HKUST 2011 iGEM Team E. trojan Boosting the effectiveness of - PowerPoint PPT Presentation

HKUST 2011 iGEM Team E. trojan – Boosting the effectiveness of antibiotics through quorum- sensing disruption

INTRODUCTION

Mistakes… Penicillin G

Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Tobramycin, Paromomycin, Loracarbef, Ertapenem, Doripenem, Imipenem, Meropenem, Cefadroxil, Cefazolin, Cefalotin, Cefalexin, Cefaclor, Cefamandole, Cefoxitin, Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren, Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime, Ceftriaxone, Cefepime, Ceftobiprole, Teicoplanin, Vancomycin, Telavancin, Clindamycin, Lincomycin, Daptomycin, Azithromycin, Clarithromycin, Dirithromycin, Erythromycin, Roxithromycin, Troleandomycin, Telithromycin, Spectinomycin, Aztreonam, Furazolidone, Nitrofurantoin, Amoxicillin, Ampicillin, Azlocillin, Carbenicillin, Cloxacillin, Dicloxacillin, Flucloxacillin, Mezlocillin, Methicillin, Nafcillin, Oxacillin, Penicillin, Piperacillin, Temocillin, Ticarcillin, Bacitracin, Colistin, Polymyxin B …

Charity

Indole Signalling Reduce Antibiotic Antibiotic-Free E. TROJAN Rearm Antibiotics Disruption (T4MO) Selection Usage

E. CRAFT – AN ALTERNATIVE TRANSFORMATION METHOD

Essential- gene- based selection method: E. CRAFT E schericia c oli R e-engineered for A ntibiotic- F ree T ransformation Deletion of an essential gene from E. CRAFT’s genome  Addiction to extragenomic source of the gene

pCarrier, a heat- insensitive analog of pDummy TRANSFORMATION TRANSFORMATION

The selection, comparison between successful and unsuccessful transformation PLASMID SHUFFLING

Development of addiction: use of the phage λ RED Recombination System Essential Gene Phage λ RED Recombinase circumvents host’s internal barriers against recombination. Presence of homologous sequences allows complementation of the 3’ overhangs of the Foreign dsDNA to the host gDNA, making possible a swap between the two DNA sequences.

How to keep the plasmid with no resistance? Mutualistic Plasmids Essential Gene

How to keep the plasmid with no resistance? Mutualistic Plasmids ANTIBIOTIC RESISTANT GENE

How to keep the plasmid with no resistance? Mutualistic Plasmids ANTIBIOTIC RESISTANT GENE Essential Gene

Use of split reporter system as indicator of completion of E. CRAFT construction

The E. trojan E. CRAFT + pCarrier with T4MO

CHARACTERIZATION OF BIOBRICK BBa_K524000

BioBrick Characterization – BBa_K524000 Heat Sensitive Origin oriR101 & repA101-ts Properties: • Derivative of pSC101 origin • oriR101 positively regulated by a heat liable protein of gene repA101-ts • Stops replication at 42 0 C # # Phillips GJ.(1995). New Cloning Vectors with Temperature-Sensitive Replication, Plasmid. 1999 Jan;41(1):78-81.

BioBrick Characterization – BBa_K524000 Heat Sensitive Origin oriR101 & repA101-ts

BioBrick Characterization – BBa_K524000 Heat Sensitive Origin oriR101 & repA101-ts FULL DILUTION DISPLAY

CULTURE TEST RESULTS

RR-1 0 μ g/ml 25 μ g/ml kanamycin gradient

RR-1 0 μ g/ml kanamycin gradient with 25 μ g/ml indole (300nM/1mM)

Comparison of MICs for indole and non-indole supplemented RR1 1.6 RR1 w/ 1mM indole 1.4 RR1 1.2 RR1 w/ 300 μ M indole 1 0.8 OD 600 0.6 0.4 0.2 0 0 2 4 6 8 10 12 14 16 -0.2 -0.4 Kanamycin concentration (µg/ml)

RFP/ RR-1 KanR 0 μ g/ml kanamycin gradient 25 μ g/ml

Ratio of RR1 to RFP in Mixed MIC 100% RR1 90% RFP 80% 70% 60% Ratio 50% 40% 30% 20% 10% 0% 0 5 7 8 10 12 14 16 18 20 25 Kanamycin concentration (µg/ml)

T4MO/ RR-1 GFP/ KanR 0 μ g/ml kanamycin gradient 25 μ g/ml

Ratio of RR1 to T4MO in Mixed MIC Ratio of RR1 to RFP in Mixed MIC 100% 100% RR1 RR1 90% 90% T4MO RFP 80% 80% 70% 70% 60% 60% Ratio Ratio 50% 50% 40% 40% 30% 30% 20% 20% 10% 10% 0% 0% 0 5 7 8 10 12 14 16 18 20 25 0 5 7 8 10 12 14 16 18 20 25 Kanamycin concentration (µg/ml) Kanamycin concentration (µg/ml)

Comparison of RFP and T4MO on Charity work 5 T4MO:RR1 RFP:RR1 4 3 Ratio 2 1 0 0 5 10 15 20 25 30 35 -1 Kanamycin concentration (µg/ml)

E. trojan • Basis for future model on other pathogenic species which also apply indole charity

HUMAN PRACTICE

• HKUST Synthetic • Synthetic Biology Biology Workshop Survey 2011

Workshop (17 th Sept 2011) 1) Introductory talk on Synthetic Biology 2) “The Life of E. coli” - An educational card game 3) Laboratory tour 4) Creative design for Synthetic Biology

Creative Design Take genes from the electric eel and put it into a mouse… Pikachu?!

Synthetic Biology Survey • A collaboration with IDC and Biofaction • Online format • 570 responses as of 1 st Oct 2011

This is just the beginning… HKUST Synthetic Synthetic Biology Biology Workshop Survey

CONCLUSION

Conclusion Klebsiella ? S. aureus ? Your Target? Salmonella ? P. aeruginosa ? Indole Signalling Antibiotic-Free E. TROJAN Disruption (T4MO) Selection Reduce Antibiotic Rearm Antibiotics Usage

Acknowledgements • IDC - Organisation for International Dialogue and Conflict Management • Biofaction • The Hong Kong Institute of Engineers (HKIE) • The Hong Kong Teachers’ Association (HKTA) • Dr Markus SCHMIDT and Dr Lei PEI • The Provost's Office, HKUST • Division of Biomedical Engineering, HKUST • Division of Life science, HKUST • Department of Social Science, HKUST • Prof. King L. CHOW, Dr Jessica Ce Mun TANG, Prof. Michelle YIK and Mr Jin ZENG • The Coli Genetic Stock Center • Mr. Kevin McClay, Cornell University • iGEM 2011 CUHK team • Life Technology, Inc. • And many others not mentioned.

Q&A SECTION