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Author
Cook López-Barrena, Daniel
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Abstract
Optogenetic expression systems consist on the use of light to control gene expression, as light provides fast and reversible responses. These systems use two proteins, a sensor and an effector. Many optogenetic expression systems have been developed in yeast and animal cells, but none have been developed for microalgae, being very innovative. Moreover, these systems are useful to be applied in microalgae as they are model organisms that are in development for industrial biotechnology and they have scarce inducible gene expression systems. In addition, it is intended to apply these systems into the chloroplasts of Chlamydomonas reinhardtii as there is a well-known mechanism of gene silencing in the nuclei.
The aim of this thesis is to design a vector containing the phyB photoreceptor from Arabidopsis thaliana for its expression in the chloroplast of Chlamydomonas reinhardtii. The phyB gene from Arabidopsis thaliana, the psaA promoter and the TrbcL terminator were selected and amplified through PCR with the use of previously designed primers. The pLM20-VapA vector was then digested with EcoRV and SmaI. The fragments were then ligated together through Gibson assembly and, Escherichia coli were transformed with the final construct. To confirm that the grown Escherichia coli colonies contained the target construct, various methods were used. First, three PCRs were done to amplify the psaA promoter, the phyB gene and the three inserts together (promoter-phyB-terminator). Then, a double digestion with SmaI and BamHI was performed. With all methods, the results assured that from the five colonies that had grown in presence of ampicillin, colonies 1, 3 and 5 contained the target vector. In the future, this vector can then be used to transform chloroplast genome of Chlamydomonas reinhardtii and express phytochrome B as a novel photosensor for optogenetic applications.
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