Introduction to Biotechnology and Processes involved

Introduction

Biotechnology is a cutting-edge field that combines biology and technology to revolutionize the way we understand and manipulate life. With advancements in DNA manipulation and genetic engineering, biotechnology has the potential to solve some of the world’s most pressing problems, from developing new treatments for diseases to improving food security and environmental sustainability.

In biotechnology, researchers use a variety of tools and techniques, including recombinant DNA, gene cloning, gene transfer, plasmids, and restriction enzymes to study and modify the genetic material of organisms. These technologies have led to major breakthroughs in the development of new medicines, improved crop yields, and more efficient and sustainable industrial processes.

If you are curious about the exciting advances in biotechnology, understanding the concepts of recombinant DNA, gene cloning, gene transfer, and more will provide you with a deeper appreciation of this rapidly evolving field and its potential impact on our day-to-day life.

Definitions of biotechnology

Biotechnology can be defined in several ways. Many people and organizations have defined biotechnology in their way. Some of the definitions are as follows:

The manipulation (as through genetic engineering) of living organisms or their components to produce useful usually commercial products (such as pest resistant crops, new bacterial strains, or novel pharmaceuticals). https://www.merriam-webster.com/dictionary/biotechnology

Biotechnology is the use of biology to solve problems and make useful products. The most prominent approach used is genetic engineering, which enables scientists to tailor an organism’s DNA at will. https://www.britannica.com/technology/biotechnology

 “Biotechnology is the integration of natural science and engineering principles in order to design and develop useful products and processes that are based on the understanding of biological systems.”        European Federation of Biotechnology

Based on the definitions provided by various organizations and scientists, Biotechnology can be defined as “the application of scientific and engineering principles to the processing of materials using biological agents, with the aim of developing useful products and processes for improving human health, agriculture, and the environment. It involves the integration of natural science and engineering and the use of living systems and organisms to create or modify products and processes.”

The basics of biotechnology

Biotechnology is a field that combines biology and technology to produce new products, processes, and innovations. It encompasses a wide range of techniques, including genetic engineering, bioprocess engineering, recombinant DNA technology, and the use of microorganisms, cells, and other biological systems to create products with various applications. The principles of biotechnology involve the manipulation of genetic material to produce desirable traits in organisms (genetic engineering), as well as the use of sterile conditions in chemical engineering processes to maintain the growth of desired microorganisms (bioprocess engineering) for the manufacture of biotechnological products.

Genetic Engineering

It is the technique of developing genetic material (DNA) with the capability to produce desired product. The process of genetic engineering involves the following steps:

  1. Identification and Isolation of the desired gene: The gene of interest is isolated from the DNA of the source organism, typically through restriction enzyme digestion or PCR amplification. 
  2. Modification of the gene: The isolated gene may be altered or modified through various techniques, such as site-directed mutagenesis or the addition of genetic elements such as promoters or enhancers.
  3. Cloning of the modified gene: The modified gene is then inserted into a plasmid vector and multiplied through bacterial fermentation. It can also be done by polymerase chain reaction (PCR) where multiple copies of a gene are synthesized in vitro.
  4. Transformation of a host organism: The recombinant plasmid is then introduced into a host organism, usually through electroporation or bacterial conjugation.
  5. Selection and screening of transformed organisms: The transformed organisms are screened for the presence of the desired recombinant plasmid and selection is performed to ensure that only organisms carrying the desired genetic modification have survived.
  6. Analysis of the genetically modified organism: The modified organism is then analyzed to confirm the success of the genetic engineering process and to assess any changes in phenotype resulting from the introduction of the recombinant DNA.

We will be discussing all these steps one-by-one in detail:

Identification and Isolation of desired gene:

The process of isolating a desired gene involves several steps:

Gene source: The gene of interest can be obtained from various sources such as a cDNA library, genomic DNA, or tissue samples.

DNA extraction: The first step is to extract the DNA from the source material using various techniques such as a salt precipitation, column purification, or a commercial kit.

