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Top 10 most innovative biotech companies in the world

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Biotechnology is multidisciplinary field that can be divided into 4 more specific areas, using color coding system. Red biotechnology is dedicated to design as much products and devices related to the medical field as possible. Green is focused on agricultural improvements and environmental protection. Blue is using ocean resources to develop various products (from food to fuels…) and white is focused on industrial processes. Out of 4 fields mentioned - red biotechnology is the most profitable: billion dollars are spent each year for research and development in medical field.

Here’s the list of 10 most innovative biotech companies and short info on their main research areas.

Life technologies

Life technologies is headquartered in Carlsbad, California. It’s founded in 2008, have ~11 000 employees and is highly profitable. 2011 revenue was 3.7 billion dollars. They are developing lab equipment for all kind of genetic testing (Applied Biosystems), products for isolation, quantification and amplifications of RNA (Ambion), biologic drug production associated materials (Gibco), DNA and biology associated products (Invitrogen), molecular probes under the same brand name, products for purification, separation and analysis of proteins (Novex), products used for gene expression experiments (TaqMan) and ion semiconductor DNA sequencing system (Ion Torrent). They have offices in more than 60 countries worldwide.

Genentech

Genentech is pioneer of biotechnology industry. Founded in 1976 with headquarter in South San Francisco. It has over 11 000 employees and as from 2009 it is wholly owned subsidiary of Roche. All scientist, researchers and post-docs are focused on 5 main research areas: oncology, neuroscience, tissue growth and repair, immunology and infectious diseases.

Bug Agentes Biologicos

Company is founded in 1999 and based in Piracicaba, Brazil. It’s focused on development of natural pesticide replacements. Main products are predatory insect eggs and parasitoids used for crop protection. Those are mainly used for soy field’s protection.

Amyris

Amyris is founded in 2003 and it’s focused on providing sustainable alternatives to petroleum derived products. Plant sugar is starting point. It undergoes industrial conversion into various hydrocarbons that will be used for renewable products development later used in cosmetic and polymer industry, for lubricants, flavors….even for jet fuel. Headquarter is in Emeryville, California.

GE (GE Healthcare)

GE Healthcare is only division of GE business that is headquartered outside the USA, in Little Chalfont, United Kingdom. It’s founded in 2004 and it’s focused on medical imaging and diagnostics (equipment they are manufacturing is ranging from X rays to magnetic resonance), drug discovery, pharmaceutical manufacturing…

Diagnostics for all

Diagnostics for all is non-profit organization founded in 2007. Idea was to help solve medical issues in developing and resource poor countries all over the world by creating a simple and cheap diagnostic device - patterned paper. Small piece of paper covered with biological and chemical assays reagent is cheap and fast way to test yourself. By applying small amount of biological fluids, assay zone is changing the color that should be compared with reference color on the device. Money they need for testing, research and manufacture is provided through public donations.

The Plant

The Plant is building in Chicago most famous for being first vertical farm. Vertical farming is becoming very popular as the number of people living in urban area is growing rapidly. The Plant produces aquaponic vegetables. Fertilizers (necessary for proper plant development) are derived from algae that are consuming waste. Idea is to create building that will have net zero energy and waste (level of produced and consumed energy and waste should be equal).

Cellular Dynamics International

Cellular Dynamics International is founded in 2004 with headquarter in Madison, Wisconsin. Company is manipulating with stem cells to produce tissues of various kinds that will be used for drug development process, for tissue engineering or organ regeneration purposes. Roche is using iCell for drug screening.

Humacyte

Humacyte is founded in 2004 in Morrisville, North Carolina. Research is focused on vascular diseases and soft tissue repair application (vein graft development). Human, extracellular matrix derived tissue would help decrease inflammation, clotting and thrombosis, foreign body response after implantation in the body and would demand fewer surgical interventions that are necessary when conventional methods are used.

Harvard Bioscience

Harvard Bioscience is founded over 100 years ago in Holliston, Massachusetts. Company is manufacturing different kind of instruments and equipment that is used in life science and regenerative medicine fields (such as molecular, cellular, and physiology research). Some of the most interesting HB products are artificial trachea, synthetic windpipe and organs made out of body’s own cells. They have 20 wholly owned subsidiaries.

Now when you know their names and research goals – you just have to choose one that suits you the best. Wink

Screening Methods for Mutants/Recombinants in Recombinant Technology

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Mutations are genetic changes or modifications caused by chemical and physical mutagens. Mutations can results from modification of a single base or few bases. However this can result in change or modification of a phenotypic character which can be used to recognize them. This feature is widely used in DNA recombinant technology. Plasmid vectors carry genes for drug resistance, toxin production which can be used to distinguish recombinants. When genes of interest are inserted into the plasmid, the reading frame for the marker genes can be altered. This results in mutants who can be identified using special chemicals/ media.

AMES test is one such method to identify mutants of Salmonella typhimurium that cannot produce Histidine. This mutant stain can be cultured only when Histidine is present in the basic medium. This is the standard culture used for testing chemical mutagens. The chemical mutagen is loaded into a well in the centre of a culture plate of inoculated with Salmonella typhimurium in a medium lacking Histidine. The chemical diffuses into the medium. A growth indicates that the chemical has induced a mutation in Histidine- stain converting it to Histidine +. Depending on the position of the colony relative to the well containing the chemical, the degree of resistance varies. Colonies growing closer to the well are quite resistant and colonies growing at the periphery indicate that the chemical even at a low concentration can induce mutation. This test has its application in pharmaceutical industry to test the effect of drugs; whether it’s a mutagen or not.

In replica plating method to screen mutants, the organism is subjected to radiation or exposed to chemical to induce the mutation. The mutants can be identified by a reduction in the colony size or change in pigmentation etc. Sub culture is made by replica plating the master culture. The sub cultured plate is subjected to mutation and incubated for growth. Then the plate subjected to mutation is replica plated onto a fresh medium and incubated to observe phenotypic variations. This method can be used to identify the dosage of radiation or chemicals required to cause mutations. If this is repeated with different dosage levels, the finally left colony will be having most resistant cells for the particular mutagen.

Gradient method is used to study the effect of chemical mutagens on bacteria. A medium containing two different concentrations are prepared separately and poured over the same plate in a slanting position. First the medium with lower concentration of the chemical is poured onto the plate in a slanting position and allowed to solidify. Then the medium with higher concentration of the chemical is poured onto the plate. The plate is inoculated and observed for growth. This is repeated till there is no growth at higher concentration to identify the effective concentration.

Blue white selection is a widely used method in screening recombinants in cloning. This is based on the gene product of lac z gene. The plasmid vectors contain this gene which produces β galactosidase enzyme. When a gene is inserted close to lac z gene, the reading frame will be distorted and the gene is inactivated. So the transformed cells will not produce this enzyme and are called competent cells. After the recombination, the bacterial cells are grown in a medium containing X gal (5-bromo-4-chloro-indolyl-β-D-galactopyranoside) and IPTG (Isopropyl β-D-1-thiogalactopyranoside). IPTG acts as the inducer for lac z gene and enhance the production of β galactosidase. When it is produced, combines with X gal to form a blue colour complex called 5,5'-dibromo-4,4'-dichloro-indigo which is insoluble. The transformed colonies will appear white in colour and non- transformed cells will appear blue in colour. This method is also called as insertional inactivation of lac z gene.

Hybridization techniques are widely used to identify recombinants. This is based on the ability of nucleic acids hybridize with complementary DNA. The transformed cells are transferred on to a nitrocellulose membrane which is subjected to cell lysis. The double stranded DNA is converted to single stranded DNA and immobilized on the membrane. Then it is treated with radiolabelled probes complementary to target DNA. If the desired DNA is present, the probes will be hybridized which can be detected by autoradiography.

Apart from these methods, immunochemical methods are used to detect protein products to screen recombinants.

Tumor Inducing Plasmids (Ti plasmid) of Agrobacterium

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Tumor inducing plasmids (Ti Plasmids) are double stranded circular DNA present in Agrobacterium tumefaciens. This article gives you complete information of these Ti Plasmids.

Agrobacterium is a gram negative soil bacterium which infects over 3000 dicots and causes crown gall disease at the collar region. This plasmid is denatured at higher temperatures and loses tumorgenic properties. Ti plasmid encode for enzymes for catabolism of opines such as permease and oxidase.

