PUC19

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Vector map of pUC19 PUC19.svg
Vector map of pUC19

pUC19 is one of a series of plasmid cloning vectors designed by Joachim Messing and co-workers. [1] The designation "pUC" is derived from the classical "p" prefix (denoting "plasmid") and the abbreviation for the University of California, where early work on the plasmid series had been conducted. [2] The pUC plasmids are all circular double stranded DNA about 2700 base pairs in length. [3] The pUC plasmids are some of the most widely used cloning vectors. [3] This is in part because cells that have successfully been transformed can be easily distinguished from those that have not based on color differences of colonies. [3] pUC18 is similar to pUC19, but the MCS region is reversed.

Contents

Features

pUC19 encodes of the N-terminal fragment of β-galactosidase ( lacZ ) gene of E. coli , also referred to as the α-peptide. [4] [3] The multiple cloning site (MCS), which contains many restriction sites, is split into codons 6-7 of the lacZ gene. [4] This allows for blue–white screening when using host strains such as E. coli JM109, which produces only the C-terminal portion of lacZ, also known as the β-polypeptide. [3] If pUC19 is inserted into E. coli JM109 and grown on agar media supplemented with IPTG and X-gal, then colonies will appear blue, as the plasmid encodes for the α-peptide required to make a functional form of β-galactosidase. If a fragment of DNA is inserted into the MCS of pUC19, however, colonies were appear white, as the plasmid will not be able to produce the α-peptide. [3]

In addition to β-galactosidase, pUC19 also encodes for an ampicillin resistance gene (ampR), via a β-lactamase enzyme that functions by degrading ampicillin and reducing its toxicity to the host. [5] Cells which have been successfully transformed with pUC19 can be differentiated from cells which have not by growing them on media with ampicillin. Only the cells with the plasmid containing ampR will survive.

The origin of replication (ori), is derived from the plasmid pMB1. [6] [1] pUC19 is a high copy number plasmid. [3] The high copy number is a result of the lack of the rop gene and a single point mutation in the ori of pMB1. [7] [8]

Mechanism

A schematic representation of the molecular mechanism involved for screening recombinant cells Blue white assay Ecoli.svg
A schematic representation of the molecular mechanism involved for screening recombinant cells

The lacZ fragment, whose synthesis can be induced by IPTG, is capable of intra-allelic complementation with a defective form of β-galactosidase enzyme encoded by host chromosome (mutation lacZDM15 in E. coli JM109, DH5α and XL1-Blue strains). [4] In the presence of IPTG in growth medium, bacteria synthesise both fragments of the enzyme. Both the fragments can together hydrolyse X-gal (5-bromo-4-chloro-3-indolyl- beta-D-galactopyranoside) and form blue colonies when grown on media where it is supplemented.

Insertion of foreign DNA into the MCS located within lacZ causes insertional inactivation of this gene at the N-terminal fragment of beta-galactosidase and abolishes intra-allelic complementation. Thus bacteria carrying recombinant plasmids in the MCS cannot hydrolyse X-gal, giving rise to white colonies, which can be distinguished on culture media from non-recombinant cells, which are blue. [9]

Use in research

Due to its extensive use as a cloning vector in research and industry, pUC19 is frequently used in research as a model plasmid. [10] For example, biophysical studies on its naturally supercoiled state have determined its radius of gyration to be 65.6 nm and its Stokes radius to be 43.6 nm.

See also

Related Research Articles

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References

  1. 1 2 Yanisch-Perron, Celeste; Vieira, Jeffrey; Messing, Joachim (1985). "Improved M13 phage cloning vectors and host strains: Nucleotide sequences of the M13mp18 and pUC19 vectors". Gene . 33 (1): 103–119. doi:10.1016/0378-1119(85)90120-9. PMID   2985470.
  2. ↑ Vieira, Jeffrey; Messing, Joachim (1982). "The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers". Gene . 19 (3): 259–268. doi:10.1016/0378-1119(82)90015-4. PMID   6295879.
  3. 1 2 3 4 5 6 7 Henkin, Tina M.; Peters, Joseph E. (2020). "Plasmids". Snyder and Champness molecular genetics of bacteria (Fifth ed.). Hoboken, NJ: John Wiley & Sons, Inc. pp. 208–209. ISBN   9781555819750.
  4. 1 2 3 Louro, Ricardo O.; Crichton, Robert R. (2013). Practical approaches to biological inorganic chemistry. Amsterdam, Oxford: Elsevier. p. 279. ISBN   9780444563590 . Retrieved 7 April 2014.
  5. ↑ Wang, Nam Sun. "Summary of Sites on pUC19". Department of Chemical & Biomolecular Engineering University of Maryland. Retrieved 27 January 2017.
  6. ↑ Helinski, Donald R. (15 December 2022). "A Brief History of Plasmids". EcoSal Plus. 10 (1). doi:10.1128/ecosalplus.esp-0028-2021.
  7. ↑ O’Hanlon Cohrt, Karen (26 November 2014). "pUC18 – Probably the Best High-Copy plasmid in the World!". BiteSize Bio.
  8. ↑ Saha, Arjun; Haralalka, Shruti; Bhadra, Rupak K. (November 2004). "A naturally occurring point mutation in the 13-mer R repeat affects the oriC function of the large chromosome of Vibrio cholerae O1 classical biotype". Archives of Microbiology. 182 (5): 421–427. Bibcode:2004ArMic.182..421S. doi:10.1007/s00203-004-0708-y. PMID   15375645. S2CID   28286917.
  9. ↑ Pasternak, Jack J. (2005). An Introduction to Human Molecular Genetics (2nd ed.). Wiley. p. 117. ISBN   978-0-471-71917-5.
  10. ↑ Störkle, Dominic (5 September 2007). "Complex Formation of DNA with Oppositely Charged Polyelectrolytes of Different Chain Topology: Cylindrical Brushes and Dendrimers". Macromolecules . 40 (22): 7998–8006. Bibcode:2007MaMol..40.7998S. doi:10.1021/ma0711689.
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