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Friday, December 28, 2012

Good Notes on oligonucleotide Yield, Resuspension, and Storage

Oligonucleotides are short stretch of single stranded DNA or RNA molecules used in genetic testing, research and forensic. These are chemically synthesized and has got applications in polymerase chain reaction (PCR),DNA sequencing etc.

Length of oligonucleotide is denoted as '-mer' for example : 18-mer, 22-mer, etc.

Nucleic  acid synthesis columns come in several scales, which are a function of the amount of the 3' nucleoside coupled to the support material. Popular column  sizes Oligonucleotide Synthesis 
range from 40 nmol  to  10umol. Popular support materials  include controlled-pore glass  (CPG) and polystyrene. The CPG columns  are  typically  supplied having pore sizes of either 500 A or 1000 A. The  scale of column used for a synthesis depends on both the quantity and the length of oligonucleotide desired. The greater the amount of 3' nucleoside  attached to the column  support,  the greater the yield of oligo. Polystyrene or large-pore CPG columns  are recommended for oligonucleotides  greater than 50.

oligonucleotides are synthesized at different scales like 0.05umol, 0.2umol, 1umol, etc. depending on the application it is used for.

The number of micromoles of an oligonucleotide synthesized on a column,  therefore, can be calculated by multiplying  the length of the oligonucleotide by the column scale.

A good explanation on calculation of oligonucleotide Yield, Resuspension, and Storage

https://www.idtdna.com/pages/docs/technical-reports/oligonucleotide-yield-resuspension-and-storage.pdf

References
Integrated DNA Technologies
Wikipedia
OligoCalc, Technical guide
Eurofins-MWG Operon,

Metric Prefixes and Scientific Notations

A metric prefix is a shorthand notation used to denote very large or vary small values of a basic unit as an alternative to expressing them as powers of 10. Basic units frequently used in the biological sciences include meters, grams, moles, and liters.Because of their simplicity, metric prefixes have found wide application in molecular biology. The following table lists the most frequently used prefixes and the values they represent. 

Metric Prefix
1 nanogram (ng) is equivalent to 1*10^-9 grams. There are, therefore, 1*10^9 nanograms per gram (the reciprocal of 1*10^-9; 1/1*10^9 = 1*10^-9). Likewise, since one microliter (uL) is equivalent to 1*10^-6 liters, there are 1 x 10^6 uL per liter.

When expressing quantities with metric prefixes, the prefix is usually chosen so that the value can be written as a number greater than 1.0 but less than 1000. For example, it is conventional to express 0.00000005 grams as 50 ng rather than 0.05 ug or 50,000 pg.

Reference:
Mathematics in molecular biology and biotechnology

Tuesday, December 25, 2012

Pipette calibration : Procedure and Calculations

Principle:
Under a constant temperature and atmospheric pressure, the density of distilled water is constant. The volume of water can be determined by weighting dispensed water. The calibration of pipette is carried out by gravimetric method. When determining the volume of water, the accuracy of measurements is effected by ambient temperature, atmospheric pressure and relative humidity. These factors are usually combined to give the Z factor, used in calculation of volume of water. Then the calculated volume of water is compared with the theoretical volume to determine the accuracy and precision of the pipette.



Pipette parts
A Micro Pipette

Material and equipment:
  • Ppipette and tips
  • 50 ml beaker and plastic medicine cup
  • Distilled water
  • Temperature meter ( ±0.1℃ )
  • Analytical balance ( ±1.0 mg )
  • Atmospheric pressure meter

