Biology Chapter 11 Biotechnology : Principles And Processes

1. Genetic engineering—Techniques to alter the makeup of genetic material (DNA and RNA), by introducing new genes into host organisms and thus change the phenotype of the host organism.

2. Maintenance of sterile (microbial contamination-free) environment in chemical engineering processes to enable growth of only the desired cell in large quantities for the manufacture of biotechnological products like antibiotics, vaccines, enzymes etc.

Genetic engineering is a technique for artificially and deliberately modifying DNA (genes) to suit human needs. It includes techniques like creating recombinant , gene cloning and gene transfer. 
Steps in genetic engineering:
1. Identification and isolation of genetic material.
2. Cutting of the DNA at specific locations using restriction enzymes.
3. Amplification of the desired gene using Polymerase Chain reaction.
4. Introduction of gene of interest  into host cell or organism.
5.The gene is cloned and the desired gene product the recombinant protein is obtained.
6. Downstream processing is done which includes processing like and separation purification
5. 
Fig. Diagram showing Various steps involved in DNA recombinant technology for the production of a recombinant protein.

1. Identification of DNA with desirable genes.

2. Introduction of the identified DNA into the host.

3. Maintenance of introduced DNA in the host and transfer of the DNA to its progeny.

1. Restriction enzymes 

2. Polymerase chain reaction

3. Cloning vectors

4. Competent host

The name is obtained using the conventaional method -:

(i) First capital letter of the name comes from the genus of the organism Escherichia is ‘E’.

(ii) Second two small letters comes from the species of cells from which they are isolated i.e. ‘co’.

(iii) Letter R is derived from the name of strain  of the bacteria i.e. Escherichia coli Ry_ 13.

(iv) Roman number indicate the order in which enzymes were isolated from that strain of bacteria .

1. The first letter of the name is taken  from the genus.

2.The next two letters comes from the name of the species of the cell from which they are isolated.

3. The next letter comes from the strain of the organism.

4. The roman number following these four letters indicate the order in which the enzymes was isolated from that strain of the bacterium.

Example :EcoR I is isolated from Escherichia coli. R Y 13 strain , and it was the first restriction enzyme isolated from E.coli.

1. Microinjection. Microinjection is the process/technique of introducing foreign genes into a host cell by injecting the DNA directly into the nucleus by using microneedle or micropipette.

2.Use of specific concentraion of divalent cations. specific concentration of divalent cations improves the efficiency of the cells to take up DNA. The divalent cation make the cells competent and thus recombinant DNA can be forced into the cell.

3. Gene Gun or biolistic is the technique of bombarding the cells with microprojectiles (gold or tungsten particles) coated with the foreign DNA with great velocity .

(a) It should be able to replicate autonomously. It should have an Origin of replication.

(b) It should have selectable markers so that it can be easily isolated .

(c) It should have recognition site preferably a single site for restriction enzymes so that introduction of DNA is easy.

Good cloning vectors have certain features -

(i) Origin of replication (Ori) so that it can replicate autonomously.

(ii) Selectable marker- so that isolation of the transformed cell is easy.

(iii) Cloning (Recognition) site so that DNA can be inserted into it.

 (iv) Small size of vector.

2. Insertion of the isolated gene in a suitable vector.

3. Introduction of vector into a suitable organism / host cell.

4. Selection of transformed host cells.

5. Multiplication or cloning of the introduced gene in the host organism.

PCR is Polymerase Chain Reaction. It is used to amplify the DNA that is make multiple copies of the desired DNA  'in vitro' using DNA polymerase and two  sets of primers.

Three main  steps of PCR are- 
1. Denaturation- where the DNA strands are heated so that the two strands separate and form single strands to which primer can attach.
2. Primer annealing - in this step the primer attach to the complementary strands on the single stranded DNA.
3. Extension of primers- DNA polymerase extends the primers and forms double stranded DNA.

The three steps of PCR:
1.Denaturation.
2. Primer annealing
3. Extension of primers.

2. It is used in genetic fingerprinting and forensics.

3. PCR can be used for DNA sequencing .

4. It is used for detecting diseases.

1. Plasmids. 
2. Bacteriophages.
3. Plant vectors like Agrobacterium tumefaciens and animal vectors likeand retroviruses.
 4.Artificial chromosomes of bacteria and yeast like BACs and YACs.

