Overview Introduction Biological model Physical and Chemical - - PowerPoint PPT Presentation

• Introduction
• Biological model
• Physical and Chemical properties of our glue
• Mathematical modeling
• Conclusions

Overview

Introduction

 Model : Caulobacter crescentus



GRAM –



Motile or sessile cell



Sessile stage : stalk

• holdfast structure
• elastic & adhesive
• Tensile strength

68N/mm2 (!) how to extract it ?

INTRO BIOLOGICAL MODEL PROPERTIES MATH MODELING CONCLUSIONS

 Holdfast synthesis pathway in C. crescentus



→ holdfast biosynthesis



→ holdfast export



→ holdfast anchoring Aim: transfer C. crescentus holdfast biosynthesis to E. coli

• E. coli has homolog proteins

hfsG & hfsH genes insertion

INTRO BIOLOGICAL MODEL PROPERTIES MATH MODELING CONCLUSIONS

• E. Toh et al. Journal of Bacteriology 2008

 GluColi was born…and stuck !

INTRO BIOLOGICAL MODEL PROPERTIES MATH MODELING CONCLUSIONS

INTRO BIOLOGICAL MODEL PROPERTIES MATH MODELING CONCLUSIONS

MATERIALS & METHODS

Biological model

 Materials and Methods



Final plasmid

INTRO BIOLOGICAL MODEL PROPERTIES MATH MODELING CONCLUSIONS

MATERIALS & METHODS



Ligations



Final plasmid

 Safety : GluColi and a free-antibiotics alternative



Antibiotics : selectable markers in industry



Why ? plasmid-free cells grow faster than plasmid-carrying cells recombinant molecules production

INTRO BIOLOGICAL MODEL PROPERTIES MATH MODELING CONCLUSIONS

SAFETY



Solution : StabyTM system → poison-antidote genes, natural plasmid maintenance → poison : CcdB protein → antidote: CcdA protein → ccdB in E. coli chromosome → ccdA in the plasmid DNA 2 bricks (ccdA) submitted

http://www.delphigenetics.com

 GluColi, engineering a biological circuit



Objective : producing the glue at a specific point



How ? Three main phases : 1. Attracting GluColi to the target point

• 2. Glue production at the leaking point
• 3. Preventing GluColi proliferation

INTRO BIOLOGICAL MODEL PROPERTIES MATH MODELING CONCLUSIONS

CIRCUIT

INTRO BIOLOGICAL MODEL PROPERTIES MATH MODELING CONCLUSIONS

CIRCUIT



Simplified model

• 1. Attracting GluColi

chemotaxis Chemotaxis

http://2008.igem.org/Team:Heidelberg

QS

• 2. Synthesizing the glue

quorum sensing

LuxR LuxR LuxR LuxR

• 3. Preventing proliferation

http://web.virginia.edu/Heidi/chapter30/chp30.htm

INTRO BIOLOGICAL MODEL PROPERTIES MATH MODELING CONCLUSIONS

CIRCUIT

Chemotaxis Quorum sensing



Detailed circuit → http://2009.igem.org/Team:ULB-Brussels

INTRO BIOLOGICAL MODEL PROPERTIES MATH MODELING CONCLUSIONS

CIRCUIT

Physical & chemical properties

 Fluorescence microscopy



Temperature incubation

• At 37°C
• → Isolated bacteria
• At 27°C

→ Higher agglomeration

INTRO BIOLOGICAL MODEL PROPERTIES MATH MODELING CONCLUSIONS



Presence of an inclusion Visible inclusion in a transformed bacterium Function: containing the glue ?

INTRO BIOLOGICAL MODEL PROPERTIES MATH MODELING CONCLUSIONS

 Wet strength

→ Inhibition of the adhesive effect

INTRO BIOLOGICAL MODEL PROPERTIES MATH MODELING CONCLUSIONS

 Wet strength

→ Inhibition of the adhesive effect → Assumptions :



Homolog hfs genes



Exogenes → transferring all the hfs genes



Anchoring proteins → transferring the hfa genes



Composition of the glue

INTRO BIOLOGICAL MODEL PROPERTIES MATH MODELING CONCLUSIONS

 Tensile strength

< 68N/mm2



Technical problems

• unappropriate materials
• humidity problems
• no purification



Lack of anchoring genes

INTRO BIOLOGICAL MODEL PROPERTIES MATH MODELING CONCLUSIONS

INTRO BIOLOGICAL MODEL PROPERTIES MATH MODELING CONCLUSIONS

Hill equations



activator



repressor Dynamical equations

Mathematical modeling

 First model → testing the logic of the circuit



LuxR + HSL quickly formed



No « cell death » Results:



3 steady states : - 2 stable states

• 1 unstable

without glue synthesis with glue synthesis



BUT : hard to regulate with IPTG

INTRO BIOLOGICAL MODEL PROPERTIES MATH MODELING CONCLUSIONS

 2nd model

→ taking into account :



complex LuxR-HSL formation



« cell death » Results :



very sensitive to IPTG

INTRO BIOLOGICAL MODEL PROPERTIES MATH MODELING CONCLUSIONS

 3rd model : toggle switch

Results :



better robustness

INTRO BIOLOGICAL MODEL PROPERTIES MATH MODELING CONCLUSIONS

Conclusions

 Production of an adhesive

BUT more experiments are necessary → strength → glue characterization

 Parts submission  Biological circuit design

controlled glue production

 Mathematical modeling explored the bistability of

the circuit but further research is necessary

INTRO BIOLOGICAL MODEL PROPERTIES MATH MODELING CONCLUSIONS

Acknowledgement

 Special thanks to



Bacterial Genetics and Physiology Laboratory, ULB



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