Brown iGEM

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Brown iGEM

• international genetically engineered machines
  competition

July Update
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What is iGEM?

• Biology
• Engineering
• Standardization

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Making it easier to engineer biology
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DNA is a language
AATGAATATCCAGATCG
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Biological Part
Promoter
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Different Parts connect together
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Gene
Promoter
Terminator
This is a device
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Different Parts connect together
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ConstitutivePromoter
Terminator
GFP
This is a device
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Biological parts are building blocks made of
genetic material
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Science

• Systematic engineering
• Standardizing biology
• Apply biological technology

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Brown iGEM
Two projects being built with biological parts

• Lead-detector
• Tri-stable Switch

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Lead Detector
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Version 1.0 Lead Detector
Fluorescent Protein
Lead Promoter
Problem Only one cell will light up!
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Version 1.1 Amplify the Signal
Fluorescent Protein
Amplifier
Lead Promoter
Problem Promoter Leakiness False Positives!
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Version 1.2 Filter False Positives

• Three Possible Solutions
• Modify the Promoter (weaker baseline)
• Tight intermediate promoter (T7)
• 3. Make amplifier less sensitive (increase
  threshold)

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Final Version The System
Fluorescent Protein
Leakiness Filter
Amplifier
Lead Promoter
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So how will this system work in the cell?
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NO LEAD
PbrR
LuxR
TetR (always on)
Transcription factors are constitutively made by
the first promoter.
LuxI
Lead Promoter
These proteins are poised to activate the Lead
Detector promoter and Message Receiver promoter
upon addition of lead.
LuxI
GFP
pLux
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LuxR
PbrR
TetR (always on)
LuxI
Lead turns on Detector promoter
Lead Promoter

LuxI
GFP
pLux
Fluorescent Protein Output
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Experimental Design

• iGEMs more than just design. This will take some
  lab work.

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Experimental Design
Three Independent System Components AHL unifies
three components with a common language to match
Inputs with Outputs.
Filter
Amplifier
Lead Receptor and Promoter
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Experimental Design
Three Independent System Components AHL unifies
three components with a common language to match
Inputs with Outputs.
Develop AHL Assay for testing all components.
STEP 1
STEP 2a and 2b
Filter
Amplifier
Lead Receptor and Promoter
STEP 3
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What is AHL?
Why and How do we measure it?
Cell Signaling Molecule
Common input and output of different devices
within our system
Acyl Homoserine Lactone
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AHL BioAssay
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AHL BioAssay
More AHL --gt More GFP Need more than 10 nM AHL
to overcome threshold
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Experimental Design
Develop AHL Assay for testing all components.
STEP 1
Amplifier
STEP 2a and 2b
Lead Receptor and Promoter
Filter
STEP 3
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Amplifier

• Chemical Transformation
• Electroporation
• Ordering from MIT
• Build it ourselves
• Measure AHL output

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Experimental Design
Develop AHL Assay for testing all components.
STEP 1
Amplifier
STEP 2a and 2b
Lead Receptor and Promoter
Filter
STEP 3
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• Lead Receptor and Promoter

Ralstonia Metallidurans CH34
Survives in metallic environments.
http//genome.jgi-psf.org/finished_microbes/ralme/
ralme.home.html
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Lead Receptor and Promoter

• We chose to examine
• Lead Receptor Protein PbrR691
• 2. Corresponding Lead Promoter

PbrR691
Lead Promoter
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Lead Receptor and Promoter

• Why?
• Incredibly Selective!
• Novel
• Successfully cloned into E Coli.

Chen, Peng, Bill Greenberg, Safiyah Taghavi,
Christine Romano, Daniel van der Lelie, and Chuan
He. An Exceptionally Selective
Lead(II)-Regulatory Protein from Ralstonia
Metallidurans Development of a Fluorescent
Lead(II) Probe. Angew. Chem. Int. Ed. 2005, 44,
2-6.
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Original Design
PbrR691
PbrR691
pTet (Constitutive On)
Amplifier
Lead Promoter
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Lead Receptor PbrR691 and Lead Promoter must be
BioBricked!
PbrR691
GACTGATCGATAGATCGAGATCGATCGATAGAGGCTCTCGAGATCGCGAG
ATATCG
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BioBrick Assembly
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How do we get PbrR691 and Lead Promoter?
PCR 2 Major Obstacles - Biobricking a promoter
adds extra bases from the restriction sites to
the ends, which may reduce promoter
efficiency. - Length of promoter very small
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Experimental Plan

• Purpose Match switch components
• PCR 12 variations of promoter and gene
• Ligate to RBS-LuxI-GFP-Term
• Test with AHL against AHL bioassay curve
• Result promoter output amplifier input

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Experimental Design
Develop AHL Assay for testing all components.
STEP 1
Amplifier
STEP 2a and 2b
Lead Receptor and Promoter
Filter
STEP 3
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Problem Leakiness

• What if the baseline is too high?
• Possible solution T7 promoter control
• Advantage strong repression (not leaky) unless
  T7 RNA polymerase is present

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T7 Filter Schematic
T7 polymerase will transcribe LuxI
T7 polymerase
pPbr
Amplifier
LuxI
T7 Promoter
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Possible Issues

• Poor sensitivity
• Poor pPbr induction
• Solution Need to test pPbr promoter as well as
  whole T7 system
• What are our choices for T7 systems?

