|
Author
Martín Rosco, Raúl
|
Abstract
Regardless the enormous contribution of classic gene expression systems, there is a need for more time-restrictive and spatial-accurate tools. Optogenetics is a discipline that control genetic circuits in living cells using light, which provides a supreme resolution in space and time. Combining a plant photoreceptor (phyB) fused to the inactive Cas9 mutant (dCas9), and an effector component containing an active phyB-binding domain (APB) fused to a transcriptional repressor, we can build an orthogonal light-switchable system to control endogenous and recombinant gene expression depending on the presence of red/far-red light in the microalgae C. reinhardtii.
The aim of this project is to design and analyse the system’s part composed by the APB fused to the transcriptional repressor. We selected 4 APB candidates from A. thaliana (P3, P6, P6A and P7) and we designed a synthetic gene, which was named OptoRepresor (OR) gene. OR gene is composed by the best APB candidate (P6.100), a western blot epitope (MYC), a transcriptional repressor (KRAB) and a nuclear localization sequence (SV40). Moreover, it is codon-optimized and has 2 interspaced introns to improve transgene expression. The 4 APB variants and the OR gene were cloned into pOptimized vector toolkit, which allow fusion of the gene of interest to a fluorescent protein, in this case, the mCerulean3. Then, C. reinhardtii transformant colonies expressing non-fused mCerulean3 protein were identified by fluorimetry and western blot as positive controls. Finally, P3, P7 and OR genes expression in C. reinhardtii were analysed by fluorimetry and western blot. No transformant colony showed detectable expression levels of anyone of these genes. In order to improve heterologous gene expression and its characterization, new screening strategies and vectors have been proposed.
|
|
