Title: Synthetic Gene Circuits
1Synthetic Gene Circuits
- Small, Middle-Sized and Huge Molecules Playing
Together - Within a Cell
2Outline
- 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
3What 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 dont work cells without
genes do not work. They work together. - Which came first the chicken or the egg?
4What 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.
5What 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).
6How genes make chemicals
- At least a two-step process
- Transcription transcribe the genes 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.
7The train-on-the-track transcription and
translation model
Rate Number of tracks x Number of trains x
Velocity of trains / Track length
8The 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)
9Sooooooo .
- 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)
10What 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 transcrip-tion
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
11Transcription FactorsIt 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.
12Transcription factors and the molecules that
activate them are crucial to determining which
genes are on.
13Transcription 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.
14Myogenesis
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!
15A caveat
- It is biological (and logical) fact that all
molecular species generated in a cell degrade.
For any intracellular species
16Unnatural Experiments
- Plasmids circles of constructed DNA that
float in bacterial cytoplasm.
- Green fluorescent protein. A reporter that
represents the integral of a cells protein
synthesis rate from mRNA.
17The repressilator
- A synthetic oscillatory network of
transcriptional regulators, Elowitz, M.,
Leibler, S., Nature 403 335-338 (20 January 2000)
18Three 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.
19Plasmid Construction
20The system looks like a negative feedback loop.
Does it have predictable stability properties?
21Repressilator Steady States
22Repressilator Simulation Results
23Repressilator Experimental Results
24Why?
- 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
25Motifs? Or in the eye of the believer?
26The engineering analysis of Gene Circuits is just
beginning.