Elemental composition of proteins

 Elemental composition of proteins

Proteins are predominantly constituted by five major elements in the following

proportion.

Carbon(C):👉       50 – 55%

Hydrogen(H):👉    06 – 7.3%

Oxygen(O): 👉       19 – 24%

Nitrogen(N):👉       13 – 19%

Sulfur(S):    👉          0 – 4%

Besides the above, proteins may also contain other elements such as P, Fe, Cu,

I, Mg, Mn, Zn etc.

Function of lipids

Functions of lipids:-

Lipids perform several important functions

1. They are the concentrated fuel reserve of the body.

2. Lipids are the constituents of membrane structure and regulate the membrane

permeability (phospholipids and cholesterol).

3. They serve as a source of fat soluble vitamins (A, D, E and K).

4. Lipids are important as cellular metabolic regulators (steroid hormones and

prostaglandins).

5. Lipids protect the internal organs, serve as insulating materials and give shape

and smooth appearance to the body

Classification of lipids

 Classification of lipids:-

Lipids are broadly classified (modified from Bloor) into simple, complex,

derived and miscellaneous lipids, which are further subdivided into different

groups

1. Simple lipids : Esters of fatty acids with alcohols. These are mainly of two

types

(a) Fats and oils (triacylglycerols) : These are esters of fatty acids with

glycerol. The difference between fat and oil is only physical. Thus, oil

is a liquid while fat is a solid at room temperature.

(b) Waxes : Esters of fatty acids (usually long chain) with alcohols other

than glycerol. These alcohols may be aliphatic or alicyclic. Cetyl

alcohol is most commonly found in waxes. Waxes are used in the

preparation of candles, lubricants, cosmotics, ointments, polishes etc.

2. Complex (or compound) lipids : These are esters of fatty acids with alcohols

containing additional groups such as phosphate, nitrogenous base,

carbohydrate, protein etc. They are further divided as follows

(a) Phospholipids : They contain phosphoric acid and frequently a

nitrogenous base. This is in addition to alcohol and fatty acids.

(i) Glycerophospholipids : These phospholipids contain glycerol as

the alcohol e.g., lecithin, cephalin.

(ii) Sphingophospholipids : Sphingosine is the alcohol in this

group of phospholipids e.g., sphingomyelin.

(b) Glycolipids : These lipids contain a fatty acid, carbohydrate and

nitrogenous base. The alcohol is sphingosine, hence they are also

called as glycosphingolipids. Glycerol and phosphate are absent e.g.,

cerebrosides, gangliosides.

(c) Lipoproteins : Macromolecular complexes of lipids with proteins.

(d) Other complex lipids : Sulfolipids, aminolipids and lipopolysaccharides

are among the other complex lipids.

3. Derived lipids : These are the derivatives obtained on the hydrolysis of group

1 and group 2 lipids which possess the characteristics of lipids. These include

glycerol and other alcohols, fatty acids, mono-and diacylglycerols, lipid (fat)soluble vitamins, steroid hormones, hydrocarbons and ketone bodies.

4. Miscellaneous lipids : These include a large number of compounds

possessing the characteristics of lipids e.g., carotenoids, squalene,

hydrocarbons such as pentacosane (in bees wax), terpenes etc.

Functions of carbohydrates

Functions of carbohydrates:-

1. They are the most abundant dietary source of energy (4Cal/g) for all

organisms.

2. Carbohydrates are precursors for many organic compounds (fats, amino

acids).

3. Carbohydrates (as glycoproteins and glycolipids) participate in the structure of

cell membrane and cellular functions such as cell growth, adhesion and

fertilization.

4. They are structural components of many organisms. These include the fiber

(cellulose) of plants, exoskeleton of some insects and the cell wall of

microorganisms.

5. Carbohydrates also serve as the storage form of energy (glycogen) to meet the

immediate energy demands of the body.

cryopreservation

 Cryopreservation

Why Preservation is important?

