CHAPTER 3
Figure 22. Tautology and contradiction. (A) Multigenic construct for the tautology ⊤LACI::GAL4AD gate. (B) Digital combination of two input inducers results in similar levels of analog gene expression outputs. (C) Construct for the contradiction ⊥UAS gate (D) Digital combination of two input inducers result in a minimal background output. The ratios between Firefly (FLuc) and Renilla (RLuc) luciferase activities were normalized using a Nopaline synthase construct. Error bars represent the SD of at least three replicates. 1x corresponds to the reference PNos:Luciferase:TNos construct. ⊤ and ⊥ denote the tautology and contradiction logic gates. P35S is CaMV 35S promote; LacI BD indicates residues 1-330 of the Y17H mutant of lac repressor; Gal4AD indicates transcription activation domain II (residues 768-881) of Saccharomyces cerevisiae Gal4; TNos is the Nopaline synthase terminator; 6xOpLacI are six closely spaced lac operators; mini35S is the minimal CaMV 35S promoter; Luciferase and Renilla are the Photinus pyralis and Renilla reniformis Luciferase genes; PAtUbq10 is the Promoter of the A. thaliana Ubiquitin 10 gene; TMtb is the S.lycopersicum Metalothionein-like protein terminator, 6xUAS is the upstream activation sequence where Gal4 binds to.
TU encoding the activator module, (ii) a reporter unit consisting of the LacIOp upstream of a minimal CaMV 35S promoter and the firefly luciferase, and (iii) the Renilla Module. The first two propositions were based on the constitutive expression of a fusion protein with three domains, which are the glucocorticoid or estradiol receptor, the LacIBD and the Gal4AD proteins. This fusion protein is primarily located in the cytoplasm forming a heteroduplex in combination with Hsp90 in the absence of ligand. It moves to the nucleus after binding the trigger molecule where it will bind the DNA and activate the transcription [157-158]. The third proposition was driven by the Arabidopsis 70B heat shock promoter to result in the expression of the modular protein LacIBD::Gal4AD. The three gates were responsive to the corresponding inducer in transient expression experiments (10 µM dexamethasone, 5 µM β-estradiol and a 2h 37ºC heat shock treatment).
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