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T7 registry parts
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Experimental Design
Develop AHL Assay for testing all components.
STEP 1
Amplifier
STEP 2a and 2b
Lead Receptor and Promoter
Filter
STEP 3
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Tri-Stable Switch
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Tristable Switch Team

• Introduction
• System Design
• Modeling
• System Tests
• 5. Labwork

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Introduction
A
B

• Stable Switch A system with 2 or more distinct
  and inducible states.

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Bistable Switch

• This is the simplest switch.
• It only involves two separate states.

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Uses for a Bistable Switch

• Drug Delivery
• Simple Logic

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Bistable Switch

• In 2000, three scientists at Boston University
  managed to create a synthetic Bistable Switch.
• They showed that you could create the Bistable
  Switch using relatively simple, standard parts.

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Bistable Switch Design

• The Bistable Switch simply consists of two
  pathways, each of which represses the other.

Pathway A
GFP
pTet
LacI
Pathway B
pLac
TetR
YFP
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Importance of Bistable Switch

• The Bistable Switch is one of the seminal
  achievements of Synthetic Biology.
• It was one of the first projects that showed that
  you could combine standard genetic parts together
  to form working circuits.

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Tristable Switch
A
B
C

• A switch with three distinct inducible states.

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Tristable Switch Design

• The design consists of three pathways, each of
  which represses the other two.
• When one of the pathways is induced it stops the
  other two from being expressed, and the system
  achieves stability.

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Tristable Switch Design

Pathway A
pTet LacI
AraC
Pathway B
pLac AraC
TetR
Pathway C
pAra TetR
LacI
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Tristable Switch Tuning

• While the design is relatively simple, the exact
  components we put into it have to be carefully
  chosen to balance the system.

pTet LacI
AraC
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Modeling
Why do we model?

• A quick and inexpensive way to quantitatively
  predict behavior
• A foundation to start testing, e.g. what
  variables do we need to test to understand our
  system

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Modeling
Why does our system lend itself to modeling?

• Sensitive system
• Future adaptations

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Variables in the Model

1. Rate of repressor production
2. Strength of repression

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Variables in the Model

• Rate of repressor production depends on
• Promoter strength (transcription)
• RibosomeBindingSite strength (translation)

RBS

• In model, a Promoter RBS
• total repressor production rate

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Variables in the Model

• Repressor strength depends on
• ß the cooperativity of repressors to promoters
• repressor the concentration of repressor

Total strength of repressor repressor?
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Variables in the Model
Graph of repressor? where ? .5, 1, 1.5, 2
ß cooperativity of repression
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Equations
For the Bi-Stable Switch
x and y repressor concentration a
repressor production rate ß cooperativity of
repression
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Equations
Bistable
Tristable
Vs.
The equations are extended to a tri stable system.
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Equations
The number of repressors correlates to the number
of terms
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The Bi Stable Region

ß cooperativity a repressor production rate
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The Tri Stable Region
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What the Model Predicts
A stable system occurs when

• ß gt 1 or larger to maximize the stable region
• a values are similar for all promoters
• a values are within the stable region

ß cooperativity a repressor production rate
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So what can we do with the modelling?
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1. Systematic Approach to Construction

• Design tests to assign values to variables in
  model
• Promoter/RBS Strength, Relative Repressor
  Cooperativity, etc
• Use these values in the model to find the right
  combination of parts.

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Alternative test, hope it works, if not, test
again.
Systematic Design is the philosophy of Synthetic
Biology
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2. Characterization of System

• It is a step towards standardization - giving
  others all the details needed to use the part.

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Testing Constructs

1. (?) Promoter/RBS Strength
2. (?) Repressor Strength
3. Inducer Strength

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Promoter/RBS Strength
Promoter RBS GFP
Because there is no way to measure strength or
concentration directly, we measure with
florescent proteins.
variable
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Repressor Strength
Variable
Inducible Promoter RBS
Repressor GFP
ß cooperativity a repressor production rate
Repressible Promoter RBS YFP
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Inducer Strength
Variable Inducer
Promoter RBS Repressor
X
Promoter RBS GFP
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Testing Restraints

• Florescent proteins not perfect read out
• Indirect measurement of gene
• a. Protein folding time
• b. Degradation Rate
• Rate of Production Repressor vs GFP
• High toll on cell machinery and resources

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What weve been up to
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KABOBS
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Mastering Cloning

• More obstacles than we thought
• Transformations, DNA concentration too low, gel
  readibility, restriction digest buffer
  compatibility, etc.
• Most kinks worked out of the way
• First ligations completed

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The Project Itself

• Looking through Modeling
• Designed Tests
• Created DNA stocks of all parts needed
• Creating a good record keeping infrastructure

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Goals
Testing Ligations
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References

• Gardner TS, Cantor CR, Collins JJ. Construction
  of a genetic toggle Switch in Escherichia coli.
  Nature 2000 Jan, 20.

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2007 Brown iGEM Team

• 7 undergraduates
• 7 grad student advisors
• 2 Faculty advisors
• 9 faculty sponsors

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Sponsors
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Special Thanks To
Office of the Dean of the CollegeOffice of the
PresidentThe Atlantic PhilanthropiesThe Center
for Computational and Molecular
BiologyDepartment of PhysicsEngineering
DepartmentDepartment of Molecular Biology, Cell
Biology, and BiochemistryDepartment of Molecular
Pharmacology, Physiology, and BiotechnologyThe
Multi Disciplinary LabPfizerLabnetNanodrop
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Thank you for listening!Questions?
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