Anita Nguyen & Harriet Gliddon

A good characterisation for a part is:“The minimum amount of information someone needs to reuse the part without any prior knowledge of it.”

Knowledge of this allows the assembly of biological systems to be predictable, reliable, efficient and inexpensive.

Information is presented in datasheets Characterisation information can be in the form

of:1. Physical characteristics2. Performance characteristics

Steady-state or dynamic? 25⁰C or 37⁰C? Minimal media or rich? What strain of E. coli? Multi-copy plasmid or integrated in the

genome?

Background information Sequencing certificate Sequence analysis (FASTA etc) Packaging type (including a protocol for the

physical linkage)

Functional description Performance measurements Environmental conditions used during testing Depend on:

◦ Chassis (host)◦ Environmental conditions

COMPLEX! To obtain a reliable and reproducible characterisation, we need to standardise the way we probe the system.

Compact, prescribed formats for communicating information from characterisation.

Some information is required from all devices, and some is specific to particular devices.

Contain information on accession number, name, brief description on function, response times, compatibility with other parts, reliability, license, and so on...

Part numbers A classic part is the gene for the receptor,

BBa_F2620◦ BB: BioBrick◦ a: the part is from the alpha release of BioBrick

standard biological parts collection◦ F: the part is involved in cell-cell signaling◦ 2620: to identify the specific device

For example, the use of:◦ Standard conditions (media, temperature, cell

density etc)◦ A standard chassis (strain/species/cell-free

system)◦ Standard measurements, including calibration

This allows you to predict a part will behave in your system

For example:1. Cell-cell communication measurements2. DNA-binding protein domain measurements3. Therapeutic bacterium measurements

Each needs different characterisation methods!

1. Steady-state and dynamic induction curves of an output promoter by different signal inputs and different cells

2. Reliability over time (mutational inactivation rate)

3. Homogeneity of induction in members of the population

4. Effect of induction on cellular growth rate5. Effect of growth phase on above function of the

device

Crystal structure Binding constraints for different DNA

sequences Toxicity of overexpression in different cells Stability in different cells Composability with different transcriptional

activation domains

Survival in different hosts Tissue localisation in different hosts Cell-type targeting efficiency in different

hosts Immune response of host to therapeutic

organism Dose-efficacy curves of therapeutic

organism Efficacy of safety measures Mutation rate

PoPS: Polymerases Per Second◦ standard unit to measure inputs and outputs of a

BioBrick Device

RiPS: Ribosomes Per Second◦ standard unit to measure translational activity of

mRNA molecule◦ RiPS per mRNA are equivalent to the rate of the

limiting step in ribosome initiation and elongation

Direct measurement in vivo:◦ Too complex

Indirect measurement:o Measure the change in protein or RNA concentration with change in timeo Alternatively, use rate of GFP synthesis as indirect measure of promoter

activity. Use quantitative model to relate GFP synthesis rate to PoPS.

GFP measurement:◦ Used as a reporter gene to allow quantification of promoter activity◦ Use RFP as an internal standard to allow one to relate cell health to

promoter activity

Using a measurement kit:◦ Use a promoter screening plasmid, a RBS screening plasmid, or a

terminator/inverter screening plasmid

Relies on the assumption that equilibrium can be established because the 30S SU of the ribosome takes longer to bind the mRNA, allowing simple hairpin structures to form, which contain the ribosome binding site (RBS)

30S SU competes with mRNA for the RBS The affinity of the 30S SU for the RBS and the number of

ribosomes present in the cell can affect the RiPS Ksu is measured and thermodynamics can be used to

calculate free energy changes etc.

Characterisation methods are necessary for predictable behaviour of parts in synthetic biology.

There are many new and emerging techniques.

Experimental constructs will hopefully be stored in a public repository and so will be accessible by everyone.

Vincent Rouilly, Imperial College London (2008) Synthetic Biology course notes

Arkin, A. (2008) Setting the standard in synthetic biology Nature Biotechnology, 26:771-774

Canton, B., Labno, A. & Endy, D. (2008) Refinement and standardization of synthetic biological parts and devices Nature Biotechnology, 26:787-793

De Smit, M.H. & van Duin, J. (2003) Translational standby sites: how ribosomes may deal with the rapid folding kinetics of mRNA Journal of Molecular Biology,331:737-743

Kelly, J. R. et al (2009) Measuring the activity of BioBrick promoters using an in vivo reference standard Journal Biological Engineering 3:4

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