Plasmid  can be used as a cloning vector in genetic engineering. Since they have the capacity to replicate autonomously (independently) and may have a high copy number , foreign DNA can be linked to it and multiple copies of the desired gene can be obtained. 

1. The bacterial cells are made competent to take up recombinant DNA by treating them with a specific concentration of calcium, that increases the efficiency with which DNA enters the cell through the pores in its cell wall. 

2. Microinjection is a method in which the recombinant DNA is directly injected into the nucleus-of the animal cell with the help of mirco-needles or micropipettes.

3. Gene gun or biolistics is a method suitable for plant cells, where plant cells are bombarded with high-velocity microparticles of gold or tungsten coated with DNA.

4. Disarmed pathogens can be  allowed to infect the host cell,  which then transfer the recombinant DNA into the host.

5. Electroporation in which electricity is applied to create transient pores in the host cell so that it takes up Recombinant DNA with ease.

Action of restriction enzyme

EcoRI cuts the DNA between bases G and A only when the sequence GAATTC is present in the DNA.

Steps in formation of recombinant DNA by action of restriction endonuclease enzyme - EcoRI

Recombined DNA technology involves the following steps :

(i) Isolation of DNA.

(ii) Cutting of DNA by restriction endonucleases.

(iii) Isolation and amplification of the gene of interest.

(iv) Ligation of the DNA fragment into a vector using DNA ligase.

(v) Transfer of DNA fragment into host cell.

(v) Culturing of host cell and extraction of the recombinant product.

(i) Denaturation in which the double-stranded DNA is denatured by using high temperatures

(ii) Primer annealing - in this step  two sets of primers are used that are complementary to the segment of DNA of interest. the lower temperature favors the attachment or annealing of primer to the DNA strands.

(iii) Extension of primers in which DNA polymerase enzyme is used to make copies of DNA . 

Features  required  in vectors that facilitate cloning  are :
1. Origin of replication (Ori) which helps in autonomous replication and also controls the copy number.
2. Selectable marker which helps in isolation of transformed cell.
3. Cloning sites which provide sites at which foreign DNA can be inserted . These sites also help in selection of recombinants.
4. Vectors like Agrobacterium tumefaciens ( in plants) and retroviruses (in animals) can be made non pathogenic and be used to fascilitate cloning.

Fig. 7.4. E. coli Cloning Vector pBR322 showing restriction sites (Hindlll, EcoRI, BamHI, Sal I, Pvu II, Pst I, Cla l), oriV and antibiotic resistance genes (ampR and tetR). Rop codes for the proteins involved in the replication of the plasmid.

(b) Importance of amp^R. It is antibiotic resistance gene for ampicillin. It serves as a cloning sites where foreign DNA can be inserted. It may also helps in selection of transformants. 

(c) Importance of rop. It codes for the proteins involved in the replication of plasmid.

Cloning vectors are used for propagation of DNA inserts in a suitable host . Examples of cloning vectors are
 pBR322 an E.coli cloning vectors and Agrobacterium tumefaciens a plant cloning vector.

Bioreactors are vessels in which raw materials are biologically converted into specific products, using microbial, plant, animal or human cells or individual enzymes.
Role. A bioreactor provides the optimal conditions for achieving the desired product by providing optimum growth conditions (temperature, pH, substrate, salts, vitamins, oxygen). They aid in the production of desired products in large quantities. One of the most commonly used bioreactor is of stirring type.


Fig.(a) Simple stirred-tank bioreactor (b) Sparged stirred-tank bioreactor through which sterile air bubbles are sparged.

(b) Bioreactor are vessels in which raw materials are biologically converted into specific products using microbial, plant or human cells. It provides optimal conditions for achieving the desired product by providing optimum growth conditions, pH, substrate salts, vitamins, oxygen, etc.

(c) Downstream processing.are the series of processes which a genetically modified product undergoes before it is ready to be marketed. It generally involves processes like  separation and  purification.

(i) They provide optimum growth conditions (temperature, pH, substrate, salts, vitamins, oxygen) to achieve the desired product.

(ii) They are cost effective .

(iii) Due to baffles, oxygen transfer rate is very high

(iv) Capacity of stirred tank is more as compared to shake flasks.

