Spirulina Platensis Into The Expression System

Spirulinaplatensis, a kind of cyanobacteriuim is famous for its rich content of protein and other sources of nutrients. In these years, it has been rapid progresses on genetic engineering of other cyanobacteria, but Spirulina platensis is extremely hostile to foreign gene transfomiation.The active endocellular nuclease of Spirulina platensis is believed to be the most serious barrier of foreign gene transfonnation. We detected plasmid pEUTISI remains after incubated with endocellular nuclease extract from SpirulinaplatensLs for two hours under 28 0C as an index of endocellular nuclease activity. Results showed that the activity of endoce]Iular nuclease could be efficiently repressed under EDTA concentration of 2mmo]IL or above for 16 hours, or cultured in Mg>-fi-ee medium for more than 72 hours. When cultured in lower temperature, such as 240C, the enzyme activity showed mild but steady decrease. This result led to a combined condition for successful transformation ofpEIJTISI and other similar plasmidsto Spindinaplatensis.By ultrasonics trealment, our research has found an efficient ~y to introduce foreign genes into the chromosome of Spirulina platensis. Gm~i in 24 0C, Combined with 2mmoL'L EDTA treatment for 16-20 hours, Spirulina platens is ~s successful transformed by plasmids pEUTISI and pEUTR.. designed forcyanobacteria gene targeting. Putative Iransfonnants SP-I and SP-R were selected by G41 8 and showed great changes on both size and shape. Results of southern-blotIIby ~r probe showed that the chromosome of SRI had been genetically reconstructed by pEUTISI. Further comflrmation was made by SDS-PAGE and western-blot Results indicated a special protein expressed in SP-I that is coherent with the expected foreign protein, Ubiquitin-Thyrnosin al, in both molecular weight and immunological characteristics.Other efforts have been made to build up cloning vectors using chloraphenicol and hygromycin as selective antibiotics respectively. }~r gene in plasrnid pUTKwas replaced with hygromycin-resistant gene in pDR2. Thus, besides hygromycin and Ampicillin-resitant genes, the final plasmid vector pAH canied both lateral homologous fragments L and R of cyanobacteria c-phycocyanin gene. Another vector pKR was built up by inserting R fragment into pKF3, '?ich canied a chloramphenicol-resistant gene. The two vectors were designed as media for further gene transformation via homologous recombination.