IDC 401:

THEORETICAL BIOLOGY

Modelling Genetic Regulatory Circuits

&
Simple Biochemical Pathways
 MODELLING BIOCHEMICAL PATHWAYS
Three complementary approaches

REVERSE ENGINEERING LARGE NETWORKS

Model existing pathways based on Construction & analysis of

information derived from –
• Genome sequences
• Protein sequences
• Biochemical & Genetic information
functionally related
pathways from large scale
gene expression and
protein interaction data
using network theory

FORWARD ENGINEERING ‘Rational Network

Design’
All designs that are not physically
forbidden are realizable, Artificial genetic and
but not all realizable designs are enzymatic networks with
functionally effective specific properties
(in relation to context and constraints constructed based on
of the system and environment). mathematical models
 MODELS OF MOLECULAR COMPUTING

Implementing a complex decision process with

multiple input cues using logic.
‘when gene A is active AND gene B is inactive
AND gene C is mutated → generate the effect’
 Forward Engineering of gene circuits
(Rational Network Design, Synthetic Biology)

Construction of desired network with specific properties predicted

from mathematical models using knowledge from biochemistry,
molecular biology, and genetics.

Boolean/Logical Circuits in Biology :

Organisms take decisions based on input signals
and give a binary (0/1) response in some cases.

Repressor

RNAP Inducer Jacob & Monod Model of the

prokaryotic operon (1961)
J. Mol. Biol., 3 (1961), pp. 318-356
Gene A Gene B Nobel Prize in 1965
Promoter Operator

“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”
 Basic digital circuits:

A A A
And C Or C Nand C
B B B

A B C A B C A B C
0 0 0 0 0 0 0 0 1
0 1 0 0 1 1 0 1 1
1 0 0 1 0 1 1 0 1
1 1 1 1 1 1 1 1 0

Digital genetic circuit: A repressible system –The NOT gate:

If repressor is input and

the gene product is output,
then if repressor is 1, gene product is 0
and vice versa.....
the input is inverted.
 Genetic Circuit Engineering Paradigm
Design - Simulate - Implement & Test

A basic assumption underlying such ‘synthetic’ biology

The properties of individual genetic components can be

used to understand and quantitatively predict circuit-level
behaviour.

“ Rational Network Design ” can -

a) engineer new cellular behaviour, and
b) improve understanding of naturally occurring networks.
DESIGN PRINCIPLES
 George Boole (1815 –1864) was an
English mathematician, educator,
philosopher and logician.
He worked in the fields of differential
equations and algebraic logic, and is
best known as the author of The Laws
of Thought (1854) which contains
Boolean algebra.
Boolean logic is credited with laying the
foundations for the information age.
Boolean algebra is named after him, as is the crater Boole on the Moon. The
keyword Bool represents a Boolean data-type in many programming languages,
though Pascal and Java, among others, both use the full name Boolean.
The library, underground lecture theatre complex and the Boole Centre for
Research in Informatics at University College Cork are named in his honour. A road
called Boole Heights in Bracknell, Berkshire is named after him.
 Logic Functions/Gates:
Mappings between multiple inputs and a single output,
where both the inputs and the output
can only take values of
0 and 1 false and true
• NOT is a horizontal bar above the number
_
0=1
_
1=0
• OR is a plus • AND is multiplication
0+0 = 0 0*0 = 0
0+1 = 1 0*1 = 0
1+0 = 1 1*0 = 0
1+1 = 1 1*1 = 1
 Buffer
• A buffer is a gate with the function F = X: X F
• In terms of Boolean function, a buffer is
the same as a connection
• Not

• And
Basic logic
• Or gates

• Nand

• Nor
 NAND Gate
• For three inputs (x, y, z), the basic NAND gate has the
following symbol : (NAND) - AND-Invert

X
Y F( X , Y, Z ) = X×Y×Z
Z

• NAND represents NOT AND, i. e., the AND function

with a NOT applied. The symbol shown is an AND-
Invert. The small circle (“bubble”) represents the
invert function.
 NOR Gate
• NOR represents NOT – OR
i.e., the OR function with a NOT applied.

• The basic NOR gate has the following symbol, for 3

inputs (x, y, z):
– OR-Invert (NOR) - The small circle (“bubble”)
represents the invert function.

X
Y F(X, Y, Z) = X +Y+ Z
Z
 TRUTH TABLES

A repressible system –The NOT gate: If repressor is input and

the gene product is output,
then if repressor is 1, gene product is 0
and vice versa.....
the input is inverted.
 Logic circuits:
Specific arrangements of logic gates (chemical
reactions) that can compute specific logic
functions.
Write the circuits for the following Boolean algebraic
expressions
__

a) x+y

x
x x+y

y
• Find the output of the following circuit

x x+y
y (x+y)y

y y

• Answer: (x+y)y
• The circuits for the following Boolean algebraic
expression:
_______

(x+y)x

x x+y
x+y (x+y)x
y
 Gene Regulatory Networks

• Gene regulatory networks (GRNs) are the

on-off switches of a cell operating at the
gene level.

