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

iGEM 2010 UPO-SEVILLA BACTERIAL CROWDING

Who are we?

Pablo de Olavide University

The simplest idea

• Bacterial Crowding
• Efficient interaction with non-diffusible targets

Bacterial Crowding Project

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

Future applications

Transport Destroy Detect

Smart drugs Bio- remediation

Bio- sensing

Gene Therapy

Division

Detecting non-diffusible signals

Prh system

• f R. solanacearum

The control Fec system

• f E. coli

Signal transduction circuits

Prh circuit Fec circuit Hybrid circuit

Signal OM sensor CM receiver ECF sigma Regulated promoter Plant cell wall signal Plant cell wall signal Fe-citrate PrhA PrhR PrhI PprhJ FecA FecR FecI PfecA FecA-PrhA FecR FecI PfecA Fur High Fe Fur High Fe

Mixed circuit

Plant cell wall signal FecR FecI PfecA Fur High Fe PrhA

Sensing devices

Prh device Fec device Hybrid device Mixed device

J23100 RBS.1 prhA prhI prhR rrnBT1-T7TE RBS.1 RBS.1 J23100 RBS.1 fecA RBS.1 fecI-fecR rrnBT1-T7TE J23100 fecA-prhA rrnBT1-T7TE fecI-fecR RBS.1 RBS.1 J23100 RBS.1 RBS.1 rrnBT1-T7TE prhA fecI-fecR

Hybrid protein

FecA PrhA

Signal peptide TonB Short Domain

N-Terminus C-Terminus

TonB-dependent Receptor Plug TonB-dependent Receptor Domain NH2-terminal extension Conserved GSGL motif

Function of Sensing circuits

Prh circuit Fec circuit Hybrid circuit

Signal OM sensor CM receiver ECF sigma Regulated promoter Plant cell wall signal Plant cell wall signal Fe-citrate PrhA PrhR PrhI PprhJ FecA FecR FecI PfecA FecA-PrhA FecR FecI PfecA Fur High Fe Fur High Fe Chemoattractant synthesis Chemoattractant synthesis Chemoattractant synthesis

Mixed circuit

Plant cell wall signal FecR FecI PfecA Fur High Fe Chemoattractant synthesis PrhA

Chemotaxis

Clockwise (“tumble”)

A A

CheY CheA CheW CheY

P

CheZ FliMNG (flagellar motor) CheA CheW

P

MCP MCP

Chemoattractant

Isochorismate synthase (ICS)

pchB pchA

Isochorismate-pyruvate lyase (IPL)

ICS IPL Chorismate Isochorismate Salicylate (chemoattractant to P. putida G7) Pyruvate

Salicylate

Aspartate-ammonia lyase (AAL)

aspA Fumarate Aspartate (chemoattractant to E. coli) AAL NH3

Aspartate

Assays

Qualitative

Soft Agar Plates Capillary Assay using Microscope

Quantitative

Capillary assays

Chemotaxis Chamber Capillary

Tip chambers Needles 10 ml micropipette’s tips 96-well PVC microplates 1 ml capillary pipettes Flow- chamber 1 ml capillary pipettes

MICROSCOPE SLIDE COVER SLIP MICROCAPILARY BACTERIAL DILUTION

Adler’s Assay

Aspartate Buffer

Adler´s capillary assay. Preparation of capillary assay to look under a microscope.

Adler’s Assay Results

Flow-Chamber Assay

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.

Buffer Aspartate

2000 4000 6000 8000 10000 12000 14000 16000 18000 20000 Channel 1 Channel 2 Channel 3

Cfu/mL

RT Assay

Buffer 10 mM asp

2000 4000 6000 8000 10000 12000 Channel 1 Channel 2 Channel 3

Cfu/mL

30ºC Assay

Buffer 10 mM asp

Buffer 10 mM Asp Asp/Buffer Average 472 8540 18,09 SD 803,71 8366,29 158,75 Buffer 10 Mm Asp Asp/Buffer Average 208,67 7533,33 36,10 SD 68,39 3438,86 9,51

Flow-Chamber Assay Results

Simbiology model

1 2 3

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
• Built devices
• Chemotaxis assays
• Simbiology model

and simulation

New parts submitted

Prh systems prhA BBa_K367000 prhR BBa_K367001 prhI BBa_K367002 PprhJ BBa_K367008 Hybrid protein BBa_K367006 Fec system PfecA BBa_K367009 fecI BBa_K367004 Glutamate synthase gltB BBa_K367011

Thanks!