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A synthetic genetic circuit to

implement a Schmitt Trigger in
E.coli
Alma Mater Studiorum - Bologna University
Cellular and Molecular Engineering Laboratory
II Faculty of Engineering - Cesena Campus
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Undergraduate Students
Silvia Tamarri • Michela Mirri
Francesca Buganè • Guido Costa
Francesco Pasqualini • Iros Barozzi
PhD student
Alice Pasini
Our team
Instructors
Silvio Cavalcanti • Francesca Ceroni
Christine Nardini
Advisors
Emanuele Giordano
Marco Tartagni
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To genetically engineer E.coli to reproduce the behaviour of the Schmitt Trigger.
• The device can switch on and off at two different thresholds (robustness).
• Hysteresis and bistability are well known features of this kind of systems.
Our Project
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In E.coli the Lac Promoter (pLac) is controlled by 2 inputs:

IPTG (lactose analog) inactivates Lac repressor and transcription starts;

glucose controls the transcription ratio.
Once induced, Lac permease increases IPTG uptake (positive feedback).
Lac Operon
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Controlled Gene Expression Trigger:
1) constant level of expression once
induced;
2) fine tuning of the expressed gene,
acting on the glucose concentration.
Glucose Sensor:
1) fluorescent output correlates with
extracellular glucose;
2) changing the reporter with proper
genes will lead to possible in vivo
medical applications.
Our Project: applications
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“Theory is when we know everything but
nothing works. Practice is when
everything work but no one know why.
Anyway we usually take both: nothing
work and no one know why”
(Albert Einstein)
Mathematical Model
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Math Model: system
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State Equations
Output Equation
Formulas
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Analytical Approach...
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Numerical Simulator
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Hysteresis
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Bistability
Bistability
Induced
Transcription On
Uninduced
Transcription Off
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10
!1
10
0
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1
10
2
30
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[Glu]
ex
− GFP
in
Static Characteristic ([IPT G]
ex
= 1 mM)
[Glu]
ex
(µM)
G
F
P
i
n
(
m
p
b
)
Glucose dependence
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• Standard conditions (no IPTG)
• IPTG induction conditions
Biodevice: functioning
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Biodevice
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• pTetR: constitutive promoter
• LacI: transcriptional repressor, binds pLac
Composite parts
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• pλ: promoter regulated by cI
• RFP: reporter
Composite parts
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• pLac: promoter regulated by LacI
• cI: transcriptional repressor, binds pλ
• LacY: lactose permease
• GFP: reporter
Composite parts
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Intermediates
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Image Acquisition System
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Images of fluorescent bacteria

Segmentation algorithm in Matlab:
1. Morphological top hat filtering
2. Watershed algorithm
3. Total fluo intensity over pixels
recognised as bacteria
4. Division by bacteria area

Output: fluorescence intensity
Image Analysis
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• Bacteria with pLac-GFP plasmid;
• no induction with IPTG @ OD=1.2;
• segmentation algorithm;
• normalized intensity value.
Protocol Validation Measures
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Promoters Characterization
Testing differences in transcription rates
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Open loop characterization
Intermediate device pTetR-LacI-pLac-GFP: open loop characterization.
Device working as the mathematical model predicted
Induction Deinduction
Glu Glu IPTG + Lactose
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Concluding Remarks
Preliminary results on the intermediates are consistent with the design
of the device.
TO DO:
• characterization of Lac permease feedback;
• enhanced characterization of pTet promoter as constitutive Lac
repressor producer.
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It was a great experience!
Thank you!
For funding thanks to:
Acknowledgements
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