(b) Restriction enzymesare  called “molecular scissors” or chemical scalpels. Restriction enzymes, synthesized by micro-organisms as a defence mechanism which can cleave double-stranded DNA at specific sites called recognition sequences. The recognition sequence is a palindrome, where the sequence of base pairs reads the same on both the DNA strands, when the orientation of reading is kept the same.

(c) Chitinase. are hydrolytic enzymes used  to degrade chitin . It is used to break the cell open to release DNA along with other macromolecules such as RNA, proteins, polysaccharides and also lipids.

(b) Difference between exonuclease and endonuclease

The technique is based on the principle that - when a charged molecule is placed in an electric field they move towards the positive or negative side according to their charge.
When electric field is applied the DNA fragments being negatively charged,  move through the gel towards anode and gets seperated.

The DNA fragments are stained with a compound known as ethidium bromide and they are then exposed to UV-radiation. the DNA can be observed as Bright orange coloured bands .

Restriction endonuclease provide protection to bacteria against bacteriophages. They are used in genetic engineering as they  cut DNA at specific sequence and form recombinant molecules.

Mechanism of their work
 Restriction endonucleases inspect the length of DNA sequence and find specific recognition sequence i.e. Palindromic nucleotide sequence in DNA. These enzymes cut the double stranded DNA a little away from centre of these sites but between the same two  bases and  leave overhanging stretches called sticky ends on each strand.

The steps to isolate DNA in purified form are -:

1.The cell is broken up to release the DNA molecules along with other macromolecules such as RNA, proteins, polysaccharides and also lipids. This is done  by treating the bacterial cells/plant or animal tissue with enzymes such as lysozyme (bacteria), cellulase (plant cells), chitinase (fungus).
2. The RNA is  removed by treatment with ribonuclease.
3. The proteins are  removed by treatment with protease.
4.Ethanol is added to precipitate the DNA.
5. The purified  DNA can be collected in the form of fine threads in the suspension.

A vector or vehicle DNA is used as carrier for transferring desired DNA into host cells.  They have the ability to replicate independently in the host cell and produce copies of the desired DNA.
Examples A plasmid can be used as vector because it can replicate automously and make several copies of itself.
Artificial chromosomes from bacteria and yeast called BACs and YACs can also be use as vectors and are more efficient for eukaryotic gene transfers. 

Fig. Plasmid and Yeast Artificial Chromosome

Bioreactors can be used to harbour cloned genes of interest and the cultures can be used for extracting the desired recombinant protein. Bioreactors  help in the large scale productionand  provide optimal conditions for achieving the desired recombinant protein by biological methods. 

C.

D.

D.

A.

D.

C.

A.

C.

C.

D.

C.

B.

DNA recombination takes place during the pachytene stage of the prophase-I of Meiosis-I.

The naming of a restriction endonuclease is done in the following way:

i)     I^st letter represents the genus of the organism from which the enzyme is derived.

ii)    II^nd and III^rd letters represent the species of the organism.

iii)  IV^th letter represents the name of the strain.

iv)  Roman number represents order in which the enzyme was isolated.

For example, In EcoRI is Derived from genus Escherichia and species coli of, strain R.
It is the 1^st to be discovered.

In RNA interference (RNAi), a complementary RNA binds to the mRNA to form a double stranded RNA that cannot translate. Hence, its expression is blocked, this phenomenon is also known as Silencing. RNAi is a method adopted to prevent infestation of roots of tobacco plants by a nematode Meloidogyne incognita.

(a)  A represents - Sticky end.

      B represents - Foreign DNA insert.

(b) The term used for A is Sticky ends, it is called so because they form hydrogen bonds with their complementary cut counterparts. Their stickiness of the ends facilitates the action of the enzyme DNA ligase.

C are called the palindromic nucleotide sequence. These are named so because the sequence of base pairs reads same on the two strands when orientation of reading is kept the same

(c) PCR stands for Polymerase Chain Reaction. PCR is a technique used to synthesize multiple copies of the gene (or DNA) of interest in vitro using two sets of primers (small chemically synthesized oligonucleotides that are complementary to the regions of DNA) and the enzyme DNA polymerase. It is extensively used in the process of gene manipulation.

Retroviruses convert their RNA into DNA by the method of reverse transcription, thus it is possible that the DNA of the infected host possesses DNA even when retroviruses do not have their own DNA.

The replication does not initiate randomly at any place in DNA it only starts at the site of origin of replication (ORI), thus it is not possible for an alien DNA to become part of a chromosome  anywhere along its length and replicate normally .

