The Basic Concept of Biotechnology

 

MEANING OF BIOTECHNOLOGY

In this section, I’m going to discuss the chapter 11 of NCERT class XII i.e., BIOTECHNOLOGY: PRINCIPLES AND PROCESSES (PAGE-192-205). This is a very important chapter from the examination point of view. I will make this chapter very simple and easy to understand. When you read the NCERT book, the content is written in brief and hard to understand. 

Considering all these problems, I have made this chapter very easy and I guarantee that after you go through the content, you’ll be able to solve all kinds of questions related to this chapter asked in various competitive examinations including NEET, ICAR, BIOTECH, UPSC, MPSC, and LIFE SCIENCE entrance exams. So, let’s start with the chapter – Biotechnology: Principles and Processes.

I would like to break BIOTECHNOLOGY word into three pieces: BIO TECHNO and LOGI. BIO means life; TECHNO means technique or technology and LOGI means study. If we want to define biotechnology from these three words, we can say that — biotechnology is the study of technique or techniques in which living organisms, cells, parts of it or molecules (DNA, RNA, proteins) are used to manufacture desired products useful to mankind. Let’s see the definition given by EFB (European Federation of Biotechnology — Spain): “Biotechnology is the integration of natural science and organisms, cells, parts thereof, and molecular analogs for products and services.

Terms used in EFB definition:

Natural science: it is the branch of science concerned with the description, prediction and understanding of natural phenomenon based on empirical evidence (observation and documentation of patterns and behavior through experimentation). Life science comes under the natural science domain.

Molecular analogues: DNA, RNA, proteins

Parts thereof: it means any part of a substance or a thing.

It is important to mention that the definition given above refers to modern biotechnology, but classical biotechnology practices were used in the past as well. For example, people would use to make curd, bread and wine using microorganisms such as bacteria and fungi. However, in modern biotechnology, advanced techniques are used even at the molecular level.

Traditional Hybridization Vs Genetic Engineering – PAGE 194 NCERT

Traditional hybridization procedures are used in plant and animal breeding. However, this method leads to the inclusion and multiplication of both desirable and undesirable genes. On the other hand, genetic engineering used for the creation of recombinant DNA, gene cloning and gene transfer overcome the limitations of traditional breeding technique. In genetic engineering technique, only the desired genes are included.

In this chapter, we’ll mostly talk about modern biotechnology. It is important to mention the name of two scientists who contributed or I would say who led the foundation of modern biotechnology. They are mentioned as follows:

Herbert Boyer: He studied a few enzymes of E. coli and their useful properties. He observed that these enzymes possess unique properties of cutting DNA strands in a particular fashion, which make sticky ends of the strands.

Stanley Cohen: He worked on bacterial plasmid and developed a method of isolating plasmids from bacterial cells and then reinserting them in other cells.

Both scientists together recombined desired DNA segments and inserted into the bacterial cells. This experiment allowed them to manufacture specific proteins from desired DNA inside the bacterial cell. This breakthrough became the basis upon which the discipline of modern biotechnology was established.

We’ll discuss biotechnology chapter under the following heading:

A. Principles of biotechnology

• Genetic engineering: It refers to techniques used to alter the chemistry of the genetic material (DNA, RNA) and then introduce the altered genetic material into the host organisms so as to change the phenotype of the host organism.

• Bioprocess engineering: It refers to the maintenance of sterile condition in chemical engineering process to enable growth of the desired microbes / eukaryotic cells in large quantities for the manufacture of biotechnological products like antibiotics, vaccines and enzymes, etc.

B. Tools of Genetic Engineering

• Enzymes
• Cloning vectors
• Competent host

C. Process of Genetic engineering

• Isolation of DNA
• Fragmentation of DNA by specific RE
• Isolation of desired DNA fragment through agarose-gel electrophoresis
• Ligation of DNA fragment into a vector treated with similar RE
• Transferring the recombinant DNA into the host
• Culturing the host cells in a medium at large scale
• Extraction of the desired product

Enzymes: Following are the list of enzymes involved in genetic engineering process.

