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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 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 of 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
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0 2 4 6 8 10 12 14 16
OD
60
0
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%
0 5 7 8 10 12 14 16 18 20 25
Rat
io
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%
0 5 7 8 10 12 14 16 18 20 25
Rat
io
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%
0 5 7 8 10 12 14 16 18 20 25
Rat
io
Kanamycin concentration (µg/ml)
Ratio of RR1 to T4MO in Mixed MIC RR1
T4MO
-1
0
1
2
3
4
5
0 5 10 15 20 25 30 35
Rat
io
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