Question

Describe the construction of a recombinant plasmid containing the gene for the green fluorescent protein (GFP) and the insertion of the plasmid into a bacterial cell by placing the steps in order.

Answer 1

A recombinant plasmid contains one or more genes of interest and different DNA elements that are used with multiples objectives, for instance, to report gene expression (like GFP protein), to stimulate transcription (i.e., promoters), etc. All these sequences are added into the plasmid vector in a series of sequential steps.

Step-by-step explanation:

The steps to design a recombinant plasmid are the following:

1- it is necessary to open the plasmid with restriction enzymes to insert the genes of interest and further DNA elements (for instance, lac promoter, transcription termination sequences, etc). In this case, we need to insert the Green Fluorescence Protein (GFP). GFP is used as a reporter gene, it means that this protein is required for detecting the expression of the gene of interest,

2- the sequences to be inserted into the plasmid are amplified by PCR (DNA cloning),

3- after insertion, the DNA molecule is closed by using DNA ligases,

4- the plasmid is inserted by transformation into the host bacterial genome. Transformation is a genetic process where DNA is exchanged between the plasmid and its host genome,

5- finally, the plasmid-carrying bacteria are grown to produce proteins of interest.