• Restriction endonucleases (molecular scissors)
• Alkaline phosphatase

When Eco RI is used as a RE, it will cut DNA at a specific site. Once the DNA is cut, the 5’ base will have phosphate group while the 3’ end will have OH group. Chances are that these P and OH group recombine. If they recombine then there would not be any benefit of cutting DNA because the GAATTC base pair will be established again. In order to prevent self ligation, phosphate needs to be removed and that is done by the enzyme alkaline phosphatase. This enzyme is either used from bacteria (BAP = Bacterial Alkaline Phosphatase) or from the calf intestine (CAP = Calf Alkaline Phosphatase).

• DNA ligase: it is used for making the phosphodiester bond. It acts as molecular glue. Generally, T4 ligase is used for this purpose (T4 is a bacteriophage).
• Reverse Transcriptase: It is used for converting RNA into DNA.
• DNA polymerases: for synthesizing a small sequence of DNA.
• Cellulase, chitinase – required to break the cell wall.

Out of the enzymes mentioned above, we’ll discuss the restriction endonuclease enzyme in detail:

Bacteria are attacked by viruses (bacteriophages). After infection, the viral DNA gets integrated to the bacterial DNA and commands bacterial DNA to form viral particles. To save bacterial DNA from damage, bacteria produce restriction endonuclease (RE) enzyme to cut viral DNA at specific points. 

In other words, the enzyme restricts the viral DNA from multiplication along with the bacterial DNA. Along with restriction endonucleases, another enzyme methylase also works in tandem because when the RE finds a similar sequence in bacterial DNA, it is methylated by the enzyme methylase to modify bacterial DNA sequence. This defense mechanism by the bacterial cell is called RESTRICTION MODIFICATION SYSTEM. Restriction endonuclease (RE) works on PALINDROMIC SEQUENCE. It is a sequence that is read similarly in both directions. For example, MALYALAM can be written from the right and left sides. Likewise the complementary sequence of GAATTC is CTTAAG and if we read both the sequences in 5’ to 3’ direction, it is read as GAATTC. Restriction endonucleases find only such palindromic sequences on the DNA.

Q. How many different classes of RE are present?

1. Class I
2. Class I, and Class II
3. Class I, II and III
4. Class I and IV

Ans: c

Class I RE requires Mg ion, ATP and S-Adenosyl Methionine. It recognizes a DNA sequence, but it does not cut that specific sequence rather it cuts DNA away from a specific sequence. In other words, when class I RE is used, it forms heterogeneous fragments of DNA.

Class II RE requires Mg ion and ATP, and it cuts DNA at a specific site. And thus it produces homogenous fragments. It means, every time we use the RE, we get the same fashion of fragments.

Class III RE requires Mg ion, ATP and S-Adenosyl Methionine. It also gives heterogeneous mixture of DNA fragments. In other words, every time we use this enzyme, the length of DNA fragments would be different.

Note: RE are generally 4-6 sequence long.

Naming of Restriction Enzyme:

Eco RI – E represents the genus, co represents the species; R represents the strain RY13 and I represents the order of discovery of RE. For example, how many different restriction enzymes have been isolated from this strain of bacteria and the first enzyme isolated was named I and II and so on. Its recognition site is —GAATTC—. EcoR I cuts DNA in a staggered manner and forms sticky ends.

The cut end of the sequence from EcoRI is sticky in nature.

Note: The restriction enzyme breaks the bond between the sugar and phosphate bond i.e., the phosphodiester bond.

Sma I = Serratia marcescens I

Sma I cuts DNA in flush cut to form blunt ends.

Sma I cuts DNA to form blunt ends.

Alu I = Arthrobactor luteus I

Bam H I = Bacillus amyloliquifaciens H I

Sal I = Streptomyces albus I

Hind II = Haemophilus influenza Rd II

Note: Hind II was the first RE to be discovered. The first RE – Hind II, whose functioning depended on a specific DNA nucleotide sequence was isolated and characterized five years later. It was found that Hind II always cut DNA molecules at a particular point by recognizing a specific sequence of six base pairs.

Note: the recognition sequence for Sal I and Hind II is the same, but their cut is at different places. In other words, two restriction enzymes could recognize the same sequence, but they cut at different places.

Note: If a restriction enzyme recognizes DNA sequence, say, GATC (4 bases), then the expected frequency of this base (4 bases) to be present in a DNA strand would have a frequency of (4)⁴ = 4x4x4x4 = 256 (i.e., the occurrence of GATC would be at every 256 in a sequence). Likewise, for 5 bases recognition sequence would present in a DNA strand at every 1024 bases.

