7.2: Introduction

A plasmid is an ideal vector for carrying DNA sequences from one organism
to another. Plasmids used in genetic engineering are equipped with (1) a promoter that enables gene transcription, (2) a sequence for the initiation of DNA replication (ori site), and (3) an antibiotic resistance gene. 

Transforming Bacteria with Recombinant Plasmid

Inserting a gene into a plasmid vector is an important first step in the gene cloning process. However, if the ultimate goal is to produce a large amount of a particular protein, the plasmid must replicate to make sure that there are many copies of the gene and the gene of interest must be expressed. Both activities can only occur inside a cell. Therefore, in this lab we will put a recombinant plasmid into E. coli bacteria through a process that is called transformation, because it changes the DNA content of the bacteria.

The plasmid will be taken up by bacteria where it replicates, and its genes will be expressed using the bacterial cellular machinery. If a gene of interest1 has been inserted into the plasmid vector, the bacteria produces the product encoded by that gene. 

 In this exercise, you will carry out the transformation of E. coli bacteria using a recombinant plasmid that contains a gene that produces colored proteins. 

Bacteria Transformation

Once a recombinant plasmid is made that contains a gene of interest, such as insulin, the plasmid can enter bacterial cells by a process called transformation. Figure 13.1 illustrates transformation.  The uptake of DNA from the environment of a bacterial cell occurs with a very low efficiency in nature. E. coli bacteria have complex plasma membranes that separate the external environment from the internal environment of the cell and carefully regulate which substances can enter and exit the cell. In addition, the cell wall is negatively charged and repels negatively charged DNA molecules. 

 Figure 13.1: Bacterial transformation

In order to increase the efficiency of DNA uptake, bacteria are treated in
two ways. First, the E. coli bacteria are placed in a solution that contains
positive calcium ions, which neutralize the negative charge on the cells’ outer membranes, enabling DNA molecules to cross the plasma membranes and enter the cell. Next, the bacteria are subjected to a heat shock, a sudden increase in temperature, which causes the pressure outside the cell to increase. This pressure difference enables the plasmid DNA to enter the bacterial cell from the outside. 

Cells treated with calcium and heat are considered competent to take up DNA more efficiently, but even with this treatment only about 1 in 10,000 bacterial cells takes up a plasmid in its environment. So how can the bacteria that have taken up the recombinant plasmid be identified? Recall that an important component of a recombinant plasmid is a gene for antibiotic resistance. If you place bacterial cells in the presence of the antibiotic, only those cells that have the recombinant plasmid will grow. 

From Plasmid DNA to Protein

Once a recombinant plasmid has entered the bacterial cell, DNA polymerase initiates replication at the ori site, and the plasmid replicates using the bacterial DNA replication enzymes. These multiple copies of plasmids can now produce the protein of interest, such as insulin, in large quantities. Usually to get the bacteria to make the protein of interest, it must be induced by adding a chemical which will encourage transcription of the gene.  In this process, the information encoded in the human DNA is transferred from DNA to protein using the transcription and translation machinery of the cell (see Figure 13.2). The protein may alter the observable traits of the organism. 

Figure 13.2: Gene expression from a plasmid in the bacterial cell

Genetic engineering is only possible because genes from different organisms can be expressed in bacteria. On Earth, all life is related, and the way that information is encoded in DNA is universal. As you may already know, proteins are made up of smaller subunits called amino acids, and a sequence of three nucleotides in DNA code for a single amino acid. These three-nucleotide sequences are called codons. For example, the codon TTG codes for the amino acid tryptophan, whereas the codon AAG codes for the amino acid lysine. In many cases, more than one codon can encode the same amino acid. For example, AAA is also a codon for lysine. In addition, there are informational codons, such as the start codon (ATG) and the stop codon (TTA), which show where in the DNA sequence the code for the protein begins and ends.