Restriction Enzyme and DNA Digestion: The extracted DNA is then cut into smaller fragments using restriction enzymes, which recognize specific sequences and cut the DNA at those sites.

  • Restriction enzymes, also known as restriction endonucleases, are enzymes that recognize specific DNA sequences and cut the DNA at those sites. They are commonly used in molecular biology for a variety of purposes, including gene cloning, DNA fingerprinting, and genome analysis.
  • Restriction enzymes are found in bacteria and function as a defense mechanism against invading bacteriophages (viruses that infect bacteria). They recognize and cut foreign DNA, preventing the bacteriophage from replicating and causing harm to the bacterium
Flow chart showing types of nucleases
  • Each restriction enzyme (type II) recognizes a specific DNA sequence, known as its restriction site.
  • The restriction site is typically 4-8 base pairs in length, and the specific recognition sequence determines the specificity of the restriction enzyme.The restriction enzymes are named based on the bacteria species they were first isolated from, for example EcoRI, HindIII, and BamHI. In EcoRI, “E” represents the genus Escherichia and “co” stands for species name coli, “R” stands for strain RY13 and the roman numeral “I” represents the order of discovery in that species.
Naming Restriction Enzyme
  • Once the restriction enzyme recognizes its restriction site, it cuts the DNA at a specific location within the site.
  • Some restriction enzymes generate blunt ends, where both ends of the cut DNA have no overhanging bases. Others generate sticky ends, where the cut DNA has single-stranded overhanging ends that are complementary to each other. These overhanging ends can then be used for ligation into a suitable vector for cloning.
Types of ends generated by RE
Restriction digestion of gene and vector

Gel Electrophoresis:

The fragmented DNA is separated by size through gel electrophoresis. This allows the desired gene fragment to be separated based on its size.

  • Gel electrophoresis is based on the movement of charged molecules in an electric field.
  • The macromolecules are loaded into an agarose gel matrix, and subjected to an electrical current. The gel acts as a sieve, and the molecules separate based on their size and electrical charge, with smaller, more highly charged molecules moving faster and farther than larger, less charged molecules.
  • After the gel has run for a specified amount of time, the separated molecules can be visualized by staining with ethidium bromide, which intercalates into the DNA and fluoresces under UV light, or by using other staining or probing methods.
Gel Electrophoresis

Gene Purification and amplification:

The desired gene fragment is then extracted from the gel and purified, typically using spin column purification. The purified gene is then amplified by the Polymerase Chain Reaction (PCR).

Gene ligation with cloning vector:

The purified DNA is then mixed with an appropriate cloning vector digested by same restriction enzymes or by enzymes which generate identical ends. This mixture is treated with enzyme ligase which seals the DNA nicks between the cloned gene and the vector

Ligation of isolated genes with vector

Cloning:

DNA cloning is the process of creating a copy of a specific segment of DNA, usually for the purpose of producing multiple copies of a particular gene or a piece of genetic material.

  • The process starts by isolating the desired DNA fragment and then inserting it into a suitable vector, such as a plasmid
  • The vector containing the inserted DNA is then introduced into a host organism, such as bacteria, where it is replicated along with the host’s genetic material.
  • The resulting colonies of host cells each contain multiple copies of the inserted DNA fragment.
  • The vector carrying the desired gene can be introduced into the host by the following methods:
  1. Chemical transformation: The recombinant DNA is inserted into a suitable host by placing bacteria in a medium containing divalent cations like Ca2+ The bacteria are given brief heat shock (at 42°C) and then placing them in the ice. It creates transient pores in the bacterial wall through which the plasmid DNA enters into the bacterium.
  2. Electroporation: In this method, a high-voltage electrical field is used to create small pores in the bacteria’s cell membrane, allowing the plasmid to enter.
  3. Bacterial conjugation: This method involves the transfer of plasmids from one bacterium to another through direct cell-to-cell contact.
  4. Lipofection: This method uses liposomes, small spherical structures composed of lipids, to deliver the plasmid into the bacteria.
  5. Microinjection: In this method, a microscopic needle is used to physically inject the plasmid into the bacteria.
  6. Biolistics or gene gun: The DNA is introduced into the cells or tissues by physically firing small inert particles (gold or platinum) coated with DNA into the target cells. This method is commonly used to transform plant cells, but can also be used to introduce DNA into animal cells and bacteria.
  7. Viral transduction: Disarmed virus is used as a vector to deliver the desired DNA into target cells. The virus infects the cells and inserts its genetic material into the host cell’s genome. This process can be manipulated so that the virus carries the DNA of interest instead of its own genetic material. The virus then infects the target cells, introducing the foreign DNA into the cells, where it can be expressed and replicated. Retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses (AAVs) are used for this purpose.

