Sunday, August 30, 2009

Futuristic use of Transgenic Animals & Transgenic Plants


Transgenic Animals:Transgenic animals are being developed for a wide variety of applications. In the near future, transgenic animals will be used increasingly in safety evaluation of new pharmaceuticals and accelerating their regulatory approval. The feasibility of producing human pharmaceutical proteins in the milk of transgenic livestock has been established.

As an alternative to cell-culture systems, such livestock appear to be appealing because of high volumetric productivity, low operating costs, capability of posttranslation modification of proteins, and potential for expansion of the producing organism. Bioprocess engineers face numerous technical challenges in converting a transgenic mammary gland system into a commercial prototype for large-scale manufacture of high-market-volume proteins, including the following:

1) Purification techniques for obtaining high-purity proteins that must be recovered and fractionated from a complex mixture of fats, proteins, sugars, and ions, some of which are in colloidal form.

2) Optimization of product stability during recovery.

3) Instrumentation to characterize posttranslation modifications made by the mammary gland "bioreactor."

4) Development of on-line sensors to monitor changes in bioactivity of products during purification.

5) Bioseparations of milk proteins.

In the longer term, transgenic animals might provide a source of tissues and organs for use in transplantation patients. Bioprocess engineering will be needed to design novel equipment to maintain, purify, and store the living tissues without affecting viability or graft response.

The hurdles to be surmounted in developing the necessary genetic tools for systematic pathway engineering are substantial, but basic research at the molecular level will continue to provide improved production strains and novel products, and continued interest in the fundamentals of bioprocessing of milk will help to define separation strategies for this complex biological fluid.

Transgenic Plants:Transgenic plants are capable of generating specialty chemicals or other bioproducts. Special bioprocessing capabilities will then also need to be developed for extracting, concentrating, and purifying such products from plant tissue. This sector of bioprocess engineering might also be important to the prospects of expanding crops or developing new varieties that are rich in fermentable carbohydrates, which are readily used as feedstocks for large-scale manufacturing of specialty and industrial chemicals.

Transgenic tobacco plants have been developed to produce monoclonal antibodies identical in function with the original mouse antibody. Other proteins produced in plants are human serum albumin and enkephalins. Processes to recover and purify proteins from plant-cell extracts will be needed if such systems are commercialized.

Tags: Bio Technology, Bio Genetics, Bio Process Engineering

Understand the Basics of Metabolic Engineering


A powerful new approach to product development is the creative application of fermentation technology and molecular biology for "metabolic engineering." Examples of metabolic engineering for heterologous-protein production include deletion of proteases that eliminate product and production of factors that facilitate product maturation and secretion. For protein production on an industrial scale, metabolic engineering could be useful in shifting metabolic flow toward a desired product, creating arrays of enzymatic activities for synthesis of novel structures, and accelerating rate-limiting steps. Metabolic engineering has recently been used to increase the efficiency of nutrient assimilation (increasing the growth rate), improve the efficiency of ATP production (decreasing nutrient demands), and reduce the production of inhibitory end products (increasing final cell densities).

Central to molecular modification of multigene pathways, such as those involved in antibiotic production, is the development of new vectors and transformation procedures and other tools of molecular biology. Another important discovery in metabolic engineering is the isolation of positive-control genes that regulate production of secondary metabolites. Positive regulators have been found in biosynthetic gene clusters for actinorhodin, bialophos, streptomycin, and undecylprodigiosin, all of which are Streptomyces products.

Genes encoding the converting enzymes D-amino acid oxidase and cephalosporin acylase were cloned from Fusarium and Pseudomonas, respectively, into the fungus Acremonium chrysogenum. Expression of this "artificial" antibiotic biosynthetic pathway was confirmed by analysis of transformants that synthesized and secreted detectable amounts of 7- aminocephalosporanic acid.

