regulation

1
regulation
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course layout

• introduction
• molecular biology
• biotechnology
• bioMEMS
• bioinformatics
• bio-modeling
• cells and e-cells
• transcription and regulation
• cell communication
• neural networks
• dna computing
• fractals and patterns
• the birds and the bees .. and ants

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introduction
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electronic pathway
5
seoul subway
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tokyo subway
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pyrimidine pathway
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(No Transcript)
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protein pathway
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from DNA to pathways
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biological information

• Two Types of Biological Information
• The genome, digital information
• Environmental, analog information

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genome information

• Two types of digital genome information
• Genes, the molecular machines of life
• Gene regulatory networks, specify the behavior of
  the genes

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what is systems biology?
Biological System
DNA
Biomodules
RNA
Cells
Networks
Proteins
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a gene network
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a gene network in a physical network
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what is a genetic circuit?

• Jacob Monod Model of the prokaryotic operon
  (1961)

Repressor
RNAP
Inducer
Gene A
Promoter
Operator
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what is a genetic circuit?

• Jacob Monod Model of the prokaryotic operon
  (1961)
• It is obvious from analysis of these bacterial
  genetic regulatory mechanisms that their known
  elements could be connected into a wide variety
  of circuits endowed with any desired degree of
  stability

A
Gene A
B
Operator
Promoter
Gene B
Promoter
Operator
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electronic circuits

• Basic electrical engineering (digital)
• A basic flip-flop memory

A
C
B
in1
out1
Stable states (with in1, in2 0) out1 out2 0
1 1 0
out2
in2
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examples

• A genetic NAND Gate
• A genetic flip-flop

out1
in1
in2
out2
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basic genetic engineering

• How do you clone a gene?

accessexcellence.com/AB/GG/plasmid.html
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genetic circuit engineering paradigm

• 1. Design
• Design genetic circuitry that demonstrates a
  rudimentary control behavior, such as
  oscillations, bistability (like the flip-flop),
  step activation, a spike, etc.
• 2. Simulate
• Build a simulation (deterministic or stochastic
  ODEs) encapsulating the design and examine its
  dynamic behavior (boundary conditions of
  different stability regimes, parameter
  sensitivity).
• 3. Implement and Test
• Use the results of this simulation to pick
  genetic parts yielding the desired behavior and
  splice them together in a plasmid. Transform the
  plasmid into bacteria and observe the behavior of
  the system. Does it match predictions from the
  simulation? -- Back to 1

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gene expression
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gene regulation mechanism

• Bacteria express only a subset of their genes at
  any given time.
• Expression of all genes constitutively in
  bacteria would be energetically inefficient.
• The genes that are expressed are essential for
  dealing with the current environmental
  conditions, such as the type of available food
  source.

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gene regulation mechanism

• Regulation of gene expression can occur at
  several levels
• Transcriptional regulation no mRNA is made.
• Translational regulation control of whether or
  how fast an mRNA is translated.
• Post-translational regulation a protein is made
  in an inactive form and later is
  activated.

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gene regulation mechanism
Transcriptional control
Translational control
Post-translational control
Lifespan of mRNA
Protein activation (by chemical
modification)
Onset of transcription
Protein
Translation rate
Ribosome
DNA
mRNA
Feedback inhibition (protein inhibits
transcription of its own gene)
RNA polymerase
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Escherichia coli
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gene regulation mechanism

• Operon
• A controllable unit of transcription consisting
  of a number of structural genes transcribed
  together. Contains at least two distinct
  regions the operator and the promoter.

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gene regulation mechanism

• Case study of the regulation of the lactose
  operon in E. coli
• E. coli utilizes glucose if it is available, but
  can metabolize other sugars if glucose is absent.

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gene regulation mechanism
Glucose Lactose
Food source
Glucose Lactose
Glucose Lactose
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11
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Second period of rapid growth with lactose as
food source
70
60
29.5
50
14.0
40
43.5
Relative density of cells
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20
26.5
Initial period of rapid growth with glucose as
food source
39.0
13.5
10
0
0
1
2
3
4
5
0
1
2
3
4
5
6
0
1
2
3
4
5
6
7
Time (hours)
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gene regulation mechanism

• Case study of the regulation of the lactose
  operon in E. coli
• Genes that encode enzymes needed to break
  other sugars down are negatively regulated.
• Example enzymes required to metabolize lactose
  are only synthesized if glucose is depleted and
  lactose is available.
• In the absence of lactose, transcription of the
  genes that encode these enzymes is repressed.
  How does this occur?

