Engineering a New SsrA-Based Degradation Tag (LAA-LAA) and a Bacterial Synthetic Oscillator

The ATP-dependent ClpXP-SspB protease complex is responsible for the degradation of intracellular proteins and is maintained at low levels in <i>Escherichia coli</i> to avoid nonspecific degradation. The rate-limiting step in the protease complex leads to proteolytic queueing, where the proteins form waiting lines, and their overall degradation rate is slowed. Synthetic biologists have leveraged proteolytic queueing to design robust synthetic circuits by tagging proteins with the SsrA tag, an 11-amino acid sequence recognized by the complex. Previous work has demonstrated the binding site of each component of the ClpXP-SspB complex to the SsrA tag. However, the precise component responsible for queueing was unknown. To identify the bottleneck in the complex, we designed different SsrA tag variants depending on the chaperone binding sequences. We further overexpressed each protein in the ClpXP-SspB complex <i>in vivo</i> to determine how an increased amount of each component affects the tagged protein levels. Based on the degradation of the SsrA variants, upon overexpression of each component of the ClpXP-SspB system, evidence supports that ClpX (the ATP-dependent chaperone) is responsible for queueing but not ClpP (the protease) or SspB (the adapter, ATP-independent chaperone). In the process, we identified LAA-LAA, a 6-amino acid ClpX-dependent tag that degraded <i>in vivo</i> faster than the original SsrA tag, AANDENYALAA. We speculated that this high degradation tag could be useful in a dynamic-synthetic circuit, so we modified the well-characterized dual-feedback oscillator by replacing its original SsrA tag with the LAA-LAA tag to form the LAA-LAA-Osc oscillator. Both population and single-cell level experiments show that the new and old oscillators have distinct frequencies. Like the original oscillator, thousands of cells containing the new oscillator could be synchronized by entrainment using an external signal. Thus, the new LAA-LAA-Osc oscillator retains the original oscillator's best characteristics (robustness to fluctuations, a steady oscillation period, and entrainment across 1000s of cells to an external signal) but oscillates at a different frequency.