Cardinale et al. show that variation in cloning strain background can affect expression of a three gene probe cassette in E. coli that is largely explainable by changes in host growth and ribosomal availability ( Figure 3A) but that when that same cassette

is passed into 88 deletion strains of E. coli BW25113 there seem to be more specific effects of each gene deletion on circuit performance ( Figure 3B) [ 55••]. Specific metabolic and signaling genes, when deleted had large positive and negative effects (respectively) on expression of all three fluorescent proteins of the probe while a couple differentially affected expression of at least one of the proteins. Key subsystems that generically and specifically affect heterologous circuit function were thereby identified and mapped to subelements of the synthetic circuit. In a complementary approach, Woodruff et al. check details [ 56] created a library of millions of overexpressed genome fragments in an ethanol production strain and subjected it to a growth selection to quantitatively map variation of host genes to improvements in ethanol tolerance and production. They identified that membrane and osmotic stress were important limiting issues for the strain and that a single host gene that when overexpressed led up to a 75% improvement

CX-5461 cost relative to the parent production strain. Other genome scale techniques for measuring macromolecular interaction and metabolic profiles will add more data that should aid in improving strain performance. Formal methods to transform these data into models of biological new parts and their interactions suitable to drive design decisions remains to be developed. Host and environmental context are intimately linked because the major (unintended) effects of environment on a heterologous circuit are likely to arise via effects on host

physiology. Sometimes, if the environment of deployment is known and static one can design or select circuits that operate well under those conditions. In metabolic engineering, there is the oft-cited problem that the biosynthetic pathways engineered in the laboratory often work poorly in the scaled-reactors that are necessary for economic production [ 57 and 58]. To demonstrate some issues, Moser et al. characterized how small synthetic circuits operate in different industrially relevant conditions and showed how changes in fermentation process affect host growth and resources thereby differentially affecting synthetic logic circuits in the host cell [ 59]. A recent industrial example of the challenge is the conversion of biosynthetic production of 1,3-propanediol, a precursor for many industrial products, from ‘specialty’ to commodity scale required the optimization of over 70 genes off-pathway before sufficient production in industrially relevant environments was achieved [ 60].