procedure pipette calibration
Pipette Calibration

Procedure:
  • Determine the water temperature and record it.
  • Place a beaker filled with distilled water into analytic balance and close the door of balance waiting for equilibrium of inner vapor.
  • Place a plastic medicine cup on the pan and adjust the weight to zero.Put a tip onto the pipette and set the volume which is to be tested.
  • Pre-rinse the tip: aspirate and dispense the setting volume three times and press the push – button on the second stop to remove any remaining liquid.
  • Press the push-button to the first positive stop. Hold the pipette vertically, immerse the tip so that 1-4 mm in the liquid and release the push button slowly and smoothly to aspirate the liquid.
  • Wait one second and withdraw the tip from the liquid.
  • Wipe any droplets away from the outside of the tip using a kimwipe.
  • Place the end of the tip against the inside wall of the plastic cup at an angle of 10-40 °.
  • Press the push-button smoothly to the first stop. Wait one second, change new site and press the push-button on the second stop.
  • Keeping the push-button press to the end, remove the pipette by drawing the tip along the inside surface of the plastic cup and release the push button.
  • Close the door of balance and record the value on the balance display after it has stabilized.
  • Repeat step (6) to (12) 14 times ( the weight display of balance should be adjusted to zero every time)
  •  Eject the tip.
  • Pipette should be calibrated every three months routinely.
Factors Affecting the Accuracy of Air Displacement Pipettes
  • Temperature 
Temperature has many effects on pipetting accuracy. The factor that has the greatest effect 
is the temperature difference between the used delivery device and liquid. The air gap (dead 
air volume) between the liquid surface and the piston experiences thermal expansion effects 
according to the case. This either reduces or increases the liquid amount aspirated into the tip 
along with other effects.
  • Density 
The density (mass/volume ratio) affects the liquid volume that is aspirated into the tip. A smaller dose of liquid with higher density than water is aspirated compared to similar operation 
with water. With lower density liquids the effect is the opposite. This is caused by the flexible 
dead air volume along with the earth gravity. The density of liquids varies also according to the 
temperature. Typically the density for water is 0.998 kg/dm3, for ethanol 0.79 kg/ dm3 and for sulfuric acid (95-98% H 2SO4) 1.84 kg/dm3 (the values apply at temperature of 0 °C).
  • Altitude
The geographic altitude affects the accuracy through air pressure.  The air pressure decreases 
in higher altitudes and the conversion factor Z decreases as well.  Also, with some liquids the 
boiling point decreases quite close to room temperature, which will increase the evaporation 
loss dramatically.
  • Pipetting position 
Pipetting position can also lead to inaccuracy.

Calculation:

Conversion of mass to volume 

V = (w + e) x Z 

V = Volume (µl)  w = Weight (mg) 
e = Evaporation loss (mg) 
Z = Conversion factor for mg/µl conversion

Accuracy (systematic error) 

Accuracy is the difference between the dispensed volume and the selected volume of a pipette.
A = V -V0

A = Accuracy 
V = Mean volume  
V0 = Nominal volume

Accuracy can be expressed as a relative value: 
A% =( 100% x A) / V0

Convert the weight unit of measured value into the volume unit of measured value using the following formula:

Volume ( ml or ul ) = Weight ( mg or ug ) x Z

Z value : conversion factor, which is conversion of density .

Mean Weight = (sample replicate 1 + sample replicate 2 + (etc.))/ Number of Replicates

Mean Volume (Corrected Mean) = Mean Weight × ZFactor

% Inaccuracy = [(Corrected Mean – True Value) ÷ True Value] × 100 

Standard deviation can be expressed as a relative value as cv. 

% CV (Coefficient of Variation) = (Standard Deviation / Corrected Mean) × 100


Calculate the average ( Mean ), accuracy, standard deviation (S.D.) and imprecision ( C.V. ) using the
following formula:

       n              n   - 2

       Σ Xi            Σ (Xi-X)

   -   i=1             i=1      1 / 2

(Mean) X ═ ---------------- ;   S.D. ═(----------------- )

       N              N

N: number of measured value



    S.D.            Measured value

C.V. = ----------- X 100% ; Accuracy = ------------------- X 100%

    Mean            Theoretical value

  • Accuracy value must be 99-101 % and C.V. value must be less than 1 %.
References
  • Y.C.Chang (Apr. 14, 2007), Laboratory manual of  Clinical  Biochem.  Res.  Lab.                                                                    Dept. of Res. & Edu.Taipei Veterans General Hospital.
  • Roger Schultz, Wisconsin State Laboratory of Hygiene, December 5, 2007
  • Thermo Pipetting Guide

Sunday, December 23, 2012

Estimation of Biomass in Plant Cell Cultures

Biomass is an important parameter which needs constant monitoring to understand the growth process in plant cell and tissue cultures. Cell biomass itself is often the final product, or even if the desired product is a secondary metabolite and the compound is intracellular one needs to have a measure of biomass for several estimations related to the secondary metabolite production.

There are larger no. of methods for estimation of biomass which differ in their degree of accuracy, rapidity, convenience, instrumental requirements etc. some methods may not be suitable for a given culture system.

