Intellectual property rights (IPR) are designed to allow novel technologies to be available so that the scientist or company receives a reward for the initiative established. Intellectual property possessions can be any codified knowledge, innovation, or anything of actual or potential economic value that has arisen from rudimentary research, analysis, and manipulation of biological systems, industrial application, or for commercial use.
The various types of biotechnological inventions may be grouped into the following-
Approaches/processes of generating useful products.
Numerous products, for example- Antibiotics, vitamins, etc.
Applications of various processes/products, for example, the use of a promoter sequence to regulate gene action.
DNA sequences and the proteins.
Strains of microorganisms, cell lines are obtained by genetic modification.
Methods for genetic modification of organisms.
Patenting of Genes and DNA Sequence–
An artificially synthesized gene is patentable in almost all countries. A patented gene holds exclusive rights to the specific DNA sequence. Once patented, the holder of the patent dictates how the gene needs to be used (whether commercially or clinically) for a minimum of 20 years from the date of the patent. In the USA, genes isolated from the organisms are patentable; gene aroA (shows glyphosate resistance) isolated from a mutant bacterium was the first to be patented. For a patent to be granted in India, it should not be covered in the negative list in Section 39 which provides an extensive list of what are not the inventions under the Indian Patents Act. The act came into force in 1972 amending the Patent act,1970.
The three conditions in order to fulfil the rules imposed by Indian patent act are :
• It should be a novel creation
• It should involve an inventive step for the mankind
• There should be various industrial applications.
Ananda Mohan Chakrabarty got the first US patent for a genetically modified organism in 1981. He discovered a method for cross-linking in such a way that it fixed all the 4 plasmids to a much stabler microbe called Pseudomonas putida capable of consuming 2-3 times faster than previous strains. Its unique characteristic was hydrocarbon degradation, therefore the name was given as “multiplasmid hydrocarbon-degrading Pseudomonas”/superbug. Prof. Chakrabarty’s momentous research has since paved the way for many patents on genetically modified micro-organisms and other life forms for the coming years.
Can Life forms be patented?
The main arguments in favouring the patent of genetically modified life forms are
-They perform novel and useful functions
-They generate economic benefits
-Their production requires large financial and technical innovative inputs
However, the chief objections are usually based on ethical, moral, and religious considerations such as
-They are products of nature and hence should not be fiddled with
-Their genetic modification does not prove an industrial invention
-The inventions cause cruelty to animals
But in 1985, a patent was granted in the USA for a maize plant by overproducing tryptophan through plant tissue culture. Later, in 1988, a genetically engineered mouse called “OncoMouse” was the first mammal to be patented. It was primarily used for cancer research. The animal designed by Philip Leder and Timothy A Stewart of Harvard University used to carry a specific gene called known as an activated oncogene. The activated oncogene increased the mouse’s resistance to cancer, and thus the mouse is a promising model for cancer research. The patenting of OncoMouse, and the extensiveness of the claims made in those patents, were well-thought-out to be unreasonable by many of their colleagues. But, amid the controversies, it was finally patented in 1992.
In the emerging field of science and technology, Biotechnology is Growing and Developing field, where new ideas and Experiments and research make this field unique and diversifying.
Biotechnology: The branch of science which uses technology with living system is biotechnology. Biotechnology uses modern system of modification of biological systems. There are many disciplines that belong to the field of Biotechnology. The development of various methods, approaches and research in this field gives a new way of approaching science and its outcomes.
Biotechnology an accidental history:
Even though we consider biotechnology to be a modern science, but it was way 1000 years back when the methods and approaches where used by our ancient people, Around 7000 years ago there was accident use of bacteria to make vinegar by Mesopotamia. Before 2,300 years Theophrastus thought that brad beans left magic in soil, but later it was concluded that some bacteria’s could fix nitrogen which enriched the soil. Development of gene banks is not a new concept, In1495 BC Queen Hatshepsut of Egypt used the concept of collecting specimens of plants which produced Frankincense (hardened gum-like material from trunk of the Boswellia sacra tree). Fermentation was always an ancient method which evolved with upcoming generation. In 19th century Sir Louis Pasteur discovered the fermenting beer using yeast. Gregor Mendel the father of genetics, was the one who believed that mathematics can be used with biology, but since his ideas and concepts were new and people considered it unbelievable was never awarded during is period.
Evolution of Biotechnology :
6000 BC :Babylonians used yeast in beer industry
320 BC : Aristotle coined the theory of inheritance from father
1630 : William Harvey explained sexual reproduction
1673: Anton van Leeuwenhoek developed Microscope., identified that these microorganism
1859: Charles Darwin Proposed Natural selection
1863 : Pasteurization discovered by Pasteur
1863 : Pasteurization discovered by Pasteur
1870: Mitosis discovered by Walter Flemming
1880: Louis Pasteur discovered weak stain of Cholera
1902: Sutton discovered that segments get transferred from Chromosomes
1906: Salvarsan was discovered by Paul Ehrlich
1907: Mutation theory by Hunt Morgan
1909:Wilhelm Johannsen Coined word genotype and phenotype
1912:William Lawrence Bragg Discovered application of X-Rays
1926: The Theory of gene by Morgan
1928: Transforming principle by Fredrick Griffith
1941: George Wells Beadle and Edward L Tatum proposed one gene one enzyme theory
1944: Selman Abraham Waksman discovered streptomycin as antibiotic
1945–1950: Animal tissue culture developed
1947: transposable elements by Barbara MacClintock
1950: Chargaff rule
1953: Double helix model by Watson and crick
1957:Crick and Gamov studied ‘central dogma
1972: First recombinant DNA molecule
1973: Ames test
1990: Human Genome Project commencement
1993: Kary Mulis developed PCR
Genentech Inc. : This Company produced somatostatin in a bacteria in 1977
Eli Lily : produced insulin using site directed Mutagenesis
Chiron crop: developed recombinant vaccine for hepatitis
Calgene Inc. : tomato polygalacturonase DNA used to synthesize antisense RNA
Novo Nordisk : focus mainly on Diabetes and hormone replacement therapy
Regeneron : Aims to develop largest gene sequencing
Alexion : develop immune-regulatory drugs
Biomarin : Develop drugs for lysosomal storage disorder
Serum Institute of India: Worlds largest vaccine manufacturer
Panacea Biotec : 3rd largest Biotech company
Scope of Biotechnology :
Since, Biotechnology shares an integrated value with many other disciplines of science , it holds a very key and vital role in the field of science. The various fields associated with biotechnology is as follows
“Biotechnology is the new brightest star in the field of techniques and Biology”- E Mithu
A biological safety cabinet is an enclosed but ventilated workspace. It is mostly used while working with contaminated pathogens. There are many levels in BSC, depending on the contaminants. There are mainly three states of protections
There are three classes for BSC based on containment capabilities
Class 1 cabinet: This is used to provide personal as well as environment protection. Biological agents should be of low to moderate risk. BSL 1,2 and 3
Class 2 cabinets: This is used to provide personnel, environment as well as product protection. Biological agents should be of low to moderate risk. BSL 1,2 and 3
Class 3 cabinet: Also known as glove box. This is used to provide personnel, environment as well as product protection .BSL 4(highly infectious agents).
