Lecture on pUC18 vector

Describes pUC18 vector and its properties
Cloning strategy of pUC18 Vector

1. pUCplasmids
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 Bengaluru North University,
K. Narayanapura, Kothanur(PO)
Bengaluru
Email: [email protected]
ORCID ID: 0000000270066334

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. Ampicillin resistance gene (blaZ)
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 lacZsequence, which encodes the promoter and the α-peptide of β-
galactosidase.
•Insertion of the MCS into the lacZfragment 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