Measurement of cell number

In very fine suspension cultures cell number may be counted directly in a haemocytometer. Most cultures usually contains aggregates and clump. In such cases cell aggregates need to broken into individual cells prior to counting the number of cells.
  1. Add two volumes of 8% w/v aqueous chromic trioxide solution to one volume of culture suspension.
  2. Heat at 70oC for 2-15 mins.
  3. After cooling, shake the culture mixture vigorously to assist the loosening of cell clusters to individual cells. 
  4. dilute appropriately and count the number of cells under microscope using haemocytometer.
  5. Express the biomass as number of cells per ml of culture after correcting for the dilution factor.
Measurement of Packed Cell Volume (PCV)
  1. Transfer 10ml of culture suspension to a 15ml graduated centrifuge tube. tubes which taper at the bottom are preferable.
  2. Centrifuge for 5 mins at 200gm.
  3. Read pellet volume and express percentage of total volume of the culture suspension.
Measurement of Fresh Weight
  1. Filter the cell suspension through a pre-weighed whatman filter paper on a buchnel funnel under slight vacuum.
  2. wash the cells with distilled water.
  3. Drain fully under vacuum.
  4. Reweigh the cells per filter.
  5. Express the weight of cell biomass as a fresh weight.
Measurement of Dry weight
  1. Filter the suspension through a pre-weighed whatman filter paper on a buchnel funnel under slight vacuum.
  2. wash the cells with distilled water.
  3. Drain fully under vacuum.
  4. Dry cells plus filter paper in a hot air oven at 60oC to a constant weight.
  5. Reweigh the cells per filter.
  6. Express the weight of cell biomass as a fresh weight.

Saturday, December 8, 2012

Generating Standard Curve to analyse the reaction optimization - Real Time qPCR, Calculating PCR Efficiency

Generating Standard Curve to analyse the reaction optimization - Real Time qPCR

A standard curve can be generated using a 10-fold dilution of a template amplified on a real-time system (Example: ABI 7500). Each dilution can assayed in triplicate(its always better to do in duplicates or in triplicates).




Below are the data generated after a Real time PCR run.

Ct
Log DNA dilution
36.47
10^-6
33.83
10^-5
29.49
10^-4
26.85
10^-3
23.11
10^-2
20.14
10^-1
17.16
10^0












The template used for this purpose can be a target with known concentration (e.g., nanograms of genomic DNA or copies of plasmid DNA) or a sample of unknown quantity (e.g., cDNA). Here plasmid is used as the template for the reaction.

The standard curve is constructed by plotting the log of the starting quantity of template (or the dilution factor, for unknown quantities) against the Ct (Cycle Threshold) value obtained during amplification of each dilution.

Below is the standard curve graph generated using the data obtained after the PCR run, here Ct value is plotted against log DNA dilution.

Standard Curve qPCR
Standard Curve qPCR

The equation of the linear regression line, along with Pearson’s correlation coefficient (r) or the coefficient of determination (R2), can then be used to evaluate whether your qPCR assay is optimized.

Real-time quantification (qPCR) is based on the relationship between initial template amount and the Ct value obtained during amplification, an optimal qPCR assay is absolutely essential for accurate and reproducible quantification of the sample.

The hallmarks of an optimized qPCR assay are:

• Linear standard curve (R^2 > 0.980 or r > |–0.990|)
• High amplification efficiency  (90–105%)
• Consistency across replicate reactions.

One should strive to achieve a PCR efficiency above 90%.

qPCR Calculations

As everyone knows in PCR one copy of the template becomes two after the first cycle. the template copy number keeps accumulating as the no of cycles proceeds. it is given by a general formula:

2^n; after 25 cycles one copy of the template becomes 2^25 = 33554432 copies or 3.35*10^7 copies.


If perfect doubling occurs with each amplification cycle, the spacing of the fluorescence curves will be determined by the equation 2^n =dilution factor, where n is the number of cycles between curves at the fluorescence threshold (in other words, the difference between the Ct values of the curves).
for example, with a 10-fold serial dilution of DNA, 2^n = 10. Therefore, n = 3.32, and the Ct values should be separated by 3.32 cycles.


if the PCR assay has 100% efficiency one copy becomes two after one cycle, so how to calculate PCR Efficiency.

Amplification efficiency denoted by E can be calculated from the below equation:

E = 10^(-1/slope);

From the standard Curve chart (y = mx + b)

where m is the slope;
b is the intercept;

slope (m) = - 3.2746

Amplification efficiency E = 10^(-1/-3.2746) = 2.02


Amplification efficiency is also frequently presented as a percentage, that is, the percent of template that was amplified in each cycle.