Ti plasmid ranges from 180-205kb in size. It has T DNA which is of 20kb and in addition it has several genes such as vir genes for virulence, ori gene for origin of replication, tra genes for transfer and genes for opine synthesis. Virulence genes are responsible for the transfer of T DNA into the host cell and integration of T DNA with host genome. Opines are derivatives of amino acids which are of two types; octopine and nopaline. Octopine is formed with two amino acids; Arginine and Alanine. Nopaline is made up of Arginine and Glutamine. Octopine and nopaline are not found in healthy plant tissues. The opines are catabolized and used as the energy source by the bacterium. A. tumefaciens is able to divert the metabolic resources of the host plant to the synthesis of opines which are of no apparent benefit to the plant. But they provide sustenance to the bacterium. During the infection through a wound, the plant cells begin to proliferate and form tumors and the plant tissues begin to synthesize opines.

Ti plasmids are classified based on the type of opines they produce in the host cell during infection. Almost all Ti plasmids have identical structure except in their sequence for opine metabolism. Octopine Ti plasmids produce Octopine ( C9H18N4O4). Tra genes encode proteins necessary for transfer of T DNA into the host. The Inc locus in octopine plasmids causes incompatibility of the plasmid in the bacterium. Shi and Roi sequences regulates shoot induction and root induction respectively. Nopaline Ti plasmids produces an opine called as nopaline ( C9H16N4O6).

T DNA is region of Ti plasmids common to both octopine and nopaline plasmids. But generally T DNA segment of Octopine plasmids is shorter than in Nopaline plasmids. In some octipine Ti plasmids, T DNA occurs in two segments. The left segment with 13kb in length and contains information for the synthesis of growth hormones and a sequence for opine synthesis. The right segment does not participate in tumor formation and maintenance.

Ti plasmids have a single repeated sequence at both ends of T DNA. This sequence is called as the bordered sequence. These two sequences act as sites during the transfer of Ti plasmid into the plant genome. It is found that removal of left bordered sequence doesn’t cause considerable change in tumor induction in the infected tissues. But the removal of right border sequence from T DNA results in failure of tumor induction. T DNA encode for the production of growth hormones like Cytokinin and Auxin which is necessary for tumor induction.
Vir region contains eight operons; Vir A, Vir B, Vir C, Vir D, Vir E, Vir F, Vir G and Vir H which together span about 40kb of DNA. This region mediates the transfer of T DNA into plant genome. The genes of vir region are not transferred by themselves, they only induce the transfer. The bordered sequences are essential for the transfer. Vir A and Vir G genes regulate Vir operon. Vir region is activated by the phenolic compounds, namely Acetosyringone and alpha hydroxyl acetosyringone produces by plant wounds. These bind to Vir A proteins activates Vir G gene by phosphorylation which in turn activates other genes. Vir D proteins together with Vir D proteins participate in the formation of conjugal tube formation between the bacterial cell and plant tissue during the infection process.

In transferring gene of interest to Ti plasmid, an intermediate vector is used such as pBR 322. T DNA portion of the Ti plasmid is separated. T DNA is inserted into the pBR 322 vector which results in formation of a shuttle vector. This shuttle vector can replicate in E. coli and in Agrobacterium.

Ti plasmids serve as ideal vectors for plant genetic engineering as it has the capability of transferring gene of interest into the target site with a high efficiency. It is easy to screen recombinant cells as marker genes are present.

Why Escherichia Coli an important tool in Biotech Industries

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The bacterium Escherichia coli (E. coli) are one among the other microorganisms constituting the micro flora of the gut of the warm blooded animals including humans. These are called as beneficial microorganisms which are harmless. But the infection caused by a particular strain of E. coli (O157:H7) masks the beneficial effect of the normal E. coli bacteria and it is always seen as an organism of threat by many. Actually speaking the unique characteristics of the E. coli bacteria makes them an important tool in biotechnology industries and it is the most preferred organism by the researchers to perform recombinant technology (gene cloning) based experiments.

Scanning the unique features of the bacterium making it important in the field of biotechnology answers the query on its significance. The simple genome of the E. coli bacteria, the rate of growth, easy to handle, complete gene sequence, the competency as a host, simple cultivation procedure and its ability to grow under both aerobic and anaerobic condition distinguishes this bacteria from others in selection procedure for an experiment.

The genome of the E. coli bacteria is very crisp and simple with only 4400 genes, easy to study and understand. The ability of the bacteria to multiply drastically producing a generation in 1200 seconds under suitable growth condition dragged the attention of the researchers. Coming to the safety in handling the organisms, except for the particular harmful strain (O157:H7), the normal organism from the flora of the gut is safe to handle under suitable microbiological environment. The first completely sequenced genome is an additional feather in the cap of E. coli making the bacteria easier to use in recombinant DNA technology which involves nothing but the ultimate expression of proteins. Also the competence of the E. coli bacteria as a host for foreign DNAs and simple laboratory procedure to cultivate and adaptability to both aerobic and anaerobic environment made the E. coli bacteria a pioneer in the field of biotechnology.

Even before the application of the rDNA technology, the first industrial application of E. coli being the production of the amino acid threonine in the year 1961 just by exposing the organism to mutagens resulting in various mutants which were screened for the desired type of mutant enabling the synthesis of the amino acid threonine and those organisms were isolated and used for large scale production.

The process of gene cloning in E. coli involves series of steps like isolation of the desired DNA and ligasing it to a suitable vector resulting in the production recombinant molecules. These molecules are screened for the expression of the desired gene and then the selected molecules are passed on to the E. coli which express the desired gene. The types of vectors used may be either a plasmid or a cosmid or a phage and the various methods which allow the vector to enter the bacteria are transformation, transfection and transduction. Thus the genetically modified E. coli bacteria made its way into industrial biotechnology.

The genetically engineered E. coli bacteria were employed in the production of human insulin by successfully introducing the human gene responsible for insulin production into the gene sequence of the E. coli bacteria and cultivating the modified bacteria under suitable laboratory environment. Then the insulin is extracted from the cells and purified and used in humans. Also human growth hormone is produced using genetically modified E. coli bacteria.

Also there is evidence of biofuel production by using the genetically modified E. coli. The ability of the modified bacteria to convert the cellulose structure into sugars is the theory behind the production of biofuel using E. coli. The ideal character of genetically modified E. coli in producing hydrogen was discovered by a Texas scientist and he applied this principle in generating a fuel cell and proving E. coli as a house of power generation.

The fully identified gene sequence and the ability to make the gene construction in the vectors directly and a very good transformation rate of E. coli are the salient features enabled scientists to develop specific antibody molecules which will be a promising field to humans on extension.

In an effort to lower the production cost, genetically engineered E. coli were used as an alternative method in the production of the amino acid tryptophan with wide medicinal application. Also scientists from Cambridge University are on their way in developing a bio sensor E. coli to detect the presence of toxic elements in water and air. Also by extending this research, they promise the use of genetically engineered E. coli (called as E. chromi) in identifying various diseases like cancer and stomach ulcer by 2039.

Proteomics - New biology in the field of Science

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In the last decade, there has been vast development in the field of science and biomedicine. The whole sequencing of the human genome is certainly a milestone in the development and progress of science. This has helped in the rapid development in the field of medicine by providing knowledge about gene therapy, individual-based treatment for various diseases, different modes of treatment for genetic diseases, etc. Proteome can be defined as the set of various proteins expressed by a particular genome. The study of the different proteins, their dynamics and their interplay and interactions constitute the study of proteomics.

Although, both Proteomics and Protein chemistry involve the identification of various proteins expressed within the body, they are totally unrelated to each other. The protein chemistry is a part of the structural biology, dealing with physical biochemistry of the various proteins from a mechanistic point of view and relates mainly to the structure and function modelling studies and the effect of one on the other. Proteomics, on the other hand, involves systems biology, dealing with multiprotein systems and characterizing the behaviour of the whole system.

