Abstract
A novel DNA sequence, derived from the antisense strand of the DNA gyrase inhibitor protein, CcdB, was toxic to E. coli. This protein (~6 kDa) decreased the growth rate of E. coli K12 by three orders of magnitude upon induction. The expressed toxic protein in E. coli K12 was soluble while it was insoluble in induced E. coli BL21. A high efficiency prokaryotic cloning/expression vector was constructed using this toxic gene sequence and gave zero background with ~100% cloning efficiency requiring no dephosphorylation. The toxic gene product also affected the survival of a ccdB resistant cell line, thus indicating a different mechanism of toxicity, other than DNA gyrase inhibition, as compared to the ccdB toxicity.
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References
Bahassi M, O’Dea M, Allali N, Messens J, Gelleert M, Couturier M (1999) Interactions of CcdB with DNA gyrase. J Biol Chem 274:10936–10944
Bernard P, Couturier M (1991) The 41 carboxy-terminal residues of the mini F plasmid CcdA protein are sufficient to antagonize the killer activity of the CcdB protein. Mol Gen Genet 226:297–304
Bernard P, Couturier M (1992) Cell killing by the F plasmid CcdB protein involve poisoning of DNA-topoisomerase II complexes. J Mol Biol 226:735–745
Bernard P, Gabant P, Bahassi EM, Couturier M (1994) Positive-selection vectors using the F plasmid ccdB killer gene. Gene 48:71–74
Curtiss R (1978) Biological containment and cloning vector transmissibility. J Infect Dis 137:668–675
Dao-Thi MH, Melderen LV, Genst ED, Afif H, Buts I, Wyns L, Loris R (2005) Molecular basis of gyrase poisoning by the addition toxin ccdB. J Mol Biol 248:1091–1102
Finbarr H (2003) Toxins-antitoxins: plasmid maintenance, programmed cell death and cell cycle arrest. Science 310:1496–1499
Ohashi-kunihiro S, Hagiwara H, Yohda M, Masaki H, Machida M (2006) Construction of a positive selection marker by lethal gene with the amber stop codon(s) regulator. Biosci Biotechnol Biochem 70:119–125
Pedersen K, Christensen SK, Gerdes K (2002) Rapid induction and reversal of a bacteriostatic condition by controlled expression of toxins and anti-toxins. Mol Microbiol 45:501–510
Sampali B, Shardul S, Anjali AD, Naganath M, Mandal G, Padmanabhan S (2009) Over expression of proteins using a modified pBAD24 vector in E. coli expression system. Biotechnol Lett 31:1031–1036
Song DD, Jacques NA (1997) Cell disruption of Escherichia coli by glass bead stirring for the recovery of recombinant proteins. Anal Biochem 248:300–301
Williams-Smith H (1978) Is it safe to use Escherichia coli in recombinant DNA experiments? J Infect Dis 137:655–660
Woodwall CA (2003) Methods in molecular biology. In: Casali N, Preston A (eds) E. coli plasmid vectors, vol 235. Humana Press, Totowa, NJ
Acknowledgements
The authors wish to thank Dr. Kamal Sharma, Managing Director, Lupin Limited, Pune, India for his constant encouragement and support. The authors also thank Rahul Nair and Dr. Shardul Salunkhe for bioreactor studies and construction of pBAD24M vector, respectively.
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Mandi, N., Kotwal, P. & Padmanabhan, S. Construction of a novel zero background prokaryotic expression vector: potential advantages. Biotechnol Lett 31, 1905–1910 (2009). https://doi.org/10.1007/s10529-009-0090-6
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DOI: https://doi.org/10.1007/s10529-009-0090-6