Renate De Smedt Eva Dhaene Tine Goovaerts Jules Haezebrouck Tim - - PowerPoint PPT Presentation

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Renate De Smedt Eva Dhaene Tine Goovaerts Jules Haezebrouck Tim - - PowerPoint PPT Presentation

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Eline De Schutter Renate De Smedt Eva Dhaene Tine Goovaerts Jules Haezebrouck Tim Hebbrecht Marlies Verschuuren Prof. Dr. ir. Marjan De Mey ir. Pieter Coussement ir. Frederik De Bruyn ir. Gert Peters Charlotte Verniers TEAM UGENT

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Eline De Schutter Renate De Smedt Eva D’haene Tine Goovaerts Jules Haezebrouck Tim Hebbrecht Marlies Verschuuren

  • Prof. Dr. ir. Marjan De Mey
  • ir. Pieter Coussement
  • ir. Frederik De Bruyn
  • ir. Gert Peters

Charlotte Verniers

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12/10/2013

IGEM UGENT TEAM

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12/10/2013

IGEM UGENT TEAM

iGEM TEAM UGENT - BIOBASE EUROPE PILOT PLANT

The team THE TEAM

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iGEM TEAM UGENT - BIOBASE EUROPE PILOT PLANT

PROBLEM

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IGEM UGENT TEAM

Plasmid-based high gene expression

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PROBLEM OUR MODEL RESULTS FUTURE

Metabolic burden Genetic instability Productivity loss

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IGEM UGENT TEAM

CIChE (Chemically inducable chromosomal evolution)

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PROBLEM OUR MODEL RESULTS FUTURE

GOI AR GOI AR

= Gene of interest = Antibiotic resistance Add antibiotics (depends on marker gene) antibiotics

n

GOI AR GOI AR

Transduction = Homologous region

n

GOI GOI AR AR

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IGEM UGENT TEAM

  • A. Yes
  • B. No

iGEM TEAM UGENT - BIOBASE EUROPE PILOT PLANT

Did you ever dwell on the use of antibiotic resistance genes during your iGEM project?

7

Yes No

54% 46%

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IGEM UGENT TEAM

What do scientists think?

Do you ever dwell on the use of antibiotic resistance genes during your research?

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PROBLEM OUR MODEL RESULTS FUTURE

Every time I use them (7%) Often (17%) Sometimes (30%) Never (33%) Other (13%) Never (33%)

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IGEM UGENT TEAM

What does the public think?

Do you worry about increasing antibiotic resistance in bacteria?

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No Yes

PROBLEM OUR MODEL RESULTS FUTURE

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ALTERNATIVES

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IGEM UGENT TEAM

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Yamaguchi et al.,2011

PROBLEM OUR MODEL RESULTS FUTURE

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IGEM UGENT TEAM

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PROBLEM OUR MODEL RESULTS FUTURE

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IGEM UGENT TEAM

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PROBLEM OUR MODEL RESULTS FUTURE

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OUR MODEL

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IGEM UGENT TEAM

Our model for CIChE

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recA ccdB T7 ccdA gfp T7 RNAP ccdA gfp λ Red

PROBLEM OUR MODEL RESULTS FUTURE

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IGEM UGENT TEAM

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ccdB T7 CcdB ccdA gfp CcdA CcdA ccdA gfp

IPTG

CcdB ccdB T7 CcdB ccdA gfp CcdA CcdB

IPTG

ccdA gfp ccdA gfp

T7 Pol T7 Pol

CcdB CcdB CcdA CcdA RecA RecA

T7 RNAP T7 RNAP recA recA

PROBLEM OUR MODEL RESULTS FUTURE

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EXPERIMENTS

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IGEM UGENT TEAM

Experiment 1

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Knock-in

gfp ccdA

PROBLEM OUR MODEL RESULTS FUTURE

T7 RNAP T7 RNAP

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IGEM UGENT TEAM

Experiment 2

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Construction ccdB plasmid

ccdB ccdB T7 PROBLEM OUR MODEL RESULTS FUTURE

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IGEM UGENT TEAM

Experiment 3

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Transformation

gfp ccdA

ccdB T7 PROBLEM OUR MODEL RESULTS FUTURE

T7 RNAP

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IGEM UGENT TEAM

Experiment 4

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CIChE

ccdB T7 gfp ccdA gfp ccdA gfp ccdA n CcdA-CcdB complex

IPTG

PROBLEM OUR MODEL RESULTS FUTURE

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IGEM UGENT TEAM

Experiment 4

CIChE

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0mM IPTG 0,01 mM IPTG 0,05 mM IPTG 0,1 mM IPTG

…

PROBLEM OUR MODEL RESULTS FUTURE

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IGEM UGENT TEAM

Experiment 5

Evaluation of strains

Optical Density (OD600) Fluorescence (F)

F/OD600

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PROBLEM OUR MODEL RESULTS FUTURE

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RESULTS

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IGEM UGENT TEAM

Results

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PROBLEM OUR MODEL RESULTS FUTURE F/OD600

Copy n° 5 Copy n° 10

IPTG IPTG IPTG F/OD600

Copy n° 15-20 Reference strain

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IGEM UGENT TEAM

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CIChE and its stumbling blocks

PROBLEM OUR MODEL RESULTS FUTURE

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IGEM UGENT TEAM

CIChE and its stumbling blocks

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PROBLEM OUR MODEL RESULTS FUTURE

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IGEM UGENT TEAM

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Gyrase will no longer be functional CcdB has lost his toxicity caused by the mutation

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IGEM UGENT TEAM

CIChE and its stumbling blocks

High mutation rate caused by:

Leaky expression ccdB pressure to mutate SOS pathway activation mutagenesis increases

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IGEM UGENT TEAM

It’s time to vote!

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IGEM UGENT TEAM

It’s time to vote!

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FUTURE

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IGEM UGENT TEAM

Further research perspectives

Assemble T7ccdB plasmid inside CcdB-survival strains Cloning T7ccdB plasmid in ccdA-strain Adding multiple ccdA copies to the ccdA-gfp construct Stronger promoter for ccdA in construct Knock out of alternative polymerases Other alternatives

Triclosan Solvents Cationic antimicrobial peptides Operator-repressor titration

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PROBLEM OUR MODEL RESULTS FUTURE

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12/10/2013

IGEM UGENT TEAM

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  • ir. Frederik De Bruyn
  • ir. Pieter Coussement
  • ir. Gert Peters
  • Prof. Dr. ir. Marjan De Mey

Charlotte Verniers

SPECIAL THANKS

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12/10/2013

Also thanks to our sponsors

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THANK YOU