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gene regulation mechanism

• Case study of the regulation of the lactose
  operon in E. coli
• All the loci required for lactose metabolism are
  grouped together into an operon.
• The lacZ locus encodes ?-galactosidase enzyme,
  which breaks down lactose.
• The lacY locus encodes galactosidase permease, a
  transport protein for lactose.
• The function of the lacA locus is unknown.
• The lacI locus encodes a repressor that blocks
  transcription of the lac operon.

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gene regulation mechanism
Regulatory function
Cleaves lactoseto glucose and galactose
Membrane transport protein-imports lactose
Regulatoryprotein
Galactosidase permease
ß-galactosidase
Lacl
LacY
LacZ
Section of E. coli chromosome
lacl
lacZ
lacY
Observations about regulation of lacZ and lacY
Glucose
(1) Lacl protein and glucose shut down
transcription of lacZ and lacY
Lactose
E. coli
Galactose
Galactosidase permease
(2) Lactose induces transcription of lacZ andlacY
Chromosome
ß-galactosidase
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gene regulation mechanism
Lac operon
lacl promoter
lacl
Operator
lacZ
lacY
lacA
Promoter
lac operon
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gene regulation mechanism

• Repression and induction of the lactose operon.
• The lac operon is under negative regulation, i.e.
  , normally, transcription is repressed.
• Glucose represses transcription of the lac
  operon.
• Glucose inhibits cAMP synthesis in the cells.
• At low cAMP levels, no cAMP is available to
  bind CAP.
• Unless CAP is bound to the CAP site in the
  promoter, no transcription occurs.

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gene regulation mechanism
When no lactose is present, the repressor binds
to DNA and blocks transcription.
NO TRANSCRIPTION
Functional repressor
lacl
lacZ
lacY
RNA polymerase blocked
Operator (binding site for repressor)
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gene regulation mechanism
Repressor plus lactose (an inducer) present.
Transcription proceeds.
Lactose
TRANSCRIPTION BEGINS
?-galactosidase
Permease
mRNA
repressor
lacl
lacZ
lacY
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gene regulation mechanism
Operons produce mRNAs that code for functionally
related proteins.
"Polycistronic" mRNA
lacZ message
lacY message
RNA polymerase binds to promoter
lacA message
lacl promoter
lacl
Promoter
Operator
lacZ
lacY
lacA
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cell programming
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programming cell communities
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programming cell communities

• Program cells to perform various tasks using
• Intra-cellular circuits
• Digital analog components
• Inter-cellular communication
• Control outgoing signals, process incoming
  signals

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programmed cell applications

• Biomedical combinatorial gene regulation with few
  inputs tissue engineering
• Environmental sensing and effecting recognize and
  respond to complex environmental conditions
• Engineered crops toggle switches control
  expression of growth hormones, pesticides
• Cellular-scale fabrication cellular robots that
  manufacture complex scaffolds

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programmed cell applications
pattern formation
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programmed cell applications
analyte source
reporter rings
analyte source detection
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biological cell programming
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biological cell programming
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cellular logic
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protein expression basics

• RNA polymerase binds to promoter
• RNAP transcribes gene into messenger RNA
• Ribosome translates messenger RNA into protein

RNA Polymerase
Z Promoter
Z Gene
DNA
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protein expression basics

• RNA polymerase binds to promoter
• RNAP transcribes gene into messenger RNA
• Ribosome translates messenger RNA into protein

RNA Polymerase
Z Promoter
Z Gene
DNA
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protein expression basics

• RNA polymerase (RNAP) binds to promoter
• RNAP transcribes gene into messenger RNA
• Ribosome translates messenger RNA into protein

Transcription
RNA Polymerase
Messenger RNA
Z Promoter
Z Gene
DNA
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protein expression basics

• RNA polymerase binds to promoter
• RNAP transcribes gene into messenger RNA
• Ribosome translates messenger RNA into protein

Translation
RNA Polymerase
Z
Protein
Transcription
Messenger RNA
Z Promoter
Z Gene
DNA
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regulation through repression