To convert E into a percentage:

% Efficiency = (E – 1) x 100%

 % Efficiency = (2.0 – 1) x 100% = 100%.


An efficiency close to 100% is the best indicator of a robust, reproducible assay. Low reaction efficiencies < 90% may be caused by poor primer design or by suboptimal reaction conditions. Reaction efficiencies >100% may indicate pipetting error in your serial dilutions or co-amplification of nonspecific products, such as primer-dimers.

When using the method described above to determine amplification efficiency, the presence of inhibitor can also result in an apparent increase in efficiency. This is because samples with the highest concentration of template also have the highest level of inhibitors, which cause a delayed Ct, whereas samples with lower template concentrations have lower levels of inhibitors, so the Ct is minimally delayed. As a result, the absolute value of the slope decreases and the calculated efficiency appears to increase. If the  reaction efficiency is <90 nbsp="" or="">105%, one should modify the assay by redesigning your primers and probes. 

Reference


PCR Application guide Bio-Rad
Mathematics in molecular biology and Biotechnology
Internet Sources

Sunday, December 2, 2012

Rapid Diagnostic Tests, Types, Strength and Weakness Applications


Rapid diagnostic tests (RDTs) are diagnostic assays designed for use at the point-of-care (POC), and can be adapted for use in low-resource settings. An RDT is low-cost, simple to operate and read, sensitive, specific, stable at high temperatures, and works in a short period of time. RDTs are already in use for several neglected diseases.

Overview

Rapid diagnostic tests (RDTs) are a type of point-of-care diagnostic, meaning that these assays are intended to provide diagnostic results conveniently and immediately to the patient while still at the health facility, screening site, or other health care provider. Receiving diagnosis at the point of care reduces the need for multiple visits to receive diagnostic results, thus improving specificity of diagnosis and the the chances the patient will receive treatment, reducing dependence on presumptive treatment, and reducing the risk that the patient will get sicker before a correct diagnosis is made. Rapid tests are used in a variety of point-of care-settings—from homes to primary care clinics or emergency rooms -- and many require little to no laboratory equipment or medical training.


Rapid Diagnostic Test


RDTs are particularly important in low-resource settings, where:

Harsh environmental conditions combined with limited access to electricity and refrigeration preclude the use of sensitive equipment
Technology, equipment, and training required for more complicated laboratory tests are lacking
Many patients cannot travel easily to the clinic to follow-up on results that take a long time

RDTs can be especially useful with patient samples that can be collected by minimally trained health personal, such as community health workers.1 Body fluids that can be collected non-invasively, such as nasal swabs, urine, saliva, and tears, are preferred as these are most amenable to collection with only minimal training. However, capillary blood collection techniques, such as those used for malaria RDTs, demonstrate that innovation in sample collection can be used to improve the utility of RDTs in low resource settings.

Rather than one specific type of technology, rapid diagnostic tests can be built in a variety of platforms, each with their own benefits and limitations. The vast majority of RDTs in use today for neglected diseases are based on immunoassay technology due to its relative simplicity. These tests generally involve the interaction of a fixed reagent of either target antigen or antibody that is linked to some type of visible detector, that then reacts with a patient sample. Other types of technologies, such as nucleic-acid amplification, may be too expensive and require too much advanced technology to be applicable as a point of care test.

Rapid diagnostic tests have particular value as epidemiological tools, in addition to use as diagnostics. They enable a rapid screening of a potentially affected population, and can be used, as is the case with lymphatic filariasis, as a test of cure to determine when a mass drug administration has been successful. RDTs are less necessary for diseases that are generally accurately diagnosed syndromically, but could prevent over-prescription of antibiotics if used to differentially diagnose fever or diarrhea, respectively.

Common RDT Platforms

There are several different platforms commonly used to build rapid diagnostic tests. The relative utility of common RDT platforms is summarized below.

Lateral flow tests are the simplest type of RDT, requiring only very minimal familiarity with the test and no equipment to perform, since all of the reactants and detectors are included in the test strip. In a lateral flow test, the sample is placed into a sample well and migrates across the zone where the antigen or antibody is immobilized. The results are read after a certain amount of time has passed. Another type of RDT, a flow-through test, obtains results even faster than lateral flow tests, but requires an added wash and buffer step, which can limit its portability and stability.