In addition to classical mutation, new tools have become available for genetic manipulation of important producers of natural products, such as Streptomyces. The ability to clone and manipulate biosynthetic genes for antibiotic production, regulatory genes for improved synthesis, and genes from primary metabolic pathways that contribute to secondary biosynthetic pathways can facilitate construction of strains that have substantially altered metabolic properties. In addition, the cloning of heterologous genes into bacterial hosts has generated strains that can produce compounds that are foreign and even
Deleterious to cell physiology.

Tags: Bio Technology, Bio Genetics, Bio Process Engineering

Know the Basics of Protein Engineering


Advances in molecular biology have provided researchers with the opportunity to develop increasingly rational approaches to the design of therapeutic drugs. This technology, when used with computer-assisted molecular modeling, is called protein engineering.

Protein engineering combines many techniques, including gene cloning, site-directed mutagenesis, protein expression, structural characterization of the product, and bioactivity analyses; it can be used to modify the primary sequence of a protein at selected sites to improve stability, pharmacokinetics, bioactivity, and serum half-life. A second application of protein engineering is the design of hybrid proteins that contain regions that aid separation and purification. That is achieved by introducing, next to the structural gene for the desired product, a DNA sequence that encodes for a specific polypeptide "tail."

The tails can be inserted at the N or C terminal of the protein to yield a fusion protein with special properties that facilitate separation. Such genetic modifications can be designed to take advantage of affinity, ion-exchange, hydrophobic, metal chelate, and covalent separations. The special properties of fusion proteins allow crude microbial extracts to be passed over an adsorbent that binds specifically to the tail, so that the desired product is retained and contaminants pass through. After elution and treatment to remove the tail, the product is purified further by standard methods, such as size-exclusion chromatography or high-performance liquid chromatography (HPLC).

Tags: Bio Technology, Bio Genetics, Bio Process Engineering

Challenges to Isolation and Purification of Proteins


Isolation generally denotes the separation of the product from the bulk of the producing organism. The disposition and state of the expressed protein affect the isolation procedure. For mammalian cells and some E. coli, Streptomyces, Bacillus, and yeast products, the protein is released from the cell into the surrounding medium, and isolation is effected by a solid-liquid separation step, usually centrifugation or microfiltration or ultrafiltration. If the product has aggregated either in the cytoplasmic or periplasmic space, isolation is more involved. Generally, the cell is first lysed by mechanical, chemical, or enzymatic treatment (or a combination). In some cases, the more dense aggregate can be separated by centrifugation from most of the soluble and insoluble cell components; in other cases, the aggregate is first solubilized while still in the soluble protein mixture.

Purification of the protein is a critical and often expensive part of the process. It might account for 50% or more of the total production cost. Purification has several objectives: to remove contaminating components from the host organism, i.e., other proteins, DNA, and lipids; to separate the desired protein (or family of proteins) from undesired variants of the desired protein; to remove and avoid the introduction of endotoxin; to inactivate viruses; to obtain required yields at acceptable cost; to avoid chemical or biochemical modification of the protein; and to make the process consistent and reliable. In some cases, the first and additional objective is to fold the protein into its desired conformation.

The most common individual operations are centrifugation, filtration, membrane separation, adsorption separation, and chromatography.

The difficulty of separation can often be decreased by changing the organism or culture conditions to produce a more uniform protein. However, it is still necessary to combine a series of purification steps each of which separates according to a different principle. Ultrafiltration steps are often used between separation steps to concentrate the protein solution or to make the buffer solution compatible with the next separation step. The final steps are designed to place the purified protein in the solution used for the product form.

The complexity of the individual purification steps and the need to be able to integrate them into a manufacturing system translate into a major opportunity for bio-processing engineering as the process moves from the bench to the plant. Research and development in purification, scaleup integration, and system design will continue to have high priority.

Tags: Bio Technology, Bio Genetics, Bio Process Engineering

Bio-Processing of Renewable Resources & its challenges


Most of the applications and potential applications of bioprocessing related to renewable and nonrenewable resources involve large-scale operations and products of relatively low value. The most abundant renewable material is lignocellulose. Wood, agricultural residue (corn stover, straw, etc.), plants grown deliberately for biomass (such as hybrid aspen), and recycled pulp fiber are the main sources of lignocellulose. Its largest industrial use is in making pulps for paper and other fiber products; second is the use of wood directly in construction.

