+ All Categories
Transcript
Page 1: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

2012 HKUST iGEM Team

The Terminator of Colon Cancer

B. hercules

Page 2: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

Background & Motive

Page 3: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

Cancer Therapy & Synthetic Biology

Background & Motive

Our  project  à  Cancer  

Page 4: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

Represent  >90%  of  colon  cancer  cases  Accessible  to  agents  in  gut  

Colon Carcinomas – A Suitable Target

Background & Motive

Page 5: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

•  Non-­‐pathogenic.  

• Well-­‐understood.  

•  Provide  engineering  op7ons.  

Our Chassis

Background & Motive

•  Gut  microbiome  

•  Lacks  virulence  

•  Integrates  DNA  

Gene  integra7on  

Page 6: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

B. hercules

Background & Motive

 Target:  colon  carcinomas      Tool:  B.  sub'lis  

Page 7: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

•  Target  Binding  

•  An>-­‐tumor  Molecule  Secre>on  

•  Regula>on  &  Control  

Background & Motive

Three Modules

Page 8: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

Module 1

Target Binding

Page 9: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

Colon  Cancer  Cell  

Module 1

RPMrel  

Tumor Recognition

Page 10: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

Bacillus  

sub'lis  

Module 1

RPMrel  

Tumor Recognition

Colon  Cancer  Cell  

Page 11: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

LytC  

Helical  Linker  

Module 1

Cell Wall Display

Page 12: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

lytC   linker   RPMrel                                      

Double    Terminator  

RBS  

             

Pveg  

Module 1

Construct

Page 13: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

Module 2

Anti-tumor Molecule Secretion

Page 14: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

Secrete  BMP2  BMP2  Module 2

Anti-tumor Drug

Page 15: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

BMP2  Signal  pep>de  

Module 2

Out  

In  

Membrane  

Secretion

Page 16: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

Module 2

Cleavage  

Signal  pep>de  

BMP2  

Secretion

Page 17: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

SPPase  

BMP2  Folding  

Module 2

Secretion

Page 18: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

RBS  xylR   Bmp2  Signal    pep>de    

   

PxylA  

Module 2

Construct

Page 19: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

0.0  

0.1  

0.2  

0.3  

0.4  

0.5  

0.6  

0.7  

0.8  

0.9  

1.0  

0.0001   0.001   0.01   0.1  

Percen

tage  of  V

iable  Ce

lls  

B.  sub'lis  Concentra>on  (OD650)  

Viability  of  HT-­‐29  Cells  Following  Co-­‐Culture  with  B.  sub'lis  

B.  sub7lis  transformed  with  empty  vector   B.  sub7lis  transformed  to  express  BMP2  

Module 2

BMP2 Functional Assay

B.  sub'lis   B.  sub'lis  

Page 20: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

Regulation & Control

Module 3

Page 21: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

Two  Regula>on  Systems  

1.  Time  Control   2.  Dosage  Regula>on  

Module 3

Regulation Systems

Page 22: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

RBS  xylR      

PxylA  

Module 3

Xylose-Inducible Promoter

Page 23: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

RBS  xylR   Bmp2  Signal    pep>de    

   

PxylA  

XylR   Inhibited  Transcrip>on  

Module 3

Xylose-Inducible Promoter

Page 24: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

RBS  xylR   Bmp2  Signal    pep>de    

   

PxylA  

XylR   Transcrip>on  Proceeds  

Module 3

xylose  

Xylose-Inducible Promoter

Page 25: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

Bmp2  Signal    

pep>de  

ydcE  RBS  

An>toxin  Toxin  

EndoA   EndoAI  

Module 3

Growth Inhibition Device

Page 26: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

Bacillus    sub'lis  

Growth  Inhibi>on  

EndoA  

(Toxin)  

EndoAI    

(An>toxin)  

Module 3

Growth Inhibition Device

Page 27: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

OD  (Average  OD595)    

Module 3

                                   RBS  

Promoter  Ac>vity  =  

   

Ptms  GFP                                      RBS  

Double  terminator  

   

J23101  GFP  

GFP  Synthesis  Rate  (Slope)  

Promoter  Ac>vity    of  Ptms  

Promoter  Ac>vity    of  Standard  Promoter  

Rela>ve  Promoter  Unit  (RPU)  of  Ptms  to  reference  standard  promoter  =

Double  terminator  

Ptms Characterization

Page 28: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

y  =  3.4x  +  3.8  

y  =  35.45x  +  225.7  

0  

50  

100  

150  

200  

250  

300  

350  

400  

450  

0   15   30   45   60  

GFP  Intensity

 (Arbitrary  Units)  

