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PUC19

From Wikipedia, in a visual modern way
Vector map of pUC19
Vector map of pUC19

pUC19 is one of a series of plasmid cloning vectors created 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] It is a circular double stranded DNA and has 2686 base pairs.[3] 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.

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Cloning vector

Cloning vector

A cloning vector is a small piece of DNA that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes. The cloning vector may be DNA taken from a virus, the cell of a higher organism, or it may be the plasmid of a bacterium. The vector contains features that allow for the convenient insertion of a DNA fragment into the vector or its removal from the vector, for example through the presence of restriction sites. The vector and the foreign DNA may be treated with a restriction enzyme that cuts the DNA, and DNA fragments thus generated contain either blunt ends or overhangs known as sticky ends, and vector DNA and foreign DNA with compatible ends can then be joined by molecular ligation. After a DNA fragment has been cloned into a cloning vector, it may be further subcloned into another vector designed for more specific use.

Joachim Messing

Joachim Messing

Joachim Wilhelm "Jo" Messing was a German-American biologist who was a professor of molecular biology and the fourth director of the Waksman Institute of Microbiology at Rutgers University.

Plasmid

Plasmid

A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; however, plasmids are sometimes present in archaea and eukaryotic organisms. In nature, plasmids often carry genes that benefit the survival of the organism and confer selective advantage such as antibiotic resistance. While chromosomes are large and contain all the essential genetic information for living under normal conditions, plasmids are usually very small and contain only additional genes that may be useful in certain situations or conditions. Artificial plasmids are widely used as vectors in molecular cloning, serving to drive the replication of recombinant DNA sequences within host organisms. In the laboratory, plasmids may be introduced into a cell via transformation. Synthetic plasmids are available for procurement over the internet.

University of California

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 its ten campuses at Berkeley, Davis, Irvine, Los Angeles, Merced, Riverside, San Diego, San Francisco, Santa Barbara, and Santa Cruz, along with numerous research centers and academic abroad centers. The system is the state's land-grant university. Major publications generally rank most UC campuses as being among the best universities in the world. Seven of the campuses, Berkeley, Davis, Santa Cruz, Irvine, Los Angeles, Santa Barbara, and San Diego are considered Public Ivies, making California the state with the most universities in the nation to hold the title. UC campuses have large numbers of distinguished faculty in almost every academic discipline, with UC faculty and researchers having won 71 Nobel Prizes as of 2021.

Recombinant DNA

Recombinant DNA

Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination that bring together genetic material from multiple sources, creating sequences that would not otherwise be found in the genome.

Components

Notably, it has an N-terminal fragment of β-galactosidase (lacZ) gene of E. coli.[4] The multiple cloning site (MCS) region is split into codons 6-7 of the lacZ gene, providing for many restriction endonucleases restriction sites.[5] 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.[6]

The ori site, or origin of replication, 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.[7] The lacZ gene codes for β-galactosidase. The recognition sites for HindIII, SphI, PstI, SalI, XbaI, BamHI, SmaI, KpnI, SacI and EcoRI restriction enzymes have been derived from the vector M13mp19.[8]

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Multiple cloning site

Multiple cloning site

A multiple cloning site (MCS), also called a polylinker, is a short segment of DNA which contains many restriction sites - a standard feature of engineered plasmids. Restriction sites within an MCS are typically unique, occurring only once within a given plasmid. The purpose of an MCS in a plasmid is to allow a piece of DNA to be inserted into that region.

Restriction site

Restriction site

Restriction sites, or restriction recognition sites, are located on a DNA molecule containing specific sequences of nucleotides, which are recognized by restriction enzymes. These are generally palindromic sequences, and a particular restriction enzyme may cut the sequence between two nucleotides within its recognition site, or somewhere nearby.

Ampicillin

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 streptococcal infection in newborns. It is used by mouth, by injection into a muscle, or intravenously. Common side effects include rash, nausea, and diarrhea. It should not be used in people who are allergic to penicillin. Serious side effects may include Clostridium difficile colitis or anaphylaxis. While usable in those with kidney problems, the dose may need to be decreased. Its use during pregnancy and breastfeeding appears to be generally safe.

Origin of replication

Origin of replication

The origin of replication 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 semiconservative replication prior to cell division to ensure each daughter cell receives the full complement of chromosomes. This can either involve the replication of DNA in living organisms such as prokaryotes and eukaryotes, or that of DNA or RNA in viruses, such as double-stranded RNA viruses. Synthesis of daughter strands starts at discrete sites, termed replication origins, and proceeds in a bidirectional manner until all genomic DNA is replicated. Despite the fundamental nature of these events, organisms have evolved surprisingly divergent strategies that control replication onset. Although the specific replication origin organization structure and recognition varies from species to species, some common characteristics are shared.

Rop protein

Rop protein

Rop is a small dimeric protein responsible for keeping the copy number of ColE1 family and related bacterial plasmids low in E. coli by increasing the speed of pairing between the preprimer RNA, RNA II, and its antisense RNA, RNA I. Structurally, Rop is a homodimeric four-helix bundle protein formed by the antiparallel interaction of two helix-turn-helix monomers. The Rop protein's structure has been solved to high resolution. Due to its small size and known structure, Rop has been used in protein design work to rearrange its helical topology and reengineer its loop regions. In general, the four-helix bundle has been extensively used in de novo protein design work as a simple model to understand the relationship between amino acid sequence and structure.