Measurement of gene expression is possible using various techniques; however, study of proteomics plays an important role considering the instability of the mRNAs preventing the formation of proteins as well as the regulatory functions of the different proteins in the biological and molecular mechanisms within the body. The gene expression can be measured by the use of cDNA as well as microarray system. Analysis of proteome is a complex task owing to the presence of posttranslational modification in proteins and also the fact that protein recognition is not based on sequence unlike oligonucleotides. Hence, special tools have been developed for proteomics such as

i)databases of protein, EST and genome sequence Mass spectrometry (MS),
ii)Mass Spectrometry (MS)
iii)collection of software that is capable of comparing the MS data with the protein sequence database, and
iv)protein separation technology

Analytical proteomics has become an emerging field in science. The identification of different proteins within the cell and their characterization is gaining importance even in medical field. The analysis of the whole protein is somewhat difficult. Hence, the tools for proteomics utilize different approaches for proper protein analysis. The protein is firstly converted to peptides and the sequence of the peptides is analysed. The sequence of the peptides is then matched with the sequence in the database to identify the proteins. The main problem in proteomics is the presence of a protein mixture in the biological sample. The protein analysis with the use of mixture is difficult; hence, the separation of the proteins is essential. The separation techniques involve the use of SDS-PAGE, which may be one-dimensional (1D) or two-dimensional (2D), High Performance Liquid Chromatography (HPLC). Proteins have also been analysed by digesting them and then carrying out separation using capillary electrophoresis or Isoelectric focusing (IEF). However, the protein separation by 2D SDS-PAGE followed by digestion into peptide fragments has emerged to be the better approach in analytical proteomics, due to numerous advantages offered by 2D SDS-PAGE. The proteins are digested using various proteases, which cleave at specific amino acids, thereby helping in the analysis with MS. Two types of instruments have been used for proteomics study: the MALDI-TOF and the ESI-tandem MS. Although, both the instruments work on completely different principles, they provide complementary information, hence both serve definite unique purpose. Peptide mass fingerprinting is a protein identification method used in high throughput proteomic study. In this method, the proteins are digested using trypsin and mass is analysed using MALDI-TOF. However, the difficulty in differentiation of homologous proteins and the similar proteins from different species limit the use of the method.

The main four applications of proteomics are:- Mining, protein expression profiling, protein network mapping and mapping of protein modifications. Mining refers to the identification of the proteins in a given sample i.e. the composition of the proteome is identified from the gene expression data from microarrays. Protein expression profiling is the identification of protein composition specific for a particular state of an organism or as a function to the effect of a drug or any other stimulus. Protein network mapping refers to the study of the interactions of various proteins in the functional network within the body and gives detailed information about the proteins in any signal transduction pathway. Mapping of protein modifications involves the study and identification of the nature and specificity of the posttranslational modification in a given protein. The development of protein arrays is under progress and if found successful, will create a great impact in the field of proteomics.

DNA to mRNA: Transcription in Eukaryotes

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According to the central dogma of molecular biology, gene expression is regulated through copying the DNA sequence into mRNA and production of encoded proteins. In this synthesizing mRNA complementary to DNA is called transcription. Transcription is generally different from prokaryotes to eukaryotes. Transcription in eukaryotes consists of initiation, elongation and termination.

Eukaryotic cells possess three different RNA Polymerases which transcribe the genes for three types of RNA’s: RNA polymerase I exclusively located in nucleolus catalyzes the synthesis of ribosomal RNA, RNA polymerase II found in nucleoplasm catalyzes the synthesis of messenger RNA and RNA polymerase III in nucleoplasm responsible for synthesis of transfer RNA. Nature of promoter recognition and initiation in transcription is different in eukaryotes than in prokaryotes.

Transcription requires the sequences on DNA that are accessible to RNA polymerase and other proteins. However, in eukaryotic cells, DNA is complexed with histone proteins in highly compressed chromatin. Therefore before transcription the chromatin structure is modified so that the DNA will come to a more open configuration and is more accessible to the transcription machinery. Enzymes involved in this modification are acetyl transferases; which adds acetyl groups to amino acids at the ends of histone proteins leading to destabilization of the nucleosome structure and makes the DNA more accessible. Chromatin remodeling proteins bind to the chromatin and displace nucleosome from promoters and other regions important for transcription.

Promoter sequences are adjacent to the genes that it regulates. Enhancer sequences are not always adjacent to the regulated genes. Promoters and enhancers are important sequences for the initiation of transcription. In eukaryotic cells, promoter recognition is carried out by accessory proteins that bind to the sequence and then recruit a specific RNA polymerase to the promoter. These comprises of general transcription factors and transcription activator proteins.

A promoter for a gene transcribed by RNA polymerase II which is the major enzyme involved typically includes one or more consensus sequences. The most common is the TATA box which has the consensus sequence TATAAA. Apart from these TFII B recognition element serve as a consensus sequence. These specific sequences in the core promoter are recognized by transcription factors that bind to them and serve as a platform for the assembly of the basal transcription apparatus. The basal transcription apparatus binds to the DNA at the start site and require for initiation. This consists of RNA polymerase, a series of general transcription factors and a complex of proteins called as mediator. General transcription factors include TFII A, TFII B, TFII D etc. These are involved in stabilizing interactions, selection of start site, active site for RNA polymerase and helicase activity to unwind DNA. Regulatory promoters are sequences located immediately upstream of the core promoter. Enhancers are involved in increasing the rate of transcription. Sometimes enhancers are act to repress transcription and these are known as silencers.

After several nucleotides have been linked together, RNA polymerase leaves the promoter and disassociates from transcription factors moving downstream. During elongation, the RNA polymerase maintains a transcription bubble in which about eight nucleotides of RNA remain base paired with DNA template.

In the course of elongation, the two strands of DNA are unwound and the Ribonucleotides that are complementary to the template strand are added to the growing 3’ end of the RNA molecule. As it funnels through the polymerase, the DNA-RNA hybrid hits the wall of amino acids, bend at almost right angle. At this bent position new nucleotides are added. The newly synthesized RNA is separated from DNA and runs through another groove before exiting from the polymerase.

Termination mechanism differs depending on the type of RNA polymerase. RNA polymerase I requires a termination factor which binds to the DNA sequence downstream of the termination site. RNA polymerase III terminates transcription after transcribing a terminator sequence that produces a string of Uracil nucleotides in the RNA molecule. Unlike the rho factor independent terminators in bacterial cells, RNA polymerase III does not require hairpin structure to proceed the string of Uracils. Research findings suggest that termination is coupled to a cleavage which is carried out by a cleavage complex that is probably associated with RNA polymerase.

At the end of transcription, a messenger RNA molecule complementary to the DNA is formed and this m RNA serve as the template for protein synthesis in translation.

Cocoa to Chocolates: Microbial Fermentation of Cocoa

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Chocolates are the wonder product comes out from cocoa. Cocoa fruits are yellow pods with a violet colour kernel. Many varieties are found in different countries such as Criollo, Trinitaro etc. Size, kernel colour, sensitivity to disease may vary depending on the variety.

After collecting the harvest, the fruits are left for few days for the attached seeds to detach from the husk. Shell and silver skin is removed when making chocolates with cocoa. Mucilage consists of nutrients, which are degraded by microbes during fermentation which results in change in flavour and aroma.

Cocoa seeds do not germinate during fermentation as microbial activity releases heat and the internal temperature rises. Inhibiting germination is one purpose of leaving the seeds for fermentation. Mucilage acts as a barrier to moisture removal. After fermentation, it can be easily removed and dried. Many biochemical reactions occur during fermentation.
The scientists have investigated cocoa fermentation and realized that the microflora were responsible for the maceration of pulp of the mucilage and killing the seeds. The seeds in a pod are sterile. When the seeds are extracted from the pods after harvesting by manual operations, they get exposed into the atmosphere where the microbes come in contact with the seeds. Fungi, Yeast and bacteria are generally involved in this fermentation process. Fungi like Aspergillus, Mucor, Penicillium, Rhizopus ; Yeasts like Saccharomyces, Pachia, Kloeckera, Candida and bacteria like lactic acid bacteria, acetic acid bacteria participate in fermentation. In first few hours of fermentation, yeast multiplication takes place. The pH of the mucilage will be around 3.6. In later stages, the environment inside the pod becomes anaerobic. Yeasts utilize the sugars in mucilage and produce Carbon dioxide. Initial temperature which was about 25 0 C rises to about 32-36 0C. Yeasts consume citric acid available in the mucilage and results in pH increase up to 4. Yeasts release pecteolytic enzymes which hydrolyzes the pectin.

In these conditions; pH 4 and temperature 320 C and anaerobic; lactic acid bacteria starts to grow. As the amount of sugars left is low, this occurs at a short period of time. These bacteria produce lactic acid. Acetic acid bacteria develop when alcohol is released into the medium as an end product by other microbes. Acetic acid bacteria convert lactic acid to acetic acid to convert energy. Small amount of acetic acid will be evaporated and the remaining penetrates into the kernel. This increases the cell wall permeability and results in further increase in pH and consequently allows biochemical reactions introducing precursors of aroma and flavour compounds. At increased pH levels, many bacteria tend to grow. In the final stages, the matrix will be rich with bacteria which break down and produce amide and some ammonical compounds. By this time, mucilage is degraded.