Selection and screening of transformed organisms:

“Selection and screening of transformed organisms” refers to the process of identifying and selecting organisms that have undergone genetic transformation, which is a process of introducing new genetic material into an organism’s DNA. The objective of this process is to identify organisms that have incorporated the desired genetic traits, and then to screen them to ensure that the transformation has been successful and the desired traits are being expressed.

  • The first step in this process is selection, which involves exposing transformed organisms to conditions that will allow only those with the desired traits to survive. For example, in crop improvement, this may involve growing plants in the presence of a herbicide that only kills the plants that do not have the desired trait of herbicide resistance. The surviving plants are then considered to be the selected population.
  • A bacterium with a gene introduced in its genome can be selected against the antibiotic resistance. Introduction of a gene in the antibiotic resistance gene will abolish its natural resistance to the antibiotic and the bacteria can be selected by exposing the bacteria to the antibiotic.
  • The next step is screening, which involves testing the selected organisms to confirm that they have indeed incorporated the desired traits. This can be done in a number of ways, such as PCR analysis to detect the presence of the introduced DNA, observation of phenotype expression, or biochemical assays to measure the activity of the desired trait.
  • Overall, the selection and screening process is crucial in ensuring that transformed organisms are viable and capable of expressing the desired traits, which is essential for applications in agriculture, medicine, and biotechnology. The process of selection and screening helps to ensure that the transformed organisms are safe, reliable, and have the desired characteristics, making them useful for various applications.
Selection of transformed bacteria

Analysis of genetically modified organism:

The genetically modified organism can be screened for the desired product. It can be tested by different methods depending upon the product. This includes laboratory studies, field trials, and environmental impact assessments. These studies evaluate the potential effects of GMOs on human health, the environment, and the economy. Such analysis is necessary to ensure the safety and efficacy of GMOs before they are released into the market.

Summary

In conclusion, biotechnology has become an increasingly important field in modern society, with the potential to address some of the world’s most pressing challenges. Advances in genetic engineering, recombinant DNA, and other technologies are paving the way for new medical treatments, sustainable agriculture, and environmentally friendly industrial processes. By understanding the key concepts of biotechnology, we can appreciate the remarkable breakthroughs and innovative solutions being developed by researchers in this field. As biotechnology continues to evolve, we can look forward to even more exciting advancements that have the potential to shape the future of our world.

References:

“Molecular Biology of the Cell” by Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter.

“Principles of Gene Manipulation and Genomics” by Sandy B. Primrose and Richard M. Twyman.

“Introduction to Biotechnology” by William J. Thieman and Michael A. Palladino.

“Biotechnology: An Introduction” by Susan R. Barnum.

“Genomes” by T.A. Brown.

“Molecular Cloning: A Laboratory Manual” by Joseph Sambrook and David Russell.

“Thank you for using our online study materials. We are a self-sustained group of individuals dedicated to creating quality educational resources for students worldwide. However, we rely on your donations to continue our work. If you have found our materials useful, please consider making a contribution to help support our mission. Your support will allow us to continue providing valuable resources to students in need. To donate, please click the ‘DONATE HERE‘ button. Thank you for your support!”

1 thought on “Introduction to Biotechnology and Processes involved”

Leave a Comment

Your email address will not be published. Required fields are marked *