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Tuesday, May 31, 2011

Biological circuits for synthetic biology

Using the tools of synthetic biology, researchers have engineered the first RNA-based regulatory system that can independently control the transcription activities of multiple targets in a single cell. This is a significant advance for the design and construction of programmable genetic networks.

Source:
http://www.sciencedaily.com/releases/2011/05/110526103006.htm

Reactive Oxygen Species (ROS) Oxidative Stress & Antioxidants

In the mitochondrial respiratory chain, oxygen is "partially reduced" to form superoxide. Superoxide is a radical, i.e. a chemical species with an unpaired electron. Radicals usually are very reactive species, because electrons like to pair up to form stable two-electron bonds. Because of its radical character, superoxide is also called a "Reactive Oxygen Species" (ROS). ROS form as a natural byproduct of the normal metabolism of oxygen and have important roles in cell signaling and homeostasis. However, during times of environmental stress (e.g., UV or heat exposure), ROS levels can increase dramatically. This may result in significant damage to cell structures. This cumulates into a situation known as oxidative stress. ROS are also generated by exogenous sources such as ionizing radiation.

Oxidative Stress

Oxidative stress is imposed on cells as a result of one of three factors:

  1. an increase in oxidant generation,

  2. a decrease in antioxidant protection, or

  3. a failure to repair oxidative damage.

Cell damage is induced by reactive oxygen species (ROS). ROS are either free radicals, reactive anions containing oxygen atoms, or molecules containing oxygen atoms that can either produce free radicals or are chemically activated by them. Examples are hydroxyl radical, superoxide, hydrogen peroxide, and peroxynitrite. The main source of ROS in vivo is aerobic respiration, although ROS are also produced by peroxisomal b-oxidation of fatty acids, microsomal cytochrome P450 metabolism of xenobiotic compounds, stimulation of phagocytosis by pathogens or lipopolysaccharides, arginine metabolism, and tissue specific enzymes. Under normal conditions, ROS are cleared from the cell by the action of superoxide dismutase (SOD), catalase, or glutathione (GSH) peroxidase. The main damage to cells results from the ROS-induced alteration of macromolecules such as polyunsaturated fatty acids in membrane lipids, essential proteins, and DNA. Additionally, oxidative stress and ROS have been implicated in disease states, such as Alzheimer's disease, Parkinson's disease, cancer, and aging.

Antioxidants

Antioxidants are scavengers of Reactive oxygen species (ROS), Antioxidants scavenge ROS before they cause damage to the various biological molecules, or prevent oxidative damage from spreading, e.g. by interrupting the radical chain reaction of lipid peroxidation. The antioxidant defense systems in the human body are extensive and consist of multiple layers, which protect at different sites and against different types of ROS.some of the enzymes which acts in clearing ROS are superoxide dismutase (SOD), catalase, or glutathione (GSH) peroxidase.

we are constantly exposed to ROS generated from endogenous and some exogenous sources. These ROS react with biological molecules, such as DNA, proteins, and lipids, causing structural and functional damage. Oxidative damage accumulates in human tissues with age and can causally contribute to a number of degenerative diseases, such as heart disease and cancer. Antioxidants, both enzymatic and non-enzymatic, limit oxidative damage to biological molecules by various mechanisms. Dietary antioxidants, such as vitamins C and E, significantly contribute to antioxidant defense systems in humans and may help protect us from certain age-related degenerative diseases.

References:

Fiers, W., et al., More than one way to die: apoptosis, necrosis and reactive oxygen damage Oncogene., 18, 7719-7730 (1999).

Nicholls, D.G., and Budd, S.L., Mitochondria and neuronal survival. Physiol. Rev., 80, 315-360 (2000).

Hayes, J.D., et al., Glutathione and glutathione-dependent enzymes represent a co-ordinately regulated defense against oxidative stress. Free Radic. Res., 31, 273-300 (1999).

