pUC19
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pUC19 is a plasmid cloning vector created by Messing and co-workers in the University of California. p in the name stands for plasmid and UC represents the University in which it was created. It is a circular double stranded DNA and has 2686 base pairs[1]. 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 β-galactosidase.
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. Further more, the transformed cells containing the plasmid with the gene of our interest can be distinguished from cell with the plasmid but without the gene of interest, just by looking at the colour of the colony they make on agar media. 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.[2]
Therefore the media used should contain ampicillin, IPTG, and X-gal.
Sequence
The recognition sites for HindIII, SphI, PstI, SalI, XbaI, BamHI, SmaI, KpnI, SacI and EcoRI restriction enzymes have been derived from the vector M13mp19 and are on the strand complementary to that shown.
The pUC19 sequence is:[3]
1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acccggggat
421 cctctagagt cgacctgcag gcatgcaagc ttggcgtaat catggtcata gctgtttcct
481 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
541 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
601 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
661 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
721 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
781 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
841 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
901 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
961 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
1021 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
1081 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
1141 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
1201 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
1261 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt
1321 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
1381 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
1441 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
1501 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
1561 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
1621 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
1681 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
1741 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
1801 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
1861 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
1921 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
1981 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
2041 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
2101 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
2161 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
2221 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
2281 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
2341 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
2401 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
2461 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
2521 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
2581 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
2641 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtc
Apparement cette sequence n'est pas la bonne : consulter le site internet "pubmed" en tappant dans "search" : cloning vector pUC19, complete sequence
See also
References
- ^ pUC19 description & restriction map
- ^ Pasternak, Jack J. (2005). "An Introduction to Human Molecular Genetics, Second Edition". Wiley-IEEE,: 512. ISBN 047171917X, 9780471719175.
{{cite journal}}: Check|isbn=value: invalid character (help); Cite journal requires|journal=(help)CS1 maint: extra punctuation (link) - ^ Sequence of pUC19