2013 TzuChiU_Formosa
Team Team Membe Members rs
Hypnoseq. Hypnos Sequence
Right before Us
Right before Us • Infections caused by resistant microorganisms often fail to respond to conventional treatment, resulting in prolonged illness, greater risk of death and higher costs. • New resistance mechanisms have emerged, making the latest generation of antibiotics virtually ineffective. － World Health Organization
1 Introduction We We ha have ve fea eatur tures es for r ev ever ery ste y step of p of th the pro e proje ject. ct.
chloramphenicol Designed four parts of Cam-antisenses － specific
Pri Primer Des mer Design ign Obtain antibiotic resistance sequence Random selection to seek high specificity regions Reverse the above sequence Add the restriction enzyme site at the end of primers
Hypno Hypnoseq. seq. RISC RISC? How doe How does it w s it work? ork? ? " Are organisms able to perform an easier mechanism to carry out our desired plan? " in Proka in Prokaryotes ryotes? Dicer? Dice r?
2 System Design 1 Introduction We We ha have ve fea eatur tures es for r ev ever ery ste y step of p of th the pro e proje ject. ct.
IPTG Target mRNA 13
2 System Design 3 1 Introduction Results We We ha have ve fea eatur tures es for r ev ever ery ste y step of p of th the pro e proje ject. ct.
Gr Group 1 oup 1 Colony PCR antisense pSB1C3 vector
Lac I PCR from pET11d (E.c .coli)
In Prog In P rogress ress pSB1C3 group Clone Lac I Construct the system Transformation Quantify the system
Gr Group 2 oup 2 antisense pET11d vector
pET11d Colony Amp
2 System Design 1 3 Introduction Results 4 Future & Application We We ha have ve fea eatur tures es for r ev ever ery ste y step of p of th the pro e proje ject. ct.
IPTG add Plating Cam add Plating
Decrease the percentage of Antibiotic or Resistance
Applications Applications • Health/Medicine ： Knockdown pathogenic genes of bacteria • New Application ： on-off switch
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)
Brand New Design ! CRISPR/Cas9 system
Conjugation CRISPR/Cas9 OriTR J01003
2 System Design 1 3 Introduction Results 4 5 Future Achieve- & ment Application We ha We have ve fea eatur tures es for r ev ever ery ste y step of p of th the pro e proje ject. ct.
Completed. Ampicillin antisense 1(BBa_K1222997) Lac operator+CamR antisense 1(BBa_K1222984) Lac promoter+LacI(BBa_K1222002) Ampicillin antisense 2(BBa_K1222996) Lac operator+CamR antisense 2(BBa_K1222983) Ampicillin antisense 3(BBa_K1222995) Lac operator+CamR antisense 3(BBa_K1222982) Ampicillin antisense 4(BBa_K1222994) Lac operator+CamR antisense 4(BBa_K1222981) GFP antisense 1(BBa_K1222998) Lac operator+GFP antisense 1(BBa_K1222987) GFP antisense 2(BBa_K1222989) Lac operator+GFP antisense 2(BBa_K1222986) GFP antisense 3(BBa_K1222988) Lac operator+GFP antisense 3(BBa_K1222985) T7 promoter+lac operator+antisense +T7 T7 promoter+lac operator+antisense+T7 terminator+Lac terminator (BBa_K1222004, BBa_K1222005) promoter+LacI(BBa_K1222000, BBa_K1222001) 28
Human Human Prac Practices tices -Spe Speech ech -Fa Facebook cebook Pag Page -SB Work SB Workshop shop
Huma Human Prac n Practic tices es
Human Human Prac Practices tices -Spe Speech ech -Fa Facebook cebook Pag Page -SB Work SB Workshop shop
Human Human Prac Practices tices -Spe Speech ech -Fa Facebook cebook Pag Page -SB Work SB Workshop shop
Acknowledgements Acknowledgements
Instruc Ins tructor tor Te Techni chnical cal Suppo Su pports rts Guang-Huey Lin Ingrid Liu Ji-Hshiung Chen Jeng-Woei Lee Shu-Jun Chiu Chun-Yao Chen Jui-Hung Yen
Advi Advisor sors Hung-Jen Chien Hsin-Jung Tien Chia-Sheng Pai Lu-Chu Ke Li-Yi Lin Kai-Chi Liang Ni-Chun Chung Yao-Hua Hew Ruo-Yu Wang Hsien-Ting Huang
Refer References ences Hale, C. R., et al. (2009). "RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex." Cell 139 (5): 945-956. Hsu, P. D., et al. (2013). "DNA targeting specificity of RNA-guided Cas9 nucleases." Nat Biotechnol 31 (9): 827-832. Huang, L., et al. (2013). "Efficient and specific gene knockdown by small interfering RNAs produced in bacteria." Nat Biotechnol 31 (4): 350-356. Wang, H., et al. (2013). "One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering." Cell 153 (4): 910-918. Wiedenheft, B., et al. (2012). "RNA-guided genetic silencing systems in bacteria and archaea." Nature 482 (7385): 331-338.
Thank you Thank you for your for your attenti attention. on.
Amp r Amp r mRNA Cam r Kan r protein protein Amp Amp
Amp r Amp r mRNA Amp r mRNA Cam r Kan r protein Anti-sense Amp r mRNA Amp IPTG
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