Question

Explain how to find whether an E. coli bacterium has transformed or not when a recombinant DNA bearing ampicillin resistant gene is transferred into it. What does ampicillin resistant gene act as in the above case?

Answer 1

Hint: The process of adding a foreign DNA fragment from a donor genome into the genome of a recipient cell is called transformation .Antibiotic resistance genes (especially E. coli) are used as selectable markers in many plant transformation systems. Although plants are eukaryotic, antibiotics can effectively inhibit protein synthesis in cellular organelles, especially chloroplasts.

Complete answer:
Transformation is the process of adding foreign DNA fragments from the donor genome to the genome of the recipient cell. The donor fragment crosses the cell membrane of the recipient cell (which may or may not belong to the same species) and is inserted into the genome of the latter by recombination. The transformation is evidenced by the presence of a new cell phenotype in the offspring of the recipient cells.
Of the large number of colonies obtained by transformation to select or screen several colonies containing recombinant plasmids, one of the simplest and most useful methods for this purpose is the use of antibiotics. The transformed cells can be placed on a selection medium containing various antibiotics.
Plasmid pBR 322 contains ampicillin resistance genes. Thus, transformants can be detected by their potential plating on media containing ampicillin. For example, the desired DNA fragment could be inserted into the antibiotic resistant genes of pBR322, in which these genes are deactivated and plasmid losses it’s antibiotics resistance due to insertion foreign DNA segment but selected from non transformants due to ampicillin resistant genes . Non-transformants grow on ampicillin containing medium but transformants do not grow on ampicillin containing medium.
2.Ampicillin resistant gene acts as a selectable marker in the above case. Antibiotic resistance genes (especially E. coli) are used as selectable markers in many plant transformation systems. Although plants are eukaryotic, antibiotics can effectively inhibit protein synthesis in cellular organelles, especially chloroplasts.

Note: Recombinant selection is a complex process because two different antibiotic dishes must be used at the same time due to antibiotic inactivation.
Recombinant and non-recombinant can also be distinguished by colour in the presence of a chromogenic substrate. Here, the recombinant DNA is inserted into the sequence encoding the β-galactosidase enzyme, which inactivates the enzyme. Thus, bacterial colonies that have inserted plasmids do not exhibit colour, whereas colonies that do not have plasmid exhibit blue colour.