Key Technical Challenges in Bioprocessing of Renewable Resources are as under:

1) To develop inexpensive cellulose pretreatment and saccharification processes effective with lignocellulosic materials on large scale with environmentally compatible methods.

2) To develop fermentations capable of converting pentoses to value-added products at yields, rates, and extents similar to those obtained for glucose with yeast and to increase product concentrations achievable in both hexose and pentose fermentations.

3) To develop more efficient separations for recovering fermentation products, sugars,and other dissolved materials from water, i.e., lower cost of separating water from product in fermentation broth.

4) To develop processes for large-scale inoculation, control, and propagation of microorganisms in surface culture (e.g., treatment of wood chips and bioremediation of soils) and solid substrate fermentation.

5) To increase knowledge of combinations of chemical, biochemical, and microbial transformations that result in value-added nonfood products from starch and cellulose.

6) To improve fractionation methods for separating oil, starch, and fiber components during corn milling to obtain higher co-product values with lower capital investment.

Tags: Bio Technology, Bio Genetics, Bio Process Engineering

Understanding Bio-Process Engineering

Bioprocess engineering is the subdiscipline within biotechnology that is responsible for translating life-science discoveries into practical products, processes, or systems capable of serving the needs of society. It is critical in moving newly discovered bioproducts into the hands of the consuming public. The bioprocess engineer has many missions. Although the most visible today is the production of biopharmaceuticals, bioprocess engineering also has a major role in the existing fermentation industries responsible for the production of ethanol, amino acids and other organic acids, antibiotics, and other specialty products.

Bioprocessing in space presents unique opportunities, particularly in bioregenerative life support and as a research platform for the study of new types of manufacturing processes.

Bioprocessing for protection and beneficiation of the environment represents another large and important opportunity. Biological processes could offer alternatives to environmentally polluting or fossil-fuel-consuming manufacturing processes and could help to remove toxic pollutants from industrial and municipal wastes. Bioremediation's promise is in its potentially lower cost, compared with other types of technology for cleaning up the environment.

Role of bio-process engineering:
The role of bio-process engineering in the successful commercialization of biotechnology is not fully understood by our national government, industrial, and academic leadership. That is in large measure because first-generation biopharmaceutical products have been successfully produced with only secondary concern for costs of manufacturing. However, products now under development will require novel techniques and more efficient and economical processes. Hence, our participation in the expanding bio-products market will necessitate an expanded role of bio-process engineering. This is all the more important because bio-process engineering could have a profound effect on the existing fermentation industry.

Bio-process development for biopharmaceuticals involves all aspects of generating a safe, effective, and stable product. It begins with the biological system, continues with product isolation and purification, and finishes when the product is placed in a stable, efficacious, and convenient form. The product is initially derived either directly or indirectly from a living organism. Thus, process development starts with the development of the biological system. It is usually a living organism that expresses the desired protein; but it might be an enzyme for protein modification or an antibody for immunoaffinity purification. RDNA and hybridoma technology allow the biological system to be optimized for maximal formation of the product, for facilitation of downstream processing, for high product quality, and for improved interaction with the production equipment. In this phase of bio-process engineering, many disciplines must be applied, including molecular biology, genetics, biochemistry, analytical chemistry, and bio-process engineering. Thus, engineers become full partners with experts trained in the bioscience disciplines in developing and scaling up manufacturing technology for biopharmaceuticals.

Tags: Bio Technology, Bio Genetics, Bio Process Engineering

Wednesday, August 5, 2009

Understanding various concerns of Human Genome Project

The importance of the Human Genome Project has raised many concerns, both biological and ethical. These questions are being addressed as the information generated by the project is being processed and used by people worldwide.

1) Privacy and confidentiality of the genetic information: Who owns the genetic information?

