Plasmid vectors: a brief study _______________________________
Presented by:
MANOJ KUMAR
SUKUMAR DANDPAT
VI YEAR__________________________________________________________________________________________________________________________
Genetic engineering stands for the transfer of DNA between hosts (or the species) by in vitro enzymatic manipulation. This means that the DNA to be transferred will be duplicated in the new host. Since most DNA fragments are incapable of self replication in E.coli or any other host cell, an additional segment of the DNA, capable of autonomous replication must be linked to the fragment to be cloned. This autonomously replicating segment is the molecular cloning vectors.
Thus vectors are self replication DNA molecules into which foreign DNA molecules are inserted (Gardner et al. 2001). Vectors have been developed and used in delivery of foreign genes into cells and chromosomes of prokaryotes and eukaryotes. Such a vector plays a central role in recombinant DNA technology.
Most cloning vectors are originally derived from naturally occurring extrachromosomal elements such as bacteriophage and pladmids.
Stanley cohen and his coworkers (1975) first reported the use of bacterial plasmids as molecular cloning vectors. Since that initial report, thousands of cloning vectors have been constructed, and their versatility in terms of cloning sites, host-range and function appears to be limited only by the deftness and imagination of inventor.
Currently specialized cloning vectors are available that enable the investigator to:
1. Identify and isolate regulatory DNA sequences such promoters and terminators;
2. Identify open translation reading frames;
3. Overproduce useful RNAs and proteins; and
4. Determine the nucleotides sequences of genes and segments of DNA.
Primarily a cloning vector is necessary as a carrier of the cloned gene. Without a vector, a DNA molecule introduced into a cell would be diluted out of the cell division and eventually get lost.
Vectors are of following types:
1. PLASMIDS,
2. BACTERIOPHAGE VECTORS,
3. COSMIDS,
4. VECTORS FOT PLANT CELLS,
5. VIRUS VECTORS FOR ANIMAL CELLS,
6. SHUTTLE VECTORS,
7. MINICHROMOSOMES (YAC VECTORS AND BAC VECTORS)
8. EXPRESSION VECTORS AND
9. GENE CARTRIDGES.
THE PRESENT LECTURE WILL BE TOTALLY CENTERED AROUND THE PLASMIDS.
PLASMIDS_____________________________________________________
Plasmids are extra-chromosomal double stranded circular DNA molecules that replicate autonomously. i.e. a plasmids is a replicon (i.e. unit of genetic material capable of independent replication) that is stably inherited (maintained without specific selection) in an extrachromosomal state. Most of the plasmids are not required for the survival of the host in which they reside. But in many cases they are essential under certain conditions, such as in the presence of an antibiotic such as chloroamphenicol, ampicillin. In rhizobium leguminosarum, the agent for nitrogen fixation and module formation are located in the plasmid.
Aspects related to the plasmids:
1. Replication of plasmids: the small sized plasmids use the DNA replicative enzymes of the host cells, while the large-sized plasmids carry genes that code for the special enzymes necessary for their replication, under certain conditions the plasmids can integrate with the bacteria chromosomes. They are called episomes or integrative plasmids.
2. Size of plasmids: the size of plasmids varies from less than 1.0 kb to more than 2000 kb. Small sized plasmids are more advantageous for the gene cloning studies.
3. Plasmid copy number: the copy number is defined as-the number of molecules of a plasmid bound in a single bacterial cell. It ranges from 1 to more than 50 per cell. This is specific for a given plasmid residing in bacteria cell.
4. Amplification of the plasmids: the process of increasing the copy number is called plasmid amplification. When the bacterial culture is at its exponential phase, the antibiotic chloramphenicol is added to the medium. This arrests chromosomal DNA replication and the cell division. The culture is then incubated to another 12 hours for the plasmid molecules to replicate (since plasmid is resistant to the chloramphenicol). This step increases the number of plasmids per cell, sometimes the plasmid copy number may reach to several thousand.
