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

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

TEAM UGENT IGEM 12/10/2013

The team THE TEAM TEAM UGENT IGEM 12/10/2013 iGEM TEAM UGENT - BIOBASE EUROPE PILOT PLANT

PROBLEM iGEM TEAM UGENT - BIOBASE EUROPE PILOT PLANT

Plasmid-based high gene expression TEAM UGENT Metabolic burden IGEM Genetic instability Productivity loss PROBLEM OUR MODEL RESULTS FUTURE 12/10/2013 iGEM TEAM UGENT - BIOBASE EUROPE PILOT PLANT 5

CIChE (Chemically inducable chromosomal evolution) TEAM = Homologous region UGENT GOI = Gene of interest AR = Antibiotic IGEM resistance AR GOI Add antibiotics (depends on marker gene) antibiotics PROBLEM OUR MODEL Transduction n n RESULTS AR AR GOI AR GOI GOI AR GOI FUTURE 12/10/2013 iGEM TEAM UGENT - BIOBASE EUROPE PILOT PLANT 6

Did you ever dwell on the use of TEAM antibiotic resistance genes during your UGENT iGEM project? 54% IGEM 46% A. Yes B. No No Yes 7 12/10/2013 iGEM TEAM UGENT - BIOBASE EUROPE PILOT PLANT

What do scientists think? TEAM UGENT Do you ever dwell on the use of antibiotic resistance genes during your research? IGEM Every time I use them (7%) Often (17%) Sometimes (30%) PROBLEM OUR MODEL Never (33%) Never (33%) RESULTS Other (13%) FUTURE 12/10/2013 iGEM TEAM UGENT - BIOBASE EUROPE PILOT PLANT 8

What does the public think? TEAM UGENT Do you worry about increasing antibiotic resistance in bacteria? IGEM No Yes PROBLEM OUR MODEL RESULTS FUTURE 12/10/2013 iGEM TEAM UGENT - BIOBASE EUROPE PILOT PLANT 9

ALTERNATIVES iGEM TEAM UGENT - BIOBASE EUROPE PILOT PLANT 10

TEAM UGENT IGEM PROBLEM OUR MODEL RESULTS FUTURE Yamaguchi et al., 2011 12/10/2013 iGEM TEAM UGENT - BIOBASE EUROPE PILOT PLANT 11

TEAM UGENT IGEM PROBLEM OUR MODEL RESULTS FUTURE 12/10/2013 iGEM TEAM UGENT - BIOBASE EUROPE PILOT PLANT 12

TEAM UGENT IGEM PROBLEM OUR MODEL RESULTS FUTURE 12/10/2013 iGEM TEAM UGENT - BIOBASE EUROPE PILOT PLANT 13

OUR MODEL iGEM TEAM UGENT - BIOBASE EUROPE PILOT PLANT 14

Our model for CIChE TEAM UGENT IGEM ccdA gfp T7 RNAP recA ccdA gfp T7 PROBLEM ccdB OUR MODEL RESULTS λ Red FUTURE 12/10/2013 iGEM TEAM UGENT - BIOBASE EUROPE PILOT PLANT 15

TEAM T7 RNAP ccdA gfp ccdA gfp UGENT recA IPTG IPTG T7 CcdA Pol CcdA T7 IGEM ccdB CcdB RecA CcdB CcdB T7 RNAP ccdA gfp ccdA gfp ccdA gfp PROBLEM recA T7 CcdA OUR MODEL Pol CcdA CcdA T7 ccdB RESULTS CcdB RecA FUTURE CcdB CcdB 12/10/2013 iGEM TEAM UGENT - BIOBASE EUROPE PILOT PLANT 16

EXPERIMENTS iGEM TEAM UGENT - BIOBASE EUROPE PILOT PLANT 17

Experiment 1 TEAM UGENT Knock-in IGEM T7 T7 RNAP RNAP gfp ccdA PROBLEM OUR MODEL RESULTS FUTURE 12/10/2013 iGEM TEAM UGENT - BIOBASE EUROPE PILOT PLANT 18