Rapid Test: Lateral Flow

An agglutination test works very simply by observation of the binding of carrier particles and target analytes into visible clumps, seen either through a microscope or with the naked eye. However, if the binding of the particles is weak, the results of the test can be inconclusive.



Dipstick format RDTs (with binding sites to test for multiple antigens) work by placing the dipstick in a sample. The dipstick is then washed and incubated to prevent non-specific analyte binding. These additional steps can limit their usability in low-resource point of care settings.

Microfluidics, or “labs on a chip” are an emerging area of rapid diagnostic development. Using electrochemical sensors, these tests would include all detectors and reactants in a single portable cassette. For more information, see the Diagnostics Innovation Map Report. The limitations and advantages of each of these test types is summarized in the table below. For more information about the mechanisms of these tests, see PATH’s RDT Info website.


Microfluidics - Rapid Diagnostic Test


Strengths and Weaknesses of RDTs and Common RDT Platforms
The general strengths of RDTs include their ease of use, minimum training requirements, rapid results, and limited need for instrumentation/infrastructure. The general weaknesses of RDTs including their subjective interpretation of readout, low throughput, often limited sensitivity relative to laboratory or reference tests, and need for quality control mechanisms.

RDTs as Non-Neglected Tropical Disease Diagnostics
Rapid diagnostic tests are commercially available for detection of multiple non-neglected diseases and conditions, including pregnancy, blood sugar in diabetic patients, and strep throat. The simplicity of these assays often allows them to be used at home or by minimally trained health care works in both the developed and developing worlds. In the United States, the use of rapid diagnostic tests by doctors is limited as current Medicaid/Medicare reimbursement schemes favor the use the centralized laboratories for diagnosis.

RDTs as Neglected Tropical Disease Diagnostics
One of the particular problems facing the production and implementation of RDTs in low-resource settings is the lack of an evaluative process to determine their real efficacy in the field. For example, the WHO recently issued a policy statement to recommend that rapid serological tests for TB not be useddue to a lack of sensitivity and specificity.4 WHO-TDR and FIND now have lot-testing programs for RDTs for neglected diseases, such as malaria and HIV, to improve standards and recommendations for use of RDTs in the field.

In order to address the ongoing challenge of quality assurance for rapid test, the WHO now independently evaluates RDTs for several diseases, including HIV and malaria, in order to evaluate them for pre-qualification. As with pre-qualification for drugs or vaccines, this designation is the WHO’s method of validating the quality of specific diagnostic assays and manufacturers for those in countries without rigorous and stringent diagnostic approval processes.

References

P. van Lode (2005). “Point-of-care immunotesting: Approaching the analytical performance of central laboratory methods”. Clinical Biochemistry 38.
Mabey et al. (2004). “Diagnostics for the Developing World.” Nature Reviews Microbiology (2)
PATH. RDT Info.
WHO News Release (2011). “WHO warns against the use of inaccurate blood tests for active tuberculosis”.
WHO. Malaria Rapid Diagnostic Tests.
FIND. Malaria RDT Product Testing.
WHO. HIV Diagnostic Test Kit.

Source:
http://www.bvgh.org/Biopharmaceutical-Solutions/Global-Health-Primer/Targets

Sunday, October 14, 2012

Composting: Vermicomposting


Composting is the biological degradation of solid organic waste into stable end products. The term compost applies to a considerable number of  composted materials produced from a variety of sources. In its broadest sense , it includes any organic material that has undergone managed aerobic microbial degradation at elevated temperatures, resulting in significant microbial, physical and chemical changes to the original material. These composts have been produced using source-separated materials derived from green wastes, kitchen wastes and wood wastes and may also include animal manure but are not derived exclusively from them.

Composting: Vermiculture
Soil Fauna - Termites and Earthworms  - play vital role in maintaining soil quality and also in managing efficient nutrient cycling


The Composting Process
Composting can be defined as the biological decomposition and stabilization of organic substrates, under conditions that allows thermophilic temperature as a result of biologically produced heat 

Aerobic Composting

•Composting with decomposers that need air (oxygen)

•The fastest way to make high quality compost

•Produces no foul odors

•Aerobic decomposers produce heat

•Active composting occurs in the temperature range of 55oF to 155oF,

Pile temperature may increase above 140oF but this is too hot for most bacteria and decomposition will slow until temperature decreases again


Vermicompost - Making compost with the use of earthworms.


