Synthetic Gene Circuits - PowerPoint PPT Presentation

1 / 26
About This Presentation
Title:

Synthetic Gene Circuits

Description:

Cells contain organelles that enable them to synthesize chemicals and structures ... of its own gene and also that of PAX-2 an activator of th WT1 promoter. ... – PowerPoint PPT presentation

Number of Views:495
Avg rating:3.0/5.0
Slides: 27
Provided by: edwardf7
Category:
Tags: circuits | gene | pax | synthetic

less

Transcript and Presenter's Notes

Title: 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.
Write a Comment
User Comments (0)
About PowerShow.com