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Project Overview

This year we want to start developing bricks for use in Chlamydomonas reinhardtii chloroplasts.

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a) Constitutive chimeric promoters for the expression of recombinant proteins

In this part of the project we aim to develop constitutive promoters that could allow for accumulation of different levels of recombinant proteins in chlamy chloroplast. We are aware that some groups around the globe have tried to determimed what is the best promoter to drive the expression of recombinant proteins that accumulate to high levels. Even though, these attempts have rendered a couple of promoter (PatpA and PpsbA ) that function reasonably well, these promoters seem to be affected by the ORF sequence at the translation level. We noticed that so far not many workd have foucued on the study of promoter/5´UTR combination so for this years proyect we aimed to circunvect this problem by construction a series of promoter+5´UTR combination tha could favour the translation of the mRNA generated. To achieved this, we have picked on of the most strong promoters in chloroplast (plants and algae), the psbA promoters. This promoter has the advantage of generating a high level of transcript accumulation but has the drawback that the translation of the transcripts is srongly regulated by light and it´s product, the D1 protein. A site in the 5´UTR of the transcript has been identified as a binding site for this protein. What we want then to construct combinations of the psbA promoter with 5´UTRs that have been identified to produce from low to very high levels of protein accumulation is to use the psbA promoter


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