Plasmid- derived cloning vectors play an important role in molecular biology and biotechnology. Naturally occurring plasmids may not possess all essential properties of a suitable cloning vector. Therefore, by inserting the genes of relaxed replication and genes for antibiotic resistance, vectors are restructured.
First major commonly used plasmid-derived cloning vector was pBR322, and a number of improved or specialized vectors have subsequently been derived from this plasmid.
The construction of plasmid pBR322 (figure 5) was carried out by recombination of three plasmids, pSC 10 1, pSF2 124, and pMB 1, and others, such as Rl and ColEl, served as intermediaries.
It was created in 1977 and named its creators, p standing for plasmid, and BR for Bolivar and Rodriguez. pBR322 is 4361 base pairs E. coli plasmid-cloning vector containing the origin of replication from pMB1 (a plasmid in the ColE1 compatibility group) and ampR gene, encoding ampicillin resistance protein (source plasmid RSF2124) and tetR gene, encoding tetracyclin resistance protein (source plasmid pSC101).
Widespread use of pBR322 has prompted numerous studies into its molecular structure and function.
pBR327 vectors are derived from pBR322, by deletion of nucleotides between 1427 to 2516. These nucleotides are deleted to reduce the size of vector and eliminate sequences that were known to interfere with the expression of the cloned DNA in eukaryotic cells. It contains genes for resistance against two antibiotics (tetracycline and ampicillin). Both pBR322 and pBR327 are very commonly used plasmid vector in molecular biology.
These are series of plasmids that are used as cloning vectors belong to pUC series (named after the place of their initial preparation i.e. University of California). These plasmids are 2700 bp long and possess (Figure 6):
- Ampicillin resistance gene
- Origin of replication derived from pBR322,
- lacZ gene derived from E. coli. Within the lac region is also found a polylinker sequence having unique restriction sites (identical to those found in phage M13), and
- Multiple cloning sites (MCS).
These plasmids when transformed into an appropriate E.coli strain having lac (e.g. JM103, JMI09), and grown in the presence of IPTG (isopropyl thiogalactoside, which behaves like lactose, and induces the synthesis of f3 galactosidase enzyme) and X-gal (substrate for the enzyme), will give rise to white or clear colonies. On the other hand, pUC having no inserts and transformed into bacteria will have an active lacZ gene and therefore will produce blue colonies, thus allowing identification of the colonies having pUC vector with cloned DNA segments (figure 7).
As discussed above, in pBR322 and pBR327, the DNA is inserted at a site located in one of the two genes for the resistance against antibiotics, so that it inactivates one of the two resistance genes.
The insert bearing plasmid can be selected by their ability to grow in a medium containing only one of the two antibiotics and by their failure to grow in a medium containing both the antibiotics. Plasmids carrying no insert on the other hand, grow in media containing one or both the antibiotics.
So, the presence of lacZ gene in pUC and resistance genes against ampicillin and tetracycline in pBR322 and pBR327 allow selection of E.coli colonies transformed with plasmids carrying the desired foreign cloned DNA segment.
pUC19 is a commonly used high copy number cloning vector. The vector encodes the N-terminal fragment of β-galactosidase (lacZa), which allows for blue/ white colony screening (i.e., a-complementation), as well as a pUC origin of replication and an ampicillin resistance gene that allow propagation and selection in E.coli.
Important feature of pUC plasmids is blue/white colony screen to detect recombinant plasmids. This screen is based upon inactivation of the lacZa peptide of beta-galactosidase, which is expressed by the vector. The cloning vectors belonging to pUC family are available in pairs with reversed orders of restriction sites relative to lacZ promoter.
pUC8 and PUC9 make one such pair. Other similar pairs include pUC12 and pUCl3 or pUC18 and pUC19.
Drawbacks of pUC Vectors
The foundation of genomic sequence analysis is large-scale cloning and sequencing from shotgun plasmid libraries obtained by assembling sequences from vast majority of clones. Gaps in shotgun libraries and unclonable DNA fragments are quite common.
Such DNA is characterized by high AT content, strong secondary structure, open reading frames, or cis-acting functions (e.g., transcriptional promoters or replication origins). In some cases, most notably AT-rich DNA, the reasons for difficulty in cloning are not well defined.
In other instances, one or more features of pUC plasmids have been shown to be incompatible with cloning or stable maintenance of the inserts, which may lead to severe difficulties in creating plasmid libraries, especially from DNA rich in AT bases (>70%).
The pGEM-3Z vector is used as a standard cloning vector, as well as for highly efficient synthesis of RNA in vitro. It is very similar to pUC vector as it carries ampicillin resistance (ampR), MCS, lacZ genes, and additionally it contains short sequences of DNA (SP6 and T7 RNA polymerase promoters) flanking MCS, each of which acts as the recognition site for the attachment of RNA polymerase enzyme.