BACTERIAL CROWDING iGEM 2010 UPO-SEVILLA Who are we? Pablo de - PowerPoint PPT Presentation

BACTERIAL CROWDING iGEM 2010 UPO-SEVILLA

Who are we? Pablo de Olavide University

The simplest idea • Bacterial Crowding • Efficient interaction with non-diffusible targets

Bacterial Crowding Project Interaction between Arabidopsis Soft agarose plate assay of chemotactic taliana and Ralstonia solanacearum response to aspartate and serine gradients by Escherichia coli

Future applications Smart Gene drugs Therapy Transport Detect Destroy Bio- Bio- sensing remediation

Division

Detecting non-diffusible signals Prh system of R. solanacearum

The control Fec system of E. coli

Signal transduction circuits Fec circuit Hybrid circuit Mixed circuit Prh circuit Plant cell wall Plant cell wall Plant cell wall Fe-citrate Signal signal signal signal FecA FecA-PrhA OM sensor PrhA PrhA FecR CM receiver PrhR FecR FecR FecI ECF sigma PrhI FecI FecI Fur Fur Fur High Fe High Fe High Fe Regulated P fecA P prhJ P fecA P fecA promoter

Sensing devices prhA prhI prhR J23100 Prh device RBS.1 RBS.1 RBS.1 rrnB T1-T7TE J23100 fecA fecI-fecR Fec device RBS.1 rrnB T1-T7TE RBS.1 J23100 fecA-prhA fecI-fecR Hybrid device RBS.1 RBS.1 rrnB T1-T7TE J23100 prhA fecI-fecR Mixed device RBS.1 RBS.1 rrnB T1-T7TE

Hybrid protein PrhA FecA NH2-terminal Conserved extension GSGL motif C-Terminus N-Terminus Signal peptide TonB-dependent TonB Short TonB-dependent Receptor Domain Domain Receptor Plug

Function of Sensing circuits Fec circuit Prh circuit Hybrid circuit Mixed circuit Plant cell wall Plant cell wall Plant cell wall Fe-citrate Signal signal signal signal FecA FecA-PrhA OM sensor PrhA PrhA PrhR FecR FecR FecR CM receiver FecI ECF sigma PrhI FecI FecI Fur Fur Fur High Fe High Fe High Fe Regulated P fecA P prhJ P fecA P fecA promoter Chemoattractant Chemoattractant Chemoattractant Chemoattractant synthesis synthesis synthesis synthesis

Chemotaxis CheW P CheA CheY MCP A FliMNG CheZ (flagellar motor) CheW P CheA A MCP CheY Clockwise (“ tumble ”)

Chemoattractant Salicylate Aspartate aspA pchB pchA Isochorismate-pyruvate lyase Isochorismate synthase Aspartate-ammonia lyase (AAL) (IPL) (ICS) ICS Fumarate Isochorismate NH 3 Chorismate AAL IPL Pyruvate Salicylate (chemoattractant Aspartate (chemoattractant to E. coli ) to P. putida G7)

Assays Soft Agar Plates Qualitative Capillary Assay using Microscope Chemotaxis Capillary Chamber Needles Tip chambers Quantitative 10 m l micropipette’s tips Capillary assays 96-well PVC 1 m l capillary pipettes microplates Flow- 1 m l capillary pipettes chamber

Adler’s Assay BACTERIAL MICROCAPILARY DILUTION COVER MICROSCOPE SLIP SLIDE Buffer Aspartate Adler´s capillary assay. Preparation of capillary assay to look under a microscope.

Adler’s Assay Results

Flow-Chamber Assay Buffer Aspartate Flow-chamber Capillary Assay . A three channel flow- chamber, which acts as chemotaxis chamber, with capillaries fixed in each end of one channel. It was observed with a fluorescence microscope.

Flow-Chamber Assay Results RT Assay 30ºC Assay 20000 12000 18000 10000 16000 14000 8000 12000 Cfu/mL Cfu/mL 10000 Buffer 6000 Buffer 8000 10 mM asp 10 mM asp 4000 6000 4000 2000 2000 0 0 Channel 1 Channel 2 Channel 3 Channel 1 Channel 2 Channel 3 Buffer 10 mM Asp Asp/Buffer Buffer 10 Mm Asp Asp/Buffer Average 208,67 7533,33 36,10 Average 472 8540 18,09 SD 68,39 3438,86 9,51 SD 803,71 8366,29 158,75

Simbiology model 1 3 2

Outputs vs inputs

Sensibility The steady-state response is quite insensitive to the variaton of parameters. The influence of the parameters is local

Sensibility The influence of the parameters on the dynamics of the system is also local

Simulation

Concluding remarks • Biobricks submitted New parts submitted Prh systems • Built devices prhA BBa_K367000 prhR BBa_K367001 prhI BBa_K367002 P prhJ BBa_K367008 • Chemotaxis assays Hybrid protein BBa_K367006 Fec system P fecA BBa_K367009 • Simbiology model fecI BBa_K367004 Glutamate synthase and simulation gltB BBa_K367011

Thanks!