A Genetic NOT Gate in Transgenic Tobacco Based on Estradiol-Induced Translational Repression

A genetic circuit implementing Boolean negation (NOT) is demonstrated in transgenic tobacco plants, using exposure to estradiol as the input signal and expression of green fluorescent protein (GFP) as the output signal. The GFP gene is controlled by a constitutive promoter, resulting in overexpression of green fluorescent protein (GFP) in the absence of estradiol. A binding site for Clostridium difficile Cas6 (CdCas6) is inserted upstream of the GFP gene, allowing for translational repression of GFP by CdCas6. The CdCas6 gene is linked with an estradiol-inducible promoter, with the selectively expressed Cas protein binding and cleaving the GFP mRNA at the corresponding site, resulting in lower levels of GFP expression in the presence of estradiol. Median levels of GFP expression are shown to reduce by 30%-45% in the presence of estradiol. Various factors affecting the performance of the circuit are discussed and possible avenues for improvements are suggested. In addition, a custom pipeline for analysis of the data is outlined, with limitations and possible improvements discussed.