RFP Green State IPTG pulse Heat shock Red State GFP cI ( ts ) Plac Prm LacI RFP

BASIC IMPROVED EPIGENETIC MINITN7 BB.CREATOR H.PRACTICES Characterization Basic flip flop time-course experiment Stability Switch 7000 Stability 6000 Basal levels 5000 Fluorescence/O.D. 4000 IPTG State 3000 2000 42 ºC State 1000 Induction stop 0 0 200 400 600 800 1000 1200 Time (minutes)

BASIC IMPROVED EPIGENETIC MINITN7 BB.CREATOR H.PRACTICES Multiagent Modeling

BASIC IMPROVED EPIGENETIC MINITN7 BB.CREATOR H.PRACTICES Mathematical Modeling REPRESSOR TRANSCRIPTION: Michaelis- DEGRADATION: Menten Kinetics + REPRESSION: Mass Action Kinetics Hill Kinetics TRANSLATION: Michaelis- Menten Kinetics (as Mass Action IPTG INDUCTION: Hill kinetics kinetics) as IPTG binding to repressor proteins

BASIC IMPROVED EPIGENETIC MINITN7 BB.CREATOR H.PRACTICES Mathematical Modeling: Basic vs Improved Basic flip-flop Improved flip-flop Mathematical models Stochastic simulations

BASIC IMPROVED EPIGENETIC MINITN7 BB.CREATOR H.PRACTICES Basic flip-flop Achievements Experimental and mathematical characterization of the transcriptional flip-flop (information included in the registry) Identification of weaknesses Solutions proposed

BASIC IMPROVED EPIGENETIC MINITN7 BB.CREATOR H.PRACTICES IMPROVED FLIP-FLOP

BASIC IMPROVED EPIGENETIC MINITN7 BB.CREATOR H.PRACTICES Improved flip-flop

BASIC IMPROVED EPIGENETIC MINITN7 BB.CREATOR H.PRACTICES Proteolysis Proteolisis is controlled by the adaptor protein levels

BASIC IMPROVED EPIGENETIC MINITN7 BB.CREATOR H.PRACTICES Proteolysis

BASIC IMPROVED EPIGENETIC MINITN7 BB.CREATOR H.PRACTICES asRNA RybB asRNA A double deletion E.coli strain obtained for the adaptor protein and the asRNA. Lambda red protocol (Datsenko & Wanner, 2000)

BASIC IMPROVED EPIGENETIC MINITN7 BB.CREATOR H.PRACTICES Induction time to achieve Stability Induction time to achieve stability

BASIC IMPROVED EPIGENETIC MINITN7 BB.CREATOR H.PRACTICES Stability over time 17h time-course experiment IPTG >> 42º (10h stop induction)>> system evolution Basic Flip-Flop Temperature State Stability Improved Flip-Flop Temperature State Stability 7000 35000 6000 Induction stop 30000 Fluorescence/O.D. 5000 25000 Fluorescence/O.D. 4000 42 ºC State 20000 3000 15000 IPTG State 2000 10000 Induction stop 1000 5000 0 0 0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200 Time (minutes) Time (minutes) Intermediary state Two clearly defined states Both states increase with time Desired state maintained

BASIC IMPROVED EPIGENETIC MINITN7 BB.CREATOR H.PRACTICES Change speed Fluorescence/O.D. Fluorescence/O.D. 160min 110min Time (minutes) Time (minutes) Merge Fluorescence/O.D. Time (minutes) Change speed decreased in 50 min (31%)

BASIC IMPROVED EPIGENETIC MINITN7 BB.CREATOR H.PRACTICES Improved flip-flop Achievements Design and construction of an improved flip-flop Characterization of the improved flip-flop (information included in the registry) Construction of two new E. coli strains by defined deletions 2 new parts submitted

BASIC IMPROVED EPIGENETIC MINITN7 BB.CREATOR H.PRACTICES EPIGENETIC FLIP-FLOP

BASIC IMPROVED EPIGENETIC MINITN7 BB.CREATOR H.PRACTICES Epigenetic flip-flop: how it works

BASIC IMPROVED EPIGENETIC MINITN7 BB.CREATOR H.PRACTICES Docking modeling approach Docking models for the TetR-Swi6 interaction The best three shape docking results are displayed in the top row, while the best shape+electrostatics docking results are shown in the row below.