The enzymes that are used for the isolation of DNA from bacterial and fungal cells for recombinant DNA technology are:

Lysozyme is used to isolate DNA from bacteria.

Chitinase is used to isolate DNA from fungus.

Each restriction endonuclease functions by ‘inspecting’ the length of a DNA sequence. Once it finds its specific recognition sequence, it binds to the DNA and cuts each of the two strands of the double helix at specific points in their sugar -phosphate backbones. Each restriction endonuclease recognizes a specific palindromic nucleotide sequences in the DNA.

Gel electrophoresis is the technique that is used to separate the DNA fragments formed by the use of restriction endonucleases.
In this technique the DNA fragments are separated on the basis of the charge , since the DNA molecule is negatively charged they are forced to move towards the anode under an electric field applied through the medium or the matrix in which the DNA is loaded. The matrix used is generally made of agarose.
i. The DNA fragments cut by the restriction enzymes, separate according to their size through the sieving effect provided by the agarose gel.
ii. The smaller the DNA fragment the faster it travels. Smaller segments travel more distance than larger DNA molecules. 
iii. The DNA which is usually stained with the EtBr dye or ethidium bromide,  is visualized under UV light. The DNA appear as orange bands. 

Grifith's experiment

i. Streptococcus pneumoniae (pneumococcus) bacteria were grown on a culture plate, some produces smooth shiny colonies (S) while others produces rough colonies (R). This was because the S strain bacteria has a mucous (polysaccharide) coat, while R strain does not.
Mice infected with the S strain (virulent) die from pneumonia infection but mice infected with the R strain do not develop pneumonia.
S strain ---> Inject into mice ----> Mice die
R strain ----> Inject into mice ----> Mice live
The virulet S strain bacteria were killed by heating them. It was observed that heat killed S strain bacteria injected into mice did not kill them.
S strain (heat-killed) ----> Inject into mice ----> Mice live
When a mixture of heat-killed S and live R bacteria was injected, the mice died.
S strain (heat-killed) + R strain (live) ----> Inject into mice ----> Mice died. Moreover, living S bacteria was recovered from the dead mice.
The experment concluded that the R strain bacteria had somehow been transformed by the heat-killed S strain bacteria and that some ‘transforming principle’, transferred from the heat-killed S strain, had enabled the R strain to become virulent. It was thought that this must be due to the transfer of the genetic material.

b.  Prior to the work of Oswald Avery, Colin MacLeod and Maclyn McCarty it was thought that the genetic material was a protein. They worked to determine the biochemical nature of ‘transforming principle’ in Griffith’s experiment. They purified biochemicals (proteins, DNA, RNA, etc.) from the heat-killed S cells to see which ones could transform live R cells into S cells.  They discovered that protein-digesting enzymes (proteases) and RNA digesting enzymes (RNases) did not affect transformation, so the transforming substance was not a protein or RNA. Digestion with DNase did inhibit transformation, suggesting that the DNA caused the transformation. They concluded that DNA is the hereditary material and that DNA alone from S bacteria caused R bacteria to become transformed.

Taq polymerase is obtained from the bacterium Thermus aquaticus. It is a thermostable DNA polymerase enzyme and remains active even remain active during the high temperature induced denaturation of double stranded DNA in a PCR reaction.

Polymerase Chain Reaction or PCR is used to amplify the gene of interest. In this method -:

Primers are designed according to the sequence of the gene of interest.

 Two sets of primers (chemically synthesized oligonucleotide stretches) that are complementary to the gene of interest, DNA polymerase enzyme, and deoxynucleotides are added to the PCR mixture.

PCR consists of 3 steps:

(a) Selectable markers in the coming vectors pBR322 :

i. Ampicillin resistance antibiotics
ii.Tetracycline
(b) The selection of recombinants due to inactivation of antibiotics is a difficult process and requires simultaneous plating on two plates having different antibiotics. Thus, to differentiate between the non-recombinants and recombinants The enzyme  - galactosidase is preferred as a selectable marker as it allows the person to differentiate the non-recombinants from recombinants. in this case when the recombinant DNA is inserted into the coding sequence of the enzyme -galactosidase. The insertion leads to the inactivation of the enzyme and hence in the presence of a chromogenic substance the recombinants do not give coloured colonies, while the non-recombinants produce blue colonies. Thus no separate plating is required. 