http://lpi.oregonstate.edu/f-w97/reactive.html

http://www.sigmaaldrich.com/life-science/cell-biology/learning-center/pathway-slides-and/oxidative-stress.html

en.wikipedia.org/wiki/Reactive_oxygen_species

Monday, May 30, 2011

Globular and Fibrous Proteins

Fibrous Proteins
  • Little or no tertiary structure.
  • Long parallel polypeptide chains.
  • Cross linkages at intervals forming long fibres or sheets.
  • Usually insoluble.
  • Many have structural roles.
  • E.g. keratin in hair and the outer layer of skin, collagen (a connective tissue).
Globular Proteins
  • Have complex tertiary and sometimes quaternary structures.
  • Folded into spherical (globular) shapes.
  • Usually soluble as hydrophobic side chains in centre of structure.
  • Roles in metabolic reactions.
  • E.g. enzymes, haemoglobin in blood.
Haemoglobin
An example of a globular protein
  • Reddish-purple oxygen carrying pigment found in red blood cells.
  • Made up of 4 polypeptide chains.
  • 2 identical alpha-chains and 2 identical beta-chains.
  • Nearly spherical - hydrophobic side chains point inwards.
  • Outward pointing hydrophilic side chains maintain solubility.
  • Each polypeptide chain contains a haem group.
  • Haem group is a prosthetic group (i.e. an important permanent part of a protein molecule which is not made from amino acids) - when combined with 4 polypeptide chains it forms a conjugated protein.
  • Haem group has an iron ion (Fe2+) at its centre.
  • The iron combines with oxygen at high oxygen concentrations and releases oxygen at low oxygen concentrations.
  • One haemoglobin molecule can carry 4 oxygen molecules.
  • Colour is bright red when combined with oxygen / purplish if not.
Collagen
An example of a fibrous protein
  • Found in skin, tendons, cartilage, bones, teeth, walls of blood vessels.
  • Structural protein in most animals.
  • Collagen molecule made of 3 polypeptide chains, each in the shape of a helix (not as tightly wound as an alpha-helix).
  • Each chain contains 1000 amino acids - every third amino acid is glycine (the smallest amino acid).
  • 3 helical chains wind around each other to form a three-stranded 'rope'.
  • Glycine allows the 3 polypeptides to lie close together to form a tight coil (any other amino acid would be too big).
  • 3 strands held together by H bonds.
  • Each 3 stranded molecule interacts with others next to it. Bonding causes fibres to form.
  • The ends of parallel molecules are staggered, preventing weak areas from running across the collagen fibre.
  • Very strong - one quarter the tensile strength of mild steel.

Thursday, May 26, 2011

First man to be cured of HIV AIDS

Timothy Ray Brown, 45, from San Francisco Bay Area, is in the news – as the first man cured of HIV- AIDS. "I think so," he calmly tells his interviewers who ask if he actually is cured.

Brown has been facing cameras, gun mikes and diagnostic kits ever since the publication of a research paper on his unique case in the journal Blood in December 2010.

The researchers led by Kristina Allers and Gero Hutter at Charite University Medicine Berlin documented what can be dubbed as a miracle.

The successful reconstitution of a set of white blood cells that the HIV eats up in Brown's body is a "very rare" occurrence, they noted.

Brown, who was tested HIV back in 1995 in Germany, was later diagnosed with another disease — leukaemia or blood cancer that involves an abnormal increase in white blood cell.

He was treated with bone marrow stem cell transplant — a cure for blood cancer. The stem cells came from a donor with a rare gene mutation that involves immunity to HIV — again a rare occurrence.
 The mechanism involved special white blood cells called CD4+ helper T cells.  When a dangerous material like a bacterium or a virus is detected in the body, immune cells immediately stimulate these special cells.

The helper T cells further activate and direct other immune cells to fight the disease. HIV specifically attacks helper T cells, making the body unable to launch a counter offensive against invaders.
Hence, AIDS patients suffer from other lethal infections. The researchers in Berlin showed that after stem cell therapy Brown's body had reconstitution of CD4+ T cells at a systemic level and specifically in his gut mucosal immune system.

"While the patient remains without any sign of HIV infection," they wrote. Brown has quit taking his HIV medication. The secret is that if the white cells could be manipulated to a state in which they are no longer infected or infectable by HIV that would mean a functional cure.

Researchers, however, have warned that though the study offers promise, it is not a surefire cure from the dreaded disease — transplants are risky, and this involved a very rare transplant. Brown is a rather lucky man. He said in a recent interview that appeared in the San Francisco media about his cure: "It makes me very happy — very, very happy."