• Repressor proteins can bind to the promoter and
  block the RNA polymerase from performing
  transcription
• The DNA site near the promoter recognized by the
  repressor is called an operator
• The target gene can code for another repression
  protein enabling regulatory cascades

RNA Polymerase
R
Transcription Translation
DNA Binding
R
R Promoter
Z Promoter Operator
Z Gene
R Gene
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transcription-based inverter

• Protein concentrations are analogous to
  electrical wires
• Proteins are not physically isolated, so unique
  wires require unique proteins

R
0
1
R
Z
1
0
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simple inverter model
Chemical Equations
Repressor Binding R O ? RO KRR (O)(R)/(RO)
Protein Synthesis O ? O Z kx
Protein Decay Z ? kdeg
Total Concentration Equations
Total Operator (OT) (O) (RO)
Total Repressor (RT) (R) (RO) ? (R) if (RT) gtgt (O)
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simple inverter model
Transfer Function Derivation
(O) (O) 1 1
(OT) (O) (RO) 1 (RO)/(O) 1 (R)/KRR
d(Z) kx (O) kdeg (Z) 0 at equilibrium
dt kx (O) kdeg (Z) 0 at equilibrium
(Z) kx (O) kx (OT)
(Z) kdeg (O) kdeg 1 (R)/KRR
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simple inverter model
Chemical Equations
Repressor Binding R O ? RO KRR (O)(R)/(RO)
Protein Synthesis O ? O Z kx
Protein Decay Z ? kdeg
Total Concentration Equations
Total Operator (OT) (O) (RO)
Total Repressor (RT) (R) (RO) ? (R) if (RT) gtgt (O)
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cooperativity

• Cooperative DNA binding is where the binding of
  one protein increases the likelihood of a second
  protein binding
• Cooperativity adds more non-linearity to the
  system
• Increases switching sensitivity
• Improves robustness to noise

RNA Polymerase
R
Transcription Translation
Cooperative DNA Binding
R
R
Z Promoter Operator
Z Gene
R Gene
R Promoter
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cooperative inverter model
Chemical Equations
Coop Binding R R O ? R2O KR2O (O)(R)2/(R2O)
Protein Synthesis O ? O Z kx
Protein Decay Z ? kdeg
Total Concentration Equations
Total Operator (OT) (O) (R2O)
Total Repressor (RT) (R) 2(R2O) ? (R) if (RT) gtgt (O)
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cooperative inverter model
Transfer Function Derivation
(O) (O) 1 1
(OT) (O) (RO) 1 (RO)/(O) 1 (R)2/KR20
d(Z) kx (O) kdeg (Z) 0 at equilibrium
dt kx (O) kdeg (Z) 0 at equilibrium
(Z) kx (O) kx (OT)
(Z) kdeg (O) kdeg 1 (R)2/KRR
Cooperative Non-Linearity
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cooperative inverter model
Chemical Equations
Coop Binding R R O ? R2O KR2O (O)(R)2/(R2O)
Protein Synthesis O ? O Z kx
Protein Decay Z ? kdeg
Total Concentration Equations
Total Operator (OT) (O) (R2O)
Total Repressor (RT) (R) 2(R2O) ? (R) if (RT) gtgt (O)
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cellular logic summary

• Current systems are limited to less than a dozen
  gates
• Three inverter ring oscillator Elowitz00
• RS latch Gardner00
• Inter-cell communication Weiss01
• A natural repressor-based logic technology
  presents serious scalability issues
• Scavenging natural repressor proteins is time
  consuming
• Matching natural repressor proteins to work
  together is difficult
• Sophisticated synthetic biological systems
  require a scalable cellular logic technology with
  good cooperativity
• Zinc-finger proteins can be engineered to create
  many unique proteins relatively easily
• Zinc-finger proteins can be fused with
  dimerization domains to increase
  cooperativity
• A cellular logic technology of only zinc-finger
  proteins should hopefully be
  easier to characterize

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in vivo logic circuits
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E. coli
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logic gates
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a genetic circuit building block
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logic circuit based on inverters

• Proteins are the wires/signals
• Promoter decay implement the gates
• NAND gate is a universal logic element
• any (finite) digital circuit can be built!

