pUC18 vector

pUC18 vector

1. pUC plasmids
Dr.Manikandan Kathirvel M.Sc., Ph.D., (NET)
Assistant Professor,
Department of Life Sciences,
Kristu Jayanti College (Autonomous),
(Reaccredited with "A" Grade by NAAC)
Affiliated to BengaluruNorthUniversity,
K. Narayanapura, Kothanur(PO)
Bengaluru 560077

2. pUCplasmids:
•pUCplasmidsaresmall,highcopynumberplasmidsofsize2686bp.
•ThisseriesofcloningvectorsweredevelopedbyMessingandco-workersinthe
UniversityofCalifornia.ThepinitsnamestandsforplasmidandUCrepresentsthe
UniversityofCalifornia.
•pUC18andpUC19vectorsareidenticalapartfromthefactthattheMCSisarranged
inoppositeorientation. 5’ 3’
3’ 5’
•pUCvectorsconsistsoffollowingelements:
►pMB1“rep”repliconregionderivedfromplasmidpBR322withsinglepoint
mutation(toincreasecopynumber).
►“bla”geneencodingβlactamasewhichprovideampicillinresistancewhichis
derivedfrompBR322.ThissiteisdifferentfrompBR322bytwopointmutations.
►LacZgenefromE.colilacoperonsystem,whichcontainsMCSrestrictionsites.
•pUCvectorscontainalacZsequenceandmultiplecloningsite(MCS)withinlacZ.
Thishelpsinuseofbroadspectrumofrestrictionendonucleasesandpermitsrapid
visualdetectionofaninsert.
•“rop”geneisremovedfromthisvectorwhichleadstoanincreaseincopynumber.
Copynumber:
500-600copiespercell

3. Multiple cloning site inserted into the gene lacZ’ coding for the enzyme β-galactosidase
•An MCS is a short DNA sequence consisting of restriction sites for many different restriction
endonucleases.
•The MCS is inserted into the lacZ’ sequence, which encodes the promoter and the α-peptide of β-
galactosidase.
•Insertion of the MCS into the lacZ’ fragment does not affect the ability of the α-peptide, while cloning DNA
fragments into the MCS does.
Clones with foreign DNA in the MCS disrupt the ability of the cells to make β-galactosidase
Plate on media with a Substrate X-gal (β-galactosidaseindicator) and
clones with intact β-galactosidaseenzyme indicate that cells containing empty vector without
insert, will produce blue colonies
Colorless (desirable) colonies indicates that cells contain the plasmid with foreign DNA
•Therefore, recombinants can be detected by blue/white screening on growth medium containing X gal in
presence of IPTG as an inducer.
Cloning of insert DNA using β -galactosidase: as reporter gene

4. Cloning in pUC18/19:
Ω-fragment
Vector DNA
GOI
GOI-GENEOF INTEREST
β-galactosidase enzyme +
chromogenic substrate (X-gal)
Blue color colonies
MCS

6. First generation plasmid cloning vectors include pBR322 and the pUC plasmids
pUC has
1 antibiotic resistance gene-Ampicillin resistance gene
Variety of unique restriction sites in lacZgenefor inserting foreign DNA
Most of these sites interrupt lacZgene making screening straightforward
Thus MCS in lacZ facilitates directional cloning.
Summary