PUC19
Encyclopedia
pUC19 is one of a series of plasmid cloning vector
s created by Messing and co-workers in the University of California
. The p in its 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. pUC19 is one of the most widely used vector molecules as the recombinant
s, 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.
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.
", 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.
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.
Therefore the media used should contain ampicillin
, IPTG
, and X-gal
.
, 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:
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
Cloning vector
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted. The insertion of the fragment into the cloning vector is carried out by treating the vehicle and the foreign DNA with a restriction enzyme that creates the same overhang, then ligating the fragments...
s created by Messing and co-workers in the University of California
University of California
The University of California is a public university system in the U.S. state of California. Under the California Master Plan for Higher Education, the University of California is a part of the state's three-tier public higher education system, which also includes the California State University...
. The p in its 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. pUC19 is one of the most widely used vector molecules as the recombinant
Recombinant DNA
Recombinant DNA molecules are DNA sequences that result from the use of laboratory methods to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms...
s, 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 (ampicillinAmpicillin
Ampicillin is a beta-lactam antibiotic that has been used extensively to treat bacterial infections since 1961. Until the introduction of ampicillin by the British company Beecham, penicillin therapies had only been effective against Gram-positive organisms such as staphylococci and streptococci...
resistance gene), and an N-terminal fragment of β-galactosidase (lac Z) gene of E. coli. The multiple cloning site
Multiple cloning site
A multiple cloning site , 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. MCSs are commonly used during...
(MCS) region is split into the lac Z gene (codons 6–7 of lac Z are replaced by MCS), where various restriction sites
Restriction sites
Restriction sites, or restriction recognition sites, are locations on a DNA molecule containing specific sequences of nucleotides, which are recognized by restriction enzymes...
for many restriction endonucleases are present.
The ori site or replicon
Replicon
*A Replicon in genetics is a region of DNA or RNA, that replicates from a single origin of replication.*Replicon is a software company providing timesheet and expense management software....
, 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
Rop protein
is a small homodimeric four-helix bundle protein formed by the antiparallel interaction of two helix-turn-helix monomers. The protein is expressed in Escherichia coli as a mechanism for regulating the gene copy numbers of plasmids. The Rop protein's structure has been solved to high resolution...
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 "transformationTransformation (genetics)
In molecular biology transformation is the genetic alteration of a cell resulting from the direct uptake, incorporation and expression of exogenous genetic material from its surroundings and taken up through the cell membrane. Transformation occurs naturally in some species of bacteria, but it can...
", 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
Ampicillin
Ampicillin is a beta-lactam antibiotic that has been used extensively to treat bacterial infections since 1961. Until the introduction of ampicillin by the British company Beecham, penicillin therapies had only been effective against Gram-positive organisms such as staphylococci and streptococci...
. 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
IPTG
Isopropyl β-D-1-thiogalactopyranoside, abbreviated IPTG, is a molecular biology reagent.This compound is used as a molecular mimic of allolactose, a lactose metabolite that triggers transcription of the lac operon...
, 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
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 Detroit, MI, in 1964. The formal chemical name is often shortened to less accurate but also less cumbersome phrases such as bromochloroindoxyl...
(5-bromo-4-chloro-3-indolyl- beta-D-galactopyranoside) and form blue colonies on media with 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 Detroit, MI, in 1964. The formal chemical name is often shortened to less accurate but also less cumbersome phrases such as bromochloroindoxyl...
.
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
Beta-galactosidase
β-galactosidase, also called beta-gal or β-gal, is a hydrolase enzyme that catalyzes the hydrolysis of β-galactosides into monosaccharides. Substrates of different β-galactosidases include ganglioside GM1, lactosylceramides, lactose, and various glycoproteins...
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 ampicillin
Ampicillin
Ampicillin is a beta-lactam antibiotic that has been used extensively to treat bacterial infections since 1961. Until the introduction of ampicillin by the British company Beecham, penicillin therapies had only been effective against Gram-positive organisms such as staphylococci and streptococci...
, IPTG
IPTG
Isopropyl β-D-1-thiogalactopyranoside, abbreviated IPTG, is a molecular biology reagent.This compound is used as a molecular mimic of allolactose, a lactose metabolite that triggers transcription of the lac operon...
, and 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 Detroit, MI, in 1964. The formal chemical name is often shortened to less accurate but also less cumbersome phrases such as bromochloroindoxyl...
.
Sequence
The recognition sites for HindIIIHindIII
HindIII is a type II site-specific deoxyribonuclease restriction enzyme isolated from Haemophilus influenzae that cleaves the palindromic DNA sequence AAGCTT in the presence of the cofactor Mg2+ via hydrolysis....
, SphI, PstI
PstI
PstI, is a Type II restriction endonuclease from Providencia stuartii. PstI recognition and cut site are as follows:...
, SalI
Sali
Sali is a village and a municipality in Croatia in the Zadar County. Sali is a small town but still the largest on the island of Dugi otok. The total population of Sali is 1,688 inhabitants...
, XbaI, BamHI
BamHI
BamHI is a restriction enzyme, derived from Bacillus amyloliquefaciens. It has the recognition site , and leaves a sticky end. One of the earlier enzymes to be used, it is popular for historical reasons, but also because digestion leaves a GATC overhang compatible with many other enzymes...
, SmaI, KpnI
KPNI
KPNI - SMU Radio is the student-run radio station of Southern Methodist University in Dallas, Texas....
, SacI
Saci
Saci may be:* An alternative spelling for the Hindu goddess Sachi* Saci , an impish mythical character of Brazilian folk tales* SacI, a restriction enzyme with the restriction site GAGCTC* Wellington Saci, Brazilian footballer...
and EcoRI
EcoRI
EcoRI is an endonuclease enzyme isolated from strains of E. coli, and is part of the restriction modification system.In molecular biology it is used as a restriction enzyme. It creates sticky ends with 5' end overhangs...
restriction enzymes have been derived from the vector M13mp19 and are on the strand complementary
Sense (molecular biology)
In molecular biology and genetics, sense is a concept used to compare the polarity of nucleic acid molecules, such as DNA or RNA, to other nucleic acid molecules...
to that shown.
The pUC19 sequence is:
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