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

HKUST 2011 iGEM Team

• E. trojan – Boosting the effectiveness
• f 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 Disruption (T4MO) Antibiotic-Free Selection

• E. TROJAN

Reduce Antibiotic Usage Rearm Antibiotics

• E. CRAFT – AN ALTERNATIVE

TRANSFORMATION METHOD

Essential- gene- based selection method:

• E. CRAFT

Eschericia coli Re-engineered for Antibiotic- Free Transformation

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

Phage λ RED Recombinase circumvents host’s internal barriers against

• recombination. Presence of homologous sequences allows complementation
• f the 3’ overhangs of the Foreign dsDNA to the host gDNA, making possible a

swap between the two DNA sequences.

Essential Gene

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

Essential Gene

ANTIBIOTIC RESISTANT 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 420C #

# 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 μg/ml 25 μg/ml kanamycin gradient

RR-1

0 μg/ml kanamycin gradient with 25 μg/ml indole

(300nM/1mM)

• 0.4
• 0.2

0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 2 4 6 8 10 12 14 16

OD600 Kanamycin concentration (µg/ml)

Comparison of MICs for indole and non-indole supplemented RR1

RR1 w/ 1mM indole RR1 RR1 w/ 300μM indole

RR-1

RFP/ KanR 0 μg/ml kanamycin gradient 25 μg/ml

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 5 7 8 10 12 14 16 18 20 25

Ratio Kanamycin concentration (µg/ml)

Ratio of RR1 to RFP in Mixed MIC

RR1 RFP

RR-1

T4MO/ GFP/ KanR

0 μg/ml kanamycin gradient 25 μg/ml

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 5 7 8 10 12 14 16 18 20 25

Ratio Kanamycin concentration (µg/ml)

Ratio of RR1 to RFP in Mixed MIC

RR1 RFP

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 5 7 8 10 12 14 16 18 20 25

Ratio Kanamycin concentration (µg/ml)

Ratio of RR1 to T4MO in Mixed MIC

RR1 T4MO

• 1

1 2 3 4 5 5 10 15 20 25 30 35

Ratio Kanamycin concentration (µg/ml) Comparison of RFP and T4MO on Charity work

T4MO:RR1 RFP:RR1

• E. trojan
• Basis for future

model on other pathogenic species which also apply indole charity

HUMAN PRACTICE

• HKUST Synthetic

Biology Workshop 2011

• Synthetic Biology

Survey

Workshop (17th 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

Take genes from the electric eel and put it into a mouse…

Creative Design

Pikachu?!

Synthetic Biology Survey

• A collaboration with

IDC and Biofaction

• Online format
• 570 responses as of

1st Oct 2011

This is just the beginning…

HKUST Synthetic Biology Workshop Synthetic Biology Survey

CONCLUSION

Conclusion

Indole Signalling Disruption (T4MO) Antibiotic-Free Selection

• E. TROJAN

Reduce Antibiotic Usage Rearm Antibiotics

Your Target?

• S. aureus?

Klebsiella? Salmonella?

• P. aeruginosa?

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