Part:BBa_K4316032
Gypsy THB1 + Su(Hw)
Our project consists in modifying a green microalgae, Chlamydomonas reinhardtii, to turn it into an iron-rich super nutritive food through the tools of synthetic biology.
Chlamydomonas reinhardtii has 12 THBs, which are homologs of truncated hemoglobins. Our research was focused on THB1, expressed in the cytosol. To improve the iron content of this microalgae, we decided to overexpress the THB1 gene. The THB1 protein is regulated by the transcription factor Nit2, which is the major transcription factor in nitrate assimilation. This hemoglobin is up-regulated by nitrate. The idea would be to use a Nit2- strain and put the THB1 transgene under a light-inducible promoter to have complete control over its expression. The growth of our algae will be done in two conditions: first, they will grow in the dark until they achieve the exponential phase and after, we will expose them to light and induce the expression of THB1. This choice of culture avoids the disruption of normal cell growth as the overexpression could cause metabolic overload.
Another part of our project will be to work with insulators. These DNA sequences allow genes to be isolated from the genomic context in eukaryotes, making them ideal for transgene expression. Insulators from a variety of eukaryote species have been tested and shown to be functional in humans or plants. Several insulator transposons have been characterized. Among these, we have decided to focus on the gypsy sequence found in the Drosophila melanogaster genome, which was shown to exhibit enhancer-blocking activity in Arabidopsis thaliana, and also to have an homolog in this plant species (Atgypsy). The gypsy insulators work with zinc finger proteins such as Su(Hw). With this system, we will have complete control on the expression of THB1.
First of all, the gypsy insulator, which is the most characterized enhancer-blocking insulator. It is found within the 5’-untranslated region of the gypsy retrotransposon from Drosophila (∼350 bp). This insulator sequence works with zinc finger proteins Su(Hw) (Suppressor of Hairy-Wing), which recruit additional factors in order to form the “insulator body”. The gypsy insulator and other non-plant insulators have been shown to be functional in plants, such as transgenic Arabidopsis thaliana. This suggests the conservation of components involved in insulator activity in plants (ex: proteins). (Kurbidaeva, Amina, and Michael Purugganan. “Insulators in Plants: Progress and Open Questions.” Genes vol. 12,9 1422. 16 Sep. 2021, doi:10.3390/genes12091422). As C. reinhardtii is one of the greatest models for superior plants, insulator sequences, such as the gypsy one could work in this organism.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1699
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1991
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 2039
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