This plasmid solves the problems of low efficiency and instability of transgenes in the genetic manipulation of microalgae, as well as offers a procedure for the expression of high amounts of the protein of interest and better transformation efficiency
The plasmid is based on the translational fusion of the gene of interest with the paromomycin resistance gene, which lacks the mutagenic effects of other antibiotics and allows, thanks to the FMDV 2A self-hydrolyzing peptide, the independent production of the protein of interest and the antibiotic resistance. It includes a region of multi-cloning, which allows the insertion of any gene of interest, through a self-hydrolyzing peptide, FMDV 2A. In addition, prior to the multi-cloning region, we have introduced an epitope readily detectable by immunochemical techniques; and the entire cassette is under the control of the Chlamydomonas RbcS2 / HSP70A hybrid promoter. The relative position of the promoters relative to the multi-cloning region, and all elements of the plasmid have been optimized by eliminating unnecessary nucleotides to improve expression and thus to increase the efficiency of the transformation up to 3 times with respect to other plasmids having the same promoter . The main originality of our plasmid against others already exists is that it avoids the use of the BLE gene, resistance to bleomycin, which has been shown to be highly mutagenic and introduces undesirable alterations to isolated transformants.
Current development status
Commercially available technologies
Desired business relationship
New technology applications