synthetic gene circuits small, middle-sized and huge molecules playing together within a cell

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

Rate = Number of tracks x Number of trains x Velocity of trains / Track length

GENE (DNA)

mRNA

Pro

tein

Pro

duct

RNA polymerase

Ribosome

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.

InducersRepressors

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

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.

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.

MyogenesisUpstream 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:

When cells are dividing and volume changes:

generation generation

rate rate

and the term becomes an "effect

generation rate

nn n n

dc dV dVV kc c c k

dt dt dt

dVk

dt

dn k ndt

ive" (larger) loss coefficient.

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?

0

Elowitz' model (6 coupled, non-linear ODE's):

loss generation - + =

rate rate 1

lacI, tetR, cI

cI, lacI, tetR

loss generation +

rate

n

i

ii

j

dmm

dt

i

j

d

dt

= rate

Notice the coupling (mRNA) and (repressor protein) in the first 3 equations.

i i

i j

m

m

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.