Time  (minutes)  

Ptms  &  J23101  GFP  Intensity  Ptms   J23101  

Module 3

Ptms  

Ptms has Low Activity

RPU:  0.046  

Page 29: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

0  

50  

100  

150  

200  

250  

300  

350  

0%   1%   2%   3%   4%   5%   10%  

GFP  Synthe

sis  R

ate/Av

erage  OD5

95  

Xylose  Concentra>on  

PxylA  Promoter  Ac>vity  Induced  With  0-­‐10%  Xylose  

Module 3

PxylA is Inducible

Page 30: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

Control  Group   Experiment  Group  

Xylose  concentra'on  from  le6  to  right  (both  groups):  0.00%,  0.05%,  0.10%,  0.15%,  0.20%,  0.25%  

Module 3

Growth Inhibition Characterization

Page 31: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

Assembly

Page 32: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

Assembly

Three Modules

•  Target  Binding  

•  An>-­‐tumor  Molecule  Secre>on  

•  Regula>on  &  Control  

Page 33: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

Assembly

Modules Assembled

Bacterial  Chromosome  

amyE  3’  amyE  5’  RPMrel  

Expression  

xylR      

PxylA   BMP2  Secre>on   Toxin   An>-­‐

toxin  

RPMrel  

LytC  

EndoA   EndoAI  

   

Ptms        

 

Page 34: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

Achievements & Future Work

Page 35: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

•  PxylA,  Ptms  func>on  as  expected.  •  Cell  growth  inhibi>on  device  func>ons.    •  RPMrel  produc>on  –  work  in  progress.  •  BMP2  produc>on  –  work  in  progress.  

Achievements & Future Work

Our Work

Page 36: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

Human Practice

Page 37: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

Presenta>on  at  Hong  Kong  Cancer  Fund

Human Practice – Presentation

Presentation

Page 38: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

Human Practice – Presentation

Objective & Role

Page 39: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

Human Practice – Presentation

Presentation

Page 40: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

Human Practice – Presentation

Positive Feedback

Synthe'c  biology  can  be  used  to  improve  cancer  treatment.    -­‐-­‐-­‐  Respondent  A

Synthe'c  biology  will  give  cancer  pa'ents  hope.  -­‐-­‐-­‐  Respondent  B

This  presenta'on  is  clear  and  professional.  -­‐-­‐-­‐  Respondent  C

Some  of  the  seeds  have  [been]  planted  which  will  allow  us  to  keep  a  close  eye  to  its  (synthe'c  biology’s)  development.  

-­‐-­‐-­‐  Ms.  HSUEH,  the  Head  of  Wellness  and  Holis7c  Health  Care

Page 41: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

Human Practice – Interviews

Interviews

Page 42: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

POLITICIAN  JOURNALIST

UNIVERSITY  STUDENT HIGH  SCHOOL  STUDENT

Human Practice – Interviews

Interviews

Page 43: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

Human Practice – Interviews

Interviews

…I  will  listen  to  their  complaints  sincerely  as  a  responsible  listener…    …I  will  tell  them  how  to  raise  this  issue  to  higher  authority…    …But  the  final  decision  should  be  made  only  by  the  majority…    

   -­‐-­‐-­‐  The  poli7cian,  Mr.  SHUM  

Page 44: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

Human Practice – Interviews

Interviews

•  Gather  informa7on  about  the  interviewees.    

•  Generate  an  interview  outline  and  ques7ons  specific  targe7ng  at  the  interviewee.  

•  Guide  your  interviewers  and  train  them  to  good  interviewers.  

Page 45: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

Human Practice

Human  Prac>ce  

Presenta>on   Interviews  

Page 46: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

Recap

Page 47: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

Cell  growth  inhibi7on  

Toxin-­‐an7toxin  casse_e  

Colon  tumor  growth  

suppression  

Chemokine  expression  

Tumor  targe7ng  

Pep7de  display  

B.  sub'lis  

Recap

Integrated  Genes  

Page 48: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

•  Office  of  the  Dean  of  Science  

•  Office  of  the  Provost  

– The  Hong  Kong  University  of  Science  &  Technology  

Acknowledgements

Page 49: B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation Presentation . Human Practice – Presentation Objective & Role . Human Practice

2012 HKUST iGEM Team

The Terminator of Colon Cancer

B. hercules


Top Related