ICE-NUCLEATION PROTEIN OF XANTHOMONAS CAMPESTRIS AND GENE THEREOF

摘要

PURPOSE: An ice-nucleation protein of Xanthomonas campestris, a gene thereof, a recombinant plasmid comprising the gene, a transformant comprising the recombinant plasmid and a culture method of the transformant are provided. CONSTITUTION: A gene cloning of ice-nucleation protein of Xanthomonas campestris(KCTC 0151 BP) is characterized by comprising the following steps: (i)after inoculating Xanthomonas campestris to LB liquid medium, culturing up to late logarithmic phase and washing the collected bio-mass twice with saline-EDTA solution and suspending in saline-Tris solution; (ii)after adding lysozyme, leaving at 37deg.C for 30 min. and dissolving the bio-mass by adding SDS and treating with proteinase K; (iii)after adding phenol and centrifuging, collecting the supernatant and adding cold ethanol as much as two times of its volume then followed by washing DNA with 70%, 80%, 90% of ethanol sequentially and dissolving in TE buffer solution; (iv)after cleaving DNA partially with restriction enzyme, performing electrophoresis and performing a ligation reaction; (v)after transforming E.coli by using mixed solution of ligation reaction, obtaining by spreading on LB agar medium; (vi)obtaining ice-nucleation transformant by inoculating on ampicillin added LB agar medium and measuring ice-nucleation activity.