BASIC IMPROVED EPIGENETIC MINITN7 BB.CREATOR H.PRACTICES Epigenetic flip-flop: how it works

BASIC IMPROVED EPIGENETIC MINITN7 BB.CREATOR H.PRACTICES Epigenetic flip-flop Achievements Design of a novel epigenetic flip-flop Docking simulations performed to check the functionality of the recombinant silencing proteins Construction of the reporter and the compacting module Registration of the first 5 parts for the yeast Schizosaccaromyces pombe model organism in the Registry

BASIC IMPROVED EPIGENETIC MINITN7 BB.CREATOR H.PRACTICES MINI-TN7 TOOL KIT

BASIC IMPROVED EPIGENETIC MINITN7 BB.CREATOR H.PRACTICES Design and function of the miniTn7 Transposase action Site-specific genome Antibiotic resistance integration removal Flp Recombinase action bacterial genome bacterial genome

BASIC IMPROVED EPIGENETIC MINITN7 BB.CREATOR H.PRACTICES miniTn7: Effects of device copy number Single copy conformation to improve the tightening of regulatory circuits

BASIC IMPROVED EPIGENETIC MINITN7 BB.CREATOR H.PRACTICES Advantages of using miniTn7 for BioBrick genome integration Industrial & Safer Tightening Stability environment Host-range organisms applications Stable Suitable for Horizontal MiniTn7 Single copy insertion at Drug selection multiple transfer insertion known not required bacterial tool minimized neutral site hosts Potential for Host-range Plasmid High copy horizontal Not fully Drug selection restricted to number transfer stable required E. coli and Vectors enterics

BASIC IMPROVED EPIGENETIC MINITN7 BB.CREATOR H.PRACTICES Tn7 attachment site conservation

BASIC IMPROVED EPIGENETIC MINITN7 BB.CREATOR H.PRACTICES miniTn7 Characterization Transformation Transposition Transposition Site-specific Delivery efficiency efficiency frequency insertions Hosts methods (transformants (transposants (transposants/ (checked by /ug DNA) /ug DNA) vible cfu) PCR) Mating NA NA 1 x 10 -4 12/12 Pseudomonas putida 6 x 10 9 7 x 10 1 Electroporation NA 10/12 Escherichia Heat-shock 1 x 10 8 4 x 10 2 NA 11/12 coli transformation

BASIC IMPROVED EPIGENETIC MINITN7 BB.CREATOR H.PRACTICES miniTn7 Characterization 6/100 drug-resistance marker excised

BASIC IMPROVED EPIGENETIC MINITN7 BB.CREATOR H.PRACTICES The tool kit

BASIC IMPROVED EPIGENETIC MINITN7 BB.CREATOR H.PRACTICES miniTn7 & flip-flops

BASIC IMPROVED EPIGENETIC MINITN7 BB.CREATOR H.PRACTICES A portable Tn7 attachment site 97/100 cfu got a miniTn7 inside its portable attTn7 after a transposition assay

BASIC IMPROVED EPIGENETIC MINITN7 BB.CREATOR H.PRACTICES miniTn7 Tool Achievements Demonstration of better tightening of regulatory circuits when the device copy number decreases Construction and characterization of a transposon-based tool for chromosome integration Study of Tn7 attachment site level of conservation, obtaining a consensus sequence New section in the Part’s Registry: Genome Integration Tool Kit 10 new plasmids submitted, including miniTn7-flip-flops A portable Tn7 transposon insertion site (submitted and characterized)

BASIC IMPROVED EPIGENETIC MINITN7 BB.CREATOR H.PRACTICES BIOBRICK CREATOR

BASIC IMPROVED EPIGENETIC MINITN7 BB.CREATOR H.PRACTICES How it works

BASIC IMPROVED EPIGENETIC MINITN7 BB.CREATOR H.PRACTICES HUMAN PRACTICE

BASIC IMPROVED EPIGENETIC MINITN7 BB.CREATOR H.PRACTICES Synthetic Biology in High Schools

BASIC IMPROVED EPIGENETIC MINITN7 BB.CREATOR H.PRACTICES Sevilla’s Science Fair

BASIC IMPROVED EPIGENETIC MINITN7 BB.CREATOR H.PRACTICES Tornillos y Genes blog http://tornillosygenes.com

BASIC IMPROVED EPIGENETIC MINITN7 BB.CREATOR H.PRACTICES Best Human Practice Advance, Europe

CONCLUSIONS Studying the existing basic flip flop and identifying its flaws. Development of an improved flip flop (proteolysis and asRNA controls). Experimental characterization of the improved flip flop, proving its advantages A new concept of flip-flop by chromatin remodelation: the epigenetic flip flop . miniTn7 tool : a BioBrick chromosome integration tool kit. A software tool: the BioBrick Creator . Human Practice : High Schools, Science Fairs, Synthetic Biology Blog. 31 new BioBricks and BioBrick compatible plasmids registered 13 physical DNA parts submitted 5 new parts characterized 1 preexisting part characterized

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