Linear Cloning Platform for Gene Assembly and Expression

We present a new platform for bacterial and eukaryotic cloning and expression of operons, repeat units, and structurally unstable DNAs. The system is based on a linear vector (“pJAZZ”) derived from the bacteriophage N15 of E. coli.  The lack of supercoiling, along with its tightly controlled transcription, circumvents numerous problems inherent to conventional circular plasmids. The pJAZZ vector tolerates a variety of elements that are typically deleted or rearranged in E. coli, and it exhibits reduced size bias for inserts of up to 40 kb. This system facilitated construction of the first comprehensive library of Plasmodium genomic DNA (80% A-T) (Welcome Trust Sanger Institute). The pJAZZ vector also is optimal for expressing multi-enzyme operons. In one application of this we developed an E. coli strain to produce isoprene by expressing an eight-gene operon for the MEP (2-C-methyl-D-erythritol 4-phosphate) pathway codon optimized for high expression and assembled by PCR. Intact clones were recovered only with the linear pJAZZ vector; none were obtained with circular vectors. The pJAZZ clone of the MEP operon produced 10 times more product (isoprene) than multiple circular plasmids expressing subsets of the genes. Mammalian versions of the vector have been used to express widely diverse targets, such as highly repetitive silk genes, multi-protein complexes, and a 40-kb tetrameric receptor gene. These vectors allow stable cloning of assemblies that include tandem arrays, deleterious coding sequences, and other difficult targets, with minimal risk of cloning bias, rearrangement, or deletion.