Fermentation Induces Instability
The bacteria’s reaction to stress during fermentation causes mobilization of IS elements and prophages, sometimes leading to cell lysis, spilling cell contents including pDNA into the medium, thus making the plasmid product difficult or impossible to recover.
Mobilization of IS elements (plasmid or chromosomal) to new locations disrupts genes in the chromosome and the resident plasmids. Any disruption that relieves the stress confers a growth advantage, enabling the resulting mutant to take over the culture.
Higher stress levels increase IS transposition and error-prone DNA replication rates. For example, plasmids with secondary structure such as viral Long Terminal Repeats (LTR) or short-hairpin RNA (shRNA) grow faster when the secondary structure (and its function) is disrupted.
Genetic instability can be triggered simply by growing a culture of commonly used bacterial strains from a cell bank. IS elements also serve as homologous targets of recombination when present in multiple copies. Deletions, insertions and large-scale rearrangements generated by IS10 have been reported.
Even the synthetic genome of Mycoplasma mycoides was found to be contaminated with IS1 from a DNA molecule grown in DH10B in an intermediate step. The extent of the IS and prophage problem in common E. coli strains is illustrated in the Table.
Transposition of some IS elements involves covalently closed active intermediates, “mini-circles”, which co-purify with plasmid DNA made by standard isolation techniques. Normally below gel detection level, they can be demonstrated by inside-out PCR, where the primers are designed to extend outwards past the end of the element.
Since the template is circular, amplimers are obtained. Unless specific methods are applied, IS transpositions generally remain undetected and their ability to cause problems has been under-appreciated. However, investigators are beginning to recognize the significance of the problem.
For example van der Heijden et al. reported that 12.85% of an HPV pDNA vaccine plasmid contained an IS2 although none had been detected in their master cell bank. They calculated that “it would have to be at a frequency below 1 in 10,000” to be undetectable. This study shows that massive expansion of IS elements from a very low starting level can be catastrophic. The authors conclude that the only way to avoid the problem is to use an IS-free host.