Question

A gene is inserted within the BamH I cloning site in pBR322. Some plasmids take up the desired gene and form recombinant DNA while some fail to take it up. This mixture of plasmids is introduced into bacteria which are first grown on a medium ‘A’ containing only ampicillin. It is then replica plated on to another medium ‘B’ in which both ampicillin and tetracycline are present. Given below are the illustrations of colonies found on the two culture media. Analyse them and identify the bacterial colonies of recombinants.

• A. 2, 3 and 5
• B. 1 and 4
• C. 1, 2, 3, 4 and 5
• D. 3, 4 and 5

Answer 1

The correct option is B 1 and 4

pBR322 is an artificial plasmid used in cloning experiments. It has two antibiotic resistance genes for ampicillin and tetracycline which are used as selectable markers. If DNA fragments are inserted in the cloning sites within these antibiotic resistance genes, the bacteria containing the plasmid will fail to grow on nutrient media containing that particular antibiotic as they will lose the resistance.
The BamH I cloning site lies within the tetracycline resistance gene in pBR322. When a gene is inserted within it, there will be insertional inactivation of the tetracycline resistant gene and the bacteria will lose resistance against the antibiotic. As a result it will not grow on a medium containing tetracycline.

In the experiment given in the question, the mixture of recombinant and non-recombinant plasmids is tried to be introduced into the bacteria. They are first grown on a medium containing ampicillin (A). The plate A is then pressed on to a sterile cloth after which plate B containing both ampicillin and tetracycline are pressed on the same cloth used earlier. This process is known as replica plating.


The colonies produced in plate B will have a spatial pattern of colonies similar to that of plate A. The following consequences can be observed:
a. Some bacteria may fail to take up the plasmid and get transformed. They will not have resistance against both ampicillin and tetracycline and thus, will not grow either on the nutrient medium A or B.
b. Some bacteria take up the plasmid without the gene of interest. They are transformed, but non-recombinants. Since the plasmids in these bacteria lack the insert, the bacteria will have resistance against both ampicillin and tetracycline and can grow on both the nutrient medium A and B.
c. Some bacteria take up the plasmid that has the gene of interest within the tetracycline resistance gene. They are transformed recombinants. They will lack resistance against tetracycline due to insertional inactivation but have resistance against ampicillin. Therefore, they can grow on the nutrient medium A but will fail to grow on nutrient medium B. Hence the colonies of recombinant bacteria are those that are found on the nutrient medium A but absent on the nutrient medium B.