Synthetic Gene Circuits

1
Synthetic Gene Circuits

• Small, Middle-Sized and Huge Molecules Playing
  Together
• Within a Cell

2
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

3
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 dont work cells without
  genes do not work. They work together.
• Which came first the chicken or the egg?

4
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.

5
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).

6
How 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.

7
The train-on-the-track transcription and
translation model
Rate Number of tracks x Number of trains x
Velocity of trains / Track length
8
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)

9
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)

10
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 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

11
Transcription 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.
12
Transcription factors and the molecules that
activate them are crucial to determining which
genes are on.
13
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.
14
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!
15
A caveat

• It is biological (and logical) fact that all
  molecular species generated in a cell degrade.
  For any intracellular species

16
Unnatural 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.

17
The repressilator

• A synthetic oscillatory network of
  transcriptional regulators, Elowitz, M.,
  Leibler, S., Nature 403 335-338 (20 January 2000)

18
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.

19
Plasmid Construction
20
The system looks like a negative feedback loop.
Does it have predictable stability properties?
21
Repressilator Steady States
22
Repressilator Simulation Results
23
Repressilator Experimental Results
24
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
25
Motifs? Or in the eye of the believer?
26
The engineering analysis of Gene Circuits is just
beginning.