2016 iGEM Jamboree Hynes Convention Center, Boston,...

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