Original Article Published in Yahoo News:
http://in.news.yahoo.com/here-is-the-first-man-to-be-cured-of-hiv--aids-.html

Culture of Yeast for the Production of Heterologous Proteins

This unit describes culture of the yeast strains Saccharomyces cerevisiae and Pichia pastoris for the production of foreign proteins. The protocols listed here for S. cerevisiae are for three widely used types of promoter: galactose-regulated (GAL1, GAL7, GAL10), glucose-repressible (e.g., ADH2), and constitutive glycolytic (e.g., PGK or GAPDH). Minor variations to each can be made depending on the selection system used. The P. pastoris expression system uses integrating vectors with the methanol-regulated AOX1 promoter and HIS4 selection marker; although transformants are stable, they are generally grown in minimal selective medium. Methods are described for small-scale S. cerevisiae and P. pastoris cultures and also for high-density fermentations with these yeasts. A simple feeding strategy based on calculated feed rates is provided for S. cerevisiae and yields cell densities of 10 to 30 g/liter. In contrast, with P. pastoris, basic fermenter equipment is used to obtain extremely high-density cultures (e.g., 130 g/liter). Finally, a Support Protocol describes small-scale preparation of protein extracts.

Link to Full Protocol: http://www.currentprotocols.com/protocol/ps0508

Thursday, May 19, 2011

'Master Switch' Gene for Obesity and Diabetes Discovered

A team of researchers, led by King's College London and the University of Oxford, have found that a gene linked to type 2 diabetes and cholesterol levels is in fact a 'master regulator' gene, which controls the behaviour of other genes found within fat in the body.

Via Science Daily

Renewable Energy: Catalyst for Splitting Water

An international team, of scientists, led by a team at Monash University has found the key to the hydrogen economy could come from a very simple mineral, commonly seen as a black stain on rocks.

Via Science Daily

Friday, May 13, 2011

Electroporation: Applications and Protocol

Molecular biology / Genetic Engineering works require a foreign gene or protein material to be inserted into a host cell. Since the plasma membrane of the cell is selectively permeable , any polar molecules, including DNA and protein, are unable to freely pass through the membrane.Many methods have been developed to surpass this barrier and allow the insertion of DNA and other molecules into the cells to be studied. These other methods include microprecipitates, microinjection, liposomes, and biological vectors, etc.One such method is electroporation.
Electroporation is technique in which cell plama membrane's permeability is increased by applying electric field which induces the formation of small pores in the membrane through which pieces of foreign materials (usually DNA, Proteins or Dyes) can be taken in to the cell.A quick voltage shock may disrupt areas of the membrane temporarily, allowing polar molecules to pass, but then the membrane may reseal quickly and leave the cell intact.

Procedure

An Image of an Electroporator

The procedure involves placing the cell suspension and the foreign material (eg: Plamsid) to be transferred into plastic or glass cuvette.The cuvettes used here are specially meant for electroporation, they have aluminium electrodes at the sides. The voltage and capacitance is set and the cuvette inserted into the electroporator. Once after electroporation, few ml medium is added , and is incubated at the the culture's (Bacteria, yeast, etc) optimal temperature for an hour or more to allow recovery of the cells.The transformed cells can be screened out using selectable markers in the plasmid (usaually antibiotic resisitance genes are used).
Advantages and Disadvantages of Electroporation
Electroporation has the following advantages and disadvantages
Advantages:
  • Versatility: Electroporation is effective with nearly all cell and species types.
  • Efficiency: A large majority of cells take in the target DNA or molecule. In a study on electrotransformation of E. coli, for example, 80% of the cells received the foreign DNA.
  • Small Scale: The amount of DNA required is smaller as compared with other methods.
Disadvantages:
  • Cell Damage: If the electric pulses are given for long time or with more intensity, some pores may become too large or fail to close after membrane discharge causing cell damage or rupture
  • Nonspecific Transport: The transport of material into and out of the cell during the time of electropermeability is relatively nonspecific. This may result in an ion imbalance that could later lead to improper cell function and cell death.
Applications
As previously mentioned, electroporation is widely used in many areas of molecular biology research and in the medical field. Some applications of electroporation include:
DNA Transfection or Transformation: This is likely the most widespread use of electroporation. Specific genes can be cloned into a plasmid and then this plasmid introduced into host cells in order to investigate gene and protein structure and function.if the host cell used is microbial then it is called Transformation and if the host cell is mammalian then it is called as Transfection.
Direct Transfer of Plasmids Between Cells: Bacterial cells already containing a plasmid may be incubated with another strain that does not contain plasmids but that has some other desireable feature. The voltage of electroporation will create pores, allowing some plasmids to exit one cell and enter another. The desired cells may then be selected by antibiotic resistance or another similar method. This type of transfer may also be performed between species. Thus, large numbers of plasmids may be grown in rapidly multiplying bacterial colonies and then transferred to yeast cells by electroporation for study .
Induced Cell Fusion: The disruption of the membrane that occurs with the quick pulse of electricity in the electroporation procedure has also been shown to induce fusion of cells..
Trans-dermal Drug Delivery: Just as electroporation causes temporary pores to form in plasma membranes, studies suggest that similar pores form in lipid bi-layers of the stratum corneum- the outermost dead layer of skin. These pores could allow drugs to pass through to the skin to a target tissue. This method of drug delivery would be more pleasant than injection for the patient (not requiring a needle) and could avoid the problems of improper absorption or degradation of oral medication in the digestive system.
Cancer Tumor Electrochemotherapy: Scientists are investigating the potential of electroporation to increase the effectiveness of chemotherapy. As in electroporation for DNA transfection, the applied electrical pulse would disrupt the membrane of the tumor cell and increase the amount of drug delivered to the site. Some studies have suggested that increased tumor reduction is seen when this method is applied to cancerous cells in animal model systems.
Gene Therapy: Much like drug delivery, electroporation techniques can allow vectors containing important genes to be transported across the skin and into the target tissue. Once incorporated into the cells of the body, the protein produced from this gene could replace a defective one and thus treat genetic disorder.

