RK2-BASED BROAD-HOST-RANGE CLONING VECTOR USEFUL FOR TRANSFER OF METAGENOMIC LIBRARIES TO A VARIETY OF BACTERIAL SPECIES

摘要

The present invention relates to a cloning vector for cloning of DNA in a broad host range of bacteria, the vector being an autonomously replicating artificial chromosome comprising: (i) the RK2 origin of replication oriV; (ii) the RK2 origin of conjugate transfer oriT; (iii) par DE from RK2; (iv) a cloning region; (v) a further origin of replication which permits replication of said vector at a copy number of no more than 1 or 2; wherein the vector is no more than 15 kb in size, does not contain trfA of RK2, and is capable of cloning inserts of at least 12 kb and wherein the content of RK2 DNA in the vector is no more than 10% of RK2. The invention also relates to host cells and a vector system having the cloning vector. Methods of cloning DNA and preparing a library and uses of the vector and the RK2 replicon in metagenomic cloning are provided.