BSL are standard level defined by Biosafety in Biomedical Laboratories (BMBL), Which mainly measures the protection needed in a laboratory setting to protect workers, environment and public. Biological risk assessment (BRA) is used to conduct each experimental protocol. BRA are assessments which mainly used to evaluate the following The infectious or toxins transmitted and can cause disease.Availability of medical treatmentsHealth checkup and training of lab employees
The main requirement for any given Biosafety level are Laboratory designPPE (Personnel protective equipment)Biosafety equipment
There are mainly 4 BSL
Biosafety Level 1: BSL1 is used for those infectious agents which is mainly not considered for causing health risk to healthy individuals. The procedures followed are mainly under the category of Standard Microbiological practices (SMP). There comes no specific requirement of special equipment or design features. The main necessities are cleaned surfaces, withstanding basic chemicals etc.
Biosafety Level 2: BSL2 focus on study of moderate risk infectious agents. The risk of getting infectious may be due to accidental inhalation, swallowing, exposed to cut skin etc. The specific requirement such as hand washing sinks, eye washing, automatic door and lock. There is basic requirement of equipment which can decontaminate lab waste, incinerator, and autoclave.
Biosafety Level 3: BSL 3 mainly focus on infectious agents which can be potentially lethal, when accidentally inhaled or exposed. For any research studies with biological agents whose spread can be lethal, BSL 3 is used. The controlled airflow or sealed enclosures are important for such agents. Easy decontamination, directional airflow, two self-closing or interlocked, doors, sealed windows, properly sealed wall surfaces, filtered ventilation etc. are basic necessities under the category of BSL2. There is basic requirement of equipment which can decontaminate lab waste, incinerator, and autoclave.
Biosafety Level 4: BSL 4 mainly focus on those agents which have high risk of aerosol transmission an may be life threatening disease, having no vaccines or therapy developed till date. It includes all BSL 3 features, along with it should be developed in an isolated zone. Significant training should be provided for the workers. Careful and controlled access. There are mainly two types of BSL4
In 1952, Joshua Lederberg invented the word ‘plasmid.’ It was initially formed from bacteria; plasmids are extrachromosomal genetic elements that can reproduce independently in most Archae, Eukarya species, and Eubacteria. Plasmids are double-stranded circular DNA molecules that are different from the chromosomal DNA of the cells.
The genetic material found within the chromosomal DNA guides the structure and function of a bacterial cell. In some instances, plasmids are usually not required for the host bacterium to survive. Although not necessary, by encoding functions that may not be defined by the bacterial chromosomal DNA, plasmids significantly contribute to bacterial genetic diversity and plasticity. Antibiotic tolerance and the expression of proteins, for example. The plasmid is also encoded by antibiotic resistance genes, allowing the bacteria to survive in an environment containing antibiotics, thereby providing the bacterium with a competitive advantage over species susceptible to antibiotics. Plasmids may be altered as a means to express the protein of interest for e.g., production of human insulin using recombinant DNA technology.
As a mechanism for gene-cloning and as a vehicle for gene-expression, plasmids have been central to modern recombinant DNA technology.
Isolation of Plasmids
In molecular biology, bacterial plasmid DNA isolation is a crucial technique and an integral step in many processes, such as cloning, DNA sequencing, transfection, and gene therapy. These manipulations need the isolation of high purity plasmid DNA. In all molecular biology procedures, cloning, such as digestion with restriction enzymes, PCR, transfection, in vitro translation, blotting, and sequencing, purified plasmid DNA can be used for immediate usage.
In molecular biology, alkaline lysis is used to separate plasmid DNA or other cell components, such as proteins, by splitting open the cells. Primarly, bacteria containing the plasmid of interest are allowed to grown and then lysed with an alkaline lysis buffer composed of a sodium dodecyl sulphate (SDS) detergent. Cellular debris is extracted, and the plasmid is separated and filtered through a series of steps, including agitation, precipitation, centrifugation, and the removal of the supernatant.
Purification of plasmid DNA from bacterial DNA using alkaline lysis is based on the differential denaturation of plasmid and chromosomal DNA. Disruption of the cellular structure to produce a lysate, separation of the plasmid from the chromosomal DNA, cell debris, and other insoluble material are the essential steps of plasmid isolation. With a lysis buffer solution, bacteria are lysed.
Materials and Equipment:
LB plate with Bacterial colonies
1.5 ml micro-centrifuge tubes
Autoclaved distilled water
1. Lysis Solution (Solution I)
2. Denaturation solution (Solution II)
3. Neutralizing solution (Solution III)
4. TE Buffer
6. Phenol: Chloroform: isoamyl alcohol
7. 70% Ethanol
Preparation of Stock solutions:
Solution I (Lysis solution): 50mM Glucose, 25mM Tris-Hcl (pH 8.0), 10mM EDTA (pH 8.0). Store at 4oC.
Solution II (Denaturation solution):1% SDS, 0.2N NaOH (pH 12.0). Freshly prepared and store at room temperature.
