pBR322 DNA is a commonly used plasmid cloning vector in E. coli (1). The molecule is a double-stranded circle 4,361* base pairs in length (2). pBR322 contains the genes for resistance to ampicillin and tetracycline, and can be amplified with chloramphenicol. The molecular weight is 2.83 x 106 daltons.

The pUC vectors offer following major advantages over pBR322 vectors: (a) High copy number of 500-600 copies per cell. (b) Easy and single step selection. (c) The unique restriction sites used for cloning are clustered within the MCS.

pBR322 is a plasmid and was one of the first widely used E. coli cloning vectors. pBR322 is 4361 base pairs in length and has two antibiotic resistance genes – the gene bla encoding the ampicillin resistance (AmpR) protein, and the gene tetA encoding the tetracycline resistance (TetR) protein.

pBR322 was the first artificial cloning vector to be constructed.

The vector pBR322 was named according to the standard rules for vector nomenclature. The “p” stands for plasmid. “BR” tells us which laboratory the vector was constructed in. This part of the vector name stands for Bolivar and Rodriguez, two of the scientists who constructed the pBR322 cloning vector in 1977.

Complete answer: (a)In the cloning vector pBR322, the selectable markers are ampicillin and tetracycline resistance genes.

e.g. Ampicillin, kanamycin are considered as useful selectable markers for E. (iii) rop in E. coli cloning vector pBR322 : rop codes for the proteins involved in the replication of the plasmid.

Answer: A plasmid without a selectable marker was chosen as vector for cloning a gene. But without the selectable marker, the selection of the transformed cells will not be possible and in the presence of antibiotics, even the transformed cells will perish. Hence, it becomes a futile exercise.

Examples of selectable markers include:

• Beta-lactamase which confers ampicillin resistance to bacterial hosts.
• Neo gene from Tn5, which confers resistance to kanamycin in bacteria and geneticin in eukaryotic cells.
• Mutant FabI gene (mFabI) from E.

Selectable markers are used to select for successful transformants, from untransformed cells, they provide a survival advantage to the cells containing exogenous DNA. Survival advantage to transformed host cells is usually conferred by the use of antibiotic-resistant genes [42].

Ti plasmid is the most commonly used natural vector for cloning genes in plants. It is present in Agrobacterium tumifecians and is used by the bacterium to cause a tumour in plants.

Transformants are cells which have undergone genetic transformation through the uptake of foreign DNA.

Transformants- these are the bacterial cells which incorporate plasmid DNA into their genome. Non transformants – these are the bacterial cells which take up the plasmid but do not incorporate the plasmid DNA into their genome.

In the Simple Cloning Lab, transformants are E. coli clones, harbouring a pGT4 plasmid, or a pGT4 derived plasmid. By a heatshock, the plasmids has been transferred into competent bacterial cells. As a result cells are ampicillin resistant, so they can be selected by growing on the antibiotic ampicillin.

Transformants refer to a cell that has undergone genetic transformation through the uptake of foreign DNA while recombinants refer to a cell that contains a combining genetic material with different origins.

Presence of more than one recognition site in the vector DNA. Presence of alien DNA into the vector DNA results into insertional inactivation of selectable marker. Antibiotic resistance gene gets inactivated due to insertion of alien DNA.

Heterologous host is the host which expresses the protein in the cell lines where they are not supposed to be produced. For example, a certain protein which is produced only in human cells can be expressed in the yeast and isolated. So yeast will then become the heterologous host for the particular protein.

Recombinant is a general name given when a piece of one DNA is combined with another DNA giving rise to a new DNA. It is an organism or cell in which genetic recombination has taken place. When there is no foreign DNA insertion in the original genome, the structure is called non recombinant.

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