(4)⁵ = 4x4x4x4x4 = 1024

Q. Arrange vectors on the basis of their insert DNA size capacity?

1. Plasmid < bacteriophage lambda < cosmid < BAC < YAC
2. Plasmid < cosmid < bacteriophage lambda < BAC < YAC
3. Cosmid < plasmid < bacteriophage lambda < BAC < YAC
4. Cosmid < plasmid < bacteriophage lambda < YAC < BAC

Ans: a

CLONING VECTORS

Q. What are the conditions for any suitable vectors?

A.

• It must have OriC site.
• It should have selectable markers: these are the genes on the basis of which we can select which one is transforming vector and which one is non transforming vector. Antibiotic resistance gene is used as selectable marker.
• Cloning recognition site: this is the site where recognition site should be there. These recognition sites must be present within the selectable marker site.

Q. Which of the following should be the features of a suitable vector?

1. The vector must contain an origin of replication so that it is independently able to replicate within the host.
2. The vector should incorporate a selectable marker, a gene whose product can identify the host cells containing the vector
3. The vector must have one unique restriction enzyme recognition site which can be used for cutting and introducing an insert
4. The vector should be small in size thereby facilitating entry / transfer into the host cell
5. All of the above

Ans: e

Types of Vectors:

1. PLASMID VECTOR: Plasmid was first discovered by William and Joshua Lederberg. Plasmids are the extra-chromosomal, self-replicating, circular genetic material. The number of copies can be 1-2 or it can range from 300-500 or more. These plasmids need to be modified before they can be used as vectors. The first plasmid vector was pBR322. The normal E.coli cells don’t carry resistance against any of these antibiotics (ampicillin, tetracycline, chloramphenicol, and kanamycin)

p- Plasmid based

B-Bolivar

R-Rodriguez

322- Code used to differentiate from other plasmid vectors

PstI, PvuI, EcoRI, ClaI, HindIII, BamHI, and SalI are restriction sites.

Rop stands for replication of plasmid

Selectable markers: Ampicillin resistance and tetracycline resistance genes.

pUC18/19: In this plasmid, the selectable marker is lacZ gene that codes for the enzyme – beta galactosidase. This selectable marker region also contains a polylinker and thus insertion of any foreign DNA into any of the restriction sites will result in an altered non-functional enzyme. During screening of recombinant plasmid containing host cells, the absence of beta-galactosidase activity is indicative of plasmids containing the insert.

Note: pBR322 and pUC vectors can replicate only in E.coli. Many of the vectors used in eukaryotic cells are constructed such that they can exist both in the eukaryotic cell and E.coli. Such vectors are known as SHUTTLE VECTORS. These vectors contain two types of origin of replication and selectable marker genes, one set which functions in eukaryotic cells (eg, yeast) and another in E.coli.  An example of shuttle vector is yeast plasmid Yep. In case of plants, a naturally occurring plasmid of bacterium Agrobacterium tumifaciens called Ti plasmid has been suitably modified to function as a vector.

2. Bacteriophage based vector: In this category, two cloning vectors are there: lambda phage and M13 phage.

Bacteriophage lambda: It is a double stranded, linear DNA genome containing 48, 514 bp, in which 12 bases on each end are unpaired and complementary (cohesive end sites) or cos sites.  An important feature of the lambda genome is that a large fragment in the central region of its genome is not essential for lytic infection of E.coli cells and thus this region can be replaced by foreign insert DNA. The linear phage DNA becomes circular due to the complementary end sequences. These phage vectors allow cloning of DNA fragments up to 23kb in size.

M13 Phage: It infects E.coli having a pilus which is selectively present in cells containing F plasmid (called F+ cells).

COMPETENT HOST

Competent host means the host cell must be capable of taking up the plasmid inside the cell. For example, if a normal host cell and plasmid is mixed together – the plasmid will not enter into the host cell until and unless the host cell is given some specific treatment. In other words, by giving a specific treatment to the host cell – the cell becomes competent to take up the DNA.

So, in this article, I have finished – (A) Principles of biotechnology and (B) Tools used in the genetic engineering. In the next section, I would finish (C) The process of genetic engineering.