Solution III (Neutralizing solution): 60 ml of 5 M potassium acetate, 11.5 ml of glacial acetic acid, and 28.5 ml of water. Store at 4oC.
TE Buffer: 10mM Tris-HCl (pH 8.0), 10mM EDTA (pH 8.0)
Phenol: Chloroform: Isoamyl alcohol -(25:24:1).
Overnight grown culture of E.coli.
Harvesting of the cells:
The single bacterial colony is transferred into a 5 ml LB medium containing appropriate antibiotic, incubated for 16 hrs at 37oC with vigorous shaking.
About 1.5 ml culture is transferred into a microfuge tube and centrifuged at 6000 rpm for 5 min at 4oC and discard the supernatant.
Isolation of Plasmid by alkyl-lysis method:
1. The bacterial pellet is re-suspended in 100μl of ice-cold solution-I by vigorous vortexing. This is essential to ensure that the bacterial pellet is wholly dispersed in the solution I.
2. Add 200μl of freshly prepared solution II. Mix the contents by inverting the tubes rapidly 4 to 5 times. A transparent and viscous solution ensured complete lysis. Do not vortex. Incubate on ice for 3 min.
3. Add 150μl of ice-cold solution III. Mix by gently inversion for about 10 seconds to disperse the solution III through the vicious bacterial lysate. Further incubate the tubes on ice for 5 min.
4. Centrifuge at10,000 rpm for 5 minutes at 4oC and transfer the supernatant to a fresh tube.
5. To this add equal volumes of phenol-chloroform, mix by vortexing. After centrifuging at 10,000 rpm 5 minutes at 4oC, transfer the aqueous phase to a fresh tube.
6. Precipitate the double-stranded DNA by adding an equal volume of isopropanol, mix gently, and allow the mixture to stand for two minutes. Then centrifuge at 10,000 rpm for 10 minutes at 4oC.
7. Remove the supernatant by gentle aspiration.
8. Then wash the pellet with 1 mL of 70% ethanol (twice). Allow the DNA pellet to air dry for 10 minutes.
9. Re-dissolve the DNA pellet in 50μl of TE (pH 8.0).
Isolation of PLASMID by spin column method:
1. Inoculate a single colony of bacterial cells from the petri plate using a sterile inoculation loop to 5ML of LB broth and allow them to grow overnight at 37°C at 200 rpm.
2. From the pre-inoculum transfer, 1.5 ML of bacteria-containing cells into 2mL centrifuge tubes and centrifuge the cells at 8000 rpm. for one minute. Then the supernatant was discarded.
3. Re-suspend the cells in 200 µL of resuspension solution. Then mix the cells thoroughly by pipetting up and down or vortexing it.
4. To this, add 200 µL of lysis solution and mix the tubes by gently inverting up and down.do not vortex it. They were then incubated for five minutes.
5. To this, add 350 µL of the neutralizing solution and, for mixing, invert the tubes 4 to 6 times. Then Centrifuge these tubes at 10,000 rpm for 10 minutes.
6. Binding column preparation: Take a new centrifuge tube and insert a new binding or spin column into it. To this, add 500 µL of column preparation solution and spin at 10,000 rpm for one minute. Then discard the solution that was collected in the centrifuge tube.
7. Then, transfer the clear lysate to the binding column and centrifuge at 10,000 rpm for one minute. Discard the flow-through that was remained in the centrifuge tube.
8. Washing: In this step, add 750 µL of washing solution to the binding column and spin the centrifuge tube for one minute. Then discard the solution that remained in the centrifuge tube. (Do the washing step twice).
9. Then, change the spin column into the new centrifuge tube. Then spin the column for one minute and incubate at room temperature for 2 – 5 minutes.
10. ELUTION: In this step, the spin column is transferred to the fresh collecting tube. Then add 70 µL of elution buffer to the spin column and spin for one minute at 10,000 rpm. Label the vial as elution I.
11. Transfer the same spin column to the fresh collecting tube and add 40 µL of elution buffer to the spin column and spin for one minute at 10,000 rpm. Label the vial as elution II.
12. Then discard the spin column, and the vials labelled as elution I and II are stored at -20oC or -80oC.
Preparation of agarose gel electrophoresis:
First, take a gel casting tray, then clean the tray with ethanol and seal the edges with transparent tape and place the comb.
Then 0.8% of agarose solution was prepared using 1X TBE. When the temperature was lowered, add a little amount of Ethidium bromide to it and mix well.
After mixing, pour the agarose solution into the gel casting trays and then allow for polymerization. Make sure with the absence of air bubbles while pouring the solution. After polymerization, remove the comb without breaking the gel.
Submerge the agarose gel into the electrophoresis tank containing 1X TBE buffer.
Electrophoresis of isolated plasmid DNA.
1. Take 3 – 4 µL of eluted sample and add 2 µL of sample buffer.
2. Load 5-6 µL of a sample into each well along with a DNA marker or ladder.
3. Run the electrophoresis and allow the plasmid DNA to run in 1X TBE buffer at a constant voltage. Keep track of the dye front.
4. Then disconnect the power electrophoresis tank and remove the gel using gloves. Place the gel in the Gel Documentation System or Transilluminator and visualize the DNA bands in the UV light.
5. Quantify the concentration of DNA from the DNA marker or ladder used.
Role of chemicals used:
Spin column: The desired nucleic acids should be bound to the column after centrifuging the lysate through the silica membrane, and impurities such as protein and polysaccharides should be in the flow-through. While plant samples are likely to contain polysaccharides and pigments, the membrane may be slightly brown or yellow in blood samples. The washing steps will remove such impurities. Usually, there are two washing measures, but this varies depending on the type of sample. A low concentration of chaotropic salts to eliminate residual proteins and pigments will also be part of the first wash. In order to extract the salts, this is often accompanied by an ethanol wash. Columns contain a resin of silica which binds to DNA/RNA selectively. By its capability to bind silica in the presence of high concentrations of chaotropic salts, the DNA of interest can be isolated.
Washing: With an alcohol-based wash, these salts are then removed, and the DNA is eluted using a low-ionic-strength solution such as TE buffer or water. Dehydration and the formation of hydrogen bonds that compete against poor electrostatic repulsion seem to be driving the binding of DNA to silica. Therefore, a high salt concentration will help push DNA adsorption to silica, and the DNA will be released at a low concentration.