Yeast Transformation Electroporation


  1. Grow 50 ml to OD 1.3-1.5. 
  2. Spin cold at 5000 rpm. 
  3. Wash twice in 25 ml cold dH2O. 
  4. Wash in 1 ml 1 M sorbitol. 
  5. Resuspend in 50 µl 1 M sorbitol. 
  6. Take 40 µl aliquot and add <5 µl desalted DNA. 
  7. Incubate on ice 5 min+. 
  8. Electroporate in 0.2 cm cuvette at 1.5 kV (for 5 milli second). 
  9. Immediately add 1 ml cold 1 M sorbitol. 
  10. Plate on selective 1 M sorbitol plate or outgrow in 1 M sorbitol YPD broth first (need 2X media and 2 M sorbitol).
References:
http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/McCord/electroporation.htm
http://en.wikipedia.org/wiki/Electroporation
www.aragonlab.com/protocols/Yeast-Transformation-Electroporation.doc

Wednesday, May 11, 2011

Mitochondria: Body’s Power Stations Can Affect Aging

Mitochondria are the body's energy producers, the power stations inside our cells. Researchers at the University of Gothenburg, Sweden, have now identified a group of mitochondrial proteins, the absence of which allows other protein groups to stabilise the genome. This could delay the onset of age-related diseases and increase lifespan.

via Science Daily

Fermentation : Production of Antibiotics



Presentation by : Saba Inayat Ali

Presentation file source: http://www.slidefinder.net/F/FERMENTATION_Saba_Inayat_Ali/25052116

Friday, May 6, 2011

Artificial Leaf :

Scientists have claimed one of the milestones in the drive for sustainable energy -- development of the first practical artificial leaf. Speaking in Anaheim, California at the 241st National Meeting of the American Chemical Society, they described an advanced solar cell the size of a poker card that mimics the process, called photosynthesis, that green plants use to convert sunlight and water into energy.

Via Science Daily

Thursday, May 5, 2011

Basics & Working of a Fermentor

Industrial processes require large vessels for fermentation which can hold huge amounts of nutritive media. These vessels are called fermenters, Industrial fermenters are designed to provide best possible growth conditions and also conditions that aid in good biosynthesis of product by microbes.

