IS-Free Plasmid Production

Plasmid DNA vaccines (pDNA) are receiving renewed interest as initial problems of low antigenicity are being solved. These vaccines are particularly attractive for their fast development cycle, temperature stability and inexpensive manufacture by E. coli fermentation. However, traditional strains of E. coli contain mobile DNA elements including up to 65 Insertion Sequences (IS) and up to 11 species of partially defective bacteriophage in their genomes Figure 1. Both phage and IS elements can be activated during production causing inconsistent fermentation results. Moreover, IS elements can transpose into the plasmid product Figure 2, changing the sequence and function of the therapeutic agent; leading to the possibility that bacterial IS elements could make their way into the mammalian genome post-administration. Fortunately, these problems can all be eliminated by the use of multiple deletion strains (MDS) of E. coli in all steps of pDNA production. These strains are precisely engineered to remove all phage and transposable sequences Figure 3.

Experimental Data
Figure 1: IS & Prophage in Popular E. coli Strains.
Figure 2: IS Elements Detected in Commercial Vectors.
Figure 3: IS-Free pDNA Vaccine Production.

Learn More: Production of DNA Vaccines Free from Mobile DNA

Learn More: Bacterial Insertion Sequence Mobilization and Transfer to Plasmids: A Common Source of Cloning Artifacts

Figure 1: IS & Prophage in popular E. coli strains. Each box shows the number of copies of the element in the genome. Note: these counts represent a snapshot in time. Strains that have been sub-cultured multiple times may differ in their IS count or contain different complements of IS elements.

Figure 2: IS Elements Detected in Commercial Vectors. Detection of IS contamination in a commercial plasmid preparation of pBR322. Inward primers (panels a-d) or outward primers (panels e-h) specific for IS1, IS2, IS3, IS5, IS10, and IS186 were used (lanes 1-6, respectively; M is 1 kb+ size standard). Panels a and e show negative controls (no DNA), while positive controls in panels b and f are the individual IS elements cloned into pBR322. Panels c and g show purchased pBR322 and panels d and h show pBR322 isolated from MDS™42. PCR amplimers generated with outward primers specific for IS1, IS2, IS3, IS5, IS10 and IS186 were ligated, cloned with selection for tetracycline or ampicillin resistance, and sequenced (data not shown). Sequencing confirmed transposition of IS1, IS2, IS5, and IS10 to pBR322 in the commercial preparation.

Figure 3: IS-Free pDNA Vaccine Production. Extra care in the first steps will ensure trouble-free production.