5. Types of plasmids____________________________________________
Plasmids are classified into following four types:
a) Conjugative or self transmissible plasmids: these plasmids carry a set of transfer (tra) genes that enable the plasmid to transfer from one bacterium to another. Thus tra genes promotes bacterial conjugation. Generally they are of high molecular weight andare present as 1-3 copies per cell
b) Non-conjugative plasmids: these plasmids lack the tra genes. They replicate autonomously but cannot transfer to another bacterium. They generally have low molecular wright and are present in multiple copies. i.e. 20-25 copies per cell.
c) Relaxed plasmids: these plasmids are maintained as multiple copies per cell.
d) Stringent plasmids: these plasmids have a limited number of copies per cell, typically 1-2 copies per cell.
6. Isolation of plasmid DNA: the isolation of the plasmid DNA include the following steps:
a) Growth of bacteria and plasmid amplification.
The bacterial strain containing the required plasmid is grown on LB (=lauria dertani) culture medium (it contains trypton(10 g/l), yeast extract (5g/l) and sodium chloride (10 g/l). the ph of the LB medium is adjusted 7.5. late long, the cultures ae then transferred to LB medium containing proper antibiotic with small amount of chloramphenicol for 12-16 hours of incubation.
b) Breaking of bacterial cells to release their contents.
The bacterial cell are subjected to gentle cell lysis, using lysozyme and then detergent..
c) Treatment of bacterial cells extracts to remove all components except the DNA.
This is followed by the clearing of lysate by centrifugation. Centrifugation tends to sediment high molecular weight DNA (predominantly chromosomal DNA) and cell debris, leaving the small plsmid molecules and RNA in subpernatant.
d) Separation of plasmids from the chromosomal DNA.
Undamaged plasmid is comparatively compact,since plasmid is supercoiled as a consequence of having very few turns of double helix per unit length. Further purification of plasmid is done by caesium chloride density gradient ultracentrifugation of nucleic acid preparation in the presence of the ethidium bromide. Ethidium bromide tends to cause unwinding of DNA as it binds to it and simultaneously decreasing its buoyant density. Since the supercoiled plasmid DNA can unwind to only a very limited extent , it will not bind the dye and have a higher density than the other types of the DNA (which binds to dye). Because of this density difference, plasmid DNA can be separated from other types of DNA (i.e., stained DNA ) by means of isopycnic ultracentrifuge.
7. Criteria for plasmid cloning: for the selection of the suitable cloning vector the following criteria are used
a. A plasmid vector should be small and should contribute as alittle as possible to the overall size of the recombinant molecule. Low molecular weight (small sized) plasmids are easy to handle, and do not get damaged during the process of their isolation.
b. The plasmid should have the ability to confer readily selectable markers (i.e. phenotypic traits on the host cells that can be used to distinguish the transformed cell from the non-transformed ones. (in the process of transformation the naked DNA molecules are taken up by the recipient cell)
c. Plasmid should be easily propagated in the desired host so that the number of recombinant DNA molecules can be amplified.
d. The plasmid should have a large number of copies.
e. A plasmid vector should have an additional genetic marker that can be inactivated by insertion of the foreign DNA. So that the inactivated gene may help in distinguishing the cell harbouring recombinant molecules on the basis of readily altered phenotype.
Design of the future molecular vectors_________________________________
a. The vector should contain dominant genetic marker that can be expressed in a wide range of hosts (e.g. CmR is a dominant genetic marker which does not restrict the vector to particular genetic background.
b. Portioning swquences such as par or cer should be also incorporated into the vector to ensure efficient segregation of plasmids to the daughter cells.
c. Method of controlling the copy number will greatly increase the versatility of the vector.
d. Since many recombinant DNA techniques requires single strands of DNA. A vector with single stranded synthesis capability is desirable.
Refrence: genetics by p.s. verma and v. k. agarwal