Elution buffer: The Elution buffer is initially used to wash away unbound proteins and release the ligand’s desired protein at a higher concentration. The elution buffer must work rapidly without altering the desired protein’s function or activity. This enables to stand within the membrane for a few minutes before centrifugation for optimum DNA elution.
Glucose: It helps in maintaining osmolarity and keeps the cells from bursting.
Tris HCL and EDTA: This helps in chelating divalent metal ions such as magnesium calcium and destabilizes the cell wall, inhibiting DNases’ action.
NaOH and SDS: In the lysis buffer or solution II, sodium hydroxide, and the detergent Sodium Dodecyl (lauryl) Sulfate (SDS) are present. SDS is used to solubilize the cell membrane. NaOH helps in the breakdown of the cell wall, but more significantly, it interferes with the hydrogen bonding between the DNA bases, turning the cell’s double-stranded DNA, including the genomic DNA and the plasmid, into single stranded DNA.
RNase: It helps incomplete digestion of unwanted RNA from the plasmid sample.
The healthcare industry is going through a massive shift in many ways and using methods. The new world of artificial intelligence and technology has left no stone unturned, including the field of biology. From personal assistants like Siri, Alexa and Google home to smart watches tracking your steps, heart rates and sleep patterns, and mobile applications directing fitness and mental health, this amalgamation of technology and biology has made its way into everyone’s life. This is just the beginning, and we have a long way to go in the exploration of applications of this unique innovation.
Various emerging Industry Applications:
Medical imaging:Many companies today are involving Artificial Intelligence – associated platforms in the medical field for scanning devices in order to improve image clarity and clinical outcomes. This is done by reducing exposure to radiation (For Example: CT scans for liver and kidney lesions).
Management of chronic diseases: Machine learning is being integrated into devices like sensors, to detect, monitor and automate the delivery of treatment. This reduces manpower and increases efficiency.
Drug discovery: A number of applications are there which involve the use of AI with drugs. This includes designing of drugs, polypharmacology, drug repurposing, screening of drugs and chemical synthesis.
Telemedicine: Electronic consultations, medicine management and analyzing medical records and other data of the patients drastically helps the doctors, nurses as well as the patients. Today’s technology has enabled telemedicine to be an emerging field and has helped millions across the globe, especially during the trying times of the Covid-19 pandemic.
Robot assisted surgeries: Data from real surgical processes are collected by cognitive surgical robotics to improve on and improve the already existing approaches to surgery. This greatly helps with minimizing the patient’s treatment time and chance or probability of error.
These are a few upcoming trends amongst various others.
Noteworthy examples of Integration of artificial intelligence in healthcare:
KENSCI is a company targeted towards AI for hospital risk prediction. Its main goal is to combine artificial intelligence and big data to predict financial, operational and clinical risk in hospitals. It takes data from different and existing sources, which can then predict who may get sick and also keeps hospital costs in check.
XTALPI (Cloud based digital drug discovery)
This unique technology combines cloud and quantum physics which results in the prediction of small molecules’ chemical and pharmaceutical properties which plays an extremely important role in drug design and development, and thus, drug discovery. This company has a polymorph prediction i.e., crystal structure prediction technology, which is able to predict complex molecular systems and structures within days, as compared to the normal time of weeks and months.
Atom wise constitutes a neural network for clinical trials, known as Atom Net. This helps to identify differing biological activity of patients, including their characteristics. The technology can screen anywhere between 10 to 20 million genetic compounds every day and has been found to deliver results 100 times faster than other competitive pharmaceutical companies in the markets. Clinical trials are essential for new discovery of pharmaceuticals and vaccines, and this practice is helping us move toward a cure for serious diseases, including Ebola and multiple sclerosis.
This is a Canadian based platform which aims at helping researchers find candidates that would be compatible with drugs that are mainly targeted towards neuromuscular and neurodegenerative disorders. Finding the right candidate can improve chances of successful clinical trials. This also aids in the revolutionizing stream of personal medicine.
Layer-like microorganism aggregations and the extracellular polymers bound to a solid surface are biofilms.
Biofilms, innate cells, are ubiquitous and are becoming more and more important in processes that are designed for pollution control including philtres that drip, rotating biological contactors and anaerobic philtres.
Biofilm processes are simple, efficient, and sustainable because natural immobilization allows excellent retention and accumulation of the biomass without the need for separate solids.
We should consider a fundamental problem concerning biofilms (indeed all aggregated systems) before designing mathematical instruments for predicting the removal of substrates by biofilm:
Here are a few possibilities:
1. Due to the advection of substrates after the film, these biofilms are exposed continuously to the fresh substrate in space. It means that if the biofilm is attached near the source of the substratum supply, the substrate concentration is higher.
2. In mandatory substratum transportation consortia or other synergistic relationships, different types of bacteria must be co-existent; for the exchange, the near juxtaposition between cells is required in a biofilm.
3. The biofilms build a more friendly internal atmosphere (for example, pH, O2, or products) than the bulk liquid. In other words, the biofilm creates special and self-created cell-beneficial micro-environments.
4. The surface itself produces a single micro-environment, for example by removing contaminants or by corrosive releases of Fe2+, a donor of electrons.
5. The surface allows the bacteria to change physiologically.
6. The cell’s strict packing modifies the physiology of the cells.
The development of biofilms can be divided into five phases:
1. Surface relation
2. Monolayer formation
3. Training in microcolony
4. Biofilm mature
The initial step, which is still reversible, is the initial touch of the moving planktonic bacteria on the surface.
The bacteria then begin to form a monolayer and create a protective “slime,” an extracellular matrix.
The matrix contains extracellular polysaccharides, structural proteins, cell scrap, and nucleic acids known as EPS.
Extracellular DNA (eDNA) predominates the initial steps of matrix development, and polysaccharides and structural proteins later take over.
At these points, microcolonies are produced that display significant growth and contact between cells such as quorum sensing.
During the last step of a new cycle of biofilm formation, some cells in mature biofilm begin to detach itself into the environment as planktonic cells.
