Biotechnology Grade 11 Life Sciences Notes, Study notes, cheat sheet, Study notes of Biology

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biotechnology:

principles & Processes

CHAPTER

• Biotechnology is the technique of using live organisms or their enzymes for products & processes useful to humans. The European Federation of Biotechnology (EFB) defines Biotechnology as ‘the integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services’.

Biotechnology deals with:

• Microbe-mediated processes(making curd, bread, wine etc).
• In vitro fertilization (test-tube baby programme).
• Synthesis and using of a gene.
• Preparation of DNA vaccine.
• Correcting a defective gene�

PRINCIPLES OF BIOTECHNOLOGY

TOOLS OF RECOMBINANT DNA

TECHNOLOGY

1. Core techniques of modern biotechnology

2. Basic steps in genetically modifying an

organism

• Genetic engineering: The technique in which genetic material (DNA & RNA) is chemically altered and introduced into host organisms to change the phenotype.
• Maintenance of sterile ambience: It is necessary is chemical engineering processes for growing desired microbe/ eukaryotic cell for the manufacture of antibiotics, vaccines, enzymes etc.

a) Identification of DNA with desirable genes: Traditional hybridisation techniques lead to inclusion and multiplication of undesirable genes along with desired genes. Genetic engineering helps to isolate and introduce only desirable genes into the target organism. b) Introduction of the identified DNA into the host: A vector DNA such as plasmid is used to deliver an alien piece of DNA into the host organism. c) Maintenance of introduced DNA in the host and transfer of the DNA to its progeny: A piece of alien DNA has no the sequence called Origin of replication (ori) needed for starting replication. So, it cannot multiply itself in the progeny cells of the organism. Hence alien DNA is integrated into the recipient genome (it has ori). It multiplies & inherits along with host DNA.

First recombinant DNA (rDNA) was produced by Stanley Cohen & Herbert Boyer (1972). They isolated an antibiotic resistance gene by cutting out a DNA piece from a plasmid. This gene was linked with a native plasmid of Salmonella typhimurium

1. Restriction Enzymes (‘molecular scissors’)

• These are the enzymes which cut DNAat specific sites into fragments.
• They belong to a class of enzymes called nucleases.
• In 1963, two enzymes responsible for restricting growth of bacteriophage in E. coli were isolated. One enzyme added methyl groups to DNA. The other (restriction endonuclease) cut DNA.
• More than 900 restriction enzymes have been isolated from over 230 strains of bacteria.

Naming of the restriction enzymes:

• First letter indicates genus and the second two letters indicate species of the prokaryotic cell from which they were isolated. E.g. EcoRI comes from E. coli RY 13 (R = the strain. Roman numbers = the order in which the enzymes were isolated from that strain of bacteria).

c. Cloning sites

• To link the alien DNA, the vector needs a single or very few recognition sites for restrictionenzymes.
• More than one recognition sites generate several fragments. It complicates the gene cloning.
• Ligation of alien DNA is carried out at a restriction site present in one of the two antibiotic resistance genes. E.g. ligation of foreign DNA at Bam H I site of tetracycline resistance gene in vector pBR322. As a result, recombinant plasmid is formed. If ligation does not occur, it is called non-recombinant plasmid.
• Restriction sites: Hind III, EcoR I, BamH I, Sal I, Pvu II, Pst I, Cla I. ori
• Antibiotic resistance genes: ampR and tetR. Rop: codes for the proteins involved in the replication of plasmid.
• The recombinant plasmids lose tetracycline resistance due to insertion of foreignDNA.
• When the plasmids are introduced into E. coli cells, 3 types of cells are obtained: o Non-transformants: They have no plasmid. So they are not resistant to either tetracycline or ampicillin. o Transformants with non-recombinant plasmid: They are resistant to both tetracycline & ampicillin. o Transformants with recombinant plasmid: They are resistant only toampicillin.
• Recombinant plasmids can be selected out from non�recombinant ones by plating transformants on ampicillin medium. Then the transformants are transferred on tetracycline medium.
• The recombinants grow in ampicillin medium but not on tetracycline medium. But, non-recombinants grow on the medium containing both the antibiotics.
• Thus, one antibiotic resistance gene helps to select the transformants. The inactivated antibiotic resistance gene helps to selectrecombinants.
• Selection of recombinants due to inactivation of antibiotics requires simultaneous plating on 2 plates having different antibiotics. Therefore, alternative selectable markers have developed to differentiate recombinants from non recombinants based on their ability to produce colour in the presence of achromogenic substrate.
• In this, a recombinant DNA is inserted within the coding sequence of an enzyme, þ -galactosidase. So, the enzyme is inactivated. It is called insertional inactivation. Such colonies do not produce any colour. These are identified as recombinant colonies.
• If the plasmid in bacteria have no an insert, it gives blue coloured colonies in presence of chromogenic substrate.