Important features of a Fermentor
  1. The vessel must be strong enough to withstand the pressures of large volumes of liquid medium.
  2. The fermentor should be made up of a material that must not corrode  & shouldnt contribute toxic ions to the medium.
  3. In aerobic fermentation process, oxygen  has to be supplied to the medium by a process called aeration.
  4. The carbon-dioxidde formed during fermentation has to be flushed out of the fermentor.
  5. The contents of the fermentor has to be stirred for a number of reasons such as equal distribution of nutrients throughout the medium, prevention of microbial cells from settling down, etc
  6. As the fermentation process proceeds along with stirring and aeration, there will be foam formation, so there should be a method to detect and control the foam formation. Foam affects product yield and also the growth of microbes.
  7. During the growth of micro-organisms, fermentator must be maintained at a predetermined temperature. so temperature control is required. Optimal Temperature is essential for the growth of microbes.
  8. The fermentor should contains aseptic means of drawing culture from the fermentor and also aseptic means of injecting nutrients and other to the fermentor.
  9. There must be a drain at the bottom of the fermentor through which the completed fermentation broth can be removed.
  10. A mechanism should be there for monitoring pH of the medium during fermentation.
Construction of Fermentor
  1. Fermentors are available in differnet sizes. The sizes are usually stated based on the total volume capacity of the fermentor. However actual operating volume of fermentor is always less than that of the total volume capacity of fermentor. There is a space above the liquid called spathe head space which allows for splashing, foaming and aeration.
  2. Small laboratory fermentors will have total volume capacity of 1-2 Lts  and medium fermentors will have volume capacity of 12-15 Ltrs. Pilot scale fermentors are in the range of 2.5 - 100 Ltrs and some times even more than that. Very large fermentors will have total  volume capacity up to 1,00,000 gallons are also in use.
  3. The material used for making fermentor construction depends on the size, small laboratory fermentors are made of glass, while large ones are made up of stainless steel.
  4. An impeller is mounted to a shaft through a bearing in the lid of the fermentor and is driven by a mortar beneath the fermentor. The impeller is fitted with impeller blades, The heights of the impeller blades are adjustable. when rotated at high speed, vigorous stirring and agitation of the medium are achieved.
  5. Stirring creates circular movements of the medium. Baffles attached to sides of the fermentor wall helps in proper mixing of the medium and microbial cells.
  6. Majority of the fermentation process are aerobic. The sparger is located at the bottom of the fermentation tank so impeller disperses air from the sparger.
  7. Actively metabolising cultures generate heat, thus can increase temperature of the medium above the optimum growth temperatures. In small fermentors this is achieved by passing cold water through jacketed walls or through coils which runs inside the tank.

Working of a Fermentor



This video has been taken during my training session at Biozeen

Copy number calculation for QPCR

A serial dilution of linearized plasmid DNA is used to generate a standard curve for QPCR.  Knowing the size of the plasmid that contains the gene of interest one can calculate the number of grams/molecule also known as copy number as follows:

Weight in Daltons (g/mol) = (bp size of plasmid+insert)(330 Da X 2 nucleotide/bp)

Ex. g/mol=(5950 bp)(330 Da X 2 nucleotide/bp)= 3927000 g/mol

Hence: (g/mol)/Avogadro's number 6.02214199 × 10^23) = g/molecule = copy number

Ex. 3927000g/mol/Avogadro's number 6.02214199 x 10^23) = 6.52 x 10^-18 g/molecule.  

Knowing the copy number for a plasmid and the concentration of the plasmid that is added to each PCR reaction, the precise number of molecule in that reaction can be determined as follows:

Concentration of plasmid (g/µl)/copy number
Ex. (3 x 10^-7 g/µl) / (6.52 x 10^-18 grams/molecules) = 4.6 x 10^10 molecules/µl

Having calculated the number of molecules in a µl of linearized plasmid solution, a series of dilutions can be made for subsequent amplification allowing one to generate a standard curve.  For the standard curve, the copy number of the unknown samples can then be derived.

More on generating standard curve and calculating amplification efficiency

Sourcehttp://www.mncf.tulane.edu/Protocols/copy%20number%20calculation%20for%20qpcr.pdf

Surface Plasmon Resonance and its Biosensor / Nanotechnology Applications

Surface plasmon resonance (SPR) is a phenomenon occurring at metal surfaces(typically gold and silver) when an incident light beam strikes the surface at a particular angle.Depending on the thickness of a molecular layer at the metal surface,the SPR phenomenon results in a graded reduction in intensity of the reflected light.Biomedical applications take advantage of the exquisite sensitivity of SPR to the refractive index of the medium next to the metal surface, which makes it possible to measure accurately the adsorption of molecules on the metal surface or on to surface of metal nanoparticles and their eventual interactions with specific ligands. It is the fundamentals behind many color based biosensor applications and different lab-on-a-chip sensors.