• Until tow decades ago the genetic resources were getting depleted owing to the continuous depredation by man.
• It was imperative therefore that many of the elite Economically important and endangered species are preserved to make them available when needed.
• The conventional methods of storage of storage failed to prevent losses caused to various reason.
• A new methodology had to be devised for long term preservation of material.

There are various methods of storage

1. Cryopreservation:- Generally involves storage in liquid Nitrogen.
2. Cold storage:- It involve storage in low and non freezing temperature.
3. Low pressure:- It involves partially reducing the atmospheric pressure of surrounding.
4. Low oxygen storage:- It involves reducing the oxygen level but maintaining the pressure.

Cryopreservation;-

• Cryo is greek word, (Kroyes -frost)
• It literally means preservation in "frozen state"

The principle:- to bring plant cells or tissue to a zero metabolism and non dividing state by reducing the temperature in the presence of cryoprotectant.

It can be done:-

• Over solid carbon dioxide (at-79°C)
• Low temperature deep freezer (at-80°C)
• In vapor phase nitrogen (at-150°C)
• In liquid nitrogen (at-196°C)

Major advantages are:-

• Once the material is successfully conserved. Particular temperature it can be preserved indefinitely.
• Once in storage no chance of new contamination of fungus or Bacteria.
•  Minimal space required.
• Minimal labor required.

Mechanism of Cryopreservation:-

 

                             There cryopreservation technique followed by the regeneration of plants involves following steps:-

1. Selection of of material.
2. Addition of Cryoprotectant.
3. Freezing
4. Storage in liquid nitrogen.
5. Thawing.
6. Washing and reculturing.
7. Measurement of viability
8. Regeneration of plants.

1. Selection of plant material:-

             Two important factors depends on it such as 

                          a. Nature and

                          b. Density

      Any tissue can be selected for this purpose e.g., meristem, embryo, ovules, seeds, etc.

The density should be high.

2.Addition of Cryoprotectant:-

• They are chemical which prevent cryodestruction.
• These are sucrose, alcohol's, glycols, Some amino acid (proline), DMSO (dimethyl sulfoxide)
• Generally tow cryoprotectant should be used together instead of single one as they are more effective.

3. Freezing:-

      

               The sensitivity of cells to low temperature depends on the plant species.

  There are four different types of Freezing:-

• Slow freezing method:- The tissue or plant material is slowly frozen at slow cooling rate. The advantage is the plant cells are partially dehydrated and survive better.
• Rapid freezing method:- It involves pluming the vials in liquid nitrogen. The temperature decreases from -300°C to -1000°C  rapidly.
• Rapid freezing method:- This is combination of both slow and rapid freezing method. The process is carried out in step wise like manner.
• Dry freezing method:- In this method dehydrated cells and seeds are stored.

4.Storage:-

• The maintenance of the frozen cells or material at specific temperature is kept -70°C to -196°C
• Prolong storage is done at temperature of -196°C in liquid nitrogen.
• To prevent damage, continuous supply of Nitrogen is done.

 

5.Thawing:-

• Usually carried out by plunging the vials. into warm water bath with vigorous swirling.
• As thawing occurs the vials are transferred to another bath at 0°C degree.

6.Washing and reculturing:-

• The preserved material is washed few times to remove the cryoprotectant.
• This material is then recultured in a fresh medium.

7.Measurement of viability

• There is possibility of death of cells due to storage stress.
• Thus viability can be found at any stage.
• It is calculated by formula:-

  No. of cells growing/no of cells thawed ✖100

8.Plant regeneration:-

• The viable seeds are cultured on non specific growth medium.
• Suitable Environment Conditions are maintained. 

                                                        Application

• It is ideal method for long term conservation of material.
• DISEASE FREE PLANT CAN BE CONSERVED AND PROPAGATE.
• Peculcitrant seeds can be maintained for long time.
• Endangered species can be maintained.
• Pollens can be maintained to increase longitivity.
• Rare germplasm and other genetic manipulations can be stored.

