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

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

Mar 19, 2023 320 likes •773 views

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

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FlashBacter

UPO-Sevilla Team

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APPLICATIONS

Biosensors Killer proteins production Multiple alleles expression And more…

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OVERVIEW

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

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BASIC FLIP-FLOP

H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC

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Basic flip-flop

Green State

GFP cI (ts) Plac Prm LacI RFP GFP cI (ts) Plac Prm LacI RFP Heat shock IPTG pulse

Red State

  • E. coli chassis

H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC

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Characterization

1000 2000 3000 4000 5000 6000 7000 200 400 600 800 1000 1200 Fluorescence/O.D. Time (minutes)

IPTG State 42 ºC State

H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC Induction stop

Basic flip flop time-course experiment

Stability Stability Basal levels Switch

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Multiagent Modeling

H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC

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TRANSCRIPTION: Michaelis- Menten Kinetics + REPRESSION: Hill Kinetics IPTG INDUCTION: Hill kinetics as IPTG binding to repressor proteins TRANSLATION: Michaelis- Menten Kinetics (as Mass Action kinetics) REPRESSOR DEGRADATION: Mass Action Kinetics

Mathematical Modeling

H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC

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Basic flip-flop Improved flip-flop Mathematical Modeling: Basic vs Improved

Stochastic simulations Mathematical models H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC

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Basic flip-flop Achievements

Experimental and mathematical characterization of the transcriptional flip-flop (information included in the registry) Identification of weaknesses Solutions proposed

H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC

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IMPROVED FLIP-FLOP

H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC

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Improved flip-flop

H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC

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Proteolysis

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

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Proteolysis

H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC

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asRNA

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

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Induction time to achieve stability

Induction time to achieve Stability

H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC

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Stability over time

1000 2000 3000 4000 5000 6000 7000 200 400 600 800 1000 1200 Fluorescence/O.D. Time (minutes)

Basic Flip-Flop Temperature State Stability

5000 10000 15000 20000 25000 30000 35000 200 400 600 800 1000 1200 Fluorescence/O.D. Time (minutes)

Improved Flip-Flop Temperature State Stability 42 ºC State IPTG State H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC

Induction stop

IPTG >> 42º (10h stop induction)>> system evolution 17h time-course experiment Intermediary state Both states increase with time Two clearly defined states Desired state maintained

Induction stop

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Change speed

H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC

110min

Merge

160min Time (minutes) Time (minutes)

Time (minutes)

Change speed decreased in 50 min (31%)

Fluorescence/O.D. Fluorescence/O.D. Fluorescence/O.D.

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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

H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC

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EPIGENETIC FLIP-FLOP

H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC

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Epigenetic flip-flop: how it works

H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC

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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. H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC

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Epigenetic flip-flop: how it works

H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC

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Epigenetic flip-flop Achievements

Design of a novel epigenetic flip-flop Docking simulations performed to check the functionality

  • f 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

H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC

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MINI-TN7 TOOL KIT

H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC

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Design and function of the miniTn7

Site-specific genome integration

Transposase action Flp Recombinase action

Antibiotic resistance removal

bacterial genome bacterial genome

H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC

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miniTn7: Effects of device copy number Single copy conformation to improve the tightening of regulatory circuits

H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC

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Tightening Safer

  • rganisms

Stability Industrial & environment applications Host-range MiniTn7 tool

Single copy insertion Horizontal transfer minimized Stable insertion at known neutral site Drug selection not required Suitable for multiple bacterial hosts

Plasmid Vectors

High copy number Potential for horizontal transfer Not fully stable Drug selection required Host-range restricted to

  • E. coli and

enterics

Advantages of using miniTn7 for BioBrick genome integration

H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC

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Tn7 attachment site conservation

H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC

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miniTn7 Characterization

Hosts Delivery methods Transformation efficiency

(transformants /ug DNA)

Transposition efficiency

(transposants /ug DNA)

Transposition frequency

(transposants/ vible cfu)

Site-specific insertions

(checked by PCR) Pseudomonas putida Mating NA NA 1 x 10-4 12/12 Electroporation 6 x 109 7 x 101 NA 10/12 Escherichia coli Heat-shock transformation 1 x 108 4 x 102 NA 11/12

H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC

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6/100 drug-resistance marker excised miniTn7 Characterization

H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC

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The tool kit

H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC

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miniTn7 & flip-flops

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A portable Tn7 attachment site

97/100 cfu got a miniTn7 inside its portable attTn7 after a transposition assay

H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC

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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)

H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC

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BIOBRICK CREATOR

H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC

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How it works

H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC

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HUMAN PRACTICE

H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC

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Synthetic Biology in High Schools

H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC

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Sevilla’s Science Fair

H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC

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Tornillos y Genes blog

http://tornillosygenes.com

H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC

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Best Human Practice Advance, Europe

H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC

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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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Thank you!!