Question

Cloning/Transformation Homework Please indicate what you would see on each media plate given the following conditions (blue colonies or white colonies) E. coli cell Nutrient agar plate, no ampicillin, no X- gal E. coli cell Nutrient agar plate with ampicillin added, no X-gal OM OM 01 01 E. coli cell with plasmid CO ОООО Nutrient agar plate with ampicillin & X- gal added E. coli cell with plasmid and correctly inserted Insulin gene in the multiple cloning site C LB agar plate with ampicillin & Xgal added

Answer 1

1. E. coli cells on nutrient agar plate (no ampicillin and no x-gal) : normal growth showing white colonies.

These colonies represent normal growth of the cells and not related to blue white colonies.

2. E.coli cells on nutrient agar plate with ampicillin but no x - gal: No growth.

Due to the absence of plasmid, E.coli doesn't have ampicillin resistance gene and so, there will be no growth of E.coli on nutrient agar medium plate containing ampicillin.

3. E.coli cells with plasmid on nutrient nagar plate containing ampicillin and x gal: Blue coloured colonies.

The plasmid present in E.coli will contain ampicillin resistance gene and LacZ gene. LacZ gene will produce best galactosidase which reacts with its substrate x-gal present in the medium. This reaction of enzyme and substrate will produce blue coloured complex forming blue coloured colonies. These cells are said to be non recombinant cells as they do not contain foreign gene.

4. E.coli cells with plasmid and correctly inserted insulin gene on lb plate containing ampicillin and x-gal: White recombinant colonies.

The insulin gene will be inserted at the place of Lacz gene by disturbing its function. Due to this replacement, there will be no production of beta galactosidase and no reaction with x-gal. So, no blue coloured complex will formed and thus, the white coloured colonies will appear which have resistance to ampicillin. These colonies will said to be the recombinant colonies.