Micro propagation

 MICROPROPAGATION:-

                                    It has been demonstrated that a variety of plant can be conveniently propagated through the techniques of cell, tissue organ culture. This is popularly described as micro propagation.

Technique of Micro propagation:-

                          In most of cases, clone propagation is achieved by placing sterilized short tips or axillary buds on to a culture medium that is sufficient to induce formation of multiple buds. 

Following stage are involved in the method of micro propagation 

  1. Stage I involves establishment of tissue in vitro.

  2. Stage II involve multiplication of shoots.

  3. Stage III concerned root formation and conditioning of propagules prior to transfer to the greenhouse. This stage requires high intensity and alteration of media for promotion of  root formation. 

   4. Stage IV involves growth in pots followed by field trials.

Advantages of Micro propagation:-   

• Rapid multiplication of superior clones and maintenance of uniformity.                                                                                                
• Multiplication of disease free plant.                                                         
• Multiplication of sexually derived sterile hybrids.                                                      
• A wide range of plants have now been regenerated through technique of tissue culture. This technique is very useful in the    commercial production of some plants like banana, potato, orchids and Chrysanthemum etc.      

Essential amino acids

 Essential or indispensable amino acids :- 

                                                                                                              The amino acids which can not be synthesized by the body and, therefore, need to be supplied through the diet are called essential amino acids. They are required for proper growth and maintenance of the individual. The ten amino acids listed below are essential for humans,

1. Arginine, 
2. Valine, 
3. Histidine, 
4. Isoleucine, 
5. Leucine, 
6. Lysine, 
7. Methionine,
8. Phenylalanine, 
9. Threonine, 
10. Tryptophan,

Nonessential amino acids

Nonessential Amino Acid or dispensable amino acids:- 

 The body can synthesize about 10 amino acids to meet the biological needs, hence they need not be consumed in the diet. These are:-

1. Glycine, 
2. Alanine, 
3. Serine,
4. Cysteine, 
5. Aspartate, 
6. Asparagine, 
7. Glutamate, 
8. Glutamine, 
9. Tyrosine and
10.  Proline,

Recombinant DNA technology

 Recombinant DNA technology:-

Basic Functions of a Fermenter

 Basic Functions of a Fermenter:-

• It should provide a controlled environment of optimum biomass yields.
• It should permit aseptic fermentation for a number of days reliably and dependably, and meet the requirements of containment regulations Containment involves prevention of escape of viable cells from a fermenter or a downstream processing equipment into the environment.
• It should provide adequate mixing and aeration for optimum growth and production, Without damaging the microorganisms.
• The power consumption should be minimum,
• It should provide easy and dependable temperature control.
• Facility for sampling should be provided.
• It should have a system for monitoring and regulating pH of the fermentation broth.
• Evaporation losses should be as low as possible.
• It should require a minimum of labour in maintenance, cleaning, Operating and harvesting operations.
• It should be suitable for a range of fermentation processes. But this range may often be restricted by containment regulations.
• It should have smooth production internal surfaces, and joints should be welded wherever possible.
• The pilot scale and production stage fermenters should have similar geometry to facilitate scale-up.
• It should be contrasted using the cheapest materials that afford satisfactory results.
• There should be adequate service provisions for individual plants.

Ti-Plasmid

                                                           Introduction

 Agrobacterium tumefaciens is a rod shaped, gram negative Bacteria, soil born motile bacterium. This bacterium infects the parts of the plant which are in contact with soil. The genus Agrobacterium inclides four species of bacteria. They are A.tumefaciens, A.radiobacter, A.rubi, A.rhizogens. They  are pathogenic in nature and cause cancerous growth in the infected plant tissues. They infect the plants and cause tumberous growth in the infected portion. The induction of tumerous growth is due to the presence of a large sized plasmid named Ti Plasmid. Accourding to Zaenen et al. (1974) almost all the strain of A.tumefaciens contain Ti plasmid.

