Synthetic biology is a useful tool to investigate the dynamics of small biological networks and to assess our capacity to predict their behavior from computational models. In this work we report the construction of three different synthetic networks in Escherichia coli based upon the incoherent feed-forward loop architecture. The steady state behavior of the networks was investigated experimentally and computationally under different mutational regimes in a population based assay. Our data shows that the three incoherent feed-forward networks, using three different macromolecular inhibitory elements, reproduce the behavior predicted from our computational model. We also demonstrate that specific biological motifs can be designed to generate similar behavior using different components. In addition we show how it is possible to tune the behavior of the networks in a predicable manner by applying suitable mutations to the inhibitory elements.

The cDNA for 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase of Arabidopsis encodes a polypeptide with an amino-terminal signal sequence for plastid import. A cDNA fragment encoding the processed form of the enzyme was expressed in Escherichia coli. The resulting protein was purified to electrophoretic homogeneity. The enzyme requires Mn 2ϩ and reduced thioredoxin (TRX) for activity. Spinach (Spinacia oleracea) TRX f has an apparent dissociation constant for the enzyme of about 0.2 m. The corresponding constant for TRX m is orders of magnitude higher. In the absence of TRX, dithiothreitol partially activates the enzyme. Upon alkylation of the enzyme with iodoacetamide, the dependence on a reducing agent is lost. These results indicate that the first enzyme in the shikimate pathway of Arabidopsis appears to be regulated by the ferredoxin/TRX redox control of the chloroplast.

The shikimate pathway proper is the common metabolic route to the three aromatic amino acids phenylalanine, tyrosine and tryptophan. These amino acids serve as building blocks for all proteins and as precursors for a large number of secondary metabolites.