The biofilm allows for:
a. tolerates antibiotic attacks;
b. Trap bacterial growth nutrients and stay in a favourable niche;
c. Adhere and avoid flushing to environmental surfaces;
d. live in close collaboration and associate with other biofilm bacteria;
e. Stop phagocytosis and attack the complementary routes of the body.
For example, Planktonic Pseudomonas aeruginosa uses polar flagella to travel by water or mucus and to reach a firm surface as mucous membranes of the body. The cell wall and pil adhesives can then be used to bind the mucous membrane epithelial cells. Attachment stimulates signalling and quorum sensing genes so that a polysaccharide alginate biofilm synthesization will eventually begin with the population of P. aeruginosa. As the biofilm forms, the bacteria lose flagellum and secrete different enzymes that allow the population to obtain nutrients from the host cells. Finally, the biofilm pillows and establishes water canals for all bacteria in the biofilm to provide water and nutrients. As the biofilm gets too crowded with bacteria, the sensing of quorum helps a few Pseudomonas to develop flagella again, escape the biofilm, and colonize a new spot.
Fig: Formation of biofilm by P. aeruginosa.
Two bacteria involved in dental caries, Streptococcus mutants and Streptococcus sobrinus, break down sucrose to glucose and fructose. Streptococcus mutans may use a dextransucrase enzyme in a sticky dextran polysaccharide, which forms a biopathic film that allows bacteria to bind to the tooth enamel and form the plaque. The pathogenicity of S bacteria. S. and mutans. In order to generate energy, sobrinus often ferments glucose. Glucose fermentations produce lactic acid, which is released on the tooth surface and causes decay. The mechanism is described in the given image.
Copper extraction from ore deposits was carried out over centuries using acidic solutions, but the involvement of bacteria was not confirmed in metal dissolution before the 1940s.
Today about 10–20% of copper mined in the United States is extracted by low-grade microbial processing. In the expansion of microbial leaching, other elements, including Uranium, Silver, Gold, Cobalt and Molybdenum, are also invested considerably.
Most microbial liquidation relies on metal sulphides’ microbial oxidation. Aqueous environments combined with mineral waste create very harsh conditions with a low pH, high metal concentrations and high temperatures that select very specialized nutrient requirements for a microbial flora.
Heap leaching is the most common method in which copper and other minerals are extracted microbially from spent ore.
The method involves arranging the spent ore fragments into a configuration of the packed bed to allow the passage of water. Acidized water (pH = 1.5-3.0) is sprayed on the porous ore bed in order to start the operation.
The solvent ferrous iron is actively oxidized, and sulphide minerals are attacked by acidophilic bacteria such as Thiobacillus iron, which can then be released from aquatic ions by releasing soluble cupric ions. In terms of the corrosion on metal surfaces, this oxidation is identical.
Biological reactions and mass transfer rates currently constrain the industrial applications of microbial fluid, but in recent years significant changes have been made to process design, and the mining industry sees the method as being promising.
Removal of Biofilms:
Traditional cleaning of biofilm has been accomplished by detergents, biocides, enzymes, and mechanical or physical methods of cleaning the biofilm.
Biofilms in sensitive locations in the food and drink manufacturing industries have grown, leading to problems such as food spoilage, production quality and other nutrient supplementation and insufficient cleaning and disinfection.
Depending on the medium temperature and relative humidity of these microorganisms, they will live longer after application. The pulp- and paper-based agent for biofilm removal is classified into three groups: chlorine, chlorine dioxide, hydrogen peroxide (hydrogen peroxide), Ozone, antioxidants, and enzymes.
The cells adhering to the staphylococcus aureus are effectively used in sodium dichloro isocyanurate, hypochlorite, iodophore, hydrogen peroxide and peracetic acid. The most powerful method of extracting adhered large amounts of L was to eliminate peracetic acid.
Monocytogenes cells remaining on chips of stainless steel after sanitization. Scanning electron microscopy has shown that on chlorine and anionic acid-treated surfaces biofilm cells and extracellular matrices remain better than iodine and ammonium-quaternary detergent sanitizers from which a viable cell is not released.
Oxidative and antioxidant biocides have long been used, although enzyme use is being studied at present.
In order to develop a quality management programme in different industries, tanks, pipes, pasteurizers, coolers, membrane filtration units and fillers must be tested, because they help to avoid microbiological hazards and severe financial losses.
Second metabolites are natural compounds that are not directly involved in normal growth, growth or metabolism. The second metabolism of the plant produces products that help the growth and development of the plant but are not necessary for the plant to survive. A common role of secondary metabolites in plants is to protect the egg that is used to fight weeds, insects and germs. In humans they plant the second most important metabolite as it is used in many fields such as medicine, flavoring agent, dye etc. The main contributors are the specific aroma, color and taste of the plant parts. The secondary metabolites play a role and play an important role in potential immune systems, especially in the chemical warfare between plants and their bacteria. Some of these compounds have also been shown to play a role in the fight against pests and pollinators, allopathic agents, and toxins, UV light protection, and signal transmission. Food, cosmetics, drugs and their important role in plant protection.
The general role of secondary metabolites
Plants to protect plants such as weed control, weed control and insecticides. For humans, plant second most important metabolites as they are used in many fields such as medicines, flavoring agents, dyes etc.
Functions of secondary plant metabolites
• Major activities include
• Competitive weapons against biological agents such as animals, plants, insects and micro-organisms.
• Metal transport agents
• Agents for compatibility with other organisms
• Reproductive agent
• They make a difference
• Biodiversity coordinators
CONSTRUCTION OF SECONDARY PLANT METABOLITE
An alkaloid is defined as a natural or synthetic product, which contains one or more nitrogen atoms, usually heterocyclic, and contains certain substances in the human or animal body, if used in small amounts. Most are based on a few amino acids. Chemicals have a ring structure and nitrogen residues. Indole alkaloids are the largest group in the family, based on tryptophan. They are widely used as medicine.