vermicompost process


  • Its a stable fine granular organic matter added to the clay soil helps loossen the soil and allows to passage for the entry of air.
  • Mucous associated with the cast can absorb water which prevents logging, improves water holding capacity.
  • Improves physical/chemical/biological properties of soil.
  • Yield of crop is increased.
Advantages
  1. Easy Management.
  2. Vermicomposting wastes as source - Profitably utilized and commercialized for economic gains.
  3. Domestic, agricultural, rural industrial organic wastes can be recycled for various usages which will help in reducing pollution.
  4. Loss of nutrients by leaching + nutrient loss is minimal.
Disadvantages
  1. Less extent of damaging crops.
  2. Casts - as ugly structures in ornamental lawns.
  3. Occasionally transmits many parasites and disease of plants and animals.

Vermiculture Process
  1. Site Selection.
  2. Availability of decomposible organic waste, daily quantity and quality, etc.
  3. Marketable outlet and requirement with future scope.
  4. Collection and study of earthworm culture.
  5. Testing.
  6. Maintenance of seed culture.
Vermicomposting Materials
  • Animal dung, Agricultural wastes
  • Industry wastes
  • City leaf litter
  • Waste paper and cotton cloth etc.
  • Biogas Slurry
Primary Treatment of Composting material
  1. Sorting, Separation.
  2. Exposure to sun for a day.
  3. Mixing of daily organic waste products.
Indoor Vemicomposting


how to do vermicompost


Moisture Content - 30 - 40%
Temperature - 20 - 30oC
  • Earthworms - Perionyx excavatus, Eudrilus eugeniae
  • Biological indicators of soil activities - used for monitoring and maintaining biological health of soil.

Saturday, October 13, 2012

Stratagies to Improve Bactrial Disease Resistance in Plants through Genetic Engineering

1. Production of Anti Bacterial Proteins of Non-Plant Origin
  • Lytic Peptides from Insects
  • Lysozymes
  • Other antibacterial Peptides
2. Inhibition of Bacterial Pathogenicity or Virulence Factors
  • Inhibition of Bacterial toxins
  • Other Approached
3. Enhancement of Natural Plant Defenses
  • Enhanced Production of Elicitors 
  • Expression of cloned resistant genes
  • Enhanced production of reactive oxygen species
  • Expression of plant defense genes 
4. Artificially Induced Programmed Cell Death at the site of infection
  • R and avr genes
  • Barnase and Barstar genes
  • Bacterio-opsin gene


Production of anti-Bacterial Proteins of Non-Plant Origin:

a) Lytic Peptides from Insects:
  •  Small Proteins with alpha helices
  • Causes pores in bacteria
    Example: Cecropins, attacins – giant silk moth 
Cecropins
  •  Synthetic analogs of cecropin gene – Potato, Tobacco. 
  •  Transgenic tobacco – bacterial wilt – delayed mortality. 
  •  Limitation: Degradation of cecropins by plant proteases. 
b) Lysozyme
  •  Lysozymes are ubiquitous enzymes. 
  • Hydrolytic bacterial cell wall containing peptidoglycan. 
Examples
  •  Transgenic tobacco- hen egg lysozyme – resistance to several species. 
  •  Transgenic potato – T4 Bacteriophage – Partial resistance to Erwinia. 
  •  Transgenic tobacco- human lysozyme – Partial resistance to P.syringae. 
c) Other antibacterial peptides

Lactoferin:
  • Iron binding glycoprotein
  • Bactericidal properties
Transgenic Tobacco
  • Human Lactoferin gene
  • Delayed symptoms on Rhizoctonia solanaceae
Trachyplesin: 
  • Codes for lytic peptides
  • Horseshoe crab.
Transgenic potato:
  • genes from horseshoe crab - partial resistance.
  • Limitation: Expressed reduced amount of tuber.

Inhibition of Bacterial Pathogenicity or Virulence Factors

a) Inhibition of bacterial toxins




Pathogen protects itself against toxin by expressing tabtoxin resistant genes (ttr), transgenic tobacco with genes show resistance towards many bacteria.

b) Other Approaches




3. Enhancement of Natural Plant Defenses

a) Enhanced Production of Elicitors


b) Expression of cloned resistant genes


c) Enhanced production of reactive oxygen species


A glucose oxidase gene from A.niger is cloned into potato plants resulted in resistance. This enzyme induce large amount of hydrogen peroxide. transgenic potato results in increased level of resistance.

d) Expression of plant defense genes


In certain cases resistance was not seen though high level of thionin is produced. This may be due to thionin is secreted into intracellular space, where bacteria are generally found.

