Part:BBa_K4316034
Sulator eGFP + 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.
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.
Two plasmids have been designed and are under construction. These are a test plasmid (pM-insulator) and a control plasmid (pM-control). The test plasmid consists of the insulator sequences, a gene encoding a fluorescence protein (mVenus) under the control of the constitutive promoter (pPSAD), and the gene encoding the Su(Hw) protein. While the control plasmid has only the fluorescence protein gene under the control of the constitutive promoter. After transformation of the uvm4 (Nit2-) strains with the two plasmids and culture in multi-well plates, the test is performed using a plate reader. The objective is to measure the fluorescence intensity of mVenus in colonies with the test plasmid and those with the control plasmid. The proof of concept is that the fluorescence intensity varies for colonies with the control plasmid and is constant for colonies with the test plasmid. Indeed, as the gypsy sequences isolate the transgene from the genomic context, the expression of the fluorescent protein should not vary.
This plasmid user the control « Sulator » sequences, which have no explicit function except act as spacers.
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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