Journal: Nature Communications
Article Title: Hierarchical control of enzymatic actuators using DNA-based switchable memories
Figure Lengend Snippet: Coupling of the translator module to an upstream INVERTER network. a Schematic illustration of the translator module coupled to a PEN-based INVERTER network. Multiplex monitoring of the dynamics of the network is performed using endogenous template βtoiα and an exogenous template αtoiβ which are 3′-end fluorescently labeled with DY530 and FAM, respectively, while the output strand σ of the translator module is measured via a MB bearing a fluorophore-quencher pair. b Results of the experiments that were conducted for 0, 2, 5, 10, 20, and 40 nM (light to dark) of translator template αtoσ in the presence of 7 nM αtoα , 20 nM of βtoiα and αtoiβ , 30 nM MB, 10 U mL −1 Bst 2.0 WarmStart DNA polymerase, 25 U mL −1 Nt. bstNBI, and 50 nM ttRecJ. The INVERTER is activated by addition of 0.5 nM α which is initially amplified until it reaches steady-state in which production by polymerase and nickase and degradation due to exonuclease are balanced. Applying a pulse of 30 nM of input β at this point initiates the production of iα , which inhibits autocatalytic production of output α . As input strand β gets degraded the system returns its pre-stimulus steady-state. Hence, the INVERTER network shows a pulse response after injection of input β , which can be characterized by its amplitude and response time which is the time needed to recover to the pre-stimulus steady-state. The charge level is the normalized fluorescence of the signal of DY530 and FAM fluorophores, which is 0 in the absence of template’s input primer and 1 at the maximal or steady-state value of primer β and α , respectively. The fluorescence of Cy5 fluorophore was converted to concentration of DNA strand σ using a standard curve (Supplementary Fig. 17 ). c Results of simulations using the heuristic model with the same concentrations of translator template as used during the experiments in b and for different values of ρ . The traces were converted to normalized units (n.u.) by normalizing α to the steady-state concentration and normalizing β to its maximum value
Article Snippet: 2.0 WarmStart DNA polymerase were obtained from NEB, while ttRecJ, a thermophilic equivalent of the RecJ enzyme from Thermus thermophilus , was obtained from André Estévez-Torres.
Techniques: Multiplex Assay, Labeling, Amplification, Injection, Fluorescence, Concentration Assay