Tzu Chi University Lilian Regina Ted Bobo Sharon Denise House - - PowerPoint PPT Presentation

▶
tzu chi university
SMART_READER_LITE
LIVE PREVIEW

Tzu Chi University Lilian Regina Ted Bobo Sharon Denise House - - PowerPoint PPT Presentation

Jan 13, 2024 328 likes •934 views

Tzu Chi University Lilian Regina Ted Bobo Sharon Denise House 1 3 Increased health care costs Increased resistance Possible side effects from antibiotic abuses 4 Elimination of Antibiotic Resistant Genes 5 7 7 CRISPR

slide-1
SLIDE 1

Tzu Chi University

slide-2
SLIDE 2

1

Lilian Regina Ted Bobo Denise House Sharon

slide-3
SLIDE 3
slide-4
SLIDE 4

3

slide-5
SLIDE 5
  • Increased health care costs
  • Increased resistance
  • Possible side effects from antibiotic abuses

4

slide-6
SLIDE 6

Elimination of Antibiotic Resistant Genes

5

slide-7
SLIDE 7
slide-8
SLIDE 8

7 7

slide-9
SLIDE 9

CRISPR

Bacteria Survive Bacteria Dead

antibiotics

Antibiotic Resistance Product Antibiotic Resistance Gene

Bacteria Dead

Create a tool to spread CRISPR system.

8

slide-10
SLIDE 10

9

slide-11
SLIDE 11

10 Smith & Scott. Methods Enzymol. 217:228, 1993

200:1

slide-12
SLIDE 12
slide-13
SLIDE 13

CRISPR

Reporter Gene Inducible Switch

Ligation

phagemid pBluescript II SK (-) Recombinant Phagemid

12

slide-14
SLIDE 14

BBa_K914003

Paris_Bettencourt 2012

CAS9

trcrRNA

CAS9

BBa_K1218011

crRNA

Stanford-Brown 2013

13

slide-15
SLIDE 15

BBa_K1218011

BBa_K914003

CAS9

trcrRNA

crRNA

BBa_K1473006

14

slide-16
SLIDE 16

trcrRNA

CAS9

BBa_K914003 crRNA

RFP

BBa_I13521

Added for signaling!

From MIT 2009

15

slide-17
SLIDE 17

Phagemid Helper phage genome

f1 ori

mutated

p15A ori

KanR

Phagemid pBluescript II SK(-)

AmpR f1

  • ri

PUC

  • ri

MCS

16

slide-18
SLIDE 18

RF gp5 gp3 gp6 gp8 gp7 gp9 gp2 F pilus

M13KO7 Helper Phage

E.coli JM101

Genome Lack f1 ori

Host

Pol.

17

slide-19
SLIDE 19

RF gp5 gp3 gp6 gp8 gp7 gp9 gp2 F pilus

M13KO7 Helper Phage

E.coli JM101

NICK

pBluescript II SK(-)

Host

Pol.

18

slide-20
SLIDE 20

CRISPR System Reporter Gene

Inducible Switch

phagemid Vector Helper Phage

Inducer

19

Use phagemid-carrying phage to spread CRISPR system

slide-21
SLIDE 21
slide-22
SLIDE 22

Spreading efficiency of CRISPR Constructed a Model Amount of phage production Percentage of Phage infection

21

slide-23
SLIDE 23

M 1 kb U uncut E EcoRI P PstI

M U E+P U E P E+P U E P E+P Clone 7 Clone 11 Control (pBluescript)

AmpR

RFP EcoRI PstI

937 bp

pBluescript (2929 bp)

1 kb 2 kb 3 kb 4 kb

937 bp 2958 bp

BBa_K1473005

22

slide-24
SLIDE 24

How can Trogene Horse produce most phagemid-carring phage? Infection Time Incubating Time M13KO7

1 x 108 pfu/ml

Kanamycin

E.coli JM101

23

slide-25
SLIDE 25

Infection Time Incubating Time

10 minutes 20 minutes 30 minutes 40 minutes

……

90 minutes 50 minutes

2 Hours 4 Hours 6 Hours 14 Hours

70 experiments

24

……

slide-26
SLIDE 26

4 x 1010 pfu/ml

plague

The Best Phage Production

25

slide-27
SLIDE 27

How can phagemid-carrying phage get the best infection rate?

MOI (Multiplicity of infection, pfu/cfu)

26

slide-28
SLIDE 28

100% MOI=2 MOI=6 MOI=1

Optimal Infection

27

slide-29
SLIDE 29

RFP

pBluescript

The Best Condition for Phage Releasing Optimal Infection Rate

CRISPR

RFP

pBluescript

Optimized Conditions

28

slide-30
SLIDE 30
slide-31
SLIDE 31
  • CRISPR only targets antibiotics resistance

genes

30

slide-32
SLIDE 32
  • The phage vector we chose can only

produce one generation of progenies

31

slide-33
SLIDE 33

32

antibiotics

Multiple Antibiotic Resistance Products

Antibiotic Resistance genes

slide-34
SLIDE 34

33

trcrRNA

Cas9

crRNA

slide-35
SLIDE 35

34

antibiotics

CRISPR CRISPR CRISPR

Target different antibiotic resistance genes

slide-36
SLIDE 36
slide-37
SLIDE 37

Workshop Conferences Promotion of iGEM Video conferences

36

slide-38
SLIDE 38

Promote iGEM and synthetic biology to local senior high school students.

37

Date : July.31st, 2014 Location : National Hualien Girl’s Senior High School A total of 52 students joined the talk.

slide-39
SLIDE 39

Make the 2015 iGEM team get prepared !!

38

Date : Sep. 30th, 2014 Location : Tzu Chi University A total of 30 students joined the talk.

slide-40
SLIDE 40

39

slide-41
SLIDE 41

40

slide-42
SLIDE 42

41

This report was generated on 10/26/14 by Google Analytics

slide-43
SLIDE 43

Skype meeting between TCU, NCTU and NYMU

42

slide-44
SLIDE 44
slide-45
SLIDE 45

44

slide-46
SLIDE 46

 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

slide-47
SLIDE 47
slide-48
SLIDE 48

47 Liu, Ingrid Chen, Ji Hshiung Lin, Min-Der Yen, Jui-Hung Woon, Peng-Yeong Ching, Yung-Hao Lin, Nien-Tsung Lin, Guang-Huey

slide-49
SLIDE 49

48 Huang, Hsien-Ting Wang, Yi-Ru Chien, Hung-Jen Chen, Jia-Wei Ke, Lu-Chu Nina, Wu

slide-50
SLIDE 50

49

slide-51
SLIDE 51

50

slide-52
SLIDE 52

iGEM , the Dream

slide-53
SLIDE 53
  • 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

slide-54
SLIDE 54

53

slide-55
SLIDE 55

54

slide-56
SLIDE 56

55

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.

slide-57
SLIDE 57

22 bp

56

slide-58
SLIDE 58

57

slide-59
SLIDE 59

58

tracrRNA crRNA