Engineering Alternative Degradation Tags for Synthetic Circuits

The goal in synthetic biology is to build robust synthetic circuits in bacteria that are dynamic, highly regulated, and result in a unified response. In the last 20 years, the synthetic biology field has effectively leveraged transcriptional (RNA production) and translational (protein production) controls. However, protein degradation plays an important role in determining the half-life of proteins and regulating biological systems. Amino acid degradation tags are exploited to avoid a reliance on cell division for protein dilution and to build dynamic circuits. The most leveraged degradation tag in E. coli is the ssrA-tag, which is an 11-amino acid sequence that primarily target proteins for degradation by the ClpXP proteolytic system. However, the use of the ssrA-tag limits scalability and complexity especially in synthetic oscillators in bacteria due to its multiple proteolytic target recognition signals. Our goal is to build orthogonal oscillators that utilize degradation tags targeting to multiple proteases with no crosstalk. In this study, we are modifying the ssrA-tag to change the substrate affinity and degradation rate and produce new synthetic oscillators. We hypothesize that changing the degradation rate can alter the output signal of the oscillator. We have tailored the ssrA-tag to reduce crosstalk between proteolytic systems to increase robustness and developed a variety of degradation tags. While many of these tags exhibited decreased or no change in degradation rates, one depicted a significant increase. We further tested interesting candidates for crosstalk between proteolytic systems and identified a tag that display minimum to no crosstalk with other proteolytic systems. We aim to build and compare the output signals of different oscillators with novel tags in batch cultures and at the single-cell level. The design and implementation of these novel degradation tags will enable development of biological building blocks for increased complexity in synthetic circuits.