Overview Introduction Biological model Physical and Chemical - PowerPoint PPT Presentation

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

I NTRO B IOLOGICAL MODEL P ROPERTIES M ATH MODELING C ONCLUSIONS 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 ?

I NTRO B IOLOGICAL MODEL P ROPERTIES M ATH MODELING C ONCLUSIONS  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

I NTRO B IOLOGICAL MODEL P ROPERTIES M ATH MODELING C ONCLUSIONS  GluColi was born … and stuck !

I NTRO B IOLOGICAL MODEL P ROPERTIES M ATH MODELING C ONCLUSIONS M ATERIALS & M ETHODS Biological model  Materials and Methods Final plasmid 

I NTRO B IOLOGICAL MODEL P ROPERTIES M ATH MODELING C ONCLUSIONS M ATERIALS & M ETHODS Ligations Final plasmid  

I NTRO B IOLOGICAL MODEL P ROPERTIES M ATH MODELING C ONCLUSIONS S AFETY  Safety : GluColi and a free-antibiotics alternative Antibiotics : selectable markers in industry  Solution : Staby TM system Why ?   → poison-antidote genes, natural plasmid maintenance plasmid-free cells grow faster than plasmid-carrying cells → poison : CcdB protein recombinant molecules production → antidote: CcdA protein → ccdB in E. coli chromosome → ccdA in the plasmid DNA 2 bricks ( ccdA ) submitted http://www.delphigenetics.com

I NTRO B IOLOGICAL MODEL P ROPERTIES M ATH MODELING C ONCLUSIONS C IRCUIT  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

I NTRO B IOLOGICAL MODEL P ROPERTIES M ATH MODELING C ONCLUSIONS C IRCUIT Simplified model  Chemotaxis 1. Attracting GluColi chemotaxis 2. Synthesizing the glue quorum sensing 3. Preventing proliferation LuxR LuxR http://2008.igem.org/Team:Heidelberg QS LuxR LuxR http://web.virginia.edu/Heidi/chapter30/chp30.htm

I NTRO B IOLOGICAL MODEL P ROPERTIES M ATH MODELING C ONCLUSIONS C IRCUIT Chemotaxis Quorum sensing

I NTRO B IOLOGICAL MODEL P ROPERTIES M ATH MODELING C ONCLUSIONS C IRCUIT Detailed circuit → http://2009.igem.org/Team:ULB-Brussels 

I NTRO B IOLOGICAL MODEL P ROPERTIES M ATH MODELING C ONCLUSIONS Physical & chemical properties  Fluorescence microscopy Temperature incubation  At 37°C • → Isolated bacteria • At 27°C • → Higher agglomeration

I NTRO B IOLOGICAL MODEL P ROPERTIES M ATH MODELING C ONCLUSIONS Presence of an inclusion  Visible inclusion in a transformed bacterium Function: containing the glue ?

I NTRO B IOLOGICAL MODEL P ROPERTIES M ATH MODELING C ONCLUSIONS  Wet strength → Inhibition of the adhesive effect

I NTRO B IOLOGICAL MODEL P ROPERTIES M ATH MODELING C ONCLUSIONS  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 

I NTRO B IOLOGICAL MODEL P ROPERTIES M ATH MODELING C ONCLUSIONS  Tensile strength < 68N/mm 2 Technical problems  unappropriate materials - humidity problems - no purification - Lack of anchoring genes 

I NTRO B IOLOGICAL MODEL P ROPERTIES M ATH MODELING C ONCLUSIONS Mathematical modeling  First model → testing the logic of the circuit LuxR + HSL quickly formed  No « cell death »  Hill equations Dynamical equations Results: activator  3 steady states : - 2 stable states  without glue synthesis with glue synthesis - 1 unstable repressor  BUT : hard to regulate with IPTG 

I NTRO B IOLOGICAL MODEL P ROPERTIES M ATH MODELING C ONCLUSIONS  2 nd model → taking into account : complex LuxR-HSL formation  « cell death »  Results : very sensitive to IPTG 

I NTRO B IOLOGICAL MODEL P ROPERTIES M ATH MODELING C ONCLUSIONS  3 rd model : toggle switch Results : better robustness 

I NTRO B IOLOGICAL MODEL P ROPERTIES M ATH MODELING C ONCLUSIONS 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

I NTRO B IOLOGICAL MODEL P ROPERTIES M ATH MODELING C ONCLUSIONS Acknowledgement  Special thanks to Bacterial Genetics and Physiology Laboratory, ULB  Sponsors : 