2) Right to use the genetic information by insurance companies, employers, courts, schools, adoption agencies, and so on: Who should have access to individual genetic information and how should it be used?

3) Psychological impact and stigma attached to an individual's genetic differences: How does personal genetic information affect an individual and society's perception of that individual? How does genomic information affect members of minority communities?

4) Reproductive issues, including informed consent for complex and potentially controversial procedures, use of genetic information in reproductive decision making, and reproductive rights: Do health-care personnel properly counsel expectant parents about the risks and limitations of genetic technology? How reliable and useful is fetal genetic testing? What are the larger societal issues raised by new reproductive technologies?

5) Clinical issues, including the education of doctors and other health service providers, patients, and the general public in genetic capabilities, scientific limitations, and social risks, including implementation of standards and quality-control measures in testing procedures: How will genetic tests be evaluated and regulated for accuracy, reliability, and utility? (Currently, there is little regulation at the federal level.) How do we prepare health-care professionals for the new information relating to genetics? How do we prepare the public to make informed choices? How do we as a society balance current scientific limitations and social risk with long-term benefits?

6) Uncertainties associated with genetic tests for susceptibilities and complex conditions (e.g., heart disease) linked to multiple genes and environmental interactions: Should testing be performed when no treatment is available? Should parents have the right to have children tested for adult-onset diseases? Are genetic tests reliable and interpretable by the medical community?

7) Conceptual and philosophical implications regarding human responsibility, free will versus genetic determinism, and concepts of health and disease: Do people's genes make them behave in a particular way? Can people always control their behavior? What is considered acceptable diversity? What is the line between medical treatment and enhancement?

8) Health and environmental issues concerning genetically modified (GM) foods and microbes: Are GM foods and other products safe to humans and the environment? How will these technologies affect developing nations' dependence on the West?

9) Commercialization of products including property rights (patents, copyrights, and trade secrets) and accessibility of data and materials: Who owns genes and other pieces of DNA? Will the patenting of DNA sequences limit their accessibility and development into useful products?

Tags: Bio Technology, Bio Genetics, Genetic Engineering

History of Human Genome Project

Scientists are still far from identifying and characterizing all the proteins in the human body. However, incredible strides have been made to provide a foundation for protein research. This reaches to the source of proteins and ultimately the source of life. This foundation is laid by deciphering the entire genome sequence, or DNA (gene) sequence of an organism. Beginning with bacteria, microscopic worms, and yeast, scientists and computational biologists have expanded DNA sequence information to include certain animals and plants. The ultimate goal of DNA sequencing is the human genome. This genome sequence would allow the understanding of the basis of human life by identifying the order of DNA nucleotides. To accomplish this goal, many groups have come together to work on the Human Genome Project.

The sequencing of the human genome, which is finding the order of the more than 3 billion nucleotides (A, T, C, and G) in the human chromosomes, is being accomplished by two independent groups of scientists. The two versions of this sequence were published in the magazines Nature and Science in February 2001. One group, formerly led by Craig Venter, is Celera Genomics, of Rockville, Maryland, a company started in 1998. The other research group is the result of a consortium of public agencies with laboratories in several countries.

The sequence of the human genome carried out by the public sector, now led by Francis Collins, has a budget of more than $3 billion. The major sponsors were the U.S. Department of Energy and the National Institutes of Health (NIH), as well as the Wellcome Foundation of England. The current map covers about 95 percent of the human genome and has been found to be 99.96 percent accurate. This work has revealed, in a surprising way, that the human genome only has about 30,000 genes instead of 70,000 to 140,000, according to previous estimates. With a DNA sequence of over 3 billion base pairs (bp) and considering the average gene size of 3,000 bp, it is estimated that only 3 percent of the human genome actually codes for some protein. This means that about 97 percent of the human genome has seemingly no coding function; that is, most of the nucleotide sequences in human DNA do not code for genes. This nonfunctional portion of DNA has, for lack of a more accurate term, been called "junk DNA," and its function and purpose have yet to be understood. More important, the data from the Human Genome Project has also revealed that each human being, independent of apparent differences, is about 99.9 percent identical to any other person.

