Steady state. This plot shows the rate of change of GFP concentration (production minus degradation) normalized over OD600 as a function of time for four representative runs. Each network tested in this paper was tracked over time to verify that the networks had reached their steady state operating points. A rate of change of near zero indicates that the synthetic networks have reached steady state (approximately after 16–20 h)

Model predictions. Steady state values of p3 for the simple generic incoherent type I feed-forward network as a function of the input p1 for the model shown in the Fig. 2. The bold line represents the ‘wildtype’ model. The dotted line represents an increase in the affinity of p2 for G2. The dashed line represents a further increase in the affinity of p2 for G2. Parameter values for the equations shown in the modeling section are set to: t1 = 5; a1 = 0.1; t2 = 1; b1 = 1; b2 = 0.1. The curves were generated by changing the affinity parameter b3 on G2 from 1 to 5 to 20, where 1 is the wild-type value. Gamma_1 and Gamma_2 were set to 0.1. Note that the curves have all been normalized with respect to the maximum peak high for each curve. This was done to emphasize the lateral change in the response curve which was measurable in our experiments. For the unnormalized curves, the peak height decreases as the inhibition strength increases as well as shifting the curve to the left

T7-metR promoter regulated system. The complete network with MetJ inhibition (Degradation steps have been omitted for clarity). (a) GFP and MetJ production are independently driven by T7-RNAP binding. MetJ inhibits by binding “met boxes” located in the regulatory region of the metR promoter driving GFP production. (b) A plot of the normalized fluorescence vs. IPTG concentration. Fluorescence was measured for each of the constructs and normalized by the optical density of each well, (●) native metR promoter region, (■) metR promoter where the center two met boxes have been mutated to the consensus sequence

T7–T7 lysozyme regulated system. Simplified representation of the T7 Lysozyme inhibition System (Degradation steps have been omitted for clarity). (a) GFP and T7 lysozyme production are independently driven by T7-RNAP binding. T7 lysozyme inhibits by binding T7-RNAP and preventing elongation complex formation. (b) A plot of the normalized fluorescence vs. IPTG concentration. The introduction of (●) lysozyme repression and (■) an 8-fold increase T7 lysozyme, available to the system through gene duplication, over the native system, was measured

The incoherent feed-forward loop type 1: The incoherent feed-forward loop type 1 was used as the basis for designing networks. Two networks were designed that fit this scheme, where X is T7 RNA polymerase and Z is GFP. (a) Y is the inhibitory element, MetJ or the reverse complement of GFP. (b) This network is a modified FFL with additional feedback, Y is T7 lysozyme

Feed-forward loop model. Interpretation of the feed-forward motif shown (Fig. 1a) in terms of a very simple genetic network. p1 is the input to the system comprising of genes G1 and G2, which produce p2 and p3, respectively. p1 activates G2 and p2 represses G2

Unregulated system. The partial networks, without inhibition, used in this work were independently characterized for IPTG dose dependent responses. (a) GFP production is driven by T7-RNAP binding approximately 70 nucleotides upstream of the GFP start codon. (b) GFP production with a met R promoter insert. A T7-RNAP binding site was introduced directly upstream of the complete met R promoter region. (c) A plot of the normalized fluorescence vs. IPTG concentration. Fluorescence was measured for each of the constructs, normalized by the optical density of each well, and scaled according to each construct’s maximal output. Increasing IPTG induces the production of T7-RNAP which in turn transcribes the downstream GFP gene: T7 promoter (●); T7-metR promoter (■). Curves were fitted to a simple Hill function using EasyGraph (Future Skill Software) and the Levenberg-Marquardt optimization algorithm. Hill coefficients for the T7 promoter (●) equaled 1.93 while for the metR promoter (■) it equaled 1.42

T7-anti-gfp regulated system. The complete network with anti-gfp inhibition (Degradation steps have been omitted for clarity). (a) The reverse complement of the GFP is transcribed by a T7 binding. The anti-gfp transcript encodes the entire gene and upstream ribosome binding site. Inhibition is through mRNA:mRNA binding of the GFP gene and its reverse complement to prevent translation. (b) A plot of the normalized fluorescence vs. IPTG concentration. The entire reverse complement of the (■) GFP gene and a (●) partial sequence were measured for the ability to inhibit

Tuning T7–T7 lysozyme regulated system. The lysozyme inhibitory network was mutated to decrease the amount of lysozyme present without changing T7-RNAP kinetics. The ribosome binding site (RBS) was altered, from strong to weak, to decrease mRNA translation into active inhibitor. The ability of the T7 lysozyme to effectively inhibit GFP production is proportional to RBS Strength. The RBS strengths are as follows: strongest (♦), strong (■), medium (●), weak (▲)