The researcher can use the method Electrophoresis (Gel electrophoresis to separate the fragments of the DNA. In this method, the DNA fragments are loaded into the well made in the gel and separated according to their size under the influence of an electric current.

The technique to obtain multiple copies of a gene is PCR - Polymerase Chain rection.
It invoves the following steps.
Denaturing – The double-stranded template DNA is heated to separate it into two single strands.

Annealing – The temperature is lowered to enable the DNA primers to attach to the template DNA.

Extending – The temperature is raised and the new strand of DNA is made by the Taq polymerase enzyme.

These three stages are repeated 20-40 times, doubling the number of DNA copies each time.

VNTR stands for Variable Number Tandem Repeat.
VNTR is a small fragment of DNA containing tandemlyy repeated sequence, whose number and length vary among chromosome and individuals. VNTR shows a high level of polymorphism and is specific for different individual, whereas Probe is a small fragment of DNA or RNA used for identification of genes in biological system. These fragments are prepared for commercial and hybridization technology either for finding complementary sequence or in the diagnosis of some diseases. 

Given image is a stirred-tank reactor.
The stirred tank reactor is used for:
1. To facilitate mixing of reactor content and oxygen.
2. It has an agitator system, an oxygen delivery system and a foam control system, a temperature control system, pH control system and sampling ports so that small volumes of the culture can be withdrawn periodically. 

A. Characteristics features of cloning vector
1. Presence of Origin of replication (ori) - The vector should have an ori which is a sequence from where replication starts. Any piece of DNA which is linked to the ori can be made to replicate within the host cells. The ori site also controls the copy number so cloning vectors having ori that support a high copy number are chosen.
2. Selectable marker – The vector should possess a selectable marker which helps to distinguish and identify the non-transformants from transformants and selectively permit the growth of transformants. Genes encoding for antibiotics like ampicillin, tetracycline, chloramphenicol or kanamycin are considered to be useful selectable markers for E.coli.
3. Cloning Sites – The cloning vector should contain a single recognition site for the restriction enzymes in order to link the alien DNA. The presence of more than one recognition sites generates several fragments and complicates the cloning process.
4. It should have a high copy number so that we obtain many copies of the DNA linked to it
5. They should be able to replicate independently.
6. They should help easy linking of alien DNA.
B. DNA cannot pass through the cell membranes as it is a hydrophilic molecule. The cell membrane is made up of lipid bilayer which makes it difficult for the hydrophilic DNA molecule to pass through it.

Bacterial cells are made competent to take up recombinant DNA from the medium in the following ways-:
1. Treating bacteria with specific concentration of divalent cation such as calcium that results in an increase in the efficiency with which DNA is taken up by the bacterial cell. Recombinant DNA can then be forced into such cells by incubating them with the recombinant DNA on ice followed by placing them briefly at 42°C (heat shock), and again back on the ice.
Some other methods available for introduction of desired DNA are: 
1. Microinjection in which the recombinant DNA is directly injected into the nucleus of an animal cell.
 
2. Biolistics or gene gun method is suitable for plant cells, where cells are bombarded with high-speed micro-particles of gold or tungsten coated with DNA.

I. Restriction enzymes - Cutting of the desired gene at a specific location is attained by using restriction endonucleases. These enzymes are specialised to cut the fragment of DNA at specific locations. Each restriction endonuclease functions by ‘inspecting’ the length of a DNA sequence. Once it finds its specific recognition sequence, it binds to the DNA and cuts each of the two strands of the double helix at specific points in their sugar -phosphate backbones. Each restriction endonuclease recognises a specific palindromic nucleotide sequence (sequence of base pairs that reads same on the two strands when the orientation of reading is kept the same) in the DNA. Restriction enzymes cut the strand of DNA a little away from the centre of the palindrome sites, but between the same two bases on the opposite strands. This leaves single-stranded portions at the ends.

II. Polymerase Chain Reaction - PCR is used to create multiple copies of the DNA being incorporated with molecular biology tools to obtain a higher copy of the desired gene. Two sets of chemically synthesised oligonucleotides and DNA polymerase are being used in vitro for the multiplication of the desired gene. 

The PCR  produces approximately billion copies of a gene in less than 20 minutes. Such higher number of the product is achieved by use of thermostable DNA polymerase (isolated from a bacterium, Thermus aquaticus. 