Plant defense mechanism is due to battery of synergetic reactions. Hence the expression of combination of heterologous genes would be more promising

4. Artificially Induced Programmed Cell Death at the site of infection




  • R and avr genes applied to fungus
  • Barnase and Barstar genes
  • Barnase - Bacterial ribonuclease gene
  • Barstar - Inhibitor of barnase
  • Bacterial opsin gene
Limitations
  • Efficacy, durability, absence of toxicity and low environmental impact
  • Gene silencing

Friday, September 28, 2012

Basics on Plant and Animal Tissue Culture

Animal Cell Culture
A wider range of ingredients needed to support survival and proliferation or differentiation. Invitro animal cell cultivation requires a complex combination of nutrients,considering glucose and glutamine as main carbon, energy and nitrogen sources.Mineral salts, amino acids and vitamins are also required; other essential nutrients, like growth factors, hormones, and receptor and transport proteins are required in small quantities as well. The pH was maintained at 7.4 and often it includes pH indicator phenol red (red at 7.4, yellow at 6.5, purple at 7.8). A typical media may or may not comprise of serum. The latter is called a serum-free media. Some of the common sources of serum can be fetal bovine serum, equine serum, calf serum etc. both the types of media have their own set of advantages and disadvantages.

The culture media is prepared in such a way that it provides
  1. The optimum conditions of factors like pH, osmotic pressure, etc.
  2. It should contain chemical constituents which the cells or tissues are incapable of synthesizing. Generally the media is the mixture of inorganic salts and other nutrients capable of sustaining cells in culture such as amino acids, fatty acids, sugars, ions,trace elements, vitamins, cofactors, and ions. Glucose is added as energy source - its concentration varying depending  on the requirement. Phenol Red is added as a pH indicator of the medium.
Basic Components in the Culture Media

Most animal cell culture media are generally having following 10 basic components and
they are as follows:

1. Energy sources: Glucose, Fructose, Amino acids
2. Nitrogen sources: Amino acids
3. Vitamins: Generally water soluble vitamins B & C
4. Inorganic salts: Na+, K+, Ca2+, Mg2+
5. Fat and Fat soluble components: Fatty acids, cholesterol.
6. Nucleic acid precursors
7. Antibiotics
8. Growth factors and hormones
9. pH and buffering systems
10. Oxygen and CO2 concentration.

Animal cell culture media vary in their complexity but most contain:

Amino acids 0.1-0.2 mM
Vitamins ca. 1 μM
Salts NaCl 150 mM
KCl 4-6 mM
CaCl 1 mM
Glucose 5-10 mM

Culture Media
A cell culture medium is composed of a number of ingredients and these ingredients vary from one culture medium to another. The nutrient media used for culture of animal cells and tissues must be able to support their survival as well as growth, i.e., must provide nutritional, hormonal factors.

The various types of media used for tissue culture may be grouped into two broad categories:

1. Natural media
2. Artificial media.

The choice of medium depends mainly on the type of cells to be cultured (normal,immortalized or transformed), and the objective of culture (growth, survival,differentiation, production of desired proteins). Non transformed or normal cells (finite life span) and primary cultures from healthy tissues require defined quantities of proteins, growth factors and hormones even in the best media developed so far. But immortalized cells (spontaneously or by transfection with viral sequences) produce most of these factors, but may still need some of the growth factors present in the serum.

In contrast, transformed cells (autonomous growth control and malignant properties) synthesize their own growth factors; in fact, addition of growth factors may even be detrimental in such cases. Buteven these cultures may require factors like insulin,transferrin, silenite, lipids, etc.

Natural Media

These media consist solely of naturally occurring biological fluids and are of the following three types:

1. Coagula or clots
2. Biological fluids
3. Tissue extracts

The natural biological fluids are generally used for organ culture. For cell cultures, artificial media with or without serum are used.

Clots
The most commonly used clots are plasma clots, which have been in use for a long time. Plasma is now commercially available either in liquid or lyophilized state. It may also be prepared in the laboratory, usually from the blood of male fowl, but blood clotting must be avoided during the preparation.