HindIII

HindIII

HindIII (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.

PstI

PstI

PstI is a type II restriction endonuclease isolated from the Gram negative species, Providencia stuartii.

XbaI

XbaI

XbaI is a restriction enzyme isolated from the bacterium Xanthomonas badrii

BamHI

BamHI

BamHI is a type II restriction endonuclease, having the capacity for recognizing short sequences of DNA and specifically cleaving them at a target site. This exhibit focuses on the structure-function relations of BamHI as described by Newman, et al. (1995). BamHI binds at the recognition sequence 5'-GGATCC-3', and cleaves these sequences just after the 5'-guanine on each strand. This cleavage results in sticky ends which are 4 bp long. In its unbound form, BamHI displays a central b sheet, which resides in between α-helices.

SacI

SacI

SacI is a restriction enzyme isolated from the bacterium Streptomyces achromogenes.

EcoRI

EcoRI

EcoRI 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 modification system. The Eco part of the enzyme's name originates from the species from which it was isolated - "E" denotes generic name which is "Escherichia" and "co" denotes species name, "coli" - while the R represents the particular strain, in this case RY13, and the I denotes that it was the first enzyme isolated from this strain.

Function

This plasmid is introduced into a bacterial cell by a process called "transformation", 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 (ampR) 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. Recombinants are white, whereas non-recombinants are blue.

Mechanism

A schematic representation of the molecular mechanism involved for screening recombinant cells
A schematic representation of the molecular mechanism involved for screening recombinant cells

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).[9] 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 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.[10]

Therefore, the media used should contain ampicillin, IPTG, and X-gal.

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Beta-galactosidase

Beta-galactosidase

β-Galactosidase, is a glycoside hydrolase enzyme that catalyzes hydrolysis of terminal non-reducing β-D-galactose residues in β-D-galactosides.

Ampicillin

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 streptococcal infection in newborns. It is used by mouth, by injection into a muscle, or intravenously. Common side effects include rash, nausea, and diarrhea. It should not be used in people who are allergic to penicillin. Serious side effects may include Clostridium difficile colitis or anaphylaxis. While usable in those with kidney problems, the dose may need to be decreased. Its use during pregnancy and breastfeeding appears to be generally safe.

X-gal

X-gal

X-gal 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 chemical name is often shortened to less accurate but also less cumbersome phrases such as bromochloroindoxyl galactoside. The X from indoxyl may be the source of the X in the X-gal contraction. X-gal is often used in molecular biology to test for the presence of an enzyme, β-galactosidase, in the place of its usual target, a β-galactoside. It is also used to detect activity of this enzyme in histochemistry and bacteriology. X-gal is one of many indoxyl glycosides and esters that yield insoluble blue compounds similar to indigo dye as a result of enzyme-catalyzed hydrolysis.

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.[11] 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.

Source: "PUC19", Wikipedia, Wikimedia Foundation, (2021, December 3rd), https://en.wikipedia.org/wiki/PUC19.

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Further Reading

Plasmid

Beta-galactosidase

Cloning vector

Isopropyl β-D-1-thiogalactopyranoside

PGLO

Multiple cloning site

Subcloning

PBR322

X-gal

Blue–white screen

Molecular cloning

In vitro recombination

Golden Gate Cloning
See also
References
  1. ^ Yanisch-Perron, C.; Vieira, J.; Messing, J. (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, J.; Messing, J. (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. ^ pUC19 description & restriction map
  4. ^ Louro, Ricardo O.; Crichton, Robert R. (2013). Practical approaches to biological inorganic chemistry. Amsterdam, Oxford: Elsevier. p. 279. ISBN 9780444563590. Retrieved April 7, 2014.
  5. ^ Louro, Ricardo O.; Crichton, Robert R. (2013). Practical approaches to biological inorganic chemistry. Amsterdam, Oxford: Elsevier. p. 279. ISBN 9780444563590. Retrieved April 7, 2014.
  6. ^ Wang, Nam Sun. "Summary of Sites on pUC19". Department of Chemical & Biomolecular Engineering University of Maryland. Retrieved 27 January 2017.
  7. ^ Saha; et al. (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. doi:10.1007/s00203-004-0708-y. PMID 15375645. S2CID 28286917.
  8. ^ Mooreland; et al. (2013). Advanced Biomolecular Genetics. Kleiske Publishing. pp. 889–932.
  9. ^ Louro, Ricardo O.; Crichton, Robert R. (2013). Practical approaches to biological inorganic chemistry. Amsterdam, Oxford: Elsevier. p. 279. ISBN 9780444563590. Retrieved April 7, 2014.
  10. ^ Pasternak, Jack J. (2005). An Introduction to Human Molecular Genetics, Second Edition. Wiley-IEEE. p. 117. ISBN 978-0-471-71917-5.
  11. ^ 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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