Large genetic engineering projects require more cistrons and consequently more strong and reliable transcriptional terminators. We have measured the strengths of a library of terminators, including 227 that are annotated in Escherichia coli--90 of which we also tested in the reverse orientation--and 265 synthetic terminators. Within this library we found 39 strong terminators, yielding >50-fold reduction in downstream expression, that have sufficient sequence diversity to reduce homologous recombination when used together in a design. We used these data to determine how the terminator sequence contributes to its strength. The dominant parameters were incorporated into a biophysical model that considers the role of the hairpin in the displacement of the U-tract from the DNA. The availability of many terminators of varying strength, as well as an understanding of the sequence dependence of their properties, will extend their usability in the forward design of synthetic cistrons.

Genetic circuits perform computational operations based on interactions between freely diffusing molecules within a cell. When transcription factors are combined to build a circuit, unintended interactions can disrupt its function. Here, we apply “part mining” to build a library of 73 TetR-family repressors gleaned from prokaryotic genomes. The operators of a subset were determined using an in vitro method and this information was used to build synthetic promoters. The promoters and repressors were screened for cross-reactions. Of these, 16 were identified that both strongly repress their cognate promoter (5- to 207-fold) and do not interact with other promoters. Each repressor:promoter pair was converted to a NOT gate and characterized. Used as a set of 16 NOR gates, there are >1054 circuits that could be built by changing the pattern of input and output promoters. This represents a large set of compatible gates that can be used to construct user-defined circuits.

The re-use of previously validated designs is critical to the evolution of synthetic biology from a research discipline to an engineering practice. Here we describe the Synthetic Biology Open Language (SBOL), a proposed data standard for exchanging designs within the synthetic biology community. SBOL represents synthetic biology designs in a communitydriven, formalized format for exchange between software tools, research groups and commercial service providers. The SBOL Developers Group has implemented SBOL as an XML/RDF serialization and provides software libraries and specification documentation to help developers implement SBOL in their own software. We describe early successes, including a demonstration of the utility of SBOL for information exchange between several different software tools and repositories from both academic and industrial partners. As a community-driven standard, SBOL will be updated as synthetic biology evolves to provide specific capabilities for different aspects of the synthetic biology workflow.Synthetic biology treats biological organisms as a new technological medium with a unique set of characteristics, such as the ability to self-repair, evolve and replicate. These characteristics create their own engineering challenges, but offer a rich and largely untapped source of potential applications across a broad range of sectors 1,2 . Applications such as biomolecular computing 3 , metabolic engineering 4 , or reconstruction and exploration of natural cell biology 5,6 commonly require the design of new genetically encoded systems. As engineers, synthetic biologists most often base their designs on previously described 'DNA segments' (see Supplementary Table 1 for definitions of selected terms) to meet their design requirements. Reuse of the DNA sequence for these segments involves their exchange between laboratories and their hierarchical composition to form devices and systems with higher level function.Every engineering field relies on a set of 'standards' 7 that practitioners follow to enable the exchange and reuse of designs for 'systems' , 'devices' and 'components' . Similarly, the representation of synthetic biology designs using computer-readable 'data standards' has the potential to facilitate the forward engineering of novel biological systems from previously characterized devices and components. For example, such standards could enable synthetic biology companies to offer catalogs of devices and components by means of computerreadable data sheets, just as modern semiconductor companies do for electronics. Such standards could also enable a synthetic biologist to develop portions of a design using one software tool, refine the design using another tool, and finally transmit it electronically to a colleague or commercial fabrication company.In order for synthetic biology designs to scale up in complexity, researchers will need to make greater use of specialized design tools and parts repositories. Seamless inter-tool communication would, for example, allow the separation of gene...

The interaction specificities of extracytoplasmic function (ECF) sigma (σ) factors with promoters and their negative regulators (anti-σs) were mapped to identify non-crossreacting parts. These orthogonal sets represent a synthetic biology toolbox of genetic switches.

Steroidogenic acute regulatory protein (StAR) appears to mediate the rapid increase in pregnenolone synthesis stimulated by tropic hormones. cDNAs encoding StAR were isolated from a human adrenal cortex library.