Biotechnology — Study Notes

1. Introduction

Biotechnology, according to the European Federation of Biotechnology, is the integration of natural science and organisms, cells, parts thereof and molecular analogues for products and services. In a broader sense, biotechnology deals with techniques of using live organisms or enzymes from organisms to produce products and processes useful to humans. The chapter focuses on the modern tools and techniques of biotechnology, which involve genetic engineering, the manipulation of DNA to create new organisms or products.

Modern biotechnology relies on two core techniques: genetic engineering (the alteration of the genetic material of an organism) and the maintenance of sterile (microbial contamination-free) conditions for growing microbes and cells in culture to enable the production of useful products. The chapter covers the principles of biotechnology, the tools required for recombinant DNA technology, and the processes involved in the production of recombinant products. This forms the basis for understanding the applications of biotechnology discussed in the next chapter.

2. Principles of Biotechnology

The two core techniques that enabled the birth of modern biotechnology are:

1. Genetic engineering: The technique of altering the genetic material (DNA/RNA) of an organism to introduce one or more new traits. This involves the insertion of a foreign gene into the genome of the host organism so that the host produces a new product.
2. Bioprocess engineering: The maintenance of sterile conditions for the growth of microbes and cells in large quantities (bioreactors) to enable the production of useful products like antibiotics, vaccines, enzymes and other biologically active substances.

These principles are applied in many fields, including the production of human insulin, vaccines, therapeutic drugs and genetically modified crops.

3. Tools of Recombinant DNA Technology

Recombinant DNA technology is the technique of combining DNA molecules from different sources into one molecule to create a new genetic combination. The tools required for this technology are:

3.1 Restriction Enzymes

Restriction enzymes (restriction endonucleases) are enzymes that cut DNA at specific nucleotide sequences. They are the molecular scissors of recombinant DNA technology. The discovery of restriction enzymes was made in the bacterium Haemophilus influenzae.

The three types of restriction enzymes are exonucleases, endonucleases and restriction endonucleases: - Exonucleases: Remove nucleotides from the ends of the DNA. - Endonucleases: Make cuts at specific positions within the DNA. - Restriction endonucleases: Cut DNA at specific recognition sequences known as restriction sites, and this cutting is essential for producing recombinant DNA.

The restriction enzyme EcoRI cuts the DNA at a specific six base pair recognition sequence (GAATTC) and produces sticky ends (single stranded overhangs), which allow the joining of DNA fragments from different sources. Some restriction enzymes produce blunt ends (no overhangs), while others produce sticky ends. The first discovered restriction enzyme used in rDNA technology was HindII. The enzyme makes two cuts, one in each strand, offset from each other, producing sticky ends.

3.2 Cloning Vectors

Cloning vectors are small pieces of DNA, such as plasmids, that can carry a foreign DNA segment into the host cell for replication and expression. Plasmids are extrachromosomal, self-replicating, circular DNA molecules present in bacteria. A cloning vector must have: - Origin of replication (ori): A sequence where replication is initiated, controlling the copy number of the linked DNA. - Selectable marker: A gene (usually for antibiotic resistance, e.g., ampicillin resistance) that allows the identification and selection of host cells that contain the vector. - Cloning sites: Recognition sites for restriction enzymes where the foreign DNA is inserted. For example, the pBR322 vector has sites for EcoRI, HindIII, BamHI, etc. - Insertional inactivation: A method to select recombinants by the inactivation of a gene due to the insertion of the foreign DNA. In the pBR322 vector, the insertion of the foreign DNA into the gene for tetracycline resistance inactivates that gene, so recombinants can be identified.

Common cloning vectors include pBR322 (a plasmid vector), the bacteriophage lambda, and yeast artificial chromosomes. The Ti plasmid (tumour inducing plasmid) of Agrobacterium tumefaciens is used as a vector to deliver genes into plants.

3.3 Competent Host

The DNA from the vector must be taken up by the host cell. Most host cells (like E. coli) do not naturally take up DNA, so they are made competent (capable of taking up DNA) by treatment with a specific concentration of a divalent cation like calcium, which increases the permeability of the cell membrane. The cells are incubated with the recombinant DNA on ice, followed by a brief heat shock (42 degrees Celsius), and then placed back on ice. This is called transformation. Alternative methods for making cells competent include biolistics (gene gun), microinjection and electroporation.

4. Processes of Recombinant DNA Technology

The main steps of recombinant DNA technology are:

4.1 Isolation of Genetic Material

The first step is the isolation of DNA from the source. The DNA is separated from the cellular components by treating the cells with lysozyme (which breaks the cell wall), followed by centrifugation. The DNA is then purified by precipitation with alcohol.