Agrobacterium is a bacterium which is used to produce transgenic plants because it contain tow kinds of vectors which are used in the transfer of genes of one plant to the genome of another plant:-

 1. Ti plasmid, 

 2. Ri plasmid,

                                                              Structure  

T-DNA carry genes for phytohormones, AND opine

Auxin:- The main function of auixn is to help plant grow. Auxin stimulates plant cell to elongate, and the apical meristem of a plant is one of the main places where auxin is produced.

Cytokinin:- Increased cell division by stimulating the process of mitosis. Increased formation of shoot and buds, as well as development of fruits and seeds.

Opin:- Nutritional source for agrobacteria and fall into different chemical grouping such as nopaline, Octopine, Mannopine, agrocinopine etc.

Vir genes:-

 Vir A:- Its a kinase portein in bacterial membrane. Act as receptor Phenolic compund relased by wounded plant.

Vir A and Vir G :- They expessed continuously respond to phenolic compund released by wounded plant.

Vir G :- Activate other vir gene (transcription gene factor)

Vir E/E2 :- Protect T-DNA against nuclease and target T-DNA to plant cell. Act as SSB protein (single standard binding protein).

Vir C :- Stimulate transfer/promotes high effciency T-stand Synthesis.

Vir D :- Responsible for virulence activity of bacterium.

Vir D1:- Topoisomerase helps virD2 to recognise and cleave within 25bp L.B sequence.

 Vir D2 :- Endonuclease ( vir D2 cut T-DNA right border, help to integration of T-DNA into host chromosome)

Vir D1 :- Prevent attack of exonuclease at 5' end of T-DNA, important for cutting phosphodiester bond.

Vir B/ Vir D4 :- ATP dependant linkage of  protein complex necessary for T-DNA transfer from bacterium of plant cell ( assemble into a secreation system which spns the inner and outer bacterial membrane.)

Vir D2/Vir E2 :- Have nuclear localization signal.

Vir F:- Plant cell cytosole, vib1protein.

                                                               Advantage

• Occure naturally in Bacteria.
• Have different restriction sites.
• Replication completely independent of Bacteria.
• Genes are easily inserted into plasmids.
• Easily transformed into bacteria.
  
  

what is bioinformatics

What is Bioinformatics:-

Translation of billions of characters in DNA sequences that make the genome into biologically meaningful information has given birth to a new field of science called "bioinformatics". The term bioinformatics has been derived by combining biology and information.

                          The key to biotechnology discoveries is locked in the genomes of organisms. The bioinformatics holds the key to unlock these data for the next generation of innovations.

intro germplam conservation

 

Germplasm Conservation:-

Germplasm broadly refers to the hereditary material transmitted to the offspring through germ cells.

Germplasm provides the raw material for the breeder to develop various crops. Thus, conservation of germplasm assumes significance in all breeding programmes.

As the primitive man learnt about the utility of plants for food and shelter, he cultivated the habit of saving selected seeds or vegetative propagules from one season to the next one. In other words, this may be regarded as primitive but conventional germplasm preservation and management, which is highly valuable in breeding programmes.

The very objective of germplasm conservation is to preserve the genetic diversity of a particular plant or genetic stock for its use at any time in future. In recent years, many new plant species with desired and improved characteristics have started replacing the primitive and conventionally used agricultural plants. It is important to conserve the endangered plants or else some of the valuable genetic traits present in the primitive plants may be lost.

A global body namely International Board of Plant Genetic Resources (IBPGR) has been established for germplasm conservation. Its main objective is to provide necessary support for collection, conservation and utilization of plant genetic resources throughout the world.

Cryobiology

 Cryobiology deals with the study of metabolic activates and their responses in plant materials and animal cells stored at low temperature (-196'C) by using liquid nitrogen in the presence of cryoprotectants. 

