You are on page 1of 26

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

1
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

2
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.

3
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).

4
Our Project: applications

Controlled Gene Expression Trigger: Glucose Sensor:


1) constant level of expression once 1) fluorescent output correlates with
induced; extracellular glucose;
2) fine tuning of the expressed gene, 2) changing the reporter with proper
acting on the glucose concentration. genes will lead to possible in vivo
medical applications.

5
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)

6
Math Model: system

7
Formulas

State Equations

Output Equation

8
Analytical Approach...

9
Numerical Simulator

10
Hysteresis

11
Bistability

Induced
Transcription On
Bistability

Uninduced
Transcription Off

12
Glucose dependence

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


)"

("

'"
GF P in (mpb)

&"

%"

$"

!! " ! #
!" !" !" !"
[Glu] ex (µM )

13
Biodevice: functioning

• Standard conditions (no IPTG)

• IPTG induction conditions

14
Biodevice

15
Composite parts

• pTetR: constitutive promoter


• LacI: transcriptional repressor, binds pLac

16
Composite parts

• pλ: promoter regulated by cI


• RFP: reporter

17
Composite parts

• pLac: promoter regulated by LacI


• cI: transcriptional repressor, binds pλ
• LacY: lactose permease
• GFP: reporter

18
Intermediates

19
Image Acquisition System

20
Image Analysis

• 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

21
Protocol Validation Measures

• Bacteria with pLac-GFP plasmid;


• no induction with IPTG @ OD=1.2;
• segmentation algorithm;
• normalized intensity value.

22
Promoters Characterization

Testing differences in transcription rates


23
Open loop characterization

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

Induction Deinduction

Glu IPTG + Lactose 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.

25
Acknowledgements

It was a great experience!


Thank you!

For funding thanks to:

26

You might also like