Presentación de PowerPoint2011.igem.org/files/presentation/UPO-Sevilla_Championship.pdf · Stability over time 0 1000 2000 3000 4000 5000 6000 7000 0 200 400 600 800 1000 1200. Time

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 FLIP-FLOP

H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC

Basic flip-flop

Green State

GFP cI (ts) Plac Prm LacI RFP

GFP cI (ts) Plac Prm LacI RFP

He

at sho

ck

IPTG

pu

lse

Red State

E. coli chassis


Characterization

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ore

sce

nce

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Time (minutes)

IPTG State

42 ºC State


Induction stop

Basic flip flop time-course experiment

Stability

Stability

Basal levels

Switch

Multiagent Modeling


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


Basic flip-flop Improved flip-flop

Mathematical Modeling: Basic vs Improved

Sto

chas

tic

sim

ula

tio

ns

Mat

hem

atic

al m

od

els


Basic flip-flop Achievements

Experimental and mathematical characterization of the transcriptional flip-flop

(information included in the registry)

Identification of weaknesses

Solutions proposed


IMPROVED FLIP-FLOP


Improved flip-flop


Proteolysis


Proteolisis is controlled by the adaptor protein levels

Proteolysis


asRNA

RybB asRNA


A double deletion E.coli strain obtained for the adaptor protein and the asRNA.

Lambda red protocol (Datsenko & Wanner, 2000)

Induction time to achieve stability

Induction time to achieve Stability


Stability over time

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Time (minutes)

Basic Flip-Flop Temperature State Stability

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25000

30000

35000

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Time (minutes)

Improved Flip-Flop Temperature State Stability

42 ºC State

IPTG State


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

Change speed


110min

Merge

160min

Time (minutes) Time (minutes)

Time (minutes)

Change speed decreased in 50 min (31%)

Flu

ore

scen

ce/O

.D.

Flu

ore

scen

ce/O

.D.

Flu

ore

scen

ce/O

.D.

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


EPIGENETIC FLIP-FLOP


Epigenetic flip-flop: how it works


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.


Epigenetic flip-flop: how it works


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


MINI-TN7 TOOL KIT


Design and function of the miniTn7

Site-specific genome

integration

Transposase action

Flp Recombinase

action

Antibiotic resistance removal

bacterial genome bacterial genome


miniTn7: Effects of device copy number

Single copy conformation to improve the tightening of regulatory circuits


Tightening

Safer organisms

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


Tn7 attachment site conservation


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


6/100 drug-resistance marker excised

miniTn7 Characterization


The tool kit


miniTn7 & flip-flops


A portable Tn7 attachment site

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


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)


BIOBRICK CREATOR


How it works


HUMAN PRACTICE


Synthetic Biology in High Schools


Sevilla’s Science Fair


Tornillos y Genes blog

http://tornillosygenes.com


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

Thank you!!

Documents

Presentación de PowerPoint2011.igem.org/files/presentation/UPO-Sevilla_Championship.pdf · Stability over time 0 1000 2000 3000 4000 5000 6000 7000 0 200 400 600 800 1000 1200. Time