Antibiotic Free Plasmid Selection

Any plasmid designed should contain elements required for maintenance and propagation in the bacterial host and for expression of the target gene in the human or animal host. The primary elements required for bacterial propagation include the origin of replication and any plasmid-encoded functions (e.g. RNA I/RNAII sequences in ColE1-type replicons) required for replication. Further, a selectable marker must be included to allow for selection of successful transformants upon initial introduction of the plasmid into the bacterial host and to create selective pressure against predominance of plasmid-free bacteria.

So far, selectable marker systems either require plasmid-encoded sequences , e.g. antibiotic resistance genes or deletion mutants to serve as a host for a plasmid, complementing the lethal deletion. Here, we describe an alternative approach, where plasmid selection depends on the host only, resulting in a minimalistic plasmid, devoid of any additional sequences. The mechanism of plasmid maintenance is based on an RNA/RNA antisense reaction provided by the plasmid origin of replication. As a choice of a replicon for plasmid replication, mostly ColE1-type origins of replication are used.

In our experimental set-up, the replication inhibitor RNA I of ColE1 based plasmids serves as an anti-sense RNA directed against the appropriately designed mRNA of a gene encoded on the host’s genome. Proof of concept is shown by using a reporter gene (green fluorescent protein). However, if replaced by a toxic or growth hampering gene, plasmid containing cells will outgrow the population without plasmid, thus, providing plasmid selection without a selectable marker on the plasmid itself. By using gfp as a model, we could show that the replication of pBR322 wild-type decreases reporter gene expression during fermentation. These results further implicate a correlation between plasmid copy number and fluorescence, thus comprising a novel “on-line” method for monitoring plasmid copy number during bacterial fermentation processes. Furthermore, by decreasing the amount of free RNA I by binding to the gfp mRNA in addition to RNA II, plasmid copy number increases at least twofold during fermentation.

Project End 2010

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  • Mairhofer J., Pfaffenzeller I., Merz D., Grabherr R.: A novel antibiotic free plasmid selection system: Advances in safe and efficient DNA therapy. Biotechnol J  2008,3:83-89.
  • Pfaffenzeller I., Mairhofer J., Striedner G., Bayer K., Grabherr R.: Using ColE1-derived RNAI for supression of a bacterially encoded gene: Implication for a novel plasmid addiction system. Biotechnol J 2006, 1:675-681.