With so much interest and emphasis on the Human Genome Project, what are the practical applications of the sequence of the human genome? The information will help in the early diagnosis of disease, an understanding of the predisposition to genetic diseases, and in genetic counseling, for example. For instance, the sequence of the human genome allows geneticists to understand why certain people have a predisposition to heart disease, and it will eventually lead to the development of new drugs specifically developed to combat the cause of disease and not the symptoms alone. Sequencing the genome will make available basic scientific knowledge for the development of gene therapies for incurable diseases, such as diabetes, muscular dystrophy, cystic fibrosis, Parkinson's disease, and Alzheimer's disease.

By the beginning of 2002, geneticists had already isolated about 13,000 human genes and learned about their functions, including those that code for eye color, circulatory proteins, and genes that when mutated cause a predisposition for developing breast cancer and prostate cancer. All this complex information is contained in each and every cell of the human body. If it were possible to stretch out the incredible amount of information contained in the DNA of all the chromosomes in a single human cell, it would reach about seven feet. If the DNA of all the cells of the human body were stretched out and aligned, it would be enough to cover the distance from Earth to the moon about 8,000 times. Incredible packaging mechanisms allow this information to be stored within each tiny cell.

Tags: Bio Technology, Bio Genetics, Genetic Engineering

Understanding Engineering Genes

Once the DNA has been obtained, it is necessary to cut the DNA into pieces to be used for the engineered gene. Restriction enzymes are used for cutting the DNA at specific sites. Most restriction enzymes cut the DNA into diametric fragments, as opposed to symmetric fragments. That cut leaves the DNA double helix with a small sequence of nonpairing bases that overhang on the end. These regions of DNA are generally used for ligation, or joining with other DNA fragments. DNA fragments cleaved with a single restriction enzyme or with complementary enzymes can be ligated to each other because the overhanging regions are complementary and will bind together. The ligation of fragments is facilitated with addition of the enzyme DNA ligases. The true art of genetic engineering is putting together the parts of puzzle, where each DNA fragment must be placed in right order and orientation so the gene is functional. As scientists know the sequences of genes encoding important traits or proteins, the information is used to engineer genes that can be used in a variety of applications.

Genetic engineers are able to manipulate tiny pieces of DNA with enzymes to create new genes and DNA sequences used in biotechnology. Relatively simple tools in a small laboratory are needed for these engineers to practice their craft. The products that result from these methods can then be used in many applications of biotechnology.

Tags: Bio Technology, Bio Genetics, Genetic Engineering

What is Central Dogma of Genetics?

The genetic material of any organism is the substance that carries the information that determines its life cycle and its characteristics. There is a procedure by which this genetic material is used in living processes; this is the central dogma of genetics. Before the development of modern genetics, it was commonly believed that the substance responsible for heredity was a protein. Once DNA was recognized as the genetic material, the central dogma was established. This states that the information contained in DNA is translated into protein through the processes of transcription and translation. The protein is then used in all life processes, from cell division to electron transport in photosynthesis. For this to occur, DNA is copied (transcribed) into mRNA, and the mRNA is used as template for production of the protein in a process called translation. The message coded by the mRNA sequence gene is translated into a sequence of amino acids, the basic components of protein. Cells cannot produce a protein by simply aligning amino acids; they need to use an RNA template. Additionally, the use of an intermediate mRNA template in protein synthesis reduces the risk of damage to the DNA that can occur from repeated use. Additionally, the central dogma postulates that the intermediate mRNA molecule, a direct copy of DNA, can be used repeatedly in protein synthesis.

Main points of the central dogma are as under:
1) Genes are made of DNA.
2) Genes carry information about structures and biological functions, coded by nucleotides (A, C, G, and T).
3) The genetic information is converted in an mRNA molecule.
4) The mRNA defines the number, type, and order of amino acids in proteins.
5) The protein structure is determined by the linear order of amino acids.
6) The three-dimensional protein structure defines its biological function.