• Two genes are connected if the expression

of one gene modulates expression of
another one by either activation or
inhibition
 Simplified Representation of GRN
• A gene regulatory network can be represented by a directed
graph;

w Node represents a gene;

w Directed edge stands for the
modulation (regulation) of one
node by another:
e.g. arrow from gene X to
gene Y means gene X affects
expression of gene Y
 Modeling Gene Regulatory Networks
• Linear Model
• Bayesian Networks
• Differential Equations
• Boolean Network

• Boolean Network
Originally introduced by Kauffman (1969)
– Boolean network is a kind of Graph
§ G(V, F) – V is a set of nodes ( genes ) as x1 , x2, …, xn
F is a list of Boolean functions f(x1 , x2, …, xn)
– Gene expression is quantized to only two level:
1 (On) and 0 (OFF)
– Every function has the result value of each node;
Basic Structure of Boolean Networks

1 means active/expressed
A B 0 means inactive/unexpressed

Boolean function
A B X
0 0 0
0 1 0
1 0 0
X
1 1 1

Two genes (A and B) together regulate gene X

In principle, any number of “input” genes are possible.

Positive/negative feedbacks are also common.
 g3

g2 g1
 *
Truth Table

Boolean Network
 State Transition Graphs

Steady State Behaviour

Non-linear Phenomenon

Periodic behaviour
Behaviour of a single
Logical States

gene g1 for the

logical states (0,1,0) or
(0,1,1):

Transition States
• At Initial states ->
g1 = 0 , g2 = 1, g3 = 0
 Forward Engineering of gene circuits
(Rational Network Design, Synthetic Biology)

Construction of desired network with specific properties predicted

from mathematical models using knowledge from biology.

“ Rational Network Design ” can -

a) engineer new cellular behaviour,
b) improve understanding of naturally occurring networks.
DESIGN PRINCIPLES

Genetic Circuit Engineering Paradigm:

Design-Simulate-Implement & Test

A basic assumption underlying such ‘synthetic’ biology

The properties of individual genetic components can be used to

understand and quantitatively predict circuit-level behaviour.
 Synthetic transcriptional regulatory networks
1. Single negative feedback: The bacteriophage l
paradigm
Induced by ATC - an analog
of tetracycline.
Two opposing negative
Becskei. & Serrano. Engineering feedback processes lead to
stability in gene networks by switch-like behaviour
autoregulation. Nature (2000).

2. Two nested negative feedback:

The Toggle:- switched
by IPTG and ATC which
induce the plac and ptet.
Construction of a genetic toggle
switch in Escherichia coli.
Gardner, Cantor & Collins.
Nature (2000)

3.Three negative feedback:

The repressilator
Oscillates due to mutual
repression of the three repressors
A synthetic oscillatory network of
transcriptional regulators. Elowitz & Leibler
Nature(2000)
 A synthetic oscillatory network of transcriptional regulators
(NATURE, VOL 403, 20 JAN 2000)

Repressilator: Daisy chain of 3 transcriptional repressors to build an oscillatory network

First repressor protein, LacI
(E.coli), inhibits the transcription of
the second repressor gene, tetR
(tetracycline-resistance transposon
Tn10), whose protein product in
turn inhibits the expression of a
third gene, cI (λ phage). Six differential equations, four
Finally, CI inhibits lacI expression, parameters (symmetric)
completing the cycle.
p & m are the respective repressor -
protein & mRNA concentrations.
α0 & α+ α0 are number of protein
copies/cell produced due to promoter
leakiness and in its absence.
β is the ratio of the protein decay
rate to mRNA decay rate;
n is a Hill co-eff.
 GFP expression in a single cell having repressilator plasmids (E. coli host strain MC4100)
[THEORY] [EXPERIMENTS]
n=2.1, α0=0
Microcolony:
Fluorescence
n=2, α0=0
Bright field

n=2, α0/α=10-3 Large

variation

GFP
Conc. of Repressor proteins with in Period and
Septation
time Amplitude of events
oscillations

Oscillatory dynamics in single cells and their siblings

Stochastic Model:
Noise in concentrations and interactions.

Negative Controls
 What is the function of this network?
Depends on transcription rate, repressor concentration, the translation rate,
and the decay rates of the protein & messenger RNA - as per theory.

The network periodically induces the synthesis of green

fluorescent protein as a readout of its state in individual
cells.

The resulting oscillations, with typical periods of hours,

are slower than the cell-division cycle, so the state of the
oscillator is transmitted from generation to generation.

This artificial clock displays noisy behaviour, possibly

because of stochastic fluctuations of its components.

Such “ Rational Network Design ” can -

a) engineer new cellular behaviours, and
b) improve understanding of naturally occurring networks.
Development of Gene Circuits The Circuit Engineering Vision
Develop a standard library of interoperable “parts” that corresponds
to various control functions
may help in controlled gene expression in gene therapy

Develop integrated computational infrastructure for

Computer Aided Design (CAD) of genetic circuits
Simulation and dynamic analysis
Build increasingly complex genetic circuits using well-characterized parts