In the cotyledon, protein breakdown, formation of complex compounds with polyphenols, sugar hydrolysis, diffusion of organic acids and increase in permeability results. Sugar breakdown products are essential precursors of chocolate aroma. Some compounds like aldehydes, pyrazines produced are directly involved in aroma development. Caffeine and theobromine which are alkaloids diffuse from kernel cells into the outside matrix decreasing the bitterness. Polyphenolic compounds and alkaloids in pigmented cells are increased by 8-18% during fermentation. These involve flavonols, anthocyanidines, hydrocinamic derivatives and coumarin. Organic acids produced include Citric acid, Acetic acid, Lactic acid, Oxalic acid and Mallic acid. After fermentation drying rate can be increased and eventually washing away the mucilage of fermented cocoa beans.

After fermentation, beans with mucilage and placenta have to be separated from the husk pieces after the mechanical breakage. This operation is often more difficult than opening the pods. Cleaned fermented cocoa beans are roasted at 160 0C for three hours which allows enhancement of aroma and flavour followed by conching. During conching, the bitterness further reduces and also reduces the level of acetic acid. Roasting step is not sufficient to remove acetic acid.

When making chocolates, as the chocolate powder particles are very fine, the surface area is enormously large and the added fat is covered around the particle. The fat layer around the fine particles adsorb flavour and aroma compounds. Therefore, flavour characteristics are improved in the final product.

Polyethylene, Polypropylene or Intelligent Packaging: Food Packaging

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Food packaging is an important step in food manufacturing operations. Food packaging ensures the shelf life of the food item protecting it from external barriers such as insect pests, microbes and also from light, moisture, oxygen etc. Some products are stored in transparent glass containers. Such products should not be sensitive to light. Products which can be degraded by light should be stored in brown glass or with packaging material having high light barrier properties. Food items with high fat contents are subjected to oxidation in the presence of oxygen. Another concern of the modern society is that the packaging should be ecofriendly and should be readily degraded. So depending on the product marketer have to select the packaging material to protect the product. Food packaging can be referred as food protection systems.

Most packaging materials traditionally used are polymers and are not easily degradable. Barrier properties of the packaging material are also important to determine the shelf life of the product. For an instance, for a fatty food Oxygen barrier properties are important. The empty space over the product in packaged food is called as head space. A proper packaging material protects preserves, promotes and informs the food item.

Polymers used for packaging include polyethylene, polypropylene, Polyvinyl chloride, Polyvinilydine chloride and Polyethylene terephthalate etc. PET and HDPE is recyclable. For respiring food items, packaging material with high barrier properties are not suitable as it leads to rotting due to moisture accumulation. Triple laminates are used to package dried products to prevent it from moisture and to prevent escape of volatile aroma compounds. Food items with pigments/dyes important for the quality of the product; are packed in light protective containers.

Apart from these traditional methods of packaging, new trends such as active packaging, intelligent packaging has scope. In active packaging, subsidiary constituents have been deliberately included in or either the packaging material or the package headspace to enhance the performance of packaging material. In this, active compounds are filled into sachets or pads which are placed inside packages or active compounds can be directly incorporated into the packaging material. These active compounds include Oxygen scavengers/absorbers that include powdered iron, ascorbic acid. Iron powder reduces the Oxygen concentration in the headspace to less than 0.01%. Water is essential for Oxygen absorbers to function. Nonmetallic absorbers include ascorbic acid, ascorbate salt and catechol. Enzymatic oxygen scavengers include glucose oxidase, ethanol oxidase incorporated into sachets, adhesive labels or immobilized onto package surface. Apart from these vacuum packaging and Nitrogen flushing is used to remove Oxygen from the package.

As Oxygen, Carbon dioxide also can be problematic to the packaged food item as it can lead to an anaerobic environment with an acidic pH. Carbon dioxide absorbers are used to remove this. Sometimes Carbon dioxide emitters are inserted into food packaging to create anaerobic conditions. Generally used Carbon dioxide emitters are Ascorbic acid with Ferrous carbonate, Ascorbic acid with Sodium bicarbonate. These chemicals absorb Oxygen and generate equivalent amount of Carbon dioxide. CO2 emitters avoid package collapse and development of a partial vacuum. Ethylene absorbers such as Potassium permanganate oxidize ethylene to Carbon dioxide and water.

Ethanol emitters show antimicrobial effects even at lower concentrations. The substance is filled into a paper copolymer sachet. Some sachets contain traces of vanilla and other aroma compounds to mask the alcohol odour. The sachet contents absorb moisture from the food and releases ethanol vapour. Water activity of the food is an important factor in this type of packaging. This is widely used in Japan to extend the shelf life of high moisture bakery products upto 20 times the normal. Moisture absorbers are also heavily used which include Polyacrylate salts, activated clay, silica gel. Moisture accumulation can result from temperature fluctuations, drip of tissue fluid from flesh and during respiration of horticultural products. This can lead to growth of molds and bacteria.

Intelligent packaging is a great advantage of food biotechnology. In this, different sensors are used in food package which indicates about the quality of the food to the consumer. These sensors are coupled with biochemical reaction taking place inside the food when deterioration of the quality of food such as lipid oxidation, rotting of fruits etc. Gas indicators are one type of intelligent packaging. Thermochromic inks which are sensitive to temperature and microwave doness indicators (MDI) which emit audible signals when the food is ready to serve are some advance applications.

Regulation of Gene Expression : LAC Operon

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In 1961, Francis Jacob and Jacques Monad described Operon model for genetic control of lactose lactose metabolism in E. coli. Operon refers to a group of genes that functions together to achieve a common task. Many bacterial genes function as operons. Vir operon in Ti plasmids is another example for a operon. Some operons are negatively induced while some are positively induced.

Lactose is one of the major carbohydrates and found in milk, known as milk sugar. It is a disaccharide consisting of Glucose and Galactose. Lactose does not easily diffuse across the E. coli cell membrane and must be actively transported into the cell by the enzyme permease. To utilize lactose as an energy source, E. coli must first break it into Glucose and Galactose, a reaction catalyzed by the enzyme β-galactosidase. This enzyme can also convert lactose into allolactose, a compound that plays an important role in regulating lactose metabolism. A third enzyme, thiogalactoside transacetylase, also is produced by lac operon, but its function in lactose metabolism is not yet known.

The lac operon is an example of a negative inducible operon. The enzymes β-galactosidase,permease and transacetylase are encoded by the structural genes in lac operon in E. coli. β-galactosidase is encoded by Lac Z gene, permease by Lac Y gene and transacetylase by Lac A gene. When lactose and glucose is absent in the medium, the rate of synthesis of all three enzymes simultaneously increases about a thousand within two or three minutes which is stimulated by a specific molecule, called as an inducer.

Although lactose appear to be the inducer here, allolactose is actually responsible for the induction. Lac Z gene, Lac Y gene and Lac A gene have a common promoter and are transcribed together. Upstream of the promoter is the regulator gene, Lac I, which has its own promoter. Lac I gene encodes a repressor protein. Each repressor protein consists of four identical polypeptides and has two binding sites; one site for binding with allolactose and the other for binding with DNA. In the absence of lactose, the repressor binds to the Lac operator site/Lac O and prevents the transcription of the lac genes by blocking binding of RNA polymerase.
When lactose is present, some of the lactose is converted into allolactose, which binds to the repressor and cause the repressor to be released from the DNA. Then the repressor is inactivated in the presence of lactose and the binding of RNA polymerase is no longer blocked. The transcription of Lac Z, Lac Y and Lac A takes place and the Lac enzymes are produced.

Repression never completely shuts down transcription of Lac operon. Even with the active repressor bound to the operator, there is low level of transcription and a few molecules of β-galactosidase, permease and transacetylase are synthesized.

When lactose appears in the medium; the permease present, transport a small amount of lactose into cell. There, the few molecules of β-galactosidase that are present, convert some of the lactose into allolactose. The allolactose then attaches to the repressor and alters its shape so that the repressor no longer binds to the operator. When the operator site is clear, RNA polymerase can bind and transcribe the structural genes of the operon.

