2016 iGEM Jamboree
Hynes Convention Center, Boston, MA
October 29, 2016
Flint, MI
Pittsburgh, PA
Washington, D.C.
Global Issue
● WHO Lead Poisoning Prevention Week
○ October 23-29th
○ Toxic
■ Children: Behavioral, Neurological
■ Adults: Liver and kidney damage
Model
Lead in
the water
Human
Absorbed
Excreted
LeadBone Blood
Simulink
Model Results: Retention of Lead in Bone and Blood
Model Results: Retention of Lead in Bone and Blood
Background
Pardee, Green, Ferrante, Cameron, DaleyKeyser, Yin, Collins. Cell.2014.
Why?
Reaction Volume Minimization
Reaction volume (μL)
52.51 10 15 25
log(F
luore
scence)
10000
1000
100
10
1
No DNA
GFP25x
DNAzyme
Toehold Switch
ReporterlacZ
Catalytic strand
Catalytic domain
Substrate strand
Lan, Furuya, & Lu. Chem. Commun. 2010.
Pardee, Green, Ferrante, Cameron, DaleyKeyser, Yin, Collins. Cell. 2014.
BBa_K2084002
Reporter Substrate
CPRG (Chlorophenol Red-β-D-galactopyranoside)
Undergoes color change when lacZ is expressed
Activation with RNA
No RNA RNA
Absorb
ance
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
Activation with DNA
8
7
Concentration
0 1 pM 1 nM 100 nM 1 μM 8 μM
Mismatch
6
5
4
3
2
1
0
Rela
tive A
bsorb
ance
2x
5x
6x
7x
8 μM
Condition
5x
No Trigger DNAzyme DNAzyme and Lead DNA Trigger
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
lacZ Expression with 2 μM LeadR
ela
tive A
bsorb
ance
5x
Comparison of Lead Concentrations
Source Concentration
(ppb)
Concentration
(nM)
EPA MCL 15 72.4
Hot Metal Switch 429 2,000
Flint, MI 13,200 63,712
DNAzyme
Toehold Switch
ReporterlacZ
DNAzyme
Toehold Switch
Reporter
T3 RNA Pol.
T3 RNA Pol.
lacZ
PT3
PT3
Amplifier
Current Project: Forward Feedback Amplification
BBa_K2084009
BBa_K2084004
BBa_K2084001
BBa_K2084007
Flexibility
Thallium
Mercury Arsenic
Lead
Future work
Human Practices
Human Practices
Human Practices
Collaborations
Collaborations
Collaborations
Collaborations
Conclusion
● Modeled the consequences of lead exposure
● Demonstrated DNA trigger of toehold switch
● Developed promising circuit with regulatory RBS
● Taught community about synthetic biology
Thank You!
Team Sponsors
Aife Ni Chochlain
Advisors and Mentors
Dr. Alexander
Deiters
Dr. Jason
Lohmueller
Dr. Lisa
Antoszewski
Dr. Cheryl Telmer Dr. Natasa
Miskov-Zivanov
Dr. Sanjeev
Shroff