pUC19 is one of a series of plasmid cloning vectors created by Joachim Messing and co-workers. The designation "pUC" is derived from the classical "p" prefix (denoting " plasmid") and the abbreviation for the

University of California The University of California (UC) is a public land-grant research university system in the U.S. state of California. The system is composed of the campuses at Berkeley, Davis, Irvine, Los Angeles, Merced, Riverside, San Diego, San Franci ...

, where early work on the plasmid series had been conducted. It is a circular double stranded DNA and has 2686 base pairs. pUC19 is one of the most widely used vector molecules as the recombinants, or the cells into which foreign DNA has been introduced, can be easily distinguished from the non-recombinants based on color differences of colonies on growth media. pUC18 is similar to pUC19, but the MCS region is reversed.

Components

Notably, it has an N-terminal fragment of β-galactosidase (''

lacZ The ''lactose'' operon (''lac'' operon) is an operon required for the transport and metabolism of lactose in ''E. coli'' and many other enteric bacteria. Although glucose is the preferred carbon source for most bacteria, the ''lac'' operon allows ...

'') gene of '' E. coli''. The

multiple cloning site A multiple cloning site (MCS), also called a polylinker, is a short segment of DNA which contains many (up to ~20) restriction sites - a standard feature of engineered plasmids. Restriction sites within an MCS are typically unique, occurring only ...

(MCS) region is split into codons 6-7 of the lacZ gene, providing for many

restriction endonucleases A restriction enzyme, restriction endonuclease, REase, ENase or'' restrictase '' is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class o ...

restriction site Restriction sites, or restriction recognition sites, are located on a DNA molecule containing specific (4-8 base pairs in length) sequences of nucleotides, which are recognized by restriction enzymes. These are generally palindromic sequences (bec ...

s. In addition to β-galactosidase, pUC19 also encodes for an

ampicillin Ampicillin is an antibiotic used to prevent and treat a number of bacterial infections, such as respiratory tract infections, urinary tract infections, meningitis, salmonellosis, and endocarditis. It may also be used to prevent group B stre ...

resistance gene (amp^R), via a β-lactamase enzyme that functions by degrading ampicillin and reducing its toxicity to the host. The ''ori'' site, or

origin of replication The origin of replication (also called the replication origin) is a particular sequence in a genome at which replication is initiated. Propagation of the genetic material between generations requires timely and accurate duplication of DNA by se ...

, is derived from the plasmid pMB1. pUC19 is small but has a high copy number. The high copy number is a result of the lack of the '' rop'' gene and a single point mutation in the ori of pMB1. The ''lacZ'' gene codes for

β-galactosidase β-Galactosidase (EC 3.2.1.23, lactase, beta-gal or β-gal; systematic name β-D-galactoside galactohydrolase), is a glycoside hydrolase enzyme that catalyzes hydrolysis of terminal non-reducing β-D-galactose residues in β-D-galactosides. β ...

. The recognition sites for

HindIII ''Hin''dIII (pronounced "Hin D Three") is a type II site-specific deoxyribonuclease restriction enzyme isolated from ''Haemophilus influenzae'' that cleaves the DNA palindromic sequence AAGCTT in the presence of the cofactor Mg2+ via hydrolysis ...

, SphI, PstI, SalI, XbaI, BamHI, SmaI, KpnI, SacI and

EcoRI ''Eco''RI (pronounced "eco R one") is a restriction endonuclease enzyme isolated from species '' E. coli.'' It is a restriction enzyme that cleaves DNA double helices into fragments at specific sites, and is also a part of the restriction modifi ...

restriction enzymes have been derived from the vector M13mp19.

Function

This plasmid is introduced into a bacterial cell by a process called "

transformation Transformation may refer to: Science and mathematics In biology and medicine * Metamorphosis, the biological process of changing physical form after birth or hatching * Malignant transformation, the process of cells becoming cancerous * Tran ...

", where it can multiply and express itself. However, due to the presence of MCS and several restriction sites, a foreign piece of DNA of choice can be introduced into it by inserting it into place in MCS region. The cells which have taken up the plasmid can be differentiated from cells which have not taken up the plasmid by growing it on media with ampicillin. Only the cells with the plasmid containing the ampicillin resistance (''amp^R'') gene will survive. Furthermore, the transformed cells containing the plasmid with the gene of interest can be distinguished from cells with the plasmid but without the gene of interest, just by looking at the color of the colony they make on agar media supplemented with IPTG and

X-gal X-gal (also abbreviated BCIG for 5-bromo-4-chloro-3-indolyl-β--galactopyranoside) is an organic compound consisting of galactose linked to a substituted indole. The compound was synthesized by Jerome Horwitz and collaborators in 1964. The formal c ...

. Recombinants are white, whereas non-recombinants are blue.

Mechanism

The ''lac Z'' 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). 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 the ''lac Z'' gene causes

insertional inactivation In molecular biology, insertional mutagenesis is the creation of mutations of DNA by addition of one or more base pairs. Such insertional mutations can occur naturally, mediated by viruses or transposons, or can be artificially created for research ...

of this gene at the N-terminal fragment of

beta-galactosidase β-Galactosidase (EC 3.2.1.23, lactase, beta-gal or β-gal; systematic name β-D-galactoside galactohydrolase), is a glycoside hydrolase enzyme that catalyzes hydrolysis of terminal non-reducing β-D-galactose residues in β-D-galactosides. β- ...

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. Therefore, the media used should contain

, IPTG, and

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. For example, biophysical studies on its naturally supercoiled state have determined its

radius of gyration ''Radius of gyration'' or gyradius of a body about the axis of rotation is defined as the radial distance to a point which would have a moment of inertia the same as the body's actual distribution of mass, if the total mass of the body were concentr ...

to be 65.6 nm and its

Stokes radius The Stokes radius or Stokes–Einstein radius of a solute is the radius of a hard sphere that diffuses at the same rate as that solute. Named after George Gabriel Stokes, it is closely related to solute mobility, factoring in not only size but also ...

to be 43.6 nm.

References

External links