Components used during PCR - Template DNA, DNA polymerase, Primers and buffer. 

Steps of PCR – • 

Denaturation - The  DNA is denatured that is the strands are separated by heating the dsDNA to 95-degree celsius. This breaks hydrogen bonds that hold DNA strands together in the helix. •

Annealing- The mixture is cooled, this allows the primer to bind to their respective complementary sequence. •

 Extension- The mixture is then heated so that DNA polymerase can synthesise new strands.

The whole cycle is repeated to create multiple copies.

During gamete formation, the homologous pair of autosomes gets separated from each other and moves to different gametes, so that each gamete receives haploid set of chromosomes.

A nematode Meloidogyne incognita infects the roots of tobacco plants and causes a great reduction in yield. A novel strategy was adopted to prevent this infestation which was based on the process of RNA interference (RNAi).

Using Agrobacterium vectors, nematode-specific genes were introduced into the host plants. The introduction of DNA was such that it produced both sense and antisense RNA in the host cells. These two RNA’s being complementary to each other formed a double-stranded (dsRNA) that initiated RNAi and thus, silenced specific mRNA of the nematode.

The consequence was that the parasite could not survive in a transgenic host expressing specific interfering RNA. The transgenic plant, therefore, got itself protected from the parasite.

Small volume cultures cannot yield appreciable quantities of products. To produce in large quantities, the development of bioreactors, where large volumes (100-100 litres) of culture can be processed, was required. Thus, bioreactors can be thought of as vessels in which raw material are biologically converted into specific products, individual enzymes, etc., using the microbial plant, animal or human cells.

The most commonly used bioreactors are of stirring type. A stirred - tank reactors is usually cylindrical or with a curved base to facilitate the mixing of the reactor contents. The stirrer facilitates even mixing and oxygen availability throughout the bioreactor. The bioreactor has an agitator system, an oxygen delivery system and a foam control system, a temperature control system. pH control system and sampling ports so that small volumes of the culture can be withdrawn periodically.

(a) Restriction enzymes :

1. Restriction enzymes belong to the class of enzymes nucleases which breaks nucleic acids by cleaving their phosphodiester bonds.
2. Since Restriction endonucleases cut DNA at specific recognition site, they are used to cut the donor DNA to isolate the desired gene.
3. The desired gene has sticky ends which can be easily ligated to cloning vector cut by same restriction enzymes having complementary sticky ends to form recombinant DNA
4. An example is EcoR1 which is obtained from E.coli bacteria “R” strain which cuts DNA at specific palindromic Recognition site.
   5‘ GAATTC 3‘
   3‘ CTTAAG 5‘

(b) Plasmids :

1. Plasmids are autonomous, extrachromosomal circular double-stranded DNA of bacteria
2. Since they are small and self replicating,they are used as cloning vectors in genetic engineering.
3. Some plasmids have antibiotic resistance genes which can be used as marker genes to identify recombinant plasmids from non-recombinant ones.
4. The plasmids are cut and ligated with desired genes and transformed into a host cell for amplification to obtain the desired products.
5. An example of artificially modified plasmids is pBR322 ( constructed by Bolivar and Rodriguez) or pUC (constructed at university at California).

D.

Hind II

Hind II is a restriction endonuclease are enzymes used for cutting DNA at specific locations.

C.

Single stranded

Plasmid is extrachromosomal double stranded circular DNA. 

D.

Thermus aquaticus

Taq polymerase is a thermostable DNA polymerase obtained from Thermus aquaticus. 

B.

polymerase chain reaction

C.

electrophoresis

DNA fragments generated by the reaction is simply DNA replication in a test tube. Restriction mapping is the process of obtaining structural information on a piece of DNA by the use of restriction enzymes, e.g., endonucleases that recognise specific 4 to 8 base regions of DNA.

C.

Algae - Methylase

In algae, the cell wall is made up of cellulose degraded by cellulase enzyme. Bacteria have lysozyme, plants cells have cell wall degraded by cellulose and fungi have chitinase.

C.

Insertional inactivation of α-galactosidase in recombinant bacteria

The colonies of recombinant bacteria appear white in contrast to blue calories of non-recombinant bacteria because of insertional inactivation of alpha-galactosidase in recombinant bacteria. Alpha-galactosidase is a glycoside hydrolase enzyme that hydrolyses the terminal alpha-galactosyl moieties from glycolipids and glycoprotein. It in encoded by theGLA gene. β-galactosidase is an exoglycosidase, which hydrolyzes the β-glycosidic bond formed between a galactose and its organic rhoiety.