Principle


The underlying physical principles of SPR are complex.Fortunately, an adequate working knowledge of the technique does not require a detailed theoretical understanding. It suffices to know that SPR-based instruments use an optical method to measure the refractive index near (within ~300 nm) a sensor surface. In the BIAcore this surface forms the floor of a small flow cell, 20-60 nL in volume , through which an aqueous solution (henceforth called the running buffer) passes under continuous flow (1-100 µL.min-1). In order to detect an interaction one molecule (the ligand) is immobilised onto the sensor surface. Its binding partner (the analyte) is injected in aqueous solution (sample buffer) through the flow cell, also under continuous flow. As the analyte binds to the ligand the accumulation of protein on the surface results in an increase in the refractive index. This change in refractive index is measured in real time, and the result plotted as response or resonance units (RUs) versus time (a sensorgram). Importantly, a response  (background response) will also be generated if there is a difference in the refractive indices of the running and sample buffers. This background response must be subtracted from the sensorgram to obtain the actual binding response. The background response is recorded by injecting the analyte through a control or reference flow cell, which has no ligand or an irrelevant ligand immobilized to the sensor surface. One RU represents the binding of approximately 1 pg protein/mm2. In practise >50 pg/mm2 of analyte binding is needed. Because is it very difficult to immobilise a sufficiently high density of ligand onto a surface to achieve this level of analyte binding, BIAcore have developed sensor   - 4 - surfaces with a 100-200 nm thick carboxymethylated dextran matrix attached. By effectively adding a third dimension to the surface, much higher levels of ligand immobilisation are possible. However, having very high levels of ligand has two important drawbacks. Firstly, with such a high ligand density the rate at which the surface binds the analyte may exceed the rate at which the analyte can be delivered to the surface (the latter is referred to as mass transport). In this situation, mass transport becomes the rate-limiting step. Consequently, the measured association rate constant (kon) is slower than the true kon. A second, related problem is that, following dissociation of the analyte, it can rebind to the unoccupied ligand before diffusing out of the matrix and being washed from the flow cell. Consequently, the measured dissociation rate constant (apparent koff) is slower than the true koff. Although the dextran matrix may exaggerate these kinetic artefacts (mass transport limitations and re-binding) they can affect all surface-binding techniques . 

Surface Plasmon Resonance stems one of the basic principles of optics, that of total internal reflectance (or TIR).
  • Occurs when a thin conducting film is placed at the interface between the two optical media.
  • At a specific incident angle, greater than the TIR angle, the surface plasmons in the conducting film resonantly couple with the light because their frequencies match.
SPR is good for:
  • Evaluation of macromolecules.
  • Equilibrium measurements (affinity and enthalpy).
  • Kinetic measurements. 
  • Analysis of mutant proteins.
SPR is not good for:

  • High throughput assays.
  • Concentration assays
  • Studying small analytes.
Some of the potential areas of application include
  • Medical diagnostics
  • Environmental monitoring
  • Agriculture pesticide and antibiotic monitoring
  • Food additive testing
  • Military and civilian airborne
  • Biological and chemical agent testing
  • Real time chemical and biological production process monitoring.
References:

Protocol for working in BIAcore can be obtained from the below link

http://users.path.ox.ac.uk/~vdmerwe/internal/spr.pdf

http://www.biacore.com/lifesciences/technology/introduction/following_interaction/index.html

http://www.surfacephysics.co.jp/project/ar/ref.html
http://www.howstuffworks.com


www.technologymind.co.nz/plasmonreferance/ref.html

Wednesday, May 4, 2011

Protein Separation Based on Different Solubility Characteristics

Proteins can be separated by exploiting differences in their solubility in aqueous solutions. The solubility of a protein molecule is determined by its amino acid sequence because this determines its size, shape, hydrophobicity and electrical charge. Proteins can be selectively precipitated or solubilized by altering the pH, ionic strength, dielectric constant or temperature of a solution. They are often used as the first step in any separation procedure because the majority of the contaminating materials can be easily removed.

Salting out

Proteins are precipitated from aqueous solutions when the salt concentration exceeds a critical level, which is known as salting-out, because all the water is "bound" to the salts, and is therefore not available to hydrate the proteins. Ammonium sulfate [(NH4)2SO4] is commonly used because it has a high water-solubility, although other neutral salts may also be used, e.g., NaCl or KCl. Generally a two-step procedure is used to maximize the separation efficiency. In the first step, the salt is added at a concentration just below that necessary to precipitate out the protein of interest. The solution is then centrifuged to remove any proteins that are less soluble than the protein of interest. The salt concentration is then increased to a point just above that required to cause precipitation of the protein. This precipitates out the protein of interest (which can be separated by centrifugation), but leaves more soluble proteins in solution. The main problem with this method is that large concentrations of salt contaminate the solution, which must be removed before the protein can be resolubilzed, e.g., by dialysis or ultrafiltration.

Isoelectric Precipitation

The isoelectric point (pI) of a protein is the pH where the net charge on the protein is zero. Proteins tend to aggregate and precipitate at their pI because there is no electrostatic repulsion keeping them apart. Proteins have different isoelectric points because of their different amino acid sequences (i.e., relative numbers of anionic and cationic groups), and thus they can be separated by adjusting the pH of a solution. When the pH is adjusted to the pI of a particular protein it precipitates leaving the other proteins in solution.

