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2007 Brown iGEM Team

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Science - Nuts and Bolts. Standardizing biology. Systematic engineering. Apply biological ... Adding New Parts and Devices. Lead Promoter and ... – PowerPoint PPT presentation

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Title: 2007 Brown iGEM Team


1
2007 Brown iGEM Team
  • 7 undergraduates
  • 7 grad student advisors
  • 2 Faculty advisors
  • 9 faculty sponsors

1/45
2
Brown iGEM
June Update
  • international genetically engineered machines
    competition

2/45
3
What is iGEM?
  • Biology
  • Engineering
  • Standardization

3/45
4
Science - Nuts and Bolts
  • Standardizing biology
  • Systematic engineering
  • Apply biological technology

4/45
5
Previous Projects
  • Bacterial Photo Film - U. Texas (published in
    Nature)

5/45
6
Previous Projects
  • Sepsis Treatment - Slovenia
  • Banana/Wintergreen Smelling Cells - MIT
  • Arsenic Water Detection - Edinburgh

5/45
7
  • Anderson, J. C., Clarke, E. J., Arkin, A. P.,
    Voigt, C. A. (2005) Environmentally Controlled
    Invasion of Cancer Cells by Engineered Bacteria,
    Journal of Molecular Biology

6/45
8
Aerobic Conditions Low Cell Density gt0.02
Arabinose OFF
Hypoxia High Cell Density lt0.02 Arabinose ON
Inv induction
INVASION
  • Anderson, J. C., Clarke, E. J., Arkin, A. P.,
    Voigt, C. A. (2005) Environmentally Controlled
    Invasion of Cancer Cells by Engineered Bacteria,
    Journal of Molecular Biology

7/45
9
Undergraduate Teams
Imagined Designed Implemented by a team of
undergraduates
8/45
10
Progress
  • Browns 2nd year
  • UTRA grants
  • Lab space in Multi Disciplinary Lab
  • Equipment sponsorship

9/45
11
Project 1 Lead Biosensor
10/45
12
Why do we need this?
  • Public health concern Lead in soil, paint,
    water, dust

11/45
13
Why do we need this?
  • Public health concern Lead in soil, paint,
    water, dust
  • Lead Poisoning is often caused by ingesting
    contaminated drinking water, or soil. It can
    cause neurological and gastrointestinal
    disorders, especially among children.

12/45
14
Why do we need this?
  • Public health concern Lead in soil, paint,
    water, dust
  • Lead Poisoning is often caused by ingesting
    contaminated drinking water, or soil. It can
    cause neurological and gastrointestinal
    disorders, especially among children.
  • The legal limit of lead in drinking water is 15
    parts per billion.

13/45
15
Why do we need this?
Current ways of testing for lead either require
expensive chemical lab analysis or involve
inaccurate home kits. So why do we want a
biosensor? - Cheap - Sensitive - Quick -
Specific
14/45
16
We have spliced together biobrick parts into
plasmids in E. Coli. The bacteria express our
genetic devices to create a lead detector.
15/45
17
General Design
Lead
Lead Detection
Lead Detection
Signal Amplification
Signal Amplification
Fluorescent Output
16/45
18
17/45
19
2 System Components
Part 1 Lead Detection
Part 2 Signal Amplification
18/45
20
Part 1 Lead Detection
Lead Receptor Protein
Lead Receptor Gene
Always On
19/45
21
Part 1 Lead Detection
Lead
Activated Lead Receptor Protein
Lead Receptor Protein
Lead Receptor Gene
Always On
20/45
22
Part 1 Lead Detection
Lead
Activated Lead Receptor Protein
Lead Receptor Protein
Activated Lead Receptor Protein
Lead Receptor Gene
Always On
Signal Amplification promoter
21/45
23
Part 1 Lead Detection
  • This is sensitive ONLY to lead other metals
    will not activate it.

