A Synthetic Genetic Circuit To Implement A Schmitt Trigger

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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Our team

Undergraduate Students Silvia Tamarri • Michela Mirri Francesca Buganè • Guido Costa Francesco Pasqualini • Iros Barozzi PhD student Alice Pasini Instructors Silvio Cavalcanti • Francesca Ceroni Christine Nardini Advisors Emanuele Giordano Marco Tartagni

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Our Project 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.

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Lac Operon

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).

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Our Project: applications

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.

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Mathematical Model

“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)

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Math Model: system

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Formulas

State Equations

Output Equation

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Analytical Approach...

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Numerical Simulator

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Hysteresis

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Bistability

Induced Transcription On

Bistability Uninduced Transcription Off

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Glucose dependence [Glu] ex − GF P in Static Characteristic ([I P T G]ex = 1 mM )

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GF P in (mpb)

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[Glu] ex (µM )

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Biodevice: functioning • Standard conditions (no IPTG)

• IPTG induction conditions

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Biodevice

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Composite parts

• pTetR: constitutive promoter • LacI: transcriptional repressor, binds pLac

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Composite parts

• pλ: promoter regulated by cI • RFP: reporter

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Composite parts

• pLac: promoter regulated by LacI • cI: transcriptional repressor, binds pλ • LacY: lactose permease • GFP: reporter 18

Intermediates

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Image Acquisition System

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Image Analysis

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Images of fluorescent bacteria

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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

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Output: fluorescence intensity

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Protocol Validation Measures • Bacteria with pLac-GFP plasmid; • no induction with IPTG @ OD=1.2; • segmentation algorithm; • normalized intensity value.

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Promoters Characterization

Testing differences in transcription rates 23

Open loop characterization Intermediate device pTetR-LacI-pLac-GFP: open loop characterization. Induction Glu

IPTG + Lactose

Deinduction Glu

Device working as the mathematical model predicted 24

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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Acknowledgements It was a great experience! Thank you! For funding thanks to:

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