Plasmid copy number

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The plasmid copy number describes the number of plasmids of a species per cell .

One distinguishes between

  • low-copy plasmid copy number between 1 and 12 per cell
  • medium-copy plasmid copy number between 15 and 20 per cell
  • high-copy plasmid copy number between 20 and 700 per cell

The differences in the number of copies arise from the different functioning of the origin of replication and the genes encoded in it. The sequence of the plasmid P15A, for example, leads to the expression of the low-copy type, that of the plasmid pBR322 to the medium-copy type. A simple point mutation in the gene for the RNA II of the oris of the pBR322 plasmid, combined with the deletion of a control protein , to the high-copy type. Other mutations can also lead to an increase in the number of copies. Copy number and growth of plasmid-containing cells influence each other. A high number of copies means a high metabolic load on the cells and, under certain circumstances, leads to significantly reduced growth. The growth rate of a cell in turn influences the number of copies. A decrease in the growth rate causes an increase in the number of copies.

For the production of recombinant proteins , plasmids of the high-copy category are mostly used. There are three rules that apply to optimizing expression performance, one after the other:

  • High number of plasmid copies leads to high gene expression, but also to higher toxicity of toxic proteins.
  • If there is no increase in the amount of expression product despite an increase in the number of copies, the second rule states that the production of expression products is subject to the limitations of cell metabolism, since the transcription and translation capacities of the cells can be exhausted.
  • The third rule states that a reduction in the number of copies can also lead to an increase in the amount of product, so that the plasmid is more stable and therefore more efficient for the desired product. Low-copy plasmids are often particularly suitable for expressing proteins that are toxic to the host .

Definition of plasmid copy number

There are different definitions of the plasmid copy number in the specialist literature. The two most common define copy number as plasmid copies per cell or plasmid copies per chromosome. Quantities such as. B. fmol plasmid per mg dry biomass, relative information that relates to a comparison plasmid, such as x times the number of copies based on a wild-type plasmid or a value for the product yield mg plasmid DNA per g cell dry weight.

Determination of the plasmid copy number

There are several direct and indirect methods of determining the plasmid copy number. When using indirect methods, it is not the plasmids that are quantified directly, but rather gene products, such as the measurement of the activity of plasmid-encoded β-lactamase. A direct proportional relationship between enzyme activity and plasmid copy number is assumed here. However, this assumption does not take into account the fact that the expression of an enzyme and the enzyme activity are influenced by other factors that are independent of the plasmid copy number. Therefore this method can only be used to a limited extent. Similar systems exist with other reporter proteins such as luciferase .

With direct methods for determining the copy number, the amount of plasmid is determined after cell disruption. In addition to the dot blot method, in which the amount of plasmid is often determined by radioactively labeled gene probes <...?> .

Most often, after the cell disruption, the plasmids are isolated or separated from the chromosomal DNA. The methods used for this are HPLC , density gradient centrifugation or gel electrophoresis and pulsed gel electrophoresis. The amount of plasmid is quantified using different methods. Something through UV absorption in HPLC or through the measurement of radioactivity in a density gradient centrifugation. For this purpose, radionuclides must be presented for incorporation prior to replication of the plasmids. A Southern blot can also be used to quantify the plasmids after gel electrophoresis , or it can be done using labeled gene probes.

The determination of plasmid copy numbers by the methods mentioned always produces an average value that can be greatly falsified by plasmid-free cells in the culture.

