Specialized plasmid cloning vectors are used for specialized applications. They are described as follows:
Low Copy Number Vectors
Most vectors have been specifically engineered to increase plasmid copy number and the amount of cloned protein that can be produced per host cell There are, however, a few instances where low copy number vectors are desirable.
This is particularly important when high-level expression of the encoded protein has a deleterious or lethal effect on the host cell. Low copy number plasmid vectors are built around replicons such as RI that controls plasmid DNA synthesis.
The first generation of low-copy vectors was designed to solve the problem of toxicity came across when foreign genes and DNA sequences were cloned in plasmid vectors. To overcome the toxicity of gene products, low copy number vectors has been developed that carry highly regulated prokaryotic promoters with a low level of basal expression.
These vectors contain MCS, ori of single-stranded bacteriophages, and T3 and T7 promoters. Although such low copy number vectors allow “lethal” proteins to be cloned, they limit the amount of protein that can be expressed.
Runaway Replication Vectors
In order to overcome the problem of low protein expression in low copy number vectors, runaway replication vectors have been developed. These vectors replicate normally at the temperatures up to 34°C. However, their copy number increases as the temperature increases till 39°C.
Low copy number vectors, such as, IncFII plasmid RI (in which an antisense RNA i.e., copA RNA negatively controls the formation of a protein that is rate-limiting for replication), have been converted to runaway replication vectors by increasing replication of repA mRNA.
The copy number of the plasmid is determined by the balance between the rates of formation of copA RNA and repA mRNA. A small increase in the rate of formation of the latter drastically reduces the rate of formation of copA RNA due to convergent transcription, which may lead to a total loss of copy number control (runaway replication). This can result in massive DNA amplification, and plasmid copy numbers up to 1000 per genome.
These are cloning vectors suitable for use in a wide range of gram-negative bacteria. For example, three naturally occurring plasmids RSF1010 (IncQ), RK2 (IncP), and pSa (IncW) have the ability to replicate in almost any species of gram-negative bacteria, and carry multiple antibiotic resistance markers that makes them attractive candidates for the development of widely applicable vectors.
These vectors require second E.coli containing tra genes for their transfer into host cells, as they are not self-transmissible.
Vectors for Gram-positive bacteria
Gram-positive bacteria, such as Bacillus spp., have a number of metabolic and physiological properties that make them interesting targets for study. Molecular understanding of these properties has necessitated the development of cloning vectors for use in gram-positive hosts.
Because of the relative rarity of plasmids encoding antibiotic resistance markers in Bacillus subtilis, small Staphylococcus aureus plasmids, such as the 2.9 kb plasmid pC194, which encodes chloramphenicol-resistance, have been used for the development of Bacillus spp. cloning vectors.
A vector constructed in such a way that it can replicate in at least two different host species (eg a prokaryote and a eukaryote) is called shuttle vector. DNA recombined into such a vector can be tested or manipulated in several cell types.
Number of shuttle vectors have been developed from E. coli and eukaryotic cells. Several eukaryotic events, such as protein transport, posttranslational modification, and RNA splicing, cannot readily be studied in E.coli.
By using shuttle vectors, DNA manipulation can be carried out in E.coli prior to the transfer of recombinants into yeasts, filamentous fungi, plants (with Agrobacterium tumifaciens as an intermediate), or mammalian hosts Plasmids for use in mammalian hosts.
A general strategy to construct these chimeric vectors is to combine a replication ori from naturally occurring plasmid with an E.coli cloning vector.
Well-designed shuttle vectors take advantage of E.coli as a host and incorporate features that facilitate cloning, such as small vector size, replaceable restriction sites, carry mobilization region, mob, to facilitate plasmid exchange by conjugation, and means to screen for recombinants.
Few shuttle vectors for bacteria other than E.coli, may also incorporate antibiotic resistance gene. Shuttle phagemids have been designed to replicate as lytic or lysogenic bacteriophage and also functions as cosmids in E.coli .
The earliest shuttle vectors were designed to shuttle between bacteria, such as, E.coli, and yeast, a simple eukaryote. Components needed to create this vector include:
- Bacterial plasmid.
- An origin of replication that works with yeast as the prokaryotic ori doesn’t work with eukaryotic ori and vice versa.
- A centromere sequence, Cen, which allows correct partition of plasmid in yeast.
- A gene to select plasmid in yeast.
Expression vectors also known as expression construct, are plasmids (generally) used to introduce desirable genes into the target cell. In order to study the properties of a protein, it is often desirable to obtain large quantities of the protein by cloning the relevant gene into a vector designed to express that gene at high levels as many proteins are expressed at low levels in vivo.
Ptac is tac promoter/operator (PTAC) and is one of the most widely used expression systems. Expression vectors are used for site directed mutagenesis in molecular biology. They also play an important role in agriculture, such as, production of golden rice.
Baculovirus expression vector system (BEVS)
Baculoviruses are the most commonly known viruses to affect the insect population. They are double-stranded, circular, supercoiled DNA molecules in a rod-shaped capsid.
More than 500 baculovirus isolates (based on hosts of origin) have been identified, most of which originated in arthropods, particularly insects of the order Lepidoptera.
Two of the most common isolates used in foreign gene expression are Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) and Bombyx mori (silkworm) nuclear polyhedrosis virus (BmNPV).
Recombinant baculoviruses are widely used to express heterologous genes in cultured insect cells and larvae. It was introduced in 1983. Now it has become one of the most versatile and powerful eukaryotic vector systems for recombinant protein expression.
As Baculovirus is safe, has high level of recombinant gene expression, accurate in post-transcriptional modifications of peptides, ideal for suspension culture and can be used in large-scale production of biologically active recombinant products, it is preferred over any other expression vector systems.