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NAND and NOT gate
x y NAND
0 0 1
0 1 1
1 0 1
1 1 0
X
XY
Y
x NOT
0 1
1 0
X
X
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logic circuit based on inverters

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why digital?

• We know how to program with it
• Signal restoration modularity robust complex
  circuits
• Cells do it
• Phage ? cI repressor Lysis or Lysogeny?Ptashne,
  A Genetic Switch, 1992
• Circuit simulation of phage ?McAdams Shapiro,
  Science, 1995
• Also working on combining analog digital
  circuitry

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why digital?
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BioCircuit CAD
SPICE
http//bwrc.eecs.berkeley.edu/classes/icbook/SPICE
/
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BioCircuit CAD
intercellular
dynamics

• BioSPICE a prototype biocircuit CAD tool
• simulates protein and chemical concentrations
• intracellular circuits, intercellular
  communication
• single cells, small cell aggregates

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genetic circuit elements
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modeling a biochemical inverter
input
repressor
promoter
output
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a BioSPICE inverter simulation
input
repressor
promoter
output
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smallest memory RS-latch flip-flop
0
1
1
0
1
0
1
0

• The output a of the R-S latch can be set to 1 by
  momentarily setting S to 0 while keeping R at 1.
• When S is set back to 1 the output a stays at 1.

• Conversely, the output a can be set to 0 by
  keeping S at 1 and momentarily setting R to 0.
• When R is set back to 1, the output a stays at 0.

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RS-latch flip-flop truth table
R

S

Q

Q

(n
1
)
(n
1
)
0

0

Q

Q

Q R Q
(n)
(n)
0

1

1

0

1

0

0

1

Q S Q
1

1

0

0


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RS-latch timing diagram
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RS-latch dangerous transition
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proof of concept in BioSPICE
RS-Latch (flip-flop)
Ring oscillator
_ R
A
_ R
_ S
A
B
time (x100 sec)
B
B
_ S
C
A
time (x100 sec)
time (x100 sec)

• They work in vivo
• Flip-flop Gardner Collins, 2000
• Ring oscillator Elowitz Leibler, 2000
• However, cells are very complex environments
• Current modeling techniques poorly predict
  behavior

Work in BioSPICE simulations Weiss, Homsy,
Nagpal, 1998
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the IMPLIES gate

• Inducers that inactivate repressors
• IPTG (Isopropylthio-ß-galactoside) ? Lac
  repressor
• aTc (Anhydrotetracycline) ? Tet repressor
• Use as a logical IMPLIES gate (NOT R) OR I

Repressor
Output
Inducer
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the IMPLIES gate
active repressor
inactive repressor
RNAP
inducer
transcription
no transcription
RNAP
gene
gene
operator
promoter
operator
promoter
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the toggle switch
pIKE lac/tet pTAK lac/cIts
Gardner Collins, 2000
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the toggle switch
Gardner Collins, 2000
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the ring oscillator
Elowitz, Leibler 2000
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the ring oscillator
The repressilator is a cyclic negative-feedback
loop composed of three repressor genes and their
corresponding promoters, as shown schematically
in the centre of the left-hand plasmid. It uses
PLlacO1 and PLtetO1, which are strong, tightly
repressible promoters containing lac and tet
operators, respectively6, as well as PR, the
right promoter from phage l. The stability of the
three repressors is reduced by the presence of
destruction tags (denoted lite'). The compatible
reporter plasmid (right) expresses an
intermediate-stability GFP variant11 (gfp-aav).
In both plasmids, transcriptional units are
isolated from neighbouring regions by T1
terminators from the E. coli rrnB operon (black
boxes).
The repressilator network
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the ring oscillator
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evaluation of the ring oscillator
Comparison of the repressilator dynamics
exhibited by sibling cells. In each case, the
fluorescence timecourse of the cell depicted in
the Fig is redrawn in red as a reference, and two
of its siblings are shown in blue and green.
Elowitz, Leibler 2000
88
evaluation of the ring oscillator

• a, Siblings exhibiting post-septation phase
  delays relative to the reference cell.
• b, Examples where phase is approximately
  maintained but amplitude varies significantly
  after division.
• c, Examples of reduced period (green) and long
  delay (blue).
• d, Two other examples of oscillatory cells from
  data obtained in different experiments, under
  conditions similar to those of ac. There is a
  large variability in period and amplitude of
  oscillations.
• e, f, Examples of negative control experiments.
• e, Cells containing the repressilator were
  disrupted by growth in media containing 50mM
  IPTG.
• f, Cells containing only the reporter plasmid.