Solvent Fractionation

The solubility of a protein depends on the dielectric constant of the solution that surrounds it because this alters the magnitude of the electrostatic interactions between charged groups. As the dielectric constant of a solution decreases the magnitude of the electrostatic interactions between charged species increases. This tends to decrease the solubility of proteins in solution because they are less ionized, and therefore the electrostatic repulsion between them is not sufficient to prevent them from aggregating. The dielectric constant of aqueous solutions can be lowered by adding water-soluble organic solvents, such as ethanol or acetone. The amount of organic solvent required to cause precipitation depends on the protein and therefore proteins can be separated on this basis. The optimum quantity of organic solvent required to precipitate a protein varies from about 5 to 60%. Solvent fractionation is usually performed at 0oC or below to prevent protein denaturation caused by temperature increases that occur when organic solvents are mixed with water.

Denaturation of Contaminating Proteins

Many proteins are denatured and precipitate from solution when heated above a certain temperature or by adjusting a solution to highly acid or basic pHs. Proteins that are stable at high temperature or at extremes of pH are most easily separated by this technique because contaminating proteins can be precipitated while the protein of interest remains in solution.


References

http://www-unix.oit.umass.edu/~mcclemen/581Proteins.html

http://www.biochem.arizona.edu/classes/bioc462/462a/NOTES/Protein_Properties/protein_purification.htm



Bacterial Transformation

1. Pipette 200ul competent cells into each of 3 ice cold Eppendorf tubes. Label the tubes Control, 1 ng, and 10 ng (1 ng is 10 -3 ug, or 10 -9 milligrams). The unknown plasmid is at a concentration of 1 ng/ul. Add 1 ng of your unknown plasmid to one tube and 10 ng to the other. Place the tubes on ice for 30 min.

2. Put the tubes at 42oC for exactly 90 seconds. Return the cells to ice for 1-2 minutes.

3. Pipette the transformation mixtures onto labeled plates containing ampicillin and spread them around using a sterilized, bent glass rod spreader. 

4. Place upside down in the 37oC incubator overnight.

5. 16 - 20 hours later, count the number of colonies on the plate with well-isolated colonies. Put parafilm around the edge of a plate and put it in a refrigerator for later use. Check the control plate to see that no colonies grew on it. 

Note: Competent Cell Preparation Procedure is given in the last post click older post to view it.

Reference

http://faculty.plattsburgh.edu/donald.slish/Transformation.html

http://www.mnstate.edu/provost/TransformationProtocol.pdf





Monday, May 2, 2011

Blue Rose

Florigene, a company based in Australia is interested in expressing proteins in flowers, not to make the protein, but to engineer in a pathway for flower pigments. For centuries, a blue rose has been the subject of fiction. It was mentioned in the Arabian Nights. There is no such thing as a blue rose however, because the key enzyme in the pathway to blue or purplish pigments is lacking in the rose family. This enzyme is a flavonoid 3'5' hydroxylase. This enzyme acts on anthocyanins that are already hydroxylated at the 3' and 4' positions to add a third hydroxyl at the 5' position of the anthocyanin B ring.This pigment is bluish or purplish in color and its specific absorption properties can be modified by pH, metal ions and copigments.

Florigene has developed methods to transform genes into carnations,chrysanthemums and roses.  They have cloned the genes for flavonoid 3'5' hydroxylase from petunia flower petals and expressed these genes in carnations.  This has led to purplish colored carnations.  They have not got all the additional factors worked out yet to get a true blue color expressed, but they are working on it.In addition, they are transforming the genes into chrysanthemums and roses.  The estimated world wide market for a blue rose is in the 3-5 billion dollar a year range, so it is worth the initial trouble to engineer this pathway into roses.  

Source:

Internet Sources

Calculating Dilutions

Dilutions can be divided into five categories:
1. To dilute a solution to an unspecified final volume in just one step.
2. To dilute a solution to a specified final volume in just one step.
3. To dilute a solution to an unspecified final volume in several steps.
4. To dilute a solution to a specified final volume in several steps.
5. Serial dilutions.

