32-mer MaSP1 geninin pBbB6c plazmid vektörüne klonlanmasi ve Escherichia coli neb 10-beta'ya transformasyonu

The main purpose of my thesis was to clone Masp1 spider silk protein encoding gene from dragline type spider into E.coli NEB 10-beta organism. The recombinant microbial production of spider silk protein and converting it into a fiber format would ultimately produce a biomaterial also called as biosteel with high toughness and elasticity whereas low density compared to Kevlar, steel and carbon fiber. For this purpose, the gene encoding the dragline spider protein (MaSP1) was cloned into E. coli NEB 10-beta using recombinant molecular methods. First, 8-mer MaSP1 was synthesized and cloned via pGSI high copy cloning vector by sticky end cutting with restriction enzymes of KpnI,Kpn2I followed by heat-shock transformation into E.coli. Second, we performed restriction of the 8-mer plasmid by NheI and Kpn2I to extract the 8-mer. Later, the restriction was performed by SpeI and Kpn2I to obtain linearized pGSI containing 8-mer Masp1. A ligation was applied to merge 8-mer and pGSI plasmid carrying 8-mer Masp1 to achieve 16-mer Masp1 containing pGSI. Again, this plasmid was heat-shock transformed into E.coli. Following the same restriction 32-mer Masp1 containing pGSI plasmid was achieved. Finally, 32-mer Masp1 fragment was cut from pGSI cloning vector and ligated to pBbB6c low copy expression plasmid followed by electroporation into E.coli. The band size of 32-mer Masp1 gene was aligned between 3 kb and 5 kb which is an agreement with the calculated size of 32-mer Masp1 gene. Future studies should focus on the expression of Masp1 and the efficient production of this valuable recombinant protein under bioreactor conditions with cutting edge bioprocessing techniques.