Tzu Chi University
Lilian Regina Ted Bobo Sharon Denise House 1
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• Increased health care costs • Increased resistance • Possible side effects from antibiotic abuses 4
Elimination of Antibiotic Resistant Genes 5
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CRISPR Antibiotic Resistance Create a tool to spread CRISPR system. Gene Bacteria Bacteria Bacteria Antibiotic Resistance Survive Dead Dead Product antibiotics 8
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200:1 Smith & Scott. Methods Enzymol. 217:228, 1993 10
CRISPR Reporter Gene Inducible Switch phagemid Recombinant Ligation pBluescript II Phagemid SK (-) 12
CAS9 BBa_K914003 Paris_Bettencourt 2012 CAS9 trcrRNA crRNA BBa_K1218011 Stanford-Brown 2013 13
BBa_K1473006 CAS9 trcrRNA crRNA BBa_K914003 BBa_K1218011 14
Added for signaling! CAS9 RFP crRNA trcrRNA BBa_K914003 BBa_I13521 From MIT 2009 15
Helper phage genome Phagemid MCS f1 ori mutated Phagemid f1 PUC pBluescript II ori ori SK(-) Amp R Kan R p15A ori 16
E.coli JM101 Host Pol. gp2 gp3 RF gp5 gp6 gp7 gp8 M13KO7 Helper Phage gp9 F pilus Genome Lack f1 ori 17
E.coli JM101 Host Pol. RF gp2 gp3 gp5 gp6 gp7 M13KO7 Helper gp8 Phage gp9 pBluescript II NICK SK(-) F pilus 18
phagemid Reporter Gene CRISPR System Vector Use phagemid-carrying phage to spread Inducible Switch CRISPR system Helper Phage Inducer 19
Spreading efficiency of CRISPR Constructed a Model Amount of Percentage of phage production Phage infection 21
Control M 1 kb (pBluescript ) Clone 7 Clone 11 U uncut M U E+P U E P E+P U E P E+P E Eco RI BBa_K1473005 P Pst I pBluescript (2929 bp) RFP 4 kb 937 bp 3 kb Eco R I Pst I 2 kb 2958 bp 1 kb Amp R 937 bp 22
E . coli M13KO7 JM101 1 x 10 8 How can Trogene Horse produce most phagemid-carring phage? Infection pfu/ml Time Kanamycin Incubating Time 23
2 Hours 10 minutes 20 minutes 30 minutes 4 Hours Infection Incubating 40 minutes Time Time 6 Hours 50 minutes …… …… 14 Hours 90 minutes 70 experiments 24
4 x 10 10 pfu/ml The Best Phage Production plague 25
How can phagemid-carrying phage get the best infection rate? MOI (Multiplicity of infection, pfu/cfu) 26
100% MOI=2 Optimal Infection MOI=6 MOI=1 27
The Best Condition for Phage Releasing Optimized Conditions Optimal Infection Rate RFP RFP CRISPR pBluescript pBluescript 28
• CRISPR only targets antibiotics resistance genes 30
• The phage vector we chose can only produce one generation of progenies 31
Antibiotic Resistance Multiple genes Antibiotic Resistance Products antibiotics 32
Cas9 trcrRNA crRNA 33
CRISPR CRISPR CRISPR Target different antibiotic resistance genes antibiotics 34
Workshop Conferences Promotion of iGEM Video conferences 36
Date : July.31st, 2014 Location : National Hualien Girl’s Senior High School A total of 52 students joined the talk. Promote iGEM and synthetic biology to local senior high school students. 37
Make the 2015 iGEM team get prepared !! Date : Sep. 30th, 2014 Location : Tzu Chi University A total of 30 students joined the talk. 38
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This report was generated on 10/26/14 by Google Analytics 41
Skype meeting between TCU, NCTU and NYMU 42
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Biobricks New: spread CRISPR system- K1473005 Improvement : pRha induced CRISPR - K1473009 Modeling Cooperation with NCTU-Formosa iGEM team Human Practice Talk Conference with several iGEM teams Promotion of iGEM Video conference 45
Woon, Peng-Yeong Ching, Yung-Hao Lin, Guang-Huey Lin, Nien-Tsung 47 Liu, Ingrid Chen, Ji Hshiung Yen, Jui-Hung Lin, Min-Der
Chen, Jia-Wei Wang, Yi-Ru Huang, Hsien-Ting Nina, Wu Chien, Hung-Jen Ke, Lu-Chu 48
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iGEM , the Dream
• Nucleic Acids Res. Dec 2006; 34(21) Eliminating helper phage from phage display • http://www.sfu.ca/biology/courses/bisc431/m13.htm • Barrow & Soothill. Trends Microbiol. 5:268, 1997 (phage therapy) • Smith & Scott. Methods Enzymol. 217:228, 1993 (phage display methods) • Russell SJ. Nature Medicine 2:276, 1996 (phage display & gene targeting) • Cell 153, 910 – 918, May 9, 2013 52
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Ingested foreign (phage M13) DNA survives transiently in the gastrointestinal tract and enters the bloodstream of mice Mol Gen Genet. 1994 Mar;242(5):495-504. 55
22 bp 56
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tracrRNA crRNA 58
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