Fungal Cells

 1. Yeast:- Yeast is an unicellular eukaryotic fungus containing a small well characterized genome. Unlike plant or animal cells, it has rather fast growth rate and itself is a non-pathogenic fungus. Most of its gene contain introns which are spliced during purification of mature mRNA. It appears that intron found in yeast contain sequences for correct splicing as they are totally absent in higher eukaryotes. Moreover, yeast can carry out post-translational modification such as removal of signal sequence from a precursor polypeptide after the secretion of cell. This reveals a major advantage of yeasts over the bacteria. Success in DNA cloning in yeast depends on uptake of foreign DNA by its spheroplast in the presence of calcium ion and polyethyleneglycol (PEG). The spheroplasts develop cell wall after the incorporation of DNA.

Properties of Good Host

 A good host have the following features:-

1. Should be easy transform,                                                                                                          
2. Should support the replication of recombinant DNA,                                                                     
3. Should be free from elements that interfere with replication of recombinant DNA,                         
4. Lack active restriction enzyme, e.g. E.coli K12 sub strain HB 101,                                   
5. Should not have Methylases since these enzyme would methylate the replicated recombinant DNA which, as a result, would become resistant to useful restriction enzymes,                                                                                                                              
6. Should be deficient in normal recombination function so that the DNA insert is not altered by recombination events.                                                                                                                                                                                                                                     E.coli support several type of  vectors, some natural, constructed, which can be grouped as follows:-                                                                                                                                                                                                                                                    1. Plasmid                                                                                                                          2. Bacteriophages (both natural)                                                                                      3. Cosmids,                                                                                                                        4. Phasmids                                                                                                                      5. Shuttle vectors,                                                                                                            6. Artificial chromosomes                                                                                                  7. Phagemids,                                

Types of Restriction Endonucleases

There are the following four distinct types of restriction endonucleases:-

Type 1st restriction endonucleases:- are complex endonucleases, and have recognition sequences of about 15 bp; they cleave the DNA about 1000 bp away from the 5'-end of the sequence "TCA" located within the recognition site, e.g, EcoK, EcoB, etc.

Type 2nd restriction endonucleases:- are remarkably stable and induce cleavage either, in most cases, than 350 different type 2nd endonucleases with over 100 different recognition sequences are known. They require Mg⁺² ions for cleavage. The first type 2nd enzyme to be isolated was HindII in 1970. Only type 2nd restriction endonucleases are used for restriction mapping and gene cloning in view of their cleavage only at specific sites.

Type 3rd restriction endonucleases:- are intermediate between the type 1st and 2nd enzymes; they cleave DNA in the immediate vicinity of their recognition sites, e.g, EcoP1, EcoP15, HindIII, ect. Type 1st and 3rd restriction enzyme are not used in gene cloning. The type 3rd enzymes recognize asymmetric target sites, and cleave the DNA duplex on one side of the recognition sequence up to 20 bp away.

Steps in GENE CLONING

Steps in Gene Cloning:-

The entire procedure of gene cloning or recombinant DNA technology may be classified into the following five steps the convenience in description and on the basis of the chief activity performed.

1. Production and isolation of the DNA fragments to cloned.                     
2. Insertion of the isolated gene in a suitable vector to obtain recombinant DNA.                                                                                  
3. Introduction of the recombinant DNA into a suitable organism/cell usually we used E. coli  called host(transformation).                                                                                 
4. Selection of the transformed host cells, and identification of the clone containing the desired gene/DNA fragment.                                   
5. Multiplication/expression of the introduced gene in the host.                    
6. Where needed, transfer and expression of the gene in the another organism.                                                                                                                                   

Autoimmune Disease

 Autoimmune Disease:-                                                                                       The immune system normally produces antibodies against g foreign proteins but not against the native proteins of body, that is, the immune system can distinguish between "self" and "non-self", However, in rare cases, individuals begin to produce antibodies against their own antigens. These antibodies are called autoantibodies and the diseases resulting from their presence are the autoimmune diseases. Among these diseases are paroxysmal cold haemoglobinuria, myasthenia gravis and systemic lupus erythematous. 