22/45
Chen P, Greenberg B, Taghavi S, Romano C, van der
Lelie D, He C (2005) An exceptionally selective
lead(II)-regulatory protein from Ralstonia
metallidurans development of a fluorescent
lead(II) probe. Angew Chem Int Ed Engl
4427152719
24
General Design
Lead
Lead Detection
Lead Detection
Signal Amplification
Signal Amplification
Fluorescent Output
23/45
25
Part 2 Signal Amplification
Activated Lead Receptor Protein
Signal Amplification promoter
24/45
26
Part 2 Signal Amplification
Message
Activated Lead Receptor Protein
Message Producer Gene
Signal Amplification promoter
25/45
27
Part 2 Signal Amplification
Message Producer Gene
Signal (GFP)
Message
Activated Lead Receptor Protein
Message Producer Gene
Signal Amplification promoter
26/45
28
Part 2 Signal Amplification
Message Producer Gene
Signal (GFP)
Message
Activated Lead Receptor Protein
Repeated activation of this promoter causes
amplification of the signal.
Message Producer Gene
Signal Amplification promoter
27/45
29
3 System Components
Lead Detection
1. Detector Sequence
2. Leakiness Filter to Eliminate False Positives
Signal Amplification
3. Positive Feedback Loop for Amplification
Weve added a Leakiness Filter as an additional
component to the system. This gives our circuit
one more level of complexity. However, the black
box diagram is generally the same.
28/45
30
NO LEAD
Lead Activator
Message Activator
Always On
Transcription factors are constitutively made by
the first promoter.
Message Maker
Filter Repressor
Lead Detector
Message Destroyer
Filter
Filter Eliminates Stray Message Molecules to
Prevent False Feedback Loop Activation
Stray Message Molecules Can Falsely Activate the
Feedback Loop.
These proteins are poised to activate the Lead
Detector promoter and Message Receiver promoter
upon addition of lead.
Message Maker
GFP Reporter
Message Receiver
29/45
31
Lead Activator
Feedback Activator
Always On
Message Maker
Filter Repressor
Lead turns on Detector promoter
Lead Detector
Message Destroyer
Leakiness Filter promoter gets turned off
Filter

Fluorescent Protein Output
Message Maker
GFP Reporter
Message Receiver
30/45
32
Lead Activator
Feedback Activator
Always On
Message Maker
Filter Repressor
Lead Detector
1
Message Destroyer
2
Filter
Message Maker
GFP Reporter
Message Receiver
3
31/45
33
PbrR
LuxR
pTet
LuxI
LacI
Pbr
1
aiiA
2
pLac
LuxI
GFP
pLux
32/45
3
34
33/45
35
How this project advances science
  • Characterization of Existing Parts
  • Adding New Parts and Devices
  • Lead Promoter and Transcription Factor
  • Amplifier Device
  • Generalize to future biosensors
  • Arsenic, Cadmium, Mercury, Zinc

34/45
36
THE TRI-STABLE SWITCH
Project 2
35/45
37
What is Tri-stability?
  • A tri-stable switch has three distinct and
    inducible states

A
B
C
36/45
38
Achieving Tri-stability
Input A
Output A
State A
Input B
Output B
State B

Input C
Output C
State C
37/45
39
The Architecture
Arabinose
RFP
pBad/Ara
tetR
lacI
IPTG
CFP
tetR
araC
pLac
Tetracycline
YFP
pTet
lacI
araC
38/45
40
Characterization
  • Characterization is an essential aspect of iGEM
  • It is a step towards standardization - giving
    others all the details needed to use the part.

39/45
41
Uses
  • Differentiation of stem cells
  • Turn on/off three different proteins in cell
  • Cellular logic
  • Tissue Engineering

40/45
42
Timeline
  • Now
  • Start cloning
  • PCR lead promoter
  • Clone tri-stable switch
  • Characterize parts
  • Test systems
  • Send back to the Registry
  • August End of lab work
  • November Jamboree at MIT

41/45
43
Why Brown?
  • Innovators
  • Entrepreneurs
  • A great place for new ideas!

42/45
44
The Future
  • Educate others about iGEM and synthetic biology
  • Synthetic Biology Course Offering in Fall 07! Led
    by Prof. Gary Wessel

43/45
45
Special Thanks To
Office of the Dean of the College Office of the
President The Atlantic Philanthropies The Center
for Computational and Molecular
Biology Department of Physics Engineering
Department Department of Molecular Biology, Cell
Biology, and Biochemistry Department of Molecular
Pharmacology, Physiology, and Biotechnology The
Multi Disciplinary Lab Pfizer Labnet Nanodrop
44/45
46
Thank you for listening! Questions?
45/45
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