Diluting a Solution to an Unspecified Volume in Just One Step

One must first calculate how many times to dilute (this is called the dilution factor) the initial material  (stock solution) to obtain the final concentration. To accomplish this type of dilution, use the following formula:

Initial Concentration (IC) / Final Concentration (FC) = Dilution Factor (DF)

For example, if you want to dilute a solution with an initial
concentration of solute of 5% down to 1%, using the above
formula gives
IC /FC  = DF 5% /  1% = 5

Thus, in order to obtain a 1% solution from a 5% solution, the latter must be diluted 5 times. This can be accomplished by taking one volume (e.g., cc, ml, liter, gallon) of the initial concentration (5%) and adding 4 volumes (e.g., cc, ml, liter, gallon) of solvent for a total of five volumes. Stated another way, 1 ml of a 5% solution + 4 ml of diluent will give a total of 5 ml, and each ml contains 1% instead of 5%.

Diluting a Solution to a Specified Volume in Just One Step

First, calculate the number of times the initial concentration must be diluted by dividing the final concentration (FC) into the initial concentration (IC)

Second, divide the number of times the initial concentration must be diluted (bottom left paragraph) into the final volume specified to determine the aliquot (or portion) of the initial concentration to be diluted.Third, dilute the aliquot of the initial concentration calculated in step 2 by the volume specified.
For example, you have a 10% solution and want a 2%solution. However, you need 100 ml of this 2% solution.

IC/FC = DF 10%/2% = 5

Divide the number of times the 10% solution must be diluted (DF) into the final volume specified:

100ml / 5 = 20ml

Dilute the portion of 10 to the volume specified:

20 ml of a 10% solution + 80 ml of diluent = 100 ml (each milliliter = 2%)

Another method for performing this type of dilution is to use the following formula:

C1/C2 = V1/V2 or C1V1 = C2V2

C1 = standard concentration available
C2 = standard concentration desired
V2 = final volume of new concentration
V1 = volume of C1 required to make the new concentration

For example, if you want to prepare 100 ml of 10% ethyl alcohol from 95% ethyl alcohol, then

C1 = 95%, C2 = 10%,
V2 = 100 ml,
V1 = x and

95/10 = 100/x

x = 1,000/95; so x = 10.5 ml

Thus, 10.5 ml of 95% ethyl alcohol + 89.5 ml of H2O = 100ml of a 10% ethyl alcohol solution.

Diluting a Solution to an Unspecified Volume in Several Steps

Frequently in the microbiology laboratory, large dilutions must be employed. They cannot be done in one step because they are too large. As a result, they must be done in several steps to conserve not only amounts of diluent to be used but also space. For example, a 0.5 g/ml solution diluted to 1 µg/ml is a 500,000-fold dilution.

0.5 g = 0.5 g × 106 µg/g
        = 500,000 µg

To obtain a solution containing 500,000 µg/ml in one step would require taking 1 ml of 0.5 gm/ml stock solution and adding 499,999 ml of diluent. As you can see, it would be almost impossible to work with such a large fluid volume.

A 500,000 times dilution can be easily performed in two steps by first taking 1 ml of the initial concentration and diluting it to 500 ml and second, by diluting 1 ml of the first dilution to 1,000 ml.
1 ml of 500,000 µg/ml + 499 ml of diluent = 1,000 µg/ml
1 ml of 1,000 µg/ml + 999 ml of diluent = 1 µg/ml
Thus, by this two-step procedure, we have cut down the volume of diluent used from 499,999 to 1,498 (499 ml + 999 ml).

Diluting a Solution to a Specified Volume in Several Steps

This type of dilution is identical to all previous dilutions with the exception that the specified final volume must be one factor of the total dilution ratio.
For example, you want a 1/10,000 dilution of whole serum (undiluted) and you need 50 ml.
Divide dilution needed by the volume:

10,000 / 50 = 200


200 (1/200 dilution) = the first step in the dilution factor;
the second is 1/50, obtained as follows:
1 ml of serum + 199 ml of diluent = 1/200 dilution.
1 ml of 1/200 dilution + 49 ml of diluent = 1/50.
To check: 50 × 200 = 10,000.


Serial Dilution : Making 10 fold Dilution

The first step in making a serial dilution is to take a known volume (usually 1ml) of stock and place it into a known volume of distilled water (usually 9ml). This produces 10ml of the dilute solution. This dilute solution has 1ml of extract /10ml, producing a 10-fold dilution. (i.e. the amount of stock in each ml of the diluted solution is 0.1ml.)
This process can be repeated to make successive dilutions.


References

Laboratory Exercises in Microbiology - Harley, Presscott
http://www.ruf.rice.edu/~bioslabs/methods/solutions/dilutions.html
http://biology.kenyon.edu/courses/biol09/tetrahymena/serialdilution2.htm