It is hard, crystalline and has a sharp melting point. Some alkaloids are amorphous compounds and some like coniine, nicotine are naturally liquid. Some alkaloids are naturally colored e.g. Betanidine shows red, Berberine shows yellow Most of the alkaloid salts are dissolved in water. Water bases only melt in water. Pseudo and proto alkaloids provide high solubility in water. Melting and alkaloid salts help the pharmaceutical industry to extract They provide the color edge with a halogenated compound
• Basic in response
• It turns neutral or acidic when active groups are associated with the release of electrons such as amide grp.
• Formation of salt by inorganic Acid – their decomposition during storage
• It contains one or more nitrogen
• Natural – can be in the form of free salt such as amine or as a salty acid or alk. N-oxide
Various kinds of fruits and flowers and spices. The word is derived from the word turpentine. Turpentine, also known as “pine resin”, is viscous, fragrant, and does not melt in flowing water when cutting or carving new bark and wood of pine species (Pinaceae). Turpentine contains “resin acids” and other hydrocarbons, formerly called terpenes. They are used in cloves, essential oils and medicines. Terpenes are mainly composed of Fusion of 5 Carbon Isoprene Units The basic elements of the structure of terpenes are sometimes called units of isoprene because terpenes can decompose at high temperatures to provide isoprene. All terpenes are sometimes called isoprenoids. Therefore, Terpenes are unused compounds made by joining together isoprene units.
Terpenes are divided into five carbon components.
• Ten terpenes, containing two units of C5, are called monoterpenes
• Three carbon terpenes (three C5 units) are sesquiterpenes
• Two carbon terpenes (four C5 units) are diterpenes.
• Large terpenes include triterpenes (30 carbons), tetraterpenes (40 carbons), and polyterpenoids
Are hydrocarbons of plant origin of the common formula (C5H8) n their oxygen, which comes from hydrogenated and water.
• Colorless liquid, soluble in natural solvents and soluble in water, active, environmentally friendly, boiling area: 150-180 ° C.
Impure Chemicals, They deal with additional reactions with hydrogen, halogen acid to form additional products such as NOCl, NOBr and hydrates. They can undergo polymerization and dehydrogenation in the ring In hot rot, terpenoids release isoprene as early as production.
Terpenoids are classified based on the number of rings present in terpenoids
• Bicyclic terpenoids (The size of the first ring is the same in all, and the size of the second ring varies). Depending on the size of the second ring, the other is divided –
1. From Bornane (Eg Camphane)
2. Based on Norbornane
• I-terriceno terriceno
• Tetracyclic terpenoids
The phenolic compounds of benzene have one or more hydroxyl groups.The plants are produced mainly to protect against stress.They are widely distributed in the plant kingdom.Many second plant metabolites. Forms 40% plant soluble protein. More than 8000 phenolics properties are known. Lignin (polyphenol) is the second most abundant compound in the plant
An emerging field of medicine called regenerative medicine or cell therapy refers to treatment derived from the idea of producing new cells that will replace malfunctioning or damaged cells as a vehicle for the treatment of diseases and injuries. The focus is on developing effective mechanisms for stem cell replacement. This is especially beneficial for age-related diseases such as Alzheimer’s disease, Parkinson’s disease, type II diabetes, heart failure, arthritis, and aging of the immune system. It is believed that replacing damaged or dysfunctional cells with full functionality could be a useful treatment strategy in the treatment of many of these diseases and conditions.
Different Types of Renewable Medicine
Each of the 200 integrated cells in the human body is derived from one cell which is the fertilized egg. As the fertilized egg grows, it forms embryonic stem cells, each of which has the potential to form the different types of cells found in adults and are organized into structures that will become tissues and organs. But some live in an inseparable state with older stem stem cells that can be transformed into a limited range of cell types.
Remedies that use patients’ cells to regenerate their organs or tissues are called spontaneous therapies. Methods include ongoing testing at two London hospitals to treat 100 heart attack patients with stem cells in their bone marrow to help repair damaged heart tissue. Therapies that use cells or tissue derived from a non-patient patient are called allogeneic therapies. Examples include the use of bone marrow or stem cells from similar donors or the use of ES cell lines established for treatment.
The term “engineering tissue” came into use in 1985, by Y. Fung, a pioneer in the field of biomechanics and bioengineering, also stated that Tertiary engineering is a multidisciplinary field that uses the principles of engineering and health science to improve biological processes that restore, maintain or improve tissue function.” Tissue engineering modifies the cells or tissues in some way in order to repair, regenerate, or regenerate tissue. Perhaps the most well-known example of this was the process of trachea formation for a patient with airways that had been severely damaged by tuberculosis. Other examples of tissue engineering include artificial skin, which is made using human cells (fibroblasts) implanted in a matrix of proteins (fibrin) and cartilage membranes to be implanted in patients who have ruptured the cartilage of the knee.
Tissue engineering at its basic level fills the scaffolding of 3D tissues (biomaterials) by cells to produce functional organ formation. Tissue engineering aims to address the latest shortage of critical organs through the formation of living organs.
Another form of self-rehabilitation therapy is to make biomedical devices that mimic the function of tissue or organ. For example, Type 2 diabetes results in the destruction of beta-producing insulin-producing cells. Patients with this type of diabetes should monitor their blood glucose levels regularly and inject the hormone insulin to keep the level normal. While some research teams are working to restore beta cell functionality they are using biomedical engineering to improve artificial limbs. Using ultra-low power electronics originally designed for mobile phones, they developed a small built-in glucose sensor chip that can be inserted into a patient. The chip would regularly monitor blood glucose levels, produce the high amount of insulin needed to maintain stable glucose levels and send wireless signals to the pump to release the right amount of insulin.
Although there are a variety of genetic therapies, the most obvious is to identify a medical condition that can be treated with a specific protein, and then introduce genetic code into that protein in the affected cells. In practice, finding genes that work in cells with the result of continuous treatment is extremely difficult. Nevertheless, in recent years there has been some progress in the use of genetic therapy to regenerate tissues, especially in the area of heart disease.
