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Introduction to Plasmid Biology

Rhonda Feinbaum¹

¹Massachusetts General Hospital, Boston, Massachusetts

Publication Name:

Current Protocols in Molecular Biology

Unit Number:

Unit 1.5

DOI:

10.1002/0471142727.mb0105s41

Online Posting Date:

May, 2001

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Abstract

An array of issues pertaining to plasmid biology are presented in this unit. Topics include replicators, an explanation of the mechanism of replication and copy-number control, plasmid incompatibility, selectable markers, cloning sites, and factors to consider when choosing a plasmid vector, whether it be for production of ssDNA, cloning of large inserts, or expression of large quantities of recombinant proteins. Considerations are discussed for the use of plasmids in yeast, cultured mammalian cells and non-E. coli bacteria. Finally, maps of many common useful plasmids are presented. This unit has recently been updated and extensively revised to include some of the latest developments in plasmid construction and experimental use.

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Unit Introduction
Replicators
Mechanism of Replication and Copy-Number Control
Plasmid Incompatibility
Selectable Markers
Cloning Site
Choosing a Plasmid Vector
Plasmid Vectors for Production of Single-Stranded DNA
Plasmid Vectors for Cloning Large Inserts
Plasmid Vectors for Expression of Large Quantities of Recombinant Proteins
Plasmid Vectors for Reporter Gene Fusions
Plasmid Vectors for Yeast
Plasmid Vectors for Expression in Cultured Mammalian Cells
Plasmid Vectors for Non–E. Coli Bacteria
Maps of Plasmids
Literature Cited
Figures
Tables

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Figures

Figure 1.5.1

ColE1 replication initiation and copy number control. ColE1 replication requires the formation of a RNA primer to initiate DNA synthesis. The RNA primer is derived from processing of a transcript, RNAII (bold), that starts upstream of the ori. Appropriate processing of the RNAII transcript is dependent on formation of a persistent hybrid between RNAII and the ori DNA template. If the proper RNAII secondary structure forms, then the RNA/DNA duplex is maintained at the ori and RNAse H can cleave the RNAII transcript to generate the primer for DNA synthesis. Processing of RNAII to form the primer is regulated by a second transcript, RNAI. RNAI is complementary to the 5¢ end of RNAII. If the RNAI transcript forms a duplex with RNAII, RNAII cannot take on the secondary structure necessary to create the persistent hybrid at the ori and maturation of the RNAII primer is inhibited. The copy number of ColE1 plasmids is determined by the balance between successful RNAII processing events and those inhibited by RNAI. Adapted from Gerhart et al. (1994) with permission from the Annual Review of Microbiology.

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Figure 1.5.2

Map of pUC19.

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Figure 1.5.3

Map of pWE15 (adapted from Wahl et al., 1987, with permission).

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Figure 1.5.4

Map of pBeloBAC11 (adapted from Shizuya et al., 1992, with permission). Abbreviation: CM, chloramphenicol.

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Figure 1.5.5

Map of pEGFP-1.

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Figure 1.5.6

Map of pRS303 (adapted from Sikorski and Heiter, 1989, with permission).

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Figure 1.5.7

Map of pcDNA3.1.

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Figure 1.5.8

Map of pRR54 (adapted from Roberts et al., 1990, with permission).

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Figure 1.5.9

Map of pBR322 (Bolivar et al. 1977; sequence in Sutcliffe, 1978).

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Figure 1.5.10

Map of pBluescript SK (+/–).

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Figure 1.5.11

Map of pTrc99A,B,C.

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Literature Cited

Literature Cited
	Amann, E., Ochs, B., and Abel, K.-J. 1988. Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli. Gene 69:301-315.
	Bolivar, F., Rodriguez, R.L., Greene, P.J., Betlach, M.C., Heynecker, H.L., and Boyer, H.W. 1977. Construction of useful cloning vectors. Gene 2:95-113.
	Chang, A.C.Y. and Cohen, S.N. 1978. Construction and characterization of amplifiable mulitcopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J. Bacteriol. 134:1141-1156.
	Gerhart, E., Wagner, H., and Simmons, R.W. 1994. Antisense RNA control in bacteria, phages, and plasmids. Annu. Rev. Microbiol. 48:713-742.
	Jackson, D.A., Symons, R.M., and Berg, P. 1972. Biochemical method for inserting new genetic information into DNA of Simian Virus 40 circular DNA molecules containing lambda phage genes and the galactose operon of Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 69:2904-2909.
	Kahn, M., Kolter, R., Thomas, C., Figurski, D., Meyer, R., Remaut, E., and Helinski, D.R. 1979. Plasmid cloning vehicles derived from plasmids ColE1, F, R6K, RK2. Methods Enzymol. 68:268-280.
	Kim, U.J., Shizuya, H., de Jong, P.J., Birren, B., and Simon, M.I. 1992. Stable propagation of cosmid- sized human DNA inserts in an F-factor based vector. NAR 20:1083-1085.
	Norrander, J., Kempe, T., and Messing, J. 1983. Construction of improved M13 vectors using oligonucleotide-directed mutagenesis. Gene 26:101-106.
	Roberts, R.C., Burioni, R., and Helinski, D.R. 1990. Genetic characterization of the stabilizing functions of a region of broad-host-range plasmid RK2. J. Bacteriol. 172:6204-6216.
	Shizuya, H., Birren, B., Kim, U.J., Mancin, V., Slepak, T., Tachiiri, Y., and Simon, M.I. 1992. A bacterial system for cloning large human DNA fragments. Proc. Natl. Acad. Sci. U.S.A. 89:8794-8797.
	Sikorski, R.S. and Hieter, P.I. 1989. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122:19-27.
	Stoker, N.G., Fairweather, N., and Spratt, B.G. 1982. Versatile low-copy-number plasmid vectors for cloning in Escherichia coli. Gene 18:335-341.
	Sutcliffe, J.G. 1978. Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harbor Symp. Quant. Biol. 43:77-90.
	Uhlin, B.E., Schweickart, V., and Clark, A.J. 1983. New runaway-replication-plasmid cloning vectors and suppression of runaway replication by novobiocin. Gene 22:255-265.
	Wahl, G.M., Lewis, K.A., Ruiz, J.C., Rothenberg, B., Zhao, J., and Evans, G.A. 1987. Cosmid vectors for rapid genomic walking, restriction mapping, and gene transfer. Proc. Natl. Acad. Sci. U.S.A. 84:2160-2164.
	Yang, T., Cheng, L., and Kain, S.R. 1996. Optimized codon usage and chromophore mutations provide enhanced sensitivity with the green fluorescent protein. Nucl. Acids Res. 24:4592-4593.