Home >Documents >B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation...

B. hercules2012.igem.org/files/presentation/HKUST-Hong_Kong.pdf · Human Practice – Presentation...

Date post:10-Aug-2020
Category:
View:1 times
Download:0 times
Share this document with a friend
Transcript:
  • 2012 HKUST iGEM Team

    The Terminator of Colon Cancer

    B. hercules

  • Background & Motive

  • Cancer Therapy & Synthetic Biology

    Background & Motive

    Our  project  à  Cancer  

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

    Colon Carcinomas – A Suitable Target

    Background & Motive

  • •  Non-‐pathogenic.  

    • Well-‐understood.  

    •  Provide  engineering  op7ons.  

    Our Chassis

    Background & Motive

    •  Gut  microbiome  

    •  Lacks  virulence  

    •  Integrates  DNA  

    Gene  integra7on  

  • B. hercules

    Background & Motive

     Target:  colon  carcinomas      Tool:  B.  sub'lis  

  • •  Target  Binding  

    •  An>-‐tumor  Molecule  Secre>on  

    •  Regula>on  &  Control  

    Background & Motive

    Three Modules

  • Module 1

    Target Binding

  • Colon  Cancer  Cell  

    Module 1

    RPMrel  

    Tumor Recognition

  • Bacillus  

    sub'lis  

    Module 1

    RPMrel  

    Tumor Recognition

    Colon  Cancer  Cell  

  • LytC  

    Helical  Linker  

    Module 1

    Cell Wall Display

  • lytC   linker   RPMrel                                      

    Double    Terminator  

    RBS  

                 

    Pveg  

    Module 1

    Construct

  • Module 2

    Anti-tumor Molecule Secretion

  • Secrete  BMP2  BMP2  Module 2

    Anti-tumor Drug

  • BMP2  Signal  pep>de  

    Module 2

    Out  

    In  

    Membrane  

    Secretion

  • Module 2

    Cleavage  

    Signal  pep>de  

    BMP2  

    Secretion

  • SPPase  

    BMP2  Folding  

    Module 2

    Secretion

  • RBS  xylR   Bmp2  Signal    pep>de    

       

    PxylA  

    Module 2

    Construct

  • 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  

  • Regulation & Control

    Module 3

  • Two  Regula>on  Systems  

    1.  Time  Control   2.  Dosage  Regula>on  

    Module 3

    Regulation Systems

  • RBS  xylR      

    PxylA  

    Module 3

    Xylose-Inducible Promoter

  • RBS  xylR  Bmp2  Signal    

    pep>de        

    PxylA  

    XylR   Inhibited  Transcrip>on  

    Module 3

    Xylose-Inducible Promoter

  • RBS  xylR  Bmp2  Signal    

    pep>de        

    PxylA  

    XylR   Transcrip>on  Proceeds  

    Module 3

    xylose  

    Xylose-Inducible Promoter

  • Bmp2  Signal    

    pep>de  

    ydcE  RBS  

    An>toxin  Toxin  

    EndoA   EndoAI  

    Module 3

    Growth Inhibition Device

  • Bacillus    sub'lis  

    Growth  Inhibi>on  

    EndoA  

    (Toxin)  

    EndoAI    

    (An>toxin)  

    Module 3

    Growth Inhibition Device

  • 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

  • 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  

  • 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

  • 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

  • Assembly

  • Assembly

    Three Modules

    •  Target  Binding  

    •  An>-‐tumor  Molecule  Secre>on  

    •  Regula>on  &  Control  

  • Assembly

    Modules Assembled

    Bacterial  Chromosome  

    amyE  3’  amyE  5’  

    RPMrel  

    Expression  

    xylR      

    PxylA   BMP2  Secre>on   Toxin   An>-‐

    toxin  

    RPMrel  

    LytC  

    EndoA   EndoAI  

       

    Ptms        

     

  • Achievements & Future Work

  • •  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

  • Human Practice

  • Presenta>on  at  Hong  Kong  Cancer  Fund

    Human Practice – Presentation

    Presentation

  • Human Practice – Presentation

    Objective & Role

  • Human Practice – Presentation

    Presentation

  • 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

  • Human Practice – Interviews

    Interviews

  • POLITICIAN  JOURNALIST

    UNIVERSITY  STUDENT HIGH  SCHOOL  STUDENT

    Human Practice – Interviews

    Interviews

  • 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  

  • 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.  

  • Human Practice

    Human  Prac>ce  

    Presenta>on   Interviews  

  • Recap

  • Cell  growth  inhibi7on  

    Toxin-‐an7toxin  casse_e  

    Colon  tumor  growth  

    suppression  

    Chemokine  expression  

    Tumor  targe7ng  

    Pep7de  display  

    B.  sub'lis  

    Recap

    Integrated  Genes  

  • •  Office  of  the  Dean  of  Science  

    •  Office  of  the  Provost  

    – The  Hong  Kong  University  of  Science  &  Technology  

    Acknowledgements

  • 2012 HKUST iGEM Team

    The Terminator of Colon Cancer

    B. hercules

Click here to load reader

Reader Image
Embed Size (px)
Recommended