Several compounds related to allolactose also can bind to the lac repressor and induce transcription of the Lac operon. One such inducer is isopropylthiogalactoside (IPTG). Although IPTG inactivated the repressor and allows the transcription of Lac Z, Lac Y and Lac A; this inducer is not metabolized by β-galactosidase.

The regulation of Lac operon is used in screening of competent cells Blue white selection. There, the mutants lack the ability to produce β-galactosidase whereas the non- transformed cells can produce β-galactosidase. The produced β-galactosidase will form a complex with X gal in the medium which appear in blue colour. IPTG acts as the inducer for the activation of lac genes.

Chromosomal Aberrations: Numerical disorders and Structural abnormalities

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The chromosomes represent genetic material of an organism and are the most stable organic compound that maintains constancy both in number and structure. However chromosomes undergo unusual changes called as aberrations which can be numerical or structural. In numerical aberrations, increase or decrease in number of chromosomes are seen. Types of numerical aberrations are:

• Euploidy- complete set of chromosomes present in multiples
• Anaploidy- partial change in chromosomes

When there is an increase in number of chromosomes compared to the chromosomal number of an organ, then the condition is called as hyperaneuploidy. It is represented as 2n+1, 2n+2 etc. Aneuploidy is also classified as Monosomy, Trisomy and Nullisomy.

Monosomy is hypoaneuploidy where one of alleles of the homologous pair is lost. Monosomy is found rarely in diploids and is commonly found in polyploidy. Depending on the chromosome number, that many types of monosomies can develop. When two different chromosomes are lost, its denoted as 2n-1-1, when 3 different chromosomes of a different homologous pair is lost it is represented as 2n-1-1-1. It is called as tri Monosomy. Trisomy is a type of hyperaneuploidy where the number of individual chromosomes is more than the number of chromosomes in an organism. Edward syndrome is caused because of a trisomy. Nullisomy is the condition where both the alleles of a gene of the same pair of homologous chromosomes are lost. It is represented as 2n-2. Usually nullisomies hardly survive.

Euploidy exists in three conditions; monoploidy, haploidy and polyploidy. Monoploidy refers to the normal condition where one set of chromosome is present. Haploidy is the presence of half the number of chromosomes in a somatic cell. Haploidy can be induced by X rays, temperature shock, colchisin and delayed pollination. Experimental methods of developing haploidy involve distant hybridization, production of androgenic plants. Haploids usually produce sterile plants. Polyploidy is the condition where the number of chromosomes present in multiple copies. Types of polyploidy include autopolyploidy, allopolyploidy and segmental alloploidy.

Structural chromosomal aberrations can be intra chromosomal or inter chromosomal. Intra chromosomal structural aberrations include deletion, duplication and inversion. Inter chromosomal aberrations include translocations. Deletions can be terminal or inter special and can be caused naturally and also by chemical mutagens and radiation. These can be identified by size of the chain, change in the position of centromere and formation of loops in pachytene stage. Deletion of a portion of a dominant allele may result in expression of a recessive character. This is called as pseudodominance.

Duplication results in structural chromosomal aberrations. Duplications occur in a lower frequency than deletions. Bar eye mutation in Drosophila results in duplication in X chromosome. Inversion is an intra-chromosomal aberration where segment of chromosomes are inverted on reversed by 180 degrees. Inversions can be paracentric, where centromere is not involved or pericentric where the centromere is involved in the inverted segments of chromosome. Translocations involve two non-homologous chromosomes and position of part of the chromosome is changed leading to change in arrangement of chromosomes. Types of arrangements in translocation include alternate, adjacent I and adjacent 2. In simple translocation, a single nick occurs and the terminal position of the chromosome gets translocated on another non-homologous chromosome. In shifted translocations, two nicks are created and interstitial chromosome segment gets translocated onto another non-homologous chromosome. In reciprocal translocation, two nicks occur on both non-homologous chromosomes and separated segments get interchanged. The translocated chromosomes show change in the size of the chromosome and in position of the centromere. During pairing of homologous chromosomes, the translocate part forms a loop. Translocation brings about new linkage groups or new variation can be linked with normal genes. Translocation in human beings can lead to leukemia.

A guide for biotech education: popular biotechnology universities in Europe

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To become successful in biotechnology, you need to get a proper education first. Here’s the list of few European schools that are providing enough knowledge for any career in biotech you want.

University of Natural Resources & Applied Life Sciences in Vienna, Austria

It’s founded in 1872. Over 10 000 students are currently enrolled in studding programs at BOKU (“Universität für Bodenkultur” – German name for the university). It is research centre for renewable resources. If you are interested in agricultural engineering and water management, food and biotechnology, landscape architecture, natural resource management and ecological engineering, environmental engineering, food science and biotechnology, organic farming, phytomedicine… - beautiful Vienna is perfect place for you. Shy

Karolinska Institute in Stockholm, Sweden

It’s one of the largest and most prestigious medical universities in Europe. One of the greatest achievements associated with this university is discovery that mature cells can be reprogrammed to become pluripotent, which is rewarded with a Nobel Prize in Physiology and Medicine in 2012. It’s founded in 1810 and has 2 campuses. For all people interested in Life science – Karolinska is excellent choice (among 20 best life science colleges in the world). You can choose some of the following departments: cell and molecular biology, environmental medicine, medical biochemistry and biophysics, medical epidemiology and biostatistics, microbiology, tumor and cell biology, neuroscience, physiology and pharmacology, molecular medicine and surgery, oncology-pathology, biosciences and nutrition, laboratory medicine, neurobiology…

ETH Zurich, Switzerland

ETH Zurich is founded in 1854. Main research areas are associated with mathematics, engineering and architecture beside natural and system-oriented sciences. It won 21 Nobel Prizes in the past and had several pretty famous students, such as Albert Einstein. Over 80 new patents are released each year and main focuses are energy supply, risk management, food security and human health. It’s one of the largest universities in Europe with over 17 000 students (from 80 different countries) and more than 400 teachers.

University of Cambridge, United Kingdom

Cambridge University is second oldest university in UK, founded in 1209. It holds a record in Nobel Prizes won – 65 so far. For the second consecutive years it’s ranked number 1, in the competition for the best university in the world, according to QS World University Rankings and U.S. News & World Report. It operates botanic garden and 8 art, scientific and cultural museums. List of famous people attending this college in the past is going all the way from Charles Darwin and Watson & Crick, to Stephen Hawking, Emma Thompson & Rachel Weisz. Graduation ceremony is unique: students wearing special academic dresses are taken by university officials to the vice-chancellor for the degree they are about to take. Praelector presents graduates and claims their knowledge using Latin language. Students are kneeling and proffering their hands to the vice-chancellor, who is clasping them and conferring degree they earned using Latin again. Nice way to conclude your college days. Wink

University of Strasbourg, France

This is the largest University in France with over 43000 students and over 4000 teachers. It’s founded in 1631. In 1970 it was divided in three separate universities: Louis Pasteur University (focused on natural sciences, technology and medicine), Marc Bloch University (dealing with humanities subjects and the social sciences) and Robert Schuman University (operating with law, politics and international relations). As from January 2009, those three fused back together and today, University of Strasbourg is among the most respectful European colleges famous for high quality research and education provided.

Norwegian University of Life Sciences, Norway

UMB (Universitetet for miljø- og biovitenskap) is located in Ås, Norway. It’s famous for his beautiful surroundings (old trees, bushes, flowers…). Although established in 1859, it received university status in 2005. It’s divided in 8 departments (animal and aquacultural sciences, biotechnology and food science, ecology and natural resource management, landscape architecture and spatial planning, plant and environmental sciences…) and 6 centers (aquaculture protein center, animal production experimental centre, centre for plant research in controlled climate, centre for continuing education, the centre for integrative genetics and Norwegian centre for bioenergy research). It collaborates with lot of worldwide institutions and have exchange agreements with 93 universities/institutions worldwide (including 44 European and 8 North American), which allow people to come and experience Norwegian style of life and get a lot of valuable knowledge from environmental & food science and biotechnology.

Those were just few universities in Europe that could provide excellent education and research options. If you are coming from different country or even continent – whole experience will be even better because you’ll have a chance to meet a lot of people and exciting new cultures that are gathered on one place with same idea: to learn and develop in biotech field. Enjoy your student days where ever you are.

Associates Degree in Biotech?

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I was thinking about getting a 2 year degree in Biotechnology.

Is this a good idea? Will there be job opportunities?

