Team:IIT-Madras/Design
DESIGN
Design of STR and GFP
The STR and GFP genes are individually preceded by the CaMV 35S promoter and a Kozak sequence (translational initiation site motif in eukaryotes). The STR sequence is followed by the CaMV 35S terminator, while the GFP sequence is followed by a portion of the NOS terminator (the rest of which is already present after the AflII site on the vector backbone). These sequences are designed for restriction cloning- the STR sequence is flanked by EcoRI (part of biobrick prefix) and PstI (part of biobrick suffix), while the GFP sequence is flanked by PstI on one side and AflII on the other. The designed sequences are also compatible with Gibson assembly.
Plasmid Design
The portion between restriction sites EcoRI and AflII, containing the GUS reporter system, was designed to be replaced with our gene of interest- STR, and a GFP reporter sequence. GFP is much easier to visualize than GUS, and hence this change in reporter system.
Cloning Strategies
Our initial approach to cloning a construct of STR (Strictosidine synthase) and GFP into the vector pCAMBIA was to construct a STR-GFP fragment first, following which this construct in cloned into the vector. However, the probability of such a process occurring is quite low with the competent cells we were using (efficiency ~10^7).
We modified our cloning strategy to a consecutive cloning approach. We planned to clone GFP into pCAMBIA, validate this part and then clone STR into this modified plasmid.
References
Schmoll M, Seibel C, Tisch D, Dorrer M, Kubicek CP. A novel class of peptide pheromone precursors in ascomycetous fungi. Mol Microbiol. 2010;77(6):1483–1501.
Seternes T, Tonheim TC, Myhr AI, Dalmo RA. A plant 35S CaMV promoter induces long-term expression of luciferase in Atlantic salmon. Sci Rep. 2016;6:25096
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Department of Biotechnology, IIT Madras.
Chennai 36, India.
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