Transcript The Dawn of Artificial Gene Circuits

Synthetic Gene Circuits
Small, Middle-Sized and Huge
Molecules Playing Together
Within a Cell
Outline:
WHY?
Background
Some things that cells can make from
genes.
 How genes make these things.
 How gene activity is controlled: gene
circuits.
 Regulatory and ‘Epigenetic’ activity
activity.
 SYNTHETIC
GENE CIRCUITS
What can genes make? (1)
 Cells contain organelles that enable them to
synthesize chemicals and structures from
instructions in genes.
 All of these organelles can reproduce
themselves – and make other chemicals and
structures – when the organelles follow the
instructions in their genes.
 Genes without cells don’t work; cells without
genes do not work. They work together.
Which came first – the chicken or the egg?
What can genes make? (2)
 Genes can make any protein, following the
genetic code (3 nucleotides emplace one amino
acid corresponding to one codon). A gene is a
one-dimensional array of nucleotides; a protein
is a one-dimensional array of amino acids.
 Using proteins as catalysts* genes can
prescribe the manufacture of all other natural
molecules – and some artificial ones as well.
A catalyst is a molecule essential to a chemical reaction but neither created nor
destroyed by the reaction.
What can genes make? (3)
The kinds of molecules that genes make is less
interesting than the functions these molecules
provide.
Concern here will be with these functions:
 gene products (transcription factors) that
directly regulate the generating gene or
another gene (intrinsic regulation).
 gene products that indirectly regulate a gene
(extrinsic regulation).
 gene products that lead to measurable
changes in a cell (reporters).
How genes make chemicals
 At least a two-step process:
 Transcription – transcribe the gene’s DNA
into a template RNA (amplification)
 Translation – translate information encoded
into the RNA into protein (more amplification)
 The protein may be the end product or
very often it may influence other reactions
that make other chemical forms.
The train-on-the-track transcription and
translation model
GE NE (DN A)
Pr
o te
in
Pr
od
uc
t
m
R
N
A
R N A polym era se
R ib oso m e
Rate = Number of tracks x Number of trains x Velocity of trains / Track length
The train-on-the-track model: implications
 Transcription and translation velocities tend
to be fixed.
 Length is determined by the gene. Thus …
 (Molar) synthesis rate for transcription is
controlled by “initiation rate” on 1 or 2 tracks
 Molar synthesis rate for translation is
determined by the number of mRNA “tracks”
 mRNA tracks is determined by balance
between synthesis and degradation:
Synthesis rate = (decay constant) [mRNA]
(first-order decay reaction)
Sooooooo ….
The initiation rate for transcription*
is of very great importance in
determining which genes are on
and which gene products are
generated
* The attachment and hence (in steady state) the
detachment rate for RNA polymerase (RNAP)
What is the RNAP “train starter”?
 Transcription factors.
Inducers
Repressors
These are protein molecules, made by genes,
that bind to a gene at an operator site, in or near
a promoter region, upstream of where transcription takes place. They often exist in two forms
inactive (or quiescent) and active. Usually a
small molecule induces the change:
Inactive factor  small molecule  active factor
Transcription Factors
It is important to remember that transcription factors are
proteins, come from genes (like all proteins), and may influence
either their predecessor gene or –often– other genes.
Summary of the structure of
the Engrailed homeodomain
bound to DNA, as revealed by
X-ray crystallography.
Cylinders represent the three
-helices of the homeodomain,
ribbons represent the sugar
phosphate backbone of the
DNA and bars symbolize the
base pairs. The recognition
helix (3) is shown in red.
Transcription factors and the
molecules that activate them are
crucial to determining which
genes are on.
Transcription of the WT1 Gene
Negative feedback: WT1 protein inhibits expression
of its own gene and also that of PAX-2 an activator of
th WT1 promoter.
Myogenesis
Upstream regulators force
differentiation to
mesodermal precursor
cells that then express
bHLH proteins that
stimulate transcription of
their own genes. They also
activate genes that make
MEF2, which further
accelerates transcription of
genes for bHLH proteins.
MEF2 and bHLH proteins
both stimulate other
muscle-specific genes.
Positive feedback!
A caveat:
It is biological (and logical) fact that all
molecular species generated in a cell degrade.
For any intracellular species:
dn  generation 

kn


dt  rate

W hen cells are dividing and volum e chang es:
V
 generation 
 generation 
dV
dV 

 

kc

c


c
k

n
n
n





dt
dt
dt 

 rate

 rate

dc n
dV 

and the term  k 
 becom es an "effect ive" (larger) loss coefficient.
dt


Unnatural Experiments
 Plasmids – circles of ‘constructed’ DNA that
float in bacterial cytoplasm.
Green fluorescent protein. A reporter that
represents the integral of a cell’s protein
synthesis rate from mRNA.
The ‘repressilator’
“A synthetic oscillatory
network of
transcriptional
regulators”, Elowitz,
M., Leibler, S.,
Nature 403 335-338
(20 January 2000)
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.
Plasmid Construction
The system looks like a negative feedback
loop. Does it have predictable stability
properties?
E low itz' m odel (6 coupled, non-linear O D E 's):
dm i
dt
 
 loss 
 generation 


 -

 + 
 = m i   
n
 1   j
 rate 
 rate



0

 i  lacI, tetR , cI 


j

cI,
lacI,
tetR


d i
dt

 loss   generation 

 +
 =    i  m i
 rate   rate


N otice the coupling m i (m R N A ) and  j (repres sor protein) in the first 3 equations.
Repressilator Steady States
Repressilator Simulation Results
Repressilator Experimental Results
Why?
 Part of a dual strategy for identifying gene
circuits:
Understand devices and low-level, device-device
interactions. Elowitz is one way to attack this
problem. It answers some questions and raises
more.
Then recognize ‘functional motifs’, identify them,
“subtract” them from a circuit diagram, and identify
the macroscopic circuit design. (Alon*)
*Shai S. Shen-Orr, Ron Milo, Shmoolik Mangan & Uri Alon
Network motifs in the transcriptional regulation network of
Escherichia coli, Nature Genetics, Published online: 22 April,
2002
Motifs? – Or in the eye of the believer?
The engineering analysis of
Gene Circuits is just
beginning.