Experiment 2 TEAM UGENT Construction ccdB plasmid IGEM T7 ccdB ccdB PROBLEM OUR MODEL RESULTS FUTURE 12/10/2013 iGEM TEAM UGENT - BIOBASE EUROPE PILOT PLANT 19

Experiment 3 TEAM UGENT Transformation IGEM T7 T7 ccdB RNAP PROBLEM gfp ccdA OUR MODEL RESULTS FUTURE 12/10/2013 iGEM TEAM UGENT - BIOBASE EUROPE PILOT PLANT 20

Experiment 4 TEAM UGENT CIChE IGEM IPTG n ccdA gfp ccdA gfp ccdA gfp PROBLEM T7 ccdB OUR MODEL RESULTS CcdA-CcdB complex FUTURE 12/10/2013 iGEM TEAM UGENT - BIOBASE EUROPE PILOT PLANT 21

Experiment 4 TEAM UGENT CIChE IGEM … PROBLEM 0,01 0,05 0,1 OUR MODEL 0mM mM mM mM IPTG IPTG IPTG IPTG RESULTS FUTURE 12/10/2013 iGEM TEAM UGENT - BIOBASE EUROPE PILOT PLANT 22

Experiment 5 TEAM UGENT Evaluation of strains IGEM Optical Density (OD 600 ) Fluorescence (F) PROBLEM F/OD 600 OUR MODEL RESULTS FUTURE 12/10/2013 iGEM TEAM UGENT - BIOBASE EUROPE PILOT PLANT 23

RESULTS iGEM TEAM UGENT - BIOBASE EUROPE PILOT PLANT 24

Results TEAM F/OD 600 UGENT Copy n° 5 Copy n° 10 IGEM IPTG IPTG F/OD 600 Copy n° 15-20 Reference strain PROBLEM OUR MODEL RESULTS FUTURE IPTG 12/10/2013 iGEM TEAM UGENT - BIOBASE EUROPE PILOT PLANT 25

CIChE and its stumbling blocks TEAM UGENT IGEM PROBLEM OUR MODEL RESULTS FUTURE 12/10/2013 iGEM TEAM UGENT - BIOBASE EUROPE PILOT PLANT 26

CIChE and its stumbling blocks TEAM UGENT IGEM PROBLEM OUR MODEL RESULTS FUTURE 12/10/2013 iGEM TEAM UGENT - BIOBASE EUROPE PILOT PLANT 27

TEAM UGENT Gyrase will no longer IGEM be functional CcdB has lost his toxicity caused by the mutation 12/10/2013 iGEM TEAM UGENT - BIOBASE EUROPE PILOT PLANT 28

CIChE and its stumbling blocks TEAM UGENT High mutation rate caused by: IGEM Leaky expression ccdB pressure to mutate SOS pathway activation mutagenesis increases 12/10/2013 iGEM TEAM UGENT - BIOBASE EUROPE PILOT PLANT 29

It’s time to vote! TEAM UGENT IGEM 12/10/2013 iGEM TEAM UGENT - BIOBASE EUROPE PILOT PLANT 30

It’s time to vote! TEAM UGENT IGEM 12/10/2013 iGEM TEAM UGENT - BIOBASE EUROPE PILOT PLANT 31

FUTURE 32

Further research perspectives TEAM UGENT Assemble T7 ccdB plasmid inside CcdB-survival strains IGEM Cloning T7 ccdB 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 PROBLEM Triclosan OUR MODEL Solvents RESULTS Cationic antimicrobial peptides FUTURE Operator-repressor titration 12/10/2013 iGEM TEAM UGENT - BIOBASE EUROPE PILOT PLANT 33

ir. Frederik De Bruyn ir. Pieter Coussement TEAM UGENT IGEM SPECIAL THANKS ir. Gert Peters Charlotte Verniers Prof. Dr. ir. Marjan De Mey 12/10/2013 iGEM TEAM UGENT - BIOBASE EUROPE PILOT PLANT 34

Also thanks to our sponsors 12/10/2013 iGEM TEAM UGENT - BIOBASE EUROPE PILOT PLANT 35

THANK YOU iGEM TEAM UGENT - BIOBASE EUROPE PILOT PLANT 36