Engineering programmable trans-splicing riboregulators for complex cellular logic computation

Abstract

Synthetic genetic circuits program the cellular input-output relationships to execute customized functions. However, efforts to scale up these circuits have been hampered by the limited number of reliable regulatory mechanisms with high programmability, performance, predictability, and orthogonality. In this thesis, I engineered a class of Split-intron ENabled Trans-splicing Riboregulators (SENTRs) based on de-novo-designed external guide sequences (EGSs). SENTR libraries provide low leakage expression, wide dynamic range, and low crosstalk at multiple component levels. SENTRs can sense RNA targets, process signals by logic computation, and transduce them into various outputs, either mRNAs or ncRNAs. SENTR represents a powerful and versatile regulatory tool at the post-transcriptional level in Escherichia coli, with broad applications in biotechnology. I subsequently demonstrated that digital logic operation with up to six inputs could be implemented by using multiple orthogonal SENTRs to regulate a single gene simultaneously and coupling SENTRs with split intein-mediated protein trans-splicing. The integration of split introns and inteins is an efficient, genetically compact, and generally applicable strategy for building multi-input processing devices to significantly increase the information processing capacity of a single regulator gene.