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