AiM  Vol.3 No.1 , March 2013
Selection Vector for Direct Cloning of Proof Reading Polymerase Chain Reaction Products Based on the Lethal ccdB Gene in Escherichia coli
ABSTRACT

Introducing PCR products into plasmids vectors is key for molecular techniques. Ideally cloning vectors are easy to construct, modify and propagate, neither require advanced techniques nor special equipment or reagents and efficiently incorporate PCR products at close to zero empty vector background. We provide an easy to engineer self-made cloning vector, neither requiring sophisticated tools or techniques nor advanced cloning knowledge. Through recombination we obtained the pUC18ccdB vector, carrying the ccdB suicide gene within the pUC18 backbone. When SmaI cleaved (within the ccdB) vector was T4 ligated with small (0.2 kbp) and intermediate (1.3 to 2.2 kbp) blunt end PCR-products and transformed into E. coli, the amount of clones with incorporated PCR product was comparable to commercial PCR-cloning kits and at a close to zero PCR product negative background. In conclusion we present a simple, versatile and cheap approach to an efficient home made PCR-cloning vector that allows integration of crude blunt end PCR products at close to zero background.


Cite this paper
P. Weibel, M. Ender, J. Madon, A. Zinkernagel and R. Schuepbach, "Selection Vector for Direct Cloning of Proof Reading Polymerase Chain Reaction Products Based on the Lethal ccdB Gene in Escherichia coli ," Advances in Microbiology, Vol. 3 No. 1, 2013, pp. 14-20. doi: 10.4236/aim.2013.31002.
References
[1]   S. N. Cohen, “Bacterial Plasmids: Their Extraordinary Contribution to Molecular Genetics,” Gene, Vol. 135, No. 1-2, 1993, pp. 67-76. doi:10.1016/0378-1119(93)90050-D

[2]   O. Tolmachov, “Designing Plasmid Vectors,” Methods in Molecular Biology, Vol. 542, 2009, pp. 117-129. doi:10.1007/978-1-59745-561-9_6

[3]   B. Guo and Y. Bi, “Cloning PCR Products. An Overview,” Methods in Molecular Biology, Vol. 192, 2002, pp. 111-119.

[4]   J. Sambrook, E. F. Fritsch and T. Maniatis, “Molecular Cloning: A Laboratory Manual,” Spring Harbor Laboratory Press, New York, 1989.

[5]   Z. G. Liu and L. M.Schwartz, “An Efficient Method for Blunt-End Ligation of PCR Products,” Biotechniques, Vol. 12, No. 1, 1992, pp. 28-30.

[6]   A. Ullmann, “Complementation in Beta-Galactosidase: from Protein Structure to Genetic Engineering,” Bioessays, Vol. 14, No. 3, 1992, pp. 201-205. doi:10.1002/bies.950140311

[7]   J. Vieira and J. Messing, “The pUC Plasmids, an M13mp7Derived System for Insertion Mutagenesis and Sequencing with Synthetic Universal Primers,” Gene, Vol. 19, No. 3, 1982, pp. 259-268. doi:10.1016/0378-1119(82)90015-4

[8]   P. Bernard, P. Gabant, E. M. Bahassi and M. Couturier, “Positive-Selection Vectors Using the F Plasmid ccdB Killer Gene,” Gene, Vol. 148, No. 1, 1994, pp. 71-74. doi:10.1016/0378-1119(94)90235-6

[9]   B. Henrich and B. Schmidtberger, “Positive-Selection Vector with Enhanced Lytic Potential Based on a Variant of Phi X174 Phage Gene E,” Gene, Vol. 154, No. 1, 1995, pp. 51-54.

[10]   P. Kast, “pKSS—A Second-Generation General Purpose Cloning Vector for Efficient Positive Selection of Recombinant Clones,” Gene, Vol. 138, No. 1-2, 1994, 109-114. doi:10.1016/0378-1119(94)90790-0

[11]   S. A. Yazynin, S. M. Deyev, M. Jucovic and R. W. Hartley, “A Plasmid Vector with Positive Selection and Directional Cloning Based on a Conditionally Lethal Gene,” Gene, Vol. 169, No. 1, 1996, pp. 131-132.

[12]   D. F. Gruber, V. A. Pieribone, B. Porton and H. T. Kao, “Strict Regulation of Gene Expression from a High-Copy Plasmid Utilizing a Dual Vector System,” Protein Expression and Purification, Vol. 60, No. 1, 2008, pp. 53-57.

[13]   P. Bernard and M. Couturier, “Cell Killing by the F Plasmid ccdB Protein Involves Poisoning of DNA-Topoisomerase П Complexes,” Journal of Molecular Biology, Vol. 226, No. 3, 1992, pp. 735-745.

[14]   R. A. Schuepbach, J. Madon, M. Ender, P. Galli and M. Riewald, “Protease Activated Receptor-1 Cleaved at R46 Mediates Cytoprotective Effects,” Journal of Thrombosis and Haemostasis, 2012. doi:10.1111/j.1538-7836.2012.04825.x

[15]   R. A. Schuepbach and M. Riewald, “Coagulation Factor Xa Cleaves Protease-Activated Receptor-1 and Mediates Signaling Dependent on Binding to the Endothelial Protein C Receptor,” Journal of Thrombosis and Haemostasis, Vol. 8, No. 2, 2010, pp. 379-388.

[16]   K. Miyazaki, “Lethal ccdB Gene-Based Zero-Background Vector for Construction of Shotgun Libraries,” Journal of Bioscience and Bioengineering, Vol. 110, No. 3, 2010, pp. 372-373.

[17]   S. Shuman, “Recombination Mediated by Vaccinia Virus DNA Topoisomerase I in Escherichia coli Is Sequence Specific,” Proceedings of National Academy of Science of USA, Vol. 88, No. 22, 1991, pp. 10104-10108. doi:10.1073/pnas.88.22.10104

 
 
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