Drug formulation in drug development process

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In the drug development process, the prospective drugs undergo a number of trials and are screened at various stages to generate the final potent drug for the treatment of various diseases. During the screening process, various properties are tested to see if the drug is suitable for the therapeutics and is non-toxic to the living system. After going through the different stages, the candidate found most suitable for the purpose is selected. Formulation of the drug plays a very important role in drug delivery to the target area within the body.

The testing of the viability of the drug within the living system is a very crucial issue in the drug discovery process. The drug must remain viable within the living system until it produces the required effect within the body after reaching the target organ or system. If the drug is very potent for the treatment but is immediately degraded after its entry into the living system due to various enzymes and the physical or biochemical environment within the living system, the administration of such a drug serves no use. Hence, the degradation and thereby clearance of the drug from the system must be avoided for proper drug action to take place. In such cases, drug formulation becomes important. Formulation of a drug has become a very important step in the drug discovery and development process. The potent drugs or biopharmaceuticals have different modes of delivery into the biological system like oral, pulmonary, nasal, transmucosal or transdermal routes depending on the characteristics of the drug, the drug target as well as the advantages of using the specific delivery route.

Formulation plays an essential role in the proper absorption of drugs within the living system. Proper formulation is necessary in solubilizing the drug in the formulation medium such that it helps in the delivery and release of the drugs to the target. Thus, formulation helps in increasing the bioavailability of the drug within the system by increasing its solubility, thus helping in improving its pharmacokinetics. The increase in the bioavailability helps in increasing the therapeutic effect of the drug shortening the time of the production of favourable effect and also in reducing the frequency of dose of the drug. It can also reduce the side effects of the drugs and the tissue specific formulations can help in the reduction of toxicity due to a particular formulation.

Different types of formulations are devised depending on the drug delivery route like for e.g. emulsion or solution for intravenous; solid, suspension or solution for oral; emulsion, suspension or solution for subcutaneous, etc. In case of insoluble compounds, various strategies have been adopted for proper formulation like:

1. the adjustment of the pH of the solution in case of ionizing compounds
as ionization increases the solubility of the compounds;

2. use of co-solvents like glycerine, ethanol, PEG, DMSO, etc to increase
its solubility, though proper care must be taken in the choice of co-
solvent in case of animal PK studies due to possible toxicity from the
co-solvent;

3. Surfactants like Tween 80, SDS, polysorbate, etc in proper dilutions
are used to help in the micellization of the drugs within the body,
which enhances the drug solubility, prevents precipitation due to
surface properties of the drugs, prevents aggregation of protein-based
drugs, thereby enhancing the stability of the drugs within the system.

Lipid based formulations are devised for the lipophilic compounds that are delivered as emulsion. The technology of the liposome delivery system has progressed in the application of oncology as well as for the treatment of viral, bacterial, and fungal infections. Its versatility is the reason, which has helped in the formulation of many classes of drugs.

The choice of proper formulation is very crucial in the PK studies, toxicity studies and in animal pharmacology studies. In the pharmacokinetic (PK) screening of the drug candidates, solubility is an important parameter and the choice of proper formulation is essential as the PK profile must remain unaffected while increasing the solubility of the drugs. The choice of proper surfactant and co-solvent is necessary such that it does not cause any form of toxicity while using the formulated drug. Optimal formulation is very important in the development of animal PK studies to demonstrate drug efficacy and activity. Recent studies have shown that formulation is being studied seriously in gene-delivery systems and in the delivery of drugs using nanoparticles.

Lactic Acid Bacteria as Probiotics and Effect of Prebiotics

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Lactic acid bacteria (LAB) are gram positive, non sporing, cocci or rods that produce lactic acid as the end product of sugar fermentation. The taxonomic boundaries of Lactic acid bacteria have been controversial. In fermented foods like yoghurt and lassi, lactic acid bacteria play a vital role in flavour development and preservation as well.

The classification of lactic acid bacteria into different genera is based on morphology, ability grow at harsh conditions, mode of glucose fermentation, chemotaxonomic markers, DNA sequence data etc. These include eight genera; Lactobacillus, Leuconostoc, Pediococcus, Streptococcus, Carnobacterium, Enterococcus, Lactococcus, Vagococcus of which the latter four genera were originally classified under Streptococci. These organisms are broadly divided into two groups as homofermenters who produce lactic acid as the major end product of glucose fermentation and heterofermenters who produce Carbon dioxide, Ethanol apart from lactic acid. Pediococcus, Lactococcus, Vagococcus belong to homofermenters and Leuconostoc, Carnobacterium and some Lactobacilli belong to heterofermenters.

Lactic acid bacteria cause some negative effects such as development of acidity in milk and spoilage of some other food such as meat etc. There is strong evidence that when consumed in sufficient quantities Lactic acid bacteria exhibit prophylactic and therapeutic effects. About one million viable cells are required to obtain these health beneficial effects.
Probiotics concept was introduced in clinical nutrition in 1980’s. This emphasized the positive physiological role of certain Lactic acid bacteria and Bifidobacteria. Probiotic lactic acid bacteria and Bifidobacteria are capable of passing through upper gastrointestinal tract and colonizing in the large intestine. Health benefits of LAB as probiotics include:

• Reduce the risk of diarrheal infections
• Enhance immune function
• Reduce the severity of lactose intolerance
• Reduce the population of harmful microorganisms in the colon
• Reduce the incidence of colon cancer
• Lower serum cholesterol level
• Lower blood pressure
• Improve mineral absorption
• Restore gut flora during or after antibiotic therapy
• Improve balance of microorganisms in the colon
• Improve bowel function by increasing stool frequency, increasing stool weight and increased production of short chain fatty acids (SCFA)

Dysbiosis is the condition where the harmful microorganisms in the gut overpopulate the beneficial bacteria. This is the major reason for many dietary diseases. The reasons for dysbiosis can be harmful pesticide residues remaining in food accumulated during a long period of time, food allergies and other disease conditions which can be overcome with the use of probiotics. Probiotic bacteria bring about these beneficial effects by competitive exclusion, production of bacteriocins and organic acids and altered absorption of the intestinal mucosa.

It was shown that incorporation of indigestible polysaccharides that escape digestion in the upper gastrointestinal tract are very effective in boosting the probiotic effects. These are known as prebiotics and increase the proportion of beneficial bacteria in the colon. Food entering the colon serves as substrate for endogenous colon bacteria, thus indirectly providing the host with energy, metabolic substrate and essential micronutrients. Prebiotic concept was introduced by Gibson et al. Cereal dietary fibre render prebiotic effects. Cereal based fermented foods carry both live bacteria and prebiotic dietary fibre. These dietary fibres should be soluble, hydrolysable and fermentable by the gut microflora. A prebiotic should,

• Be neither hydrolyzed nor absorbed in the upper Gastrointestinal tract
• Selectively stimulate the growth of potentially beneficial bacteria in the colon

End products of these fibres include short chain fatty acids ( acetic acid, propionic acid, butyrate acid etc.) , Carbon dioxide, Hydrogen Sulphide, Methane and Hydrogen. Gases are either excreted or metabolized. Short chain fatty acids are quickly absorbed in to the blood. Especially propionates and acetates have been reported to lower hepatic cholesterol synthesis. Resistant starch, unabsorbable sugars, oligo and polysaccharides (soybean oligosaccharides-stachyose and raffinose), β glucan. Arabinoxylans are few examples for prebiotics. These are also called as ‘colonic food’.

Synbiotics are the metabolites produced by the synergistic action of prebiotics and probiotics such as short chain fatty acids, amino acids, peptides, growth factors, signal molecules etc.

Role of Microbes in Fermented Food

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Fermentation is the process of controlling microbes (bacteria, yeast, and moulds) to modify food, producing a desired product.

Often we talk about the negative issues caused by microorganisms in food such as food spoilage. But they can be used as efficient live factories to produce health beneficial food. Without some microorganisms, production of certain food items may not be possible or may be costly. Microbes are cheap resources that consist of numerous enzymes which can convert complex chemical structures into simple digestible molecules with a high efficiency. These microorganisms use the nutrients in the food as the substrate to produce energy and other required precursors for their growth in which a fermented food results. There are numerous examples for fermented food.

Fish sauce
In production of fish sauce, uneviscerated fish is mixed with salt and placed in fermented tanks to allow liquefaction for about six months. The collected liquid is further ripened for few more months. Halophillic microbes are involved in this fermentation process. Streptococcus, Micrococcus and Bacillus species predominate. This product is dark coloured with a distinct aroma.