d. Vectors for cloning genes in plants & animals

• Genetic tools of some pathogens can be transformed into useful vectors for delivering genes to plants & animals. E.g. Agrobacterium tumifaciens (a pathogen of many dicot plants) can deliver a piece of DNA (T-DNA) to transform normal plant cells into a tumor. These tumor cells produce the chemicals required by the pathogen. The tumor inducing (Ti) plasmid of A. tumifaciens is modified into a cloning vector which is not pathogenic to the plants but is able to use the mechanisms to deliver genes of interest into plants. Retroviruses in animals can transform normal cells into cancerous cells. So, they are used to deliver desirable genes into animal cells.�

3. Competent Host (For Transformation with

Recombinant DNA)

• DNA is a hydrophilic molecule. So, it cannot pass through cell membranes.
• To avoid this problem, bacterial cells are treated with a specific concentration of a divalent cation (e.g. calcium). So, DNA enters the bacterium through pores in cell wall.
• Such cells are incubated with recombinant DNA on ice. Then they are placed briefly at 420C (heat shock) and put them back on ice. This enables the bacteria to take up recombinant DNA.

PROCESSES OF RECOMBINANT

DNA TECHNOLOGY

Other methods to introduce alien DNA into host cells

• Micro-injection : In this, recombinant DNA is directly injected into the nucleus of an animal cell.
• Biolistics (gene gun) : In this, cells are bombarded with high velocity micro-particles of gold or tungsten coated with DNA. This method issuitable for plants.
• Disarmed pathogen’ vectors : They infect the cell and transfer the
  recombinant DNA into the host.

1. Isolation of the Genetic Material (DNA)

2. Cutting of DNA at Specific Locations

3. Amplification of Gene of Interest using PCR

4. Insertion of Recombinant DNA into HostCell

5. Obtaining the Foreign Gene Product

• The bacterial cells/plant or animal tissue are treated with enzymes like lysozyme (bacteria), cellulase (plants), chitinase (fungus) etc. The cell is broken releasing DNA & other macromolecules (RNA, proteins, polysaccharides and lipids).
• RNA is removed by treating with ribonuclease. Proteins are removed by treatment with protease. Other molecules are removed by appropriate treatments.
• When chilled ethanol is added, purified DNA precipitates out as a collection of fine threads in the suspension.
• Purified DNA is incubated with the restriction enzyme at optimal conditions. As a result, DNA digests.
• Agarose gel electrophoresis is employed to check the progression of a restriction enzyme digestion. DNA is negatively charged. So it moves towards the anode. The DNA fragments separate according to their size through sieving effect of the agarose gel (a polymer extracted from sea weeds). The smaller sized fragment moves farther.
• The process isrepeated with the vector DNAalso.
• After cutting the source DNA and vector DNA, the cut-out gene of interest from source DNA and cut vector aremixed and ligase is added. It creates recombinant DNA.
• Polymerase Chain Reaction (PCR) is the synthesis of multiple copies of the gene of interest in vitro using 2 sets of primers & the enzyme DNA polymerase.
• Primers are small chemically synthesized oligo nucleotides that are complementary to the regions of DNA.
• The enzyme extends the primers using the nucleotides and genomic DNA (template). Through continuous replication , the DNA segment is amplified up to 1 billion copies.
• For amplification, a thermostable DNA polymerase (isolated from a bacterium, Thermus aquaticus) is used. It remains active in high temperature during the denaturation of double stranded DNA.
• The amplified fragment can be used to ligate with a vector for further cloning.
• Using any methods, the ligated DNA is introduced into recipient cells. They take up DNA from its surrounding.
• If a recombinant DNA bearing ampicillin resistant gene is transferred into E. coli cells, the host cells become ampicillin-resistant cells.
• If the transformed cells are spread on agar plates containing ampicillin, only transformants will grow. Untransformed recipient cells will die.
• The aim of recombinant DNA technology is to produce a desirable protein.
• If a protein encoding foreign gene is expressed in a heterologous host, it is called a recombinant protein.
• The cells with foreign genes can be grown in laboratory. The cultures are used to extract the desired protein and purify it by using separation techniques.
• The cells can also be multiplied in a continuous culture system. Here, the used medium is drained out from one side while fresh medium is added from the other. It maintains the cells more physiologically active and so produces a larger biomass. It yields more desired protein.