Genetic Engineering and Transformation Background—Lesson 1

An important method in biotechnology is genetic engineering (GE), which includes all techniques and technologies of modifying and manipulating the genetic information in cells. Genetic engineering encompasses any deliberate change made in the genetic code of an organism. The engineering can be as simple as modifying a single nucleotide in a DNA sequence or transferring a single gene to a cell, to being an expansive as changing large sections of chromosomes that code for multiple characteristics. Many changes in genetic information can lead to new or improved products.

The specifics of each genetic modification procedure are unique to the particular organism being engineered and to the characteristics of the desired product. In general through, the steps of genetic engineering to produce a protein product use the process of recombinant DNA technology, transformation, cloning, and protein purification (if a protein product is the end goal).

General Protocol- to produce a protein product

1. Recombinant DNA Technology – the genetic code (DNA) for the desired characteristic or protein is identified and isolated from a donor cell, confirmed by RFLP and or sequenced, and pasted into a vector, producing a recombinant DNA [rDNA] vector (usually a plasmid), that can carry the desired DNA code into a recipient.

2. Transformation – Genetically engineered cells are produced when the recombinant vector carrying the gene of interest is transferred into new host cells. If the cells express the new DNA, transcribing and translating it into a new (or recombinant) protein, the cells are then said to be transformed. Assays confirm the transformation has happened by testing for the gene product.

3. Cloning - The transformed cells, producing their recombinant DNA and proteins, are grown in culture (cloning).

4. Purification – Finally, the recombinant protein product, being produced in manufacturing, must be isolated and purified from the cells and other proteins in cell culture.

Students will use a plasmid synthesized in the laboratory which contains a jellyfish gene, Green Fluorescent Protein (GFP) expressed by a promoter, as well as a gene for antibiotic resistance, beta-lactamase (bla) expressed by its own promoter. :

Transformation requires good sterile technique and the ability to manipulate bacteria. The concept of cloning, increasing copies of identical cells and therefore DNA they contain as well as the proteins which they express..

Before beginning the transformation of E. coli (bacteria) to enable it to produce a fluorescent protein from Jellyfish students may need to review what a gene is and understand how gene expression is accomplished. Students should remember that a gene is transcribed into mRNA by RNA polymerase, and that the mRNA of interest is then translated into protein by ribosomes. In addition students should review how gene expression is regulated in cells, specifically how bacteria regulate gene, which differs from how eukaryotes regulate gene expression. Kahn Academy has a resource on gene expression in bacteria (including videos on left side for Try and Lac operons):

https://www.khanacademy.org/science/biology/gene-regulation/gene-regulation-in-bacteria/a/overview-gene-regulation-in-bacteria