Introduction to Plasmid Biology

Rhonda Feinbaum1

1 Massachusetts 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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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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Table of Contents

  • 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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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.
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