structure of antibody

 Structure of antibody:-

What is sterilization and Types of sterilization

 Sterilization:-

All the materials, e.g., vessels, instruments, medium, plant material, etc., used in culture work must be freed from microbes. This is achieved by one of the following approaches:

1. Dry heat,

2. Flame sterilization,

3. Autoclaving,

4. Filter sterilization,

5. Wiping with 70% ethanol,

6. Surface sterilization,

                                                            1.Dry Heat

Glassware and Teflon plastic ware, and instruments may be sterilized by dry heat an oven at 160-180°C for 3 hours. But most workers prefer to autoclave glassware and plastic ware etc. And flame sterilize instruments like forceps, etc. More recently, glass bead sterilizers (300°C) are being employed for the sterilization of forceps, scalpels, etc. these devices use dry heat.

                                     

                                                2.Flame Sterilization

Instrumental like forceps, scalpels, needles, etc. Are ordinary flame sterilized by dipping them in 95% alcohol followed by flaming. These instruments are repeatedly sterilized during the operation to avoid contamination. It is customary to flame the mouth of culture vessels prior to inoculation.

                                                  3.Autoclaving

Culture vessels, etc. Are generally sterilized by heating in an autoclave or a pressure cooker to 121°C at 15p.s.i. (pounds per square inch 1.06kg/cm²) for 15 to 40 minutes, The time being longer for larger medium volumes. Sterilization during autoclaving depends mainly on temperature. Certain types of plastic ware and some instruments, e.g., micropipettes, etc. Are also autoclave. Care should be taken to properly stopper all the vessels and to open the autoclave only its pressure gauge indicates zero pressure. 

                                                4. Filter Sterilization

Some growth regulators, e.g., GA3, zeatin, ABA (abscisic acid), urea, certain vitamins, and enzymes are heat labile. Such compounds are filter sterilized by passing their solution through a membrane filter of 0.45μ or lower pore size. The membrane filter is held in a suitable assembly, the  assembly together with the filter is sterilized by autoclaving before use. Filter sterilized heat labile compounds are added to autoclaved and cooled media, in case of agar medium, they are added when the medium has cooled to about 40°C and is still liquefied.

Laminar flow cabinets are used to create an aseptic working space by blowing filter-sterilized air through an enclosed space. The air is first filtered through a coarse profiteer to remove large particles, it is then passed through a HEPA (high efficiency particulate air) filter, which filters out all particles larger than 0.3μm. This sterilized air blows through the cabinet at 1.8 km/hr. which is sufficient to keep the enclosed working area aseptic. 

        

                               5. Wiping with 70% Ethanol 

The surfaces that can not be sterilized by other techniques, e.g., platform of the laminar flow cabinet, hands of the operator, are sterilized by wiping them thoroughly with 70% ethyl alcohol is allowed to dry.

                                    6. Surface Sterilization

 All the plant materials to be used for culture are treated with an appropriate sterilizing agent to inactive the microbes present on their surface, This is called surface sterilization. Surface sterilization protocol will depend mainly on the source and the type of tissue of the explant is the excised piece of tissue or organ used for culture.

The sterilizing agent used for surface disinfection are calcium hypochlorite (9-10%), sodium hypochlorite (2%), mercuric chloride (0.1-1%), silver nitrate (1%), Bromine water (1-2%), H2O2 (10-12%) and antibiotics (4-50 mg/l). Of these, calcium or sodium hypochlorite and HgCl2 (satisfactory results) are the most commonly used. The duration of treatment varies form 15-30min. Since these agents are also toxic to plant tissues, the duration and the concentration used should be such as to cause minimum tissue death, and the rinsing after treatment should remove them as completely as possible.

Thanks For reading............................,