4.2 Cutting of DNA by Restriction Enzymes

The isolated DNA is cut into fragments at specific sites by restriction enzymes. The same restriction enzyme used to cut the source DNA is used to cut the vector DNA, producing compatible ends. The DNA fragments are separated by gel electrophoresis, in which DNA moves towards the positive electrode because it is negatively charged, and the smaller fragments move faster.

4.3 Amplification of Gene of Interest Using PCR

The polymerase chain reaction (PCR) is used to amplify (make multiple copies of) the gene of interest. PCR requires a DNA template, two primers, deoxynucleotides (dNTPs) and a thermostable DNA polymerase called Taq polymerase, which is isolated from the bacterium Thermus aquaticus. The three steps of PCR are: - Denaturation: Heating the DNA to separate the two strands (about 95 degrees Celsius). - Annealing: Cooling to allow the primers to bind to the complementary sequences (about 55 degrees Celsius). - Extension: Raising the temperature so that Taq polymerase extends the primers, synthesising new DNA strands (about 72 degrees Celsius).

Each cycle of PCR doubles the amount of DNA, so a small amount of DNA can be amplified enormously in a few hours.

4.4 Insertion of Recombinant DNA into the Host

The recombinant DNA (vector with the gene of interest) is inserted into the host cell. If the gene of interest is linked to the appropriate vector and promoter, it can be expressed in the host. The host with the recombinant DNA is called a transformed cell.

4.5 Obtaining the Foreign Gene Product

After insertion, the host cell expresses the foreign gene and produces the desired product. If the product is to be used in medicine, it must be purified and, for human use, it must be in the native conformation. There may be problems of protein folding, where the protein may fold into a non-functional form; in such cases, the protein must be recovered and refolded into its native form. Recombinant proteins produced in bacterial hosts may need modification, and in such cases eukaryotic hosts may be used.

4.6 Downstream Processing

Downstream processing involves the separation and purification of the product, followed by its formulation with suitable preservatives. It is the collection of the product, its separation from the cells, purification and formulation.

Quick Revision Tables

Table 1: Tools of Recombinant DNA Technology

Table 2: Steps of Recombinant DNA Technology

Table 3: PCR Steps

Mind Map

Important Diagrams (SVG)

Common Mistakes

1. Students confuse exonucleases with restriction endonucleases; exonucleases remove nucleotides from the ends, while restriction endonucleases cut within the DNA at specific sequences.
2. EcoRI is said to produce blunt ends; EcoRI produces sticky ends with single stranded overhangs, which facilitate joining of different DNA fragments.
3. The Ti plasmid is said to come from a bacterium of the gut; it is the tumour inducing plasmid of the plant pathogen Agrobacterium tumefaciens.
4. Taq polymerase is said to be a normal DNA polymerase; it is a thermostable polymerase isolated from Thermus aquaticus, which survives the high temperature of PCR.
5. The order of PCR steps is said to be extension, denaturation, annealing; the correct order is denaturation, annealing, extension.
6. Competent cells are said to naturally take up DNA; most host cells are made competent by calcium treatment followed by heat shock.
7. The ori (origin of replication) is said to control gene expression; it controls the initiation of replication and the copy number of the linked DNA.
8. In insertional inactivation, the foreign gene insertion is said to activate the marker gene; it inactivates the marker gene (e.g., tetracycline resistance), allowing identification of recombinants.

Exam Tips

1. Remember the tools of rDNA technology: restriction enzymes, cloning vectors, competent host, and their functions.
2. Learn the EcoRI recognition sequence (GAATTC) and the concept of sticky ends vs blunt ends.
3. For cloning vectors, remember the required features: ori, selectable marker, cloning sites and insertional inactivation, with pBR322 as the classic example.
4. PCR: know the three steps with their temperatures, the requirements (template, primers, dNTPs, Taq polymerase) and that Taq comes from Thermus aquaticus.
5. The order of rDNA processes: isolation, cutting, amplification, insertion, product, downstream processing.
6. Remember transformation and the methods to make cells competent: calcium chloride, biolistics, microinjection, electroporation.
7. The Ti plasmid for plants and its source (Agrobacterium) is a high-frequency question.

Conclusion

Biotechnology, with its twin pillars of genetic engineering and bioprocess engineering, has given humanity unprecedented power to manipulate living systems for useful products. The tools of recombinant DNA technology, the restriction enzymes that cut DNA at precise sequences, the cloning vectors that carry foreign genes, and the competent hosts that take them up, combine in a systematic process to create recombinant organisms. The polymerase chain reaction amplifies genes of interest enormously, while downstream processing ensures that the final product is pure and functional. The principles and processes described in this chapter are the practical toolkit of modern biology, and they find direct application in medicine, agriculture and industry. This foundation prepares the student for the next chapter, which surveys the remarkable applications of biotechnology, from genetically modified crops to gene therapy.