Presentación de PowerPoint2011.igem.org/files/presentation/UPO-Sevilla_Championship.pdf ·...
Transcript of Presentación de PowerPoint2011.igem.org/files/presentation/UPO-Sevilla_Championship.pdf ·...
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
H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC
Characterization
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ore
sce
nce
/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
Multiagent Modeling
H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC
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
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
H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC
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
IMPROVED FLIP-FLOP
H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC
Improved flip-flop
H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC
Proteolysis
H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC
Proteolisis is controlled by the adaptor protein levels
Proteolysis
H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC
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)
Induction time to achieve stability
Induction time to achieve Stability
H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC
Stability over time
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ore
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Time (minutes)
Basic Flip-Flop Temperature State Stability
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35000
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ore
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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
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%)
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
H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC
EPIGENETIC FLIP-FLOP
H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC
Epigenetic flip-flop: how it works
H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC
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
Epigenetic flip-flop: how it works
H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC
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
H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC
MINI-TN7 TOOL KIT
H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC
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
miniTn7: Effects of device copy number
Single copy conformation to improve the tightening of regulatory circuits
H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC
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
H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC
Tn7 attachment site conservation
H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC
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
6/100 drug-resistance marker excised
miniTn7 Characterization
H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC
The tool kit
H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC
miniTn7 & flip-flops
H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC
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
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
BIOBRICK CREATOR
H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC
How it works
H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC
HUMAN PRACTICE
H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC
Synthetic Biology in High Schools
H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC
Sevilla’s Science Fair
H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC
Tornillos y Genes blog
http://tornillosygenes.com
H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC
Best Human Practice Advance, Europe
H.PRACTICES BB.CREATOR MINITN7 EPIGENETIC IMPROVED BASIC
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!!