  The construction of synthetic biochemical circuits is an essential step for
developing quantitative understanding of information processing in natural
organisms. Here, we report construction and analysis of an in vitro circuit
with positive autoregulation that consists of just four synthetic DNA strands
and three enzymes, bacteriophage T7 RNA polymerase, Escherichia coli
ribonuclease (RNase) H, and RNase R. The modularity of the DNA switch template
allowed a rational design of a synthetic DNA switch regulated by its RNA output
acting as a transcription activator. We verified that the thermodynamic and
kinetic constraints dictated by the sequence design criteria were enough to
experimentally achieve the intended dynamics: a transcription activator
configured to regulate its own production. Although only RNase H is necessary
to achieve bistability of switch states, RNase R is necessary to maintain
stable RNA signal levels and to control incomplete degradation products. A
simple mathematical model was used to fit ensemble parameters for the training
set of experimental results and was then directly applied to predict
time-courses of switch dynamics and sensitivity to parameter variations with
reasonable agreement. The positive autoregulation switches can be used to
provide constant input signals and store outputs of biochemical networks and
are potentially useful for chemical control applications.
