PUC plasmids:
PUC plasmids are small, high copy number plasmids of size 2686 bp.
This series of cloning vectors were developed by Messing and co-workers in the University of California. The P in its name stands for plasmid and UC represents the University of California.
PUC vectors contain a lacz sequence and multiple cloning site (MCS) within lacz. This helps in use of broad spectrum of restriction endonucleases and permits rapid visual detection of an insert.
pUC18 and pUC19 vectors are identical apart from the fact that the MCS is arranged in opposite orientation.
PUC vectors consists of following elements:
PMB1 "rep" replicon region derived from plasmid pBR322 with single point mutation (to increase copy number).
"bla" gene encoding ẞ lactamase which provide ampicillin resistance which is derived from p8R322. This site is different from PBR322 by two point mutations
E.coli lac operon system.
About.
"rop" gene is removed from this vector which leads to an increase in copy number
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 to mediate complementation,while cloning the DNA fragment into MCS does.
Therefore, recombinants can be detected by blue or white screening on growth medium containing X-gall in presence of IPTZ as an inducer.
The native lac-Z promoter, (Plac) is situated just upstream of the cloned gene, allowing expression of genes on inserts that are correctly oriented. Most of the non-essential DNA has been removed to provide the ability to clone larger fragments. An ampicillin marker is included, for selection of transformants.
Recombinant selection with PUC vectors: Alpha complementation/blue-white screening/Insertional inactivation
The lacZ gene product (𝛃-galactosidase) is a tetramer and each monomer is made up of two parts - lacZ-alpha and lac-Z-omega.
If the Alpha fragment was deleted the Omega fragment is non functional ; however Alpha fragment functionality can be restored in transformation via plasmid. Hands the name Alpha complementation.
The E Coli enzyme is a homo tetramer of the protein product of the lacZ gene.
Certain mutations in the 5’ regions of lacZ present subunit association.
Because monomers lack enzyme activity, the failure to assemble leads to a Lac phenotype.
The activity of the enzyme ẞ-galactosidase is easily monitored by inducing in the growth medium the chromogenic substrate 5-bromo-4-chloro-3-indolyl-B-galactoside (Xgal).
This compound is colorless, but on cleavage, releases a blue indolyl derivative.
On solid medium, colonies that are expressing active B-galactosidase are blue in colour, while those without the activity are white in colour.
This is often referred to as blue/white screening.
pUC Plasmids:
Small and High Copy Number: pUC plasmids are small and can replicate many times in a cell, with a size of 2686 base pairs (bp).
Development: These cloning vectors were created by Messing and colleagues at the University of California. "p" stands for plasmid and "UC" stands for the University of California.
lacZ and MCS: They include a lacZ gene and a Multiple Cloning Site (MCS) within lacZ, allowing easy insertion of foreign DNA and simple detection of successful cloning.
pUC18 and pUC19: These two vectors are the same except for the direction of the MCS.
Key Elements of pUC Vectors:
pMB1 "rep": This is a replication origin derived from plasmid pBR322, altered by a single mutation to increase the number of copies.
"bla" gene: This gene provides resistance to ampicillin (an antibiotic) and is also derived from pBR322 but modified by two mutations.
E. coli lac operon: Contains parts of the lac operon system from E. coli bacteria.
rop gene Removal: The rop gene, which controls plasmid replication, is removed to further increase the number of plasmid copies in the cell.
This makes pUC plasmids very useful tools for cloning and gene expression studies.
Multiple Cloning Site (MCS) and pUC Plasmids:
MCS Definition: An MCS is a short DNA sequence with many restriction sites for different restriction enzymes.
Location: The MCS is inserted into the lacZ gene, which encodes the promoter and the α-peptide of β-galactosidase.
Function: The insertion of the MCS does not affect the function of the α-peptide, but inserting DNA into the MCS will disrupt this function.
Detection: Recombinant DNA (cloned fragments) can be detected by blue/white screening on growth medium with X-gal and IPTG. Blue colonies do not have inserts, while white colonies do.
lacZ Promoter (Plac): The natural lacZ promoter is located just before the inserted gene, allowing proper expression. Non-essential DNA is removed to allow the cloning of larger fragments. An ampicillin resistance marker helps select cells with the plasmid.
This setup helps easily insert and identify foreign DNA in cloning experiments.
Recombinant Selection with pUC Vectors: Alpha Complementation and Blue-White Screening
β-galactosidase Structure: The lacZ gene makes β-galactosidase, a protein that functions as a tetramer (four parts). Each part has two segments: lacZ-alpha and lacZ-omega.
Alpha Complementation:
If the alpha fragment is missing, the omega fragment alone is non-functional.
The alpha fragment can be restored by a plasmid, allowing the enzyme to function. This is called alpha-complementation.
Blue-White Screening: The β-galactosidase enzyme from E. coli forms a tetramer to work. Certain mutations in the beginning (5' region) of lacZ prevent these parts from coming together.
Blue Colonies: No insert; the enzyme is active and converts X-gal to a blue colour.
White Colonies: Insert present; the enzyme is inactive, and colonies remain white.
lacZ Phenotype: If the monomers (individual parts) of the enzyme cannot come together due to mutations or insertional inactivation, the bacteria will show a Lac⁻ phenotype, meaning they cannot break down lactose.
This system helps identify cells that have successfully taken up foreign DNA.
To check if the enzyme β-galactosidase is working, we use a chemical called X-gal.
X-gal is colorless, but when β-galactosidase breaks it down, it turns blue.
On a solid medium, colonies with active β-galactosidase turn blue, and those without it stay white.
This method is known as blue/white screening.