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evaluation of the ring oscillator

• Reliable long-term oscillation doesnt work yet
• Will matching gates help?
• Need to better understand noise
• Need better models for circuit design

Elowitz, Leibler 2000
90
three repressors

• LacI is a repressor protein made from the lacI
  gene, the lactose inhibitor gene of E. coli.
• TetR is a repressor protein made from the tetR
  gene.
• CI is a repressor protein made from the cI gene
  of ? phage.
• Each one of these, with its cognate promoter,
  will stop production of whatever gene is
  downstream from the promoter.

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ring oscillator with mismatched inverters
A original cI/?P(R) B repressor binding 3X
weaker C transcription 2X stronger
92
device physics in steady state
Ideal inverter

• Transfer curve
• gain (flat,steep,flat)
• adequate noise margins

gain
output
0
1
input

• Curve can be achieved with certain dna-binding
  proteins
• Inverters with these properties can be used to
  build complex circuits

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measuring a transfer curve

• Construct a circuit that allows
• Control and observation of input protein levels
• Simultaneous observation of resulting output
  levels

inverter
R
YFP
CFP
drive gene
output gene

• Also, need to normalize CFP vs YFP

94
flow cytometry (FACS)
95
drive input levels by varying inducer
lacI high
IPTG (uM)
YFP
0 (Off)
P(lacIq)
P(lac)
0
IPTG
100
lacI
P(lacIq)
IPTG
1000
YFP
P(lac)
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measuring a transfer curve
for lacI/p(lac)
tetR
?P(R)
YFP
P(lac)
aTc
P(Ltet-O1)
lacI
CFP
97
transfer curve data points
0?1
1?0
undefined
1 ng/ml aTc
10 ng/ml aTc
100 ng/ml aTc
98
lacl/p(lac) transfer curve
gain 4.72
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evaluating the transfer curve

• Noise margins

• Gain / Signal restoration

high gain

• note graphing vs. aTc (i.e. transfer curve of 2
  gates)

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applications
101
some possibilities

• Forward Engineering as a means of learning
  about natural genetic regulation.
• Biotechnology
• Experimental systems
• Validation of models

102
forward engineering

• Reductionism Simulation reverse engineering.
• Main Difficulty system is WAY to complex
• reductionism will never be finished
• when it is, models/ parameter-space will be too
  huge
• we dont have much intuition for parallelism,
  processes interacting at different scales...
• Possible modes of attack
• Engineering math sensitivity analysis, control
  theory
• Complex Systems analysis

103
forward engineering

• Forward Engineering Approach
• We learned more about how birds fly from trying
  to build airplanes than from studying structural
  anatomy of birds. - ?(ai)
• Try to build something that has same
  functionality as system under study. Learn what
  some of the critical component requirements are,
  what the main design challenges.
• Generate testable hypotheses about how natural
  genetic regulation functions.
• Use forward and reverse engineering techniques in
  parallel.

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biotechnology

• Genetic engineering applications
• production of antibiotics and other drugs
• production of proteins for detergents, solvents,
  aminos
• bioremediation
• Metabolic Control Analysis, directed evolution
  and other techniques used to optimize design of
  metabolic pathways for given task.
• Genetic circuit engineering could yield finer
  more sophisticated control.
• Genetic circuits as sensors.

105
experimental systems

• Perhaps genetic circuits can be used as clever
  assays/probes, similar to the Yeast Two-Hybrid
  system used to detect interacting proteins.
• A Transcription Factor
• Fuse domains to putative interacting proteins
• Is TF active?
• Or Genetic circuits could be used to examine a
  systems response to complex controllable inputs.

DNA Binding
Activation
bait
fish
DNA Binding
Activation
GFP
106
validation of modeling techniques

• Many competing techniques for modeling
  biochemical systems kinetics-based, stochastic
  kinetics, graph theoretical, discrete-event
• Ultimate gold-standard would be to design a
  system using a simulation technique, build it,
  and verify predictions of model.