Biological Fluids
Of the various biological fluids used as culture medium, serum is the most widely used.
Serum is one of the very important components of animal cell culture which is the source of various amino acids, hormones, lipids, vitamins, polyamines, and salts containing ions such as calcium, ferrous, ferric, potassium etc. It also contains the growth factors which promotes cell proliferation, cell attachment and adhesion factors. Serum may be obtained from adult human blood, placental cord blood, horse blood or
calf blood (foetal calf serum, newborn calf serum, and calf serum); of these foetal calf serum is the most commonly used. Serum is the liquid exuded from coagulating blood.Different preparations of serum differ in their properties; they have to be tested for sterility and toxicity before use.

Tissue Extracts
Tissue or organ extracts and/or hydrolysates (e.g., bovine pituitary extract (BPE), bovine brain extract,chick embryo extract and bovine embryo extract), and animal-derived lipids and fatty acids, peptones, Excyte, sterols (e.g., cholesterol) and lipoproteins (e.g., high-density and low-density lipoproteins(HDLs and LDLs, respectively) are used in culturing of animal cells. Tissue extracts for example, Embryo extracts—Other biological fluids used as natural media include amniotic fluids,ascetic and pleural fluids, aqueous humour (from eye), serum ultra filtrate, insect haemolymph etc. Chick embryo extract is the most commonly used tissue extract, but bovine embryo extract is also used. Other tissue extracts that have been used are spleen, liver, bone marrow, etc. extracts. Tissue extracts can often be substituted by a mixture of amino acids and certain other organic compounds.

Artificial Media
Different artificial media have been devised to serve one of the following purposes:
1. Immediate survival (a balanced salt solution, with specified pH and osmotic pressure is adequate),
2. Prolonged survival (a balanced salt solution supplemented with serum, or with suitable formulation of organic compounds),
3. Indefinite growth
4. Specialized functions.

The various artificial media developed for cell cultures may be grouped into the following four classes:
(i) Serum containing media
(ii) Serum free media
(iii) Chemically defined media
(iv) Protein free media.

SERUM
Liquid yellowish, clear content left over after fibrin and cells are removed from the blood is known as serum. Calf (bovine), foetal bovine, or horse are used, in some cases human. Fetal bovine serum (FBS)(10-20% v/v) is the most commonly applied supplement in animal cell culture media. Normal growth media often contain 2-10% of serum.  These supplements provide carriers or chelators for labile or water-insoluble nutrients; bind and neutralize toxic moieties; provide hormones and growth factors,protease inhibitors and essential, often unidentified or undefined low molecular weight nutrients; and  protect cells from physical stress and damage. Thus, serum and/or animal extracts are commonly used as relatively low-cost supplements to provide an optimal culture medium for the cultivation of animal cells. The role for all constituents (more than 200) is not clear proteins, peptides, special factors released during platelet aggregation e.g., PDGF, TGF-β, lipids, lipid transport proteins, carbohydrates,micronutrients such as minerals, etc.

Chemically Defined Media:  These media contain contamination free  ultra pure inorganic and organic constituents, and may contain pure protein additives, like insulin, epidermal growth factor, etc. that have been produced in bacteria or yeast by genetic engineering with the addition of vitamins, cholesterol, fatty acids and specific amino acids. The CHO cell lines are widely used for being highly stable expression systems for
heterologous genes (those from a different organism), and for its relatively simple adaptation to adherence-independent growth in serum and protein free media.

Protein-Free Media:  In contrast, protein free media do not contain any protein; they only contain non-protein constituents necessary for culture of the cells. The formulations MEM, DME, RPMI-1640,etc. are protein free; where required, protein supplementation is provided.

APPLICATIONS OF ANIMAL CELL CULTURE
The animal cell cultures are used for a diverse range of research and development.These areas are:
(a) Production of antiviral vaccines, which requires the standardization  of cell lines for the multiplication and assay of viruses.
(b) Cancer research, which requires the study of uncontrolled cell division in cultures.
(c) Cell fusion techniques.
(d) Genetic manipulation, which is easy to carry out in cells or organ cultures.
(e) Production of monoclonal antibodies requires cell lines in culture.
(f ) Production of pharmaceutical drugs using cell lines.
(g) Chromosome analysis of cells derived from womb.
(h) Study of the effects of toxins and pollutants using cell lines.
(i) Use of artificial skin.
(j) Study the function of the nerve cells.
(k) Many commercial proteins have been produced by animal cell culture and there medical application is being evaluated. Tissue Plasminogen activator (t-PA) was the first drug that was produced by the mammalian cell culture by using rDNA technology. The recombinant t-PA is safe and effective for dissolving blood