Plasmid vectors

//Plasmid vectors

Plasmid vectors

Vectors can be described as an autonomously replicating DNA sequence that can be used to carry foreign DNA fragments. Commonly used vectors are bacteriophages and plasmids. A vector that is used predominantly for reproducing DNA segment is referred to as cloning vectors and vectors used for expressing a gene contained within the cloned DNA is referred to as expression vectors.

Characteristics of a good Vector

  • Ability to self-replicate.
  • Selectable characteristic so that transformed cells can be recognized from non-transformed cells.
  • Should have origin of replication.
  • Selectable markers.
  • Small size.
  • Plasmid high copy number.
  • Restriction site.

Cloning Vectors

E.coli Plasmids : E.coli plasmid  is most commonly used as vector in almost all cases (even with eukaryotes) because of its following advantages

  • Easy to grow in sample, inexpensive growth medium.
  • Has rapid doubling time of about 20-30 minute during log-phase growth.
  • Its genetics is well understood.
  • Has fully mapped and sequenced genome.
  • Extra-chromosomal copies of DNA can be exploited to carry foreign DNA fragments.
  • It is non-toxic.

F-Gene These genes exist on a circular piece of DNA that replicates independently from the bacterial chromosome, or they can be integrated into chromosome. F stands for fertility. There are three manifestations of fertility factor

  • F+ also called F-episome: Large circular dsDNA molecule that carries only fertility genes.
  • Hfr The F element has become integrated into E.coli genome. When conjugation occurs, the F-gene start traveling across the pilus, dragging rest of genome behind.
  • F- Large circular dsDNA molecule that contains the fertility genes and a few other genes. These genes are transferred very efficiently from one bacterium to another.

Plasmid

These are naturally occurring extra-chromosomal circular DNA that are stably inherited from one generation to another in extra-chromosomal state. Closed circular ds DNA molecule that confer particular phenotype onto bacterial cell in which they are replicated. Plasmids often carry a gene that codes resistance to either antibiotics or heavy metals, or that produces DNA restriction and modification enzyme, that bacterium normally does not produce. Plasmids may be:

  1. Single copy plasmids: one plasmid DNA per cell
  2. Multi-copy plasmids:  10-12 plasmid per cell
  3. Plasmids under relaxed replication control, which can increase their copy number in the host cell up to 100 copies.

 pSC101. The selection is done by selectable markers called antibiotic resistance genes. When foreign gene is inserted in the gene, insertional inactivation occurs. The disadvantage of this vector is that its selection is not easy.

pBR322  In PBR322,  p indicates plasmid; BR represents Bolivar and Rodriguez (Scientist who discovered the plasmid pBR322) and 322 represents the number of plasmid within their stock collection. Components of the plasmid

Ori Origin of replication,  which encodes a restrictor of plasmid copy number.

Antibiotic Resistance Genes PBR322  has two antibiotic resistance genes; ampicillin and tetracycline. Each marker includes restriction sites used for cloning.

Restriction Sites Plasmid carries unique restriction sites for restriction enzymes. These are located in Antibiotic resistance genes.

Selection There is a direct selection of recombinants in the process called insertional inactivation. If we want to insert DNA fragment into BamH1, the insert will disrupt the gene responsible for tetracycline resistance but ampR will not be altered. Transformed cells are first grown on ampicillin plates to kill all cells that do not contain a plasmid. Cells that grow on amp plates are then replica plated onto medium containing amp and tet. Cells that grow in ampicillin presence but dies under tetracycline selection, contains plasmids that have foreign DNA inserts. The insertion of foreign DNA fragment into an antibody resistance gene inactivates gene production and leads to antibody sensitivity.

pBR327 It is constructed by removing 1089 bp from pBR322. It has different conjugative and replicative properties. It can carry larger size of DNA because of its small size. It has high copy number; advantageous because more the copy number more will be the copies present of a cloned gene and more likely the effect of a cloned gene on host cell will be detected. Deletion also destroys the conjugative ability of pBR322 making pBR327 a non-conjugative plasmid that cannot direct its own transfer to other E.coli cells.

pUC8 It is derived from pBR322 with only the replicate origin and the ampR gene. Nucleotide sequence of ampR gene has been changed and it no longer contains unique restriction site and LacZ gene carries all the cloning sites.

High copy number Mutation within ori can produce 500-600 copies of plasmid/cell without any chloramphenicol amplification.

Identification of recombinant cells can be achieved in a single step process. It can be done by plating onto agar medium containing ampicillin + X-gal. LacZ gene codes for β-galactosidase.

Multiple cloning site or a polylinker It is a synthetic piece of DNA that harbors the sequence of several unique restriction enzyme recognition sequence inserted in LacZ gene (Vector portion encoding β-galactosidase).

Advantages of pUC8 over pBR322:

  1. DNA fragments can be cloned into a variety of restriction enzyme sites and recombinants are rapidly screened.
  2. It requires only a small gene to be carried onto the plasmid.
  3.  Its selection is done by blue-white screening.

 

By |2018-04-17T12:21:29+00:00April 17th, 2018|Molecular Biology|Comments Off on Plasmid vectors

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