Stem cells and Regenerative medicines
Stem cells have are able to develop into many types of cells in the body. They can act as a kind of immune system they can also differentiate without limit to fill other cells. When a stem cell divide, the new cell obtained has the ability to reside as a stem cell or into another type of cell which are having specific functions it can turn out to be like a muscle cell, a red blood cell, or a brain cell. There two types of stem cells: embryonic stem cells and adult stem cells. Embryonic stem cells are found in embryos and the sources of adult cells are the umbilical cord, menstrual blood, muscles, tendons, adipose tissue, bone marrow etc. a group of adult stem cells can be incorporated in vitro or in vivo to separate osteoblasts, chondrocytes, adipocytes, tenocytes, myotubes, neural cells and stroma supporting hematopoietic. The sheer strength of these cells, their easy separation from culture, and their extremely high ex vivo power make these cells an attractive therapeutic tool in reconstructive medicine.
Recent Advancements in Regenerative Medicine
Direction of cell expansion and differentiation, which explains the processes of how tissues and organ grow.
Development of techniques for assembly of cells into large, three dimensional tissue-like structures, which will lead to the physical creation of three dimensional organs.
Custom-designed biomaterials to serve as structural templates for tissue development, which helps scientists build organs.
Automated bioreactor culture vessels, which allow scientists to mass produce cells and tissues.
1.Recombinant DNA or RDNA technology is defined as the procedure of joining DNA molecules of two different species together and inserted into the host organism to produce a variety of new genetic combinations. This is also known as Genetic engineering.
2. The DNA fragments are selected from two different species and combined. This technique was developed by two scientists namely Boyer and Cohen in 1973.
3. The DNA molecule which is inserted into another DNA molecule is called a VECTOR. The recombinant vector is then introduced into a host cell where it replicates itself, and the new gene is produced. This is the basic principle behind Recombinant DNA technology.
TOOLS OF THE RECOMBINANT DNA TECHNOLOGY:
Restriction endonucleases: These are used to cut DNA molecules at specific sequences into many smaller DNA fragments.
Plasmids: These are extrachromosomal circular DNA present in the bacteria, which can replicate independently. During cloning, these plasmids carry drug resistance genes that are used for selection. Foreign DNA can be placed into a plasmid and it is replicated further.
DNA ligase: This enzyme is used to join the two pieces of DNA together.
Foreign DNA: This is also known as passenger DNA, which contains desired gene sequences.
Vector: It is a vehicle used to insert the desired DNA into the host cell. Some of the vectors used are Plasmid DNA, Bacteriophage DNA, Yeast DNA, Viral DNA, Bacterial DNA, etc.
GOALS OF RDNA TECHNOLOGY:
To isolate and characterize a gene or DNA from an organism.
To eliminate undesirable phenotypic characters.
To combine the needy and beneficial traits of two or more organisms.
To make desired alterations in one or more isolated genes or DNA
Inserting the altered genes or DNA into the host cell of another organism.
To synthesize new genes using artificial methods.
To alter the genome of the organism
Understanding the diseases which transmit due to heredity.
Understanding the treatment for heredity related disorders.
To create new gene combinations.
PROCEDURE TO PREPARE RDNA:
1 Isolation of DNA from the organism: The cells are lysed using detergent mixtures, which creates pores in the plasma membrane. Then the mixture of cell contents is treated with protease and RNAase enzymes. The enzyme protease destroys the proteins present in the mixture and the enzyme RNAase destroys the RNA molecules present in the mixture. Then the mixture is centrifuged and the supernatant containing the DNA is transferred into a clean test tube and the DNA precipitated with the addition of ethanol.
2. Insertion of foreign genes into vectors: By using plasmid as a vector, isolated from the bacterial cell and treated with restriction enzymes and target DNA is obtained and it is placed into a vector to produce recombinant DNA.
3. Insertion of Recombinant DNA into host cell: The plasmid containing the foreign DNA is placed into a bacterial or host cell for multiplication.
4.Transformation: The vector is used as a vehicle to transport the gene to host cell, bacterium or other living cells are used as vectors. The vector is multiplied in the host cell and produces many identical copies, which are similar to both DNA and gene present in the DNA.
5. Cloning: After the division of the host cell the rDNA copies produced are transmitted to the progeny and further vector replication takes place in the progeny cell, with the continuous division of cells, a clone of identical host cells is formed. Each clone contains one or more copies of the rDNA molecule. Later the identical host cells are lysis and rDNA molecules are separated from the host cells.
APPLICATIONS OF RDNA TECHNOLOGY:
This technology helps to grow crops which are resistant diseases and pesticides, crops of our choice, fruits, and flowers of attractive colors.
This technique is employed in the production of artificial insulin and to deliver the drugs to target sites.
Used in Molecular diagnosis of diseases.
Used in Gene therapy.
Employed in DNA fingerprinting.
Used in the production of vaccines and pharmaceutical products.
Deals with insertion of desirable characteristics into plants through genetic modifications for the purpose of creating beneficial plants. Plants which are modified genetically are termed as Transgenic plants. Transgenic plants usually are created by modifying their DNA to serve different purposes.
The principle foundation of plant biotechnology was through the theories of cellular Totipotency( totipotency is the ability of single cell to divide and differentiate) and Genetic transformation. This theories led to the development of modifications in plants. Genetic transformation theory was proposed by Griffith, while cell theory was given by Schleiden and Theodore Schwann.
The plant breeding process has a rich history and developments in plants began as early as 12000 years ago. It is known that, the bread making wheat was started in Egypt during 4000-5000bc where it was grown in Egypt and cultivated in China. Selective breeding techniques were known to be used first by Babylonians for date palm cultivation.(875bc). The term biotechnology in the year 1919 was first given by Karoly Ereky. Demonstration of plant tissue culture technique for the first time was given by Haberlandt.
Methods in Plant biotechnology:
Tissue and cell culture:
This technique helps to maintain cells and tissues for a longer duration of time in an artificial medium. Cells are isolated and grown in a medium which is called as tissue culture medium that contains all the necessary nutrients required for the plant growth and it’s proliferation.
Selection of a suitable explant and than sterilizing it using disinfectant.
Preparation of an appropriate culture medium which suits the explant selected.
The prepared medium is than sterilized by using autoclave.
Inoculation of the sterilized explant and is than transferred to the culture medium in aseptic conditions.
