Recombinant DNA technology is a method of joining DNA of two species and inserting it into a host organism, to produce new genetic combinations. The laboratory process used to produce recombinant DNA is molecular cloning. PCR replicates the desired DNA fragment that is inserted into a plasmid. The recombined plasmid is transformed into a host organism to produce a large number of copies of the recombined plasmid. Bacteria is the host organism used in recombinant DNA technology and there are several reasons for the use of them as the host.
Key Areas Covered
1. What is Recombinant DNA Technology
– Definition, Steps of the Process
2. Why are Bacteria Used in Recombinant DNA Technology
– Reasons for the Use of Bacteria as a Host Organism
Key Terms: Bacteria, Molecular Cloning, Growth Rate, Recombinant DNA Technology, PCR, Plasmids, Selection
What is Recombinant DNA Technology
Recombinant DNA technology is a molecular biology technique used to produce recombinant DNA molecules that carry the desired characteristic to a particular organism. Molecular cloning is the laboratory technique used to produce a large copy number of recombinant DNA coupled with PCR. The process of molecular cloning consists of seven steps as described below.
- Choice of host organism and cloning vector – The host organism is mainly bacteria. The choice of cloning vector depends on the choice of the host organism, the size of the foreign DNA fragment, and the level of expression.
- Preparation of vector DNA – The cloning vector is digested with restriction enzymes to make compatible ends with the foreign DNA fragment.
- Preparation of DNA to be cloned – The desired DNA fragment to be cloned can be amplified by PCR and digested with the restriction enzymes to generate compatible ends with the cloning vector.
- Creation of recombinant DNA – The digested cloning vector and the PCR fragment are ligated by treating with DNA ligase.
- Introduction of recombinant DNA into the host organism – The recombined DNA molecules are transformed into bacteria to obtain a large number of copies.
- Selection of transformed organisms – a selectable marker such as antibiotic resistance can be used to select the transformed bacteria in a culture.
- Screening for clones with desired DNA – The blue-white screening system, PCR, restriction fragment analysis, nucleic acid hybridization, DNA sequencing, and antibody probes can be used to screen the clones with desired DNA fragment.
The steps of recombinant DNA technology is shown in figure 1.
Why are Bacteria Used in Recombinant DNA Technology
Bacteria become ‘factories’ that produce a large number of copies of the recombinant DNA. There are several reasons for the use of bacteria as the host in the recombinant DNA technology. They are;
- Bacterial cells are easy to grow, maintain, and manipulate in a laboratory. The growth requirements are simple in bacteria and can be supplied in a petri dish. The growth conditions can be provided easily inside an incubator. They can also tolerate foreign DNA inside the cell.
- They multiply rapidly. Since bacteria are small organisms, they grow rapidly than complex cell types. Their rates of cell division are high.
- The extrachromosomal elements of bacteria known as plasmids can be manipulated and can be used as carriers of recombinant DNA into cells. Plasmids can be isolated from bacteria to insert foreign DNA and then transformed back into bacteria.
- The cloned, recombinant plasmids can be readily isolated from bacteria. Plasmid DNA can be isolated by easy laboratory processes through bacterial cell lysis.
The use of bacteria in recombinant DNA technology is shown in figure 2.
E. coli is the widely-used type of bacteria due to several reasons:
- E. coli genome is well-studied and is relatively simple. It carries only 4, 400 genes. Furthermore, it remains haploid throughout the lifetime. Therefore, protein engineering is easy with E. coli as a single gene copy is there to be masked by site-directed mutagenesis.
- The growth rate of E. coli is high. It replicates rapidly within 20 minutes. Therefore, it is easy to obtain the log phase (mid-way to maximum density).
- Many E. coli strains are safe to handle with reasonable hygiene.
- The preparation of competent cells (the cells that are capable of uptaking foreign DNA) and the transformation of recombinant molecules are easy with E. coli.
Recombinant DNA technology is used to introduce desired characteristics to organisms. Bacteria are used as models in the recombinant DNA technology due to many reasons such as easy growth and manipulation, rapid cell division, simplicity, ability to select and screen transformants.
1.Griffiths, Anthony JF. “Making recombinant DNA.” An Introduction to Genetic Analysis. 7th edition., U.S. National Library of Medicine, 1 Jan. 1970, Available here.
2.Phillips, Theresa. “Top 6 Reasons E. coli Is Used for Gene Cloning.” The Balance, Available here.