A.

Restriction enzymes

Restricition enzymes are DNA cutting enzymes found in bacteria. A restriction enzyme recognizes and cuts DNA only at a particular sequence of nucleotides. For example, the bacterium Haemophilus aegyptius produces an enzyme named Hae III that cuts DNA wherever, it encounters the sequence.
5'-G G C-3'
3'-C C G G-5'

A.

Infection of the plant by agrobacterium tumefaciens

Agrobacterium tumefaciens, a pathogen of several dicot plants is able to deliver a piece of DNA known as tDNA to transform normal plant cells into a tumour and direct these tumour cells to produce the chemicals required by the pathogen. 

B.

Vector

The DNA molecule to which the gene of interest is integrated for cloning is called vector. It is a DNA molecule used as a vehicle to artifically carry foreign genetic material into another cell, A vector containing foreign DNA is termed as recombinant DNA.

D.

amp^R , tet^R -antibiotic resistance genes

Ori represents the site of origin of replication, rop represents the proteins that take part in the replication of plasmid. Hind III, Eco RI are the recognition sites of restriction endonucleases. amp^R and tet^R are the antibiotic resistant gene parts. 

B.

Phellogen

The cork cambium or phellogen cells divide periclinally cutting off cells towards the outside and inside. The cells cut off towards the outside become suberized and dead. These are compactly packed in radial rows without intercellular spaces and form cork or phellem. Cork is impervious to water due to suberin and provides protection to underlying tissues. The cells cutt off from cork cambium towards inside add to the cortex and are called secondary cortex cells or phelloderm cells.

B.

Vector DNA - Site for tRNA synthesis

A vector is a DNA molecule used as a vehicle to transfer foreign genetic material into desired cell.
the tRNA is synthesised in the nucleus on a DNA molecule template. Only 0.025% of total DNA content codes for tRNA.

D.

gold or tungsten

Biolistics or gene gun is a direct or vectorless way used to introduce alien DNA into host cells. In this method of gene transfer, high velocity micro-particles of gold or tungsten, coated with DNA are bombarded on the plant cells. 

B.

Transformation of plant cells

In biolistic or gene gun method 1-2 g tungsten or gold particle (called microprojectile) coated with DNA to be used for transformation are accelerated to velocities which enable their entry into plant cells/nuclei. This technique has general applicability to plant species and can be used to deliver DNA into virtually all tissues.

B.

to select healthy vectors

The integration of selectable marker gene along with a gene of interest is helpful for identifying transformed cells. However, the presence of selectable marker for antibiotic resistance in a genetically modified organism that is released into environment is not desirable.

B.

5´ - GAATTC - 3
3´ - CTTAAG - 5´

In a palindrome, the base sequence in the second half of a DNA strand is the mirror image of sequence in first half. But in palindrome with rotational symmetry the base sequence in first half of one strand is mirror image of second half of complementary strand, e.g., 
5´ - GATACC - 3´
3´ - CCTAAG - 5´

C.

Genetic Engineering Approval Committee

Genetic engineering Approval Committee regulates reserach and safety of Genetically Modified (GM)  organisms for public services in India. This committee is governed by the Ministry of Environment, Forest and Climate Change (MOEFC).

C.

Palindromic sequence of base pairs

C.

coli

Restriction endonuclease recognizes a particular palindromic sequence and degrades the same. It was so, called because it restricted the growth of bacteriophase in the bacterium (e.g. E. coli). The convention for naming these enzymes is the first letter of the name comes from the bacterial genus; the second two letters come from the species, and the fourth letter from strain, e.g., numbers following the names indicate the order in which the enzymes were isoalted.

C.

gel electrophoresis

Gel electrophoresis is a technique to separate fragments of DNA. Since DNA fragments are negatively charged molecules they can be separated by forcing them to move towards the anode under an electric field through a medium/matrix. Now-a-days the most commonly used matrix is agarose which is a natural polymer extracted from seaweeds (e.g., Gelidium, Gracilaria, Gigartina, etc.).

C.

A - Lecithin - a component of cell membrane

Lecithin is a phospholipid composed of choline and inositol. It is found in all living cells as a major component of cell membrane.

