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

Transcription

1

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

3 Introduction Model : Caulobacter crescentus GRAM Motile or sessile cell Sessile stage : stalk holdfast structure elastic & adhesive Tensile strength 68N/mm 2 (!) how to extract it?

4 Holdfast synthesis pathway in C. crescentus holdfast biosynthesis holdfast export holdfast anchoring E. Toh et al. Journal of Bacteriology 2008 Aim: transfer C. crescentus holdfast biosynthesis to E. coli E. coli has homolog proteins hfsg & hfsh genes insertion

5 GluColi was born and stuck!

6 MATERIALS & METHODS Biological model Materials and Methods Final plasmid

7 MATERIALS & METHODS Ligations Final plasmid

8 SAFETY Safety : GluColi and a free-antibiotics alternative Antibiotics : selectable markers in industry Why Solution? : Staby TM system plasmid-free poison-antidote cells genes, grow faster natural than plasmid-carrying maintenancecells poison recombinant : CcdB protein molecules production antidote: CcdA protein ccdb in E. coli chromosome ccda in the plasmid DNA 2 bricks (ccda) submitted

9 CIRCUIT 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

10 CIRCUIT Simplified model Chemotaxis 1. Attracting GluColi 2. Synthesizing chemotaxis the glue 3. Preventing quorum proliferation sensing LuxR LuxR QS LuxR LuxR

11 CIRCUIT Chemotaxis Quorum sensing

12 CIRCUIT Detailed circuit

13 Physical & chemical properties Fluorescence microscopy Temperature incubation At 37 C Isolated bacteria At 27 C Higher agglomeration

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

15 Wet strength Inhibition of the adhesive effect

16 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

17 Tensile strength < 68N/mm 2 Technical problems - unappropriate materials - humidity problems - no purification Lack of anchoring genes

18 Mathematical modeling First model testing the logic of the circuit LuxR + HSL quickly formed No «cell death» Hill Dynamical equations equations Results: activator 3 steady states : - 2 stable states without glue synthesis with glue synthesis - 1 unstable repressor BUT : hard to regulate with IPTG

19 2 nd model taking into account : complex LuxR-HSL formation «cell death» Results : very sensitive to IPTG

20 3 rd model : toggle switch Results : better robustness

21 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

22 Special thanks to Acknowledgement Bacterial Genetics and Physiology Laboratory, ULB Sponsors :

23