Individual evidence

  1. ↑ Mayer MP (1995): A new set of useful cloning and expression vectors derived from pBluescript. Gene 163 41-46.
  2. ↑ I. Boros, G. Pósfai, P. Venetians: high-copy-number derivative of the plasmid cloning vector pBR322. In: Genes. Volume 30, Numbers 1-3, October 1984, pp. 257-260, PMID 6096220 .
  3. ^ AK Müller, F. Rojo, JC Alonso: The level of the pUB110 replication initiator protein is autoregulated, which provides an additional control for plasmid copy number. In: Nucleic acids research. Volume 23, Number 11, June 1995, pp. 1894-1900, PMID 7596815 , PMC 306960 (free full text).
  4. ↑ C. Anthony Mason, James E. Bailey: Effects of plasmid presence on growth and enzyme activity of Escherichia coli DH5 ?. In: Applied Microbiology and Biotechnology. 32, 1989, doi : 10.1007 / BF00164823 .
  5. ↑ Jung Hyun Lee, Kye Joon Lee: Analysis of the expression rate of cloned β-lactamase gene in a recombinant of Streptomyces lividans. In: Journal of Biotechnology. 52, 1996, p. 161, doi : 10.1016 / S0168-1656 (96) 01633-1 .
  6. ↑ Kramer W., Mattanovitch D., Elmecker G., R. Weik, Liege C., Bayer K., Katinger H. (1994): Regulable expression system for the optimization of recombinant protein production in Escherichia coli. Prog. Biotechnol. 9 827-830.
  7. ↑ C. French, JM Ward: Production and modification of E. coli transketolase for large-scale biocatalysis. In: Annals of the New York Academy of Sciences. Volume 799, October 1996, pp. 11-18, PMID 8958067 .
  8. ↑ MP Mayer: A new set of useful cloning and expression vectors derived from pBlueScript. In: Genes. Volume 163, Number 1, September 1995, pp. 41-46, PMID 7557476 .
  9. ↑ V. Nacken, T. Achstetter, E. Degryse: Probing the limits of expression levels by varying promoter strength and plasmid copy number in Saccharomyces cerevisiae. In: Genes. Volume 175, Number 1-2, October 1996, pp. 253-260, PMID 8917107 .
  10. ↑ Jeong-Yoon Kim, Dewey DY Ryu: The effects of plasmid content, transcription efficiency, and translation efficiency on the productivity of a cloned gene protein in Escherichia coli. In: Biotechnology and Bioengineering. 38, 1991, p. 1271, doi : 10.1002 / bit.260381103 .
  11. ^ S. La Fontaine, SD Firth, PJ Lockhart, JA Paynter, JF Mercer: Eukaryotic expression vectors that replicate to low copy number in bacteria: transient expression of the Menkes protein. In: plasmid. Volume 39, number 3, 1998, pp. 245-251, doi : 10.1006 / plas.1997.1334 , PMID 9571140 .
  12. ↑ J. Müller, JM van Dijl, G. Venema, S. Bron: Cloning of heterologous genes specifying detrimental proteins on pUC-derived plasmids in Escherichia coli. In: Molecular & general genetics: MGG. Volume 252, Number 1-2, August 1996, pp. 207-211, PMID 8804394 .
  13. ^ Leipold RJ, Krewson CE, Dhurjati P. (1994): Mathematical model of temperature-sensitive plasmid replication. Plasmid 32 131-167.
  14. ↑ Kiewiet RJ, Kok JF Seegers ML, Venema G., Bron. S. (1993): The mode of replication is a major factor in segregational plasmid instability in Lactococcus lactis. Appl. Environ. Microbiol. 59 358-364.
  15. ↑ Schendel FJ, Baude EJ, Flickinger. MC (1989): Determination of protein expression and plasmid copy number from cloned genes in Escherichia coli by flow injection analysis using an enzyme indicator vector. Biotechnol. Bioeng. 34 1023-1036.
  16. ^ Austin SJ, Eichorn BG (1992): Random diffusion can account for topA-dependent suppression of partition defects in low-copy-number plasmids. J. Bacteriol. 174 5190-5195.
  17. ↑ Schmidt T., K. Friehs, Flaschel E. (1996): Rapid determination of plasmid copy number. J. Biotechnol. 49 219-229.
  18. ^ Miller CA, Cohen SN (1993): The partition par locus of pSC101 is an enhancer of plasmid incompatibility. Mol. Microbiol. 9 695-702.
  19. ↑ Betenbaugh MJ, di Pasquantonio VM, Dhurjati P. (1987): Growth kinetics of Escherichia coli containing temperature-sensitive plasmid pOU140. Biotechnol. Bioeng. 29 1164-1172.
  20. ^ Coronado C., Vazquez ME, Cebolla A., Palomares AJ (1994): Use of firefly luciferase gene for plasmid copynumber determination. Plasmid 32 336-341.
  21. ↑ Hinnebusch J., Barbour AG (1992): linear and circular-plasmid copy numbers in Borrelia burgdorferi. J. Bacteriol. 174 5251-5257.
  22. ↑ Coppella SJ, Acheson CM, Dhurjati P. (1987): Measurement of copy number using HPLC. Biotechnol. Bioeng. 29 646-647.
  23. ↑ Weaver KE, Clewell DB, An F. (1993): Identification characterization and nucleotide sequence of a region of Enterococcus faecalis pheromone-responsive plasmid pAD1 capable of autonomous replication. J. Bacteriol. 175 1900-1909.
  24. ↑ Projan SJ, Carleton S., Novick RP (1983): Determination of plasmid copy number by fluorescence densitometry. Plasmid 9 182-190.
  25. ^ Ward AC, Hillier AJ, Davidson BE, Powell IB (1993): Stability analysis of the Lactococcus lactis DRC1 lactose plasmid using pulsed-field gel electrophoresis. Plasmid 29 70-73.
  26. ↑ Olsson T., K. Ekwall, T. Rusla. (1993): The silent P mating type locus in fission yeast contains two autonomously replicating sequences. Nucleic Acids Res. 21855-861.