A.

mice 

animals that have had their DNA manipulated to possess and express an extra (foreign) gene are known as transgenic animals. e.g, rats,rabbits, pig, sheep, cows, fish etc. Over 95% of all eisting transgenic animals are mice.

A.

Escherichia Coil

The bacterium E. coil is a prokaryote.

D.

heterosis

The superiority of hybrids over either of the parents (dominant or recessive is called hybrid vigour (G. shull). Heterosis is equivalent of hybrids vigour.

A.

binds with epithelial cells of midgut of the insect pest ultimately killing it

Soil bacterium Bacillus thuringuensis (Bt) possesses is a gene family cry gene (1-40) over its plasmid, which forms endotoxic proteins, cry protein (protein crystals) during a particular phase of its growth. These crystals contain a toxic insecticidal protein.This toxin, however, does not kill the Bacillus.Actually, the BT toxin protein exists as an inactive toxin, it is converted into an active form of toxin due to the alkaline pH of the gut which solubilizes the crystals. The activated toxin binds to the surface of midgut epithelial and lysis, and eventually cause the death of the insect.

C.

bioinformatic 

Human Genome Project (HGP) was closely associated with  rapid development of the new area in biology called a Bioinformatics, the science of collecting and analysing complex biological data such as genetic codes. 

A.

insect - resistance

The genetically modified brinjal or Bt brinjal has the same Cry - LAC gene from Bacillus thuringiensis as cotton. The gene from Bacillus thuringiensis as cotton. The gene is supposed to make the plant tolerant to the shoot and fruit borer insect, which attacks it throughout its life cycle.

A.

make cuts at specific positions within the DNA molecule

Restriction endonucleases recognise a specific DNA base sequence (recognition sequence of the recognition site, restriction sequence or restriction site) and cleave both the strands of DNA at or near the site. The enzyme cuts are DNA, generating restriction fragments wit overhanging ends or blunt ends.

D.

availability of oxygen throughout the process

The most common type of aerobic bioreactor in use today is the stirred tank- reactor, which may feature a specific internal configuration designed to provide a specific circulation pattern. The stirred tank bioreactor have been designed for the availability of oxygen throughout the processes.

A.

The inactive protoxin gets converted into active form in the insect gut

Bacillus thuringiensis toxin is an inactive protoxin, which gets converted into an active form in the insect gut. It works as an insecticide.

B.

UAG, UGA - stop

The group of nucleotides that specifies one amino acid is a code word or condo. The nucleotides of mRNA are arranged as a linear sequence of codons, each London consisting of three successive nitrogenous bases. Three codons UAG, UAA and UGA are the chain stop or termination codons. They do not code for any of the amino acids.
In most organisms AUG codon is the start or initiation codon, They do not code or any of the amino acids.
In most organisms AUG codon is the start or initiation codon, ie, the polypeptide chain starts either with methionine or N-formylmethionine.
Leucine - UUA, UUG, CUU, CUC, CUA, CUG

Alanine - GUC, GCC, GCA, GCG
GUU - Valine
UCA - Serine

D.

A: Perilymph
B: Tectorial membrane
C: Endolymph

B.

certain types of bacteria

Lysozyme is an enzyme that breaks down bacterial cell walls and provides protection against bacterial invasion in the skin, mucous membranes and many body fluids. It is found in tears, sweat and saliva.

B.

cuts the DNA molecule at specific sites

A.

Selectable marker

D.

Ethidium bromide

Ethidium bromide is used to stain the DNA fragments and will appear as orange coloured bands under UV light.

C.

Oxidative phosphorylation takes place in outer mitochondrial membrane

Oxidative phosphorylation takes place in inner mitochondrial membrane.

B.

Biopiracy

Biopiracy is a term used for or refers to the use of bioresources by multinational companies and other organisation without proper authorisation from the countries and people concerned with compensatory payment.

C.

Genetic Engineering Appraisal Committee (GEAC)

Indian Government has setup organisation such as GEAC (Genetic Engineering Appraisal Committee) which will make decisions regarding the validity of GM research and safety of introducing GM-organism for public services.

C.

Denaturation, Annealing, Extension

PCR is based on three steps:
(i) Denaturation: denaturation of the template inot single strands.
(ii) Annealing: annealing of primers to each original strand for new strand synthesis.
(iii) Extension:extension of new DNA strands from the primers.