PUC19
pUC19 is one of a series of plasmid cloning vectors created by 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 colour differences of colonies on growth media. pUC18 is similar to pUC19, but the MCS region is reversed.
Components
It has one ampR gene (ampicillin resistance gene), and an N-terminal fragment of β-galactosidase (lac Z) gene of E. coli. The multiple cloning site (MCS) region is split into the lac Z gene (codons 6–7 of lac Z are replaced by MCS), where various restriction sites for many restriction endonucleases are present.
The ori site or replicon, rep is derived from pMB1 vector. pUC vector is small but has a high copy number. The high copy number of pUC plasmids is a result of the lack of the rop gene and a single point mutation in rep of pMB1. The lac Z gene codes for α-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.
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 our interest can be distinguished from cells with the plasmid but without the gene of interest, just by looking at the colour of the colony they make on agar media supplemented with IPTG and X-gal. Recombinants are white, whereas non-recombinants are blue in colour. This is the most notable feature of pUC19.
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 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 on media with X-gal.
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.[4]
Therefore the media used should contain ampicillin, IPTG, and X-gal.
See also
- Plasmid
- Vector (molecular biology)
- Blue white screen
- Antibiotic resistance
- Transformation (genetics)
- Restriction enzymes
References
- ↑ 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.
- ↑ 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.
- ↑ pUC19 description & restriction map
- ↑ Pasternak, Jack J. (2005). An Introduction to Human Molecular Genetics, Second Edition. Wiley-IEEE,. p. 512. ISBN 0-471-71917-X.