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 characteristics 2.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...

 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 measurements 2.DNA-binding protein domain measurements 3.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 rate 5.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 time o 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  K su 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:  Canton, B., Labno, A. & Endy, D. (2008) Refinement and standardization of synthetic biological parts and devices Nature Biotechnology, 26:  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:  Kelly, J. R. et al (2009) Measuring the activity of BioBrick promoters using an in vivo reference standard Journal Biological Engineering 3:4  OpenWetWare wiki