Engineering a library of orthogonal genetic NAND gates from intein-split transcriptional repressors
Living cells are natural computing systems that exhibit complex behaviours such as decision making, signal processing and spatial organisation. Engineered genetic circuits take advantage of this capability to produce organisms that can respond to environmental stimulus and perform functions to aid biotechnological advancements in agriculture, manufacturing and therapeutics. However, such advancements have been hindered by the lack of orthogonal, biological parts and difficulty achieving precise gene expression levels. In this thesis, I tried to solve such issues by building a library of orthogonal, intein-split TetR-family repressors that limit cross-reactivity between regulators and produce varying gene expression levels. This was achieved by splitting repressors and attaching split intein terminals, with the aim of reconstituting a functional repressor after posttranslational trans-splicing of the intein when both, independently controlled, termini are expressed together. This controlled behaviour is akin to a NAND gate logical operation if both termini are orthogonal to each other. By using structural knowledge of TetR-family repressors and inteins’ splicing criteria, a library of post-translational repressors was created. An initial mutagenesis study of 5 TetR-family repressors was conducted to compare repression capability of wild-type and mutated, splicing optimised repressors. Results show that in many cases, loss of repression dynamic range of mutated repressors when compared to wild-type repressors. Based on results from the initial study, I assembled and characterised 40 2-input based repressors, with 3 showing orthogonal NAND gate like behaviour. From here, I focused on optimising gene expressions at the transcription level via inducible promoter selection and at the translation level using 3 different ribosome binding site sequences denoting different binding strengths. This lead to 44 more post-translational repressors. Finally, I demonstrate that intein mediated repressors can be rationally layered together to produce combinatorial NOT and AND gates. Building on the work carried out in this thesis, it is possible to build other Boolean logical operators such as OR and NOR gates using the same rational design method and our library of 2-input repressors. Additionally, this work provides the foundation to continue studying DNA-protein interactions, build more orthogonal NAND gates and produce more advanced, multi-layered logical behaviours.