Incubation of cultures in a culture room with proper temperature, moisture and humidity conditions.
Next step is, sub culturing where the cultures are transferred to a fresh nutrient medium to obtain plantlets.
After acclimatising plantlets to environmental conditions, they are transferred to green house or can be grown in pots.
By using this technique it is possible to cultivate any plant species by using a variable explant, for example: pedicle, stem segment, leaf segment, petiole, anther, etc. Plant tissue culture can be carried out in both solid or liquid media depending on the plant specie and requirement of conditions.
Recombinant DNA technology:
Technology in which DNA from one genome is inserted to the other. This approach is best suited for recombination between unrelated species. With this technique, foreign gene is introduced to the plant genome artificially and the resultant plant is genetically engineered or modified.
Three main components involved with genetic engineering:
Isolation of foreign gene from a suitable source.
Suitable vector carrying the gene.
Various means to introduce the vector into the host genome.
Genetic Engineering has following steps:
Isolation of gene of interest to be cloned from desired organism.
Transfer of the isolated gene to create recombinant DNA molecule. By cutting the DNA molecules at specific sites by using Restriction Enzymes, recombinant DNA molecules can be created.
Restriction enzymes cut DNA at specific locations. Restriction enzymes are designated as I, II, III, and IV. This enzymes varies in their structure, site of cleavage and some cofactors (substance which is required for enzyme activity).
By the action of RE, DNA is left with sticky ends (short portion of unpaired bases). Same restriction enzymes is used to cut the plasmid, so that the plasmid also has the similar sticky ends and can base pair.
The plasmid and the isolated gene are joined together by an enzyme called DNA ligase.
The two pieces of DNA with same sticky ends (as cut by same RE) are being linked together by DNA ligase and forms a single, unbroken molecule.
Than the genetically engineered plasmid is inserted into bacterial cell (Transformation). The gene is transferred by means of Vectors. Selection of vectors is specific and will depend on the type of gene to be inserted.
When the bacteria reproduce, the plasmid will get copied and this recombinant plasmid spreads as bacteria multiply and expresses the gene and makes a human protein.
By using recombinant DNA technology, transgenic plants can be prepared.
Plants with their genome modified by either addition of a foreign gene or removal of damaged gene are called as Transgenic plants. Transgenic plants are created based on the need. For example plants can be modified to resist pests, pathogens, insects and environmental conditions.
Example: Bt cotton is a genetically modified crop. It is modified to combat bollworms.
Production of transgenic plants using Ti plasmids:
Agrobacterium tumifaecins, is a soil bacterium that causes crown gall disease by incorporating the T-DNA region of Ti(tumour inducing plasmid) into the host cells. Thus, Ti plasmids can be used to prepare genetically engineered plants by modifying the T-DNA region of Ti plasmid. Steps are as follows:
Ti plasmid is genetically engineered at T-DNA region by inserting an antibiotic resistant gene.(Kan R- kanamycin) as well as a foreign gene of interest.
The plant cells in culture containing this cointegrated Ti plasmid transfers the foreign DNA into host cell.
Thus, when foreign DNA is integrated, it disrupts the tumour formation, and only those plant cells can grow which are resistant to Kan-R.
Plants are than regenerated from the culture through calluses.
Foreign gene is usually expressed by the adult transgenic plants.
This method is also called as variation breeding.
In this method the seeds are exposed to radiation or chemicals to generate mutants.
This technique generally helps to improve disease resistance to plants.
It helps to improve specific characteristics of high yielding varieties. For example- Jiahezazhan Rice.
Mutation can be induced by both physical and chemical agents.
This type of breeding is more suitable to improve one or two specific traits. (Selective traits)
Crossing between 2 different plants to produce hybrids is called as hybrid breeding. Hybrid breeding is generally used to create hybrids that are completely different product as they are made from different parent lines.
Advantages of hybrid breeding:
Fast growing plants, so it is advantageous for farmers as they can reap more crop and can earn larger profit.
More disease resistant than parent plant.
Can withstand abiotic stresses.
Disadvantages of hybrid breeding:
Heterosis effect( progeny exhibits greater fertility, biomass, growth than parents) lasts only for one generation.
Application and Potential of Plant biotechnology:
Large scale plant species can be raised by this method.
Meristem is used in this method and is cultured in basal medium containing hormones, nutrients, carbohydrates and nitrogen sources.
Technique is employed to eliminate pathogens and viruses.
Examples of plants: Sugarcane and potato are micro-propagated commercially to prevent virus and pathogen free plants for better yield and profit.
Herbicide resistant plants:
Weeds often ground the crop plants and reduces the yield. To control weeds, farmers uses herbicides to destroy weeds.
But generally, it has been observed that herbicides used can cause side effects to plants. For this reason genes for some enzymes have been genetically engineered to provide resistance to various herbicides. Table below is attached showing the gene strategy used and the plants benefitted through it:
Resistant to abiotic stresses:
Conditions like drought, salinity(high salt concentration), flooding, heat and freezing leads to poor harvest of the crop.
Protective proteins or enzymes from other plants or organisms are genetically engineered to fight against such adverse conditions.
saturation levels of membrane fatty acids are usually altered. Other than this Osmolytes, osmoprotectants and rate of reactive oxygen intermediate is also changed.
Abiotic stress tolerant genes, when introduced into plants can provide tolerance to abiotic stresses.
Example- Enzyme choline dehydrogenase from E.coli when injected into potato and tobacco plants, produces glycine betaine, which resistance to salts and freezing.
Insect pests and disease resistance:
Insects destroys plants and leads to economic loss. Traditional insecticides can kill the pests but simultaneously it also causes damage to useful insects and leads to soil damage.
Many strategies have been adopted to engineer disease resistant gene in plants. One common example is Bt cotton, where resistant gene is introduced to kill cotton bollworms.
Genes conferred with protease inhibitors, alpha amylases are introduced which interacts with insect metabolism and confers resistance by destroying it.
Apart from these, plant biotechnology is also used to improve nutritional quality of food and to enhance its nutritional value. Nutritional value of plants can be increased by altering the amino acid composition of plants proteins and by introducing transgene with desired traits.