Hysteretic Genetic Circuit for Detection of Proteasomal Degradation in Mammalian Cells.
Yimeng ZengBachhav BhagyashreeWenting ZhaoTram NguyenLaura SegatoriPublished in: ACS synthetic biology (2019)
Synthetic hysteretic mammalian gene circuits generating sustained cellular responses to transient perturbations provide important tools to investigate complex cellular behaviors and reprogram cells for a variety of applications, ranging from protein production to cell fate decisions. The design rules of synthetic gene circuits with controlled hysteretic behaviors, however, remain uncharacterized. To identify the criteria for achieving predictable control of hysteresis, we built a genetic circuit for detection of proteasomal degradation (Hys-Deg). The Hys-Deg circuit is based on a tetracycline-controlled transactivator (tTA) variant engineered to interface with the ubiquitin proteasome system (UPS). The tTA variant activates its own expression, generating a positive feedback loop that is triggered by expression of another tTA gene that is constitutively regulated. Guided by predictive modeling, we characterized the hysteretic response of the Hys-Deg circuit. We demonstrated that control of the hysteretic response is achieved by modulating the ratio of expression of constitutive to inducible tTA. We also showed that the system can be finely tuned through dosage of the inducer tetracycline to calibrate the circuit for detection of the desired levels of UPS activation. This study establishes the design rules for building a hysteretic genetic circuit with an autoregulatory feedback loop and provides a synthetic memory module that could be easily integrated into regulatory gene networks to study and engineer complex cellular behaviors.
Keyphrases
- genome wide
- copy number
- poor prognosis
- transcription factor
- genome wide identification
- dna methylation
- loop mediated isothermal amplification
- binding protein
- cell fate
- real time pcr
- induced apoptosis
- signaling pathway
- label free
- oxidative stress
- gene expression
- cell proliferation
- brain injury
- cell cycle arrest
- blood brain barrier
- amino acid
- quantum dots
- sensitive detection