Tags: Bio Technology, Bio Genetics, Genetic Engineering

How do we Manipulate Genes?

Before beginning any genetic engineering project, it is necessary to obtain a reasonable amount of relatively pure DNA, which is then cut and ligated to build the new gene. Today, several companies make DNA extraction and purification kits, making the genetic engineering process much simpler. The basic procedure requires the releasing of the DNA from the cells and purification of the DNA to be used in the experiments.

In a typical extraction and purification procedure for plasmid DNA, bacterial cells with the desired plasmid are lysed (broken up) under alkaline conditions and the crude lysate (remains of the cells) is purified using either filters or centrifugation. The lysate is then loaded onto an apparatus where plasmid DNA selectively binds under appropriate low-salt and pH conditions. RNA, proteins, metabolites, and other low-molecular-weight impurities are removed by a medium-salt wash, then plasmid DNA is released in high-salt buffer. The DNA can then be concentrated and desalted for genetic engineering uses.

General Steps for Gene Manipulation are as under:
1) Grow the bacteria in liquid culture.
2) Centrifuge the bacterial suspension to concentrate the bacteria.
3) Discard the supernatant (the liquid part that remains above the pellet).
4) Resuspend the bacteria pellet in a solution with RNAse (enzyme that degrades RNA).
5) Add a buffer to promote an alkaline lyse of the bacteria.
6) Neutralize and adjust the saline conditions of the suspension with the buffer.
7) Centrifuge to separate proteins and other impurities.
8) Adsorb the plasmid DNA onto a membrane by filtration.
9) Rinse the membrane with a solution containing ethanol.
10) Elute plasmid DNA from the membrane with EDTA, a chemical substance that preserves the integrity of DNA.

Tags: Bio Technology, Bio Genetics, Genetic Engineering

What are Viruses?

Viruses are microorganisms in the gray area of what is living and nonliving. Viruses are made of a protein envelope, which surrounds the genetic material (DNA or RNA). Although viruses have their own genetic material like many other living organisms, they do not possess the capacity to reproduce by themselves. For that, they need to use the machinery of living cells to produce a new virus.

The viruses that live in bacteria are called bacteriophage. They inject their DNA into bacterium, leaving their protein envelope outside. Inside of the bacterium, the virus is a filament of nucleic acid that contains coded information for the synthesis of new virus particles, which can be released with the bacteria lyses. The genetic engineering came about from the observation of how viruses use cells of other organisms or bacteria to express their own genes. In that sense, the viruses can be considered genetic engineers. One of the first experiments of genetic engineering was carried out using a bacteriophage as a true Trojan horse, to introduce DNA from other organisms into the bacterium.

One of the requirements for genetic engineering experiments is the production of DNA fragments that contain the desired information. At the beginning of the molecular biology era, DNA used to be cleaved with vibration by ultrasound waves. One of the difficulties that scientists faced in those experiments was the random fashion in which the DNA fragmented. In 1970, however, Dr. W. Arber discovered that bacteria themselves possess a mechanism to specifically cut DNA at certain sequences. Bacteria produce proteins called restriction enzymes that cleave the DNA at specific recognition sites. It was only after the discovery of the restriction enzymes that genetic engineering became a reality. The restriction enzymes were developed as a defense mechanism of bacteria against viruses.

Viruses inject DNA into bacteria and use their bacteria as a mechanism for reproduction. The bacteria, however, develop a mechanism that fragments the exogenous DNA using restriction enzymes. The restriction enzymes recognize the foreign DNA by means of certain specific nucleotide sequences. Different enzymes recognize and cut the DNA at different sites. Using this knowledge, restriction enzymes became essential tools for the genetic engineer to cut DNA into fragments and build new genes. Hundreds of different restriction enzymes exist, many of which are frequently used in biotechnology.

Tags: Bio Technology, Bio Genetics, Genetic Engineering, Virus