Sauerkraut
This refers to fermented cabbage. Normal microflora in cabbage is involved in the fermentation process under anaerobic conditions. Leuconostoc mesenteroides and Lactobacillus plantarum is involved. Temperature is a crucial factor in the control of fermentation. If the temperature is below 21 degrees Celcius, Lactobacilli outgrow. L. mesenteroides require a lower temperature below 21 0C. Acidity created by Lactobacilli prevent the growth of L. mesenteroides.
Pickels
Pickels consist of vegetables like cucumber, onions, chilies etc. Lactic acid bacteria such as Leuconostoc mesenteroides, P. cerevisiae, L. brevis, L. plantarum are involved in the fermentation process These bacteria also take part in fermentation of olives.

Soy sauce
In production of soy sauce, a mixture of soybean and wheat flour is inoculated with Aspergillus oryzae and Aspergillus soyae. These fungi digest complex starch and produce sugars which facilitate the growth of bacteria. Anaerobic bacteria carry out fermentation to produce soy sauce.

Beer and Ale
Malted beverages are produced by brewing. Mainly the yeasts are involved in the process. Yeasts convert fermentable sugars to ethanol and carbon dioxide. As yeasts do not produce enough amylases to hydrolyze starch in barley grains, they are germinated prior to brewing. Hops which are added for bitterness have an inhibitory effect on gram positive bacteria. Saccharomyces carlsbergensis is the principle organism used. This species is subjected to various genetic modifications to increase the efficiency of the fermentation. In addition to ethanol and carbon dioxide, yeasts produce a small amount of glycerol, acetic acid and aromatic esters. Ale is a top fermented beverage with Saccharomyces cerevisiae.

Wine
Wine is made from grape juice in large scale. Yests; Saccharomyces cerevisiae var. ellipsoideus is the culture used in wine fermentation. High temperature is not suitable for this fermentation as yeasts die while low temperature allows the growth of lactic acid bacteria.

Environmental factors such as acidity, pH, oxygen level, moisture level, temperature, sugar content are important for this fermentation processes. In most food commodities, acidity is developed by microorganisms. This developed acidity is important for preserving the food. Proteolytic action may bring down the pH to a higher value. Some yeasts produce alkaline by products such as ammonia in their regular metabolism which is encouraged in the production of limburger cheese.

Alcohol, which is a byproduct of many fermentation pathways also have a preservative action. Alcohol content produced will depend on the sugar content, type of yeast involved in fermentation, temperature and the oxygen level. Most yeast can’t tolerate high alcohol levels.
Temperature has a direct effect on microbial fermentation which in turn affects the final quality of the product. Sauerkraut production is an ideal example to show the importance of temperature in fermentation process.
Microorganisms have different oxygen requirements for their growth and fermentation. In wine production, yeasts grow best under aerobic conditions. In baking, anaerobic conditions favour the quality of the final product. Vinegar production involves anaerobic as well as aerobic fermentation.

What are some hot biotech/biology research techniques being developed or used in labs

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At the DNA or RNA level, please. For example, the method of RNAi.

Thank you!

Recombinant Insulin - Successful Application of Genetic Engineering

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Insulin is a protein hormone secreted from the specialized cells of the islet of Langerhans in pancreas. Only 2% of the pancreas has endocrine function secreting various types of hormones regulating the metabolism of glucose. Of them, the β cells constitute about 65-80% and they are responsible for the secretion of insulin. It is polypeptide in nature and affects almost all the cells and organs within the body. It affects the metabolism of carbohydrates, fats and proteins and is responsible for various anabolic reactions, which help in lowering the blood sugar level in the blood. Diabetes mellitus has become a very common disease in the present times after cancer and cardiovascular diseases. Hence, the role of insulin and its various aspects have become a topic for serious study and research.

Insulin is a simple polypeptide hormone consisting of 51 amino acids. It is made of two polypeptide chains A and B. A chain consists of 30 amino acids while the B chain consists of 21 amino acids and the two chains are linked by a disulfide bond. The insulin structure and composition varies in different species as the sequence of the amino acids in human insulin is highly conserved. Diabetes mellitus if of two types: Type I, which is an autoimmune disorder characterized by lower insulin levels in the body and Type II in which insulin levels are normal but the body develops peripheral resistance to it resulting in high blood sugar levels. Type I form is a very common disease; hence, different types of treatment to maintain insulin level in the body are being developed. It was seen that the administration of purified animal insulin could not be done as it resulted in immune response. Hence, two forms of treatment was followed, which are: artificial synthesis of insulin using amino acids, which is an expensive and long drawn process and the replacement of amino acids in the purified animal insulin to represent human insulin, known as semi-synthetic insulin. The synthesis of semi-synthetic insulin also faced problems in the form of limited availability of porcine insulin, which was most suitable for the purpose. In this situation, the research on the synthesis of recombinant insulin was carried out to meet the increasing demands for insulin.

Recombinant insulin is the first commercial biotech product. The advances in RDT (Recombinant DNA Technology) with the discovery of various restriction enzymes, cloning strategies, etc has helped in the successful formation of recombinant insulin. The use of recombinant insulin offers two main advantages: unlimited availability of insulin to meet increasing demands of diabetic patients and the identical similarity of human and recombinant insulin. The RDT has used both bacteria and yeast for the recombinant insulin production. The production uses two types of strategies:

i) The synthesis of the two chains of insulin and joining them with disulfide linkage using ideal conditions, and
ii) The synthesis of proinsulin, which then forms mature Insulin after the cleavage of C-peptide and formation of disulfide bond between the two chains.

In the first method, two DNA fragments based on the sequence of the amino acids in the A and B chains were synthesised. These fragments were inserted into plasmids, which were transformed into the bacteria. The two chains were then isolated and mixed under ideal conditions for the formation of disulfide links between them, producing functional Insulin. The other method involved the formation of proinsulin in bacteria, which then formed mature Insulin by a single enzymatic reaction. This was found to be a better procedure due to the lesser number of steps involved in the production and purification of mature insulin. After the formation of recombinant insulin from bacteria, purification is a very essential step as contamination with the C-peptide, proinsulin, etc may cause ineffective result in the use of recombinant insulin. In order to minimise the purification procedure, which may result in the loss of recombinant insulin, the latest technology involves the use of yeast for the production of proinsulin. The enzymes required for the formation of mature insulin like the ones present within the human body are present within the yeast yielding a better formation and isolation of the formed mature insulin from the media, where recombinant yeast is cultured.

Insulin is the first genetically engineered hormone opening the field of research for the generation of other important hormones and proteins for the treatment of different fatal diseases. The advance in genetic engineering technology is becoming the main gateway for the progress of medical science and discovery of helpful drugs for the welfare of living beings.

Development of DNA chip technology

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The great advancement in the field of molecular biology has been the Human Genome Project, whereby the whole human genome was sequenced using DNA sequencing method. This project has marked a turning point and has opened the gate for various medical discoveries and technology. The advent of DNA chip technology or DNA microarray technology has ushered in a new era in the field of Systems biology, which has helped in studying the transcriptional behaviour of different genes. The DNA chip actually helps in knowing the expression of mRNA in its steady state. The DNA chips have proved to be very useful in the study of different diseases and the modification of the gene expression in the development of the disease.

Various breakthroughs in the field of technology have helped in the development of DNA or gene chips. The technology of DNA chip is the result of the combination of molecular biology and microfabrication technology. Although gene expression could be known by sequencing method, but the complete idea about the levels of gene expression or the activity of the genes could be known only after the study of mRNA transcribed from the genes as all the genes present are not transcribed into mRNA. The differences in the amount and nature of mRNA transcribed by the DNA of a particular organ or cell gives clear idea about the specific proteins transcribed from the specific organ or tissue or cell within the body and their effect on various diseases.

DNA chip technology works on the hybridization principle. There are mainly two types of chips: cDNA array and oligonucleotide array. The cDNA array consists of long DNA fragments purified by PCR, which are immobilised on glass or plastic wafer with the use of robots. They are generally used for the study of expression and screening. The oligonucleotide array consists of short fragments of DNA that are synthesised chemically or conventionally and are then immobilised on the glass substrate. They are generally used for the study of mutations, monitoring of gene expression, discovery and mapping. For the genomic analysis, both the types of arrays are used simultaneously. The probes used for the development of DNA chips are manufactured using a number of techniques such as light induced chemical synthesis, or the use of spraying solution, which deposits chemical probes on the chip substrate, or the use of robots, which deposit the probes on the substrate. The main idea is to miniaturize the whole technology of genomic analysis with the use of DNA chips. The level of the mRNA transcription can be studied with the use of DNA chips. The total mRNA is first isolated, purified and reverse transcribed to cDNA and then with the use of probes, it is hybridised on the immobilised DNA on the chip substrate and finally the level of its expression can be known.

