Remarkable stability of an instability-prone lentiviral vector plasmid in Escherichia coli Stbl3.
Large-scale production of plasmid DNA to prepare therapeutic gene vectors or DNA-based vaccines requires a suitable bacterial host, which can stably maintain the plasmid DNA during industrial cultivation. Plasmid loss during bacterial cell divisions and structural changes in the plasmid DNA can dramatically reduce the yield of the desired recombinant plasmid DNA. While generating an HIV-based gene vector containing a bicistronic expression cassette 5'-Olig2cDNA-IRES-dsRed2-3', we encountered plasmid DNA instability, which occurred in homologous recombination deficient recA1 Escherichia coli strain Stbl2 specifically during large-scale bacterial cultivation. Unexpectedly, the new recombinant plasmid was structurally changed or completely lost in 0.5 L liquid cultures but not in the preceding 5 mL cultures. Neither the employment of an array of alternative recA1 E. coli plasmid hosts, nor the lowering of the culture incubation temperature prevented the instability. However, after the introduction of this instability-prone plasmid into the recA13E. coli strain Stbl3, the transformed bacteria grew without being overrun by plasmid-free cells, reduction in the plasmid DNA yield or structural changes in plasmid DNA. Thus, E. coli strain Stbl3 conferred structural and maintenance stability to the otherwise instability-prone lentivirus-based recombinant plasmid, suggesting that this strain can be used for the faithful maintenance of similar stability-compromised plasmids in large-scale bacterial cultivations. In contrast to Stbl2, which is derived wholly from the wild type isolate E. coli K12, E. coli Stbl3 is a hybrid strain of mixed E. coli K12 and E. coli B parentage. Therefore, we speculate that genetic determinants for the benevolent properties of E. coli Stbl3 for safe plasmid propagation originate from its E. coli B ancestor.
Department of Medical Genetics, Faculty of Medicine, Umm Al-Qura University, Al-Abedia Campus, P. O. Box 715, Makkah, 21955, Saudi Arabia. fallaf@hotmail.com.|Division of Clinical Sciences, Faculty of Medicine, Institute of Reproductive and Developmental Biology, Imperial College London, London, W12 0NN, UK. fallaf@hotmail.com.
Faisal A
FA
Al-Allaf
Faculty of Medicine, National Heart and Lung Institute, Sir Alexander Fleming Building, Imperial College London, London, SW7 2AZ, UK.
Oleg E
OE
Tolmachov
Division of Clinical Sciences, Faculty of Medicine, Institute of Reproductive and Developmental Biology, Imperial College London, London, W12 0NN, UK.
Lia Paola
LP
Zambetti
Division of Clinical Sciences, Faculty of Medicine, Institute of Reproductive and Developmental Biology, Imperial College London, London, W12 0NN, UK.
Viktoria
V
Tchetchelnitski
Division of Clinical Sciences, Faculty of Medicine, Institute of Reproductive and Developmental Biology, Imperial College London, London, W12 0NN, UK.
Huseyin
H
Mehmet
Escherichia coli; Lentiviral vector; Plasmid instability; Stbl2; Stbl3