The use of DNA chips have many advantages such as they can be used to monitor many genes at the same time, producing fast results, comparing diseased and normal cells according to the expression of various genes and also help in categorizing the subgroups of a particular disease with slight variations in the different genes. However, there are some disadvantages also of the technique. The technology is quite expensive and the production of too many results at a time requires long time for analysis, which is quite complex in nature. The reproducibility of the results is also a question as the results are not always quantitative. The DNA chips do not have very long shelf life, which proves to be another major disadvantage of the technology.

The applications of the DNA chip technology has gained much importance in the present time and will pave way for further innovative developments in molecular biology in future. The technology has found application in not only the study of mRNA expression but also the DNA replication in bacteria, yeasts, etc., the study of histone proteins, their modification, and the different mRNA binding proteins and even in the study of different mechanisms in cell biology. The development of DNA chip technology or nanoarray technology can be used to detect different diseases like Alzheimer’s disease, cystic fibrosis, different forms of cancer, etc. The DNA chip has also found application in the detection of Single Nucleotide Polymorphisms (SNPs) in the genome and also in the research related to effect of different drugs on the different cells of the body and drug efficacy.

Overview of Pharmacogenetics and Pharmacogenomics in drug discovery process

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In the drug research process, a number of candidates fail to become therapeutic agents due to a number of reasons. In the drug discovery and development process, the aspect of the effect of genes has become a major issue in the present times. The discovery of a number of genetic diseases, the role of genes in regulating the pharmacokinetics of the drugs within the body and also the role of gene expression in determining the drug efficacy has resulted in the study of pharmacogenetics and pharmacogenomics largely.

Although the terms Pharmacogenetics and Pharmcogenomics are used interchangeably, there is considerable difference between the two. Pharmacogenetics identifies with the study of single gene mutations and its effect on the response of the drug within the body, while Pharmacogenomics has wider aspect covering the whole genome and the effect of different genes and their expression on the different aspects of drug response and efficacy within the body. The study of the pharmacogenomic profile of the patients helps in the determination of the target, its specificity and the whole drug development process, in general. It has been suggested that Pharmacogenetics may help in the differentiation between the patients while studying about a drug, while Pharmacogenomics may help in the differentiation and screening of the compounds.

It is seen that the medicines are being administered on the patients based on the symptoms and evidence after collecting the data of the clinical trials on the population as a whole. This general administration of the drug based on statistical data may give rise to differential response in the patient and may also result in possible toxicity. Hence, the study of the pharmacogenomic profile of the patient has become very important, in order to carry out proper treatment of the patient without any toxicity. The study of the SNPs (Single Nucleotide Polymorphisms) has become very essential as the occurrence of the SNP in the genome of an individual is high, which are also inherited resulting in the haplotype of an individual. Hence, in future the scanning of the genome for SNPs and the haplotype may help in the determination of the nature of drug response in a better way. Other Pharmacologic technologies such as DNA chips and Proteomics have also helped in the drug development process to a large extent.

Pharmacogenetics and Pharmacogenomics play important role in the target specification and pharmacokinetics of the drugs. The targets are polymorphic in nature as the targets are mainly protein in nature. The appropriate target selection is very essential for the pre-clinical trials of the prospective drugs as it gives a clear idea about drug efficacy. The polymorphic nature of the drug metabolizing enzymes, receptors, and transporters affects the pharmacokinetics of the drugs drastically. Hence, the knowledge about the pharmacogenomics of an individual is very essential. The study of the SNPs helps in knowing the safety profile of the drugs, which is helpful in avoiding toxic reactions in patients during the pre-clinical trials. The Pharmacogenomic profile gives a very clear idea about the variation in the genes responsible for the formation of the disease, thus giving an idea about the predisposition of people to develop particular diseases. This can help in further research related to the discovery of drugs and therapeutics like gene therapy for the treatment of the disease. The non-responsiveness to some drugs or development of adverse reactions for some drugs can also be known with the help of the pharmacogenomic profile of the patients.

Thus, it can be said that Pharmacogenomics has a very important role in the pharmaceutical industry. Genotype-guided therapies are becoming very crucial as patients respond to a drug based on their genetic factors. Pharmacogenomics helps in treating the right patient with the right drug and at right dose. This is the major implication of the study. However, it has to be kept in mind that Pharmacogenomics is important in healthcare and its study will be included as a part of the pre-clinical trial of the prospective drugs in near future. However, it is not important for all drugs, in general as its study is important for the drugs, which have narrow therapeutic index. The successful application of the knowledge of Pharmacogenomics in the drug discovery and development process will thus help in the reduction in the overall cost in the process by the reduction of the number of failures in the process.

Transformation, Transduction and Transfection –Gene transfer methods

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The three very effective modes of gene transfer Transformation, Transduction and Transfection observed in bacteria fascinated the scientist leading to the development of molecular cloning. The basic principle applied in molecular cloning is transfer of desired gene from donor to a selected recipient for various applications in the field of medicine, research, gene therapy with an ultimate aim of beneficial to the mankind.

Transformation: Transformation is the naturally occurring process of gene transfer which involves absorption of the genetic material by a cell through cell membrane causing the fusion of the foreign DNA with the native DNA resulting in the genetic expression of the received DNA. Transformation is usually a natural method of gene transfer but as a result of technological advancement originated the artificial or induced transformation. Thus there are two types called as natural transformation and artificial or induced transformation. In natural transformation, the foreign DNA attaches itself to the host cell DNA receptor and with the help of the protein DNA translocase it enters the host cell. The presence of nucleases restricts the entry of two strands of the DNA, destroys a single strand thus allowing only one strand to enter the host cell. This single stranded DNA mingles with the host genetic material successfully.

The artificial or induced method of transformation is done under laboratory condition which is either a chemical mediated gene transfer or done by electroporation. In the chemical mediated gene transfer, the cold conditioned cells in calcium chloride solution are exposed to sudden heat which increases the permeability of the cell membrane allowing the foreign DNA. The electroporation method as the name indicates, pores are made in the cell by exposing it to suitable electric field, allowing the entry of the DNA. The opened up portions of the cell are sealed by the ability of the cell to repair.

Transduction: In transduction, a media like virus is required between two bacterial cells in transferring genes from one cell to the other. Researchers used virus as a tool to introduce foreign DNA from the selected species to the target organism. Transduction mode of gene transfer follows either a lysogenic phase or lytic phase. In the lysogenic phase, the viral (phage) DNA once joining the bacterial DNA through transduction stays dormant in the following generations. The induction of lysogenic cycle by an external factor like UV light results in lytic phase. In lytic phase, the viral or phage DNA exists a s a separate entity in the host cell and the host cell replicates viral DNA mistaking it for its own DNA.As a result many phages are produced within the host cell and when the number exceeds it causes the lysis of the host cell and the phages exits and infects other cells. As this process involves existence of both the genome of the phage and the genome of the bacteria in the same cell, it may result in exchange of some genes between the two DNA. As a result, the newly developed phage leaving the cell may carry a bacterial gene and transfer it to the other cell it infects. Also some of the phage genes may be present in the host cell. There are two types of transduction called as generalized transduction in which any of the bacterial gene is transferred via the bacteriophage to the other bacteria and specialized transduction involves transfer of limited or selected set of genes.

Transfection: One of the methods of gene transfer where the genetic material is deliberately introduced into the animal cell in view of studying various functions of proteins and the gene. This mode of gene transfer involves creation of pores on the cell membrane enabling the cell to receive the foreign genetic material. The significance of creating pores and introducing the DNA into the host mammalian cell contributed to different methods in transfection. Chemical mediated transfection involves use of either calcium phosphate or cationic polymers or liposomes. Electroporation, sonoporation, impalefection, optical transfection, hydro dynamic delivery are some of the non chemical based gene transfer. Particle based transfection uses gene gun technique where a nanoparticle is used to transfer the DNA to host cell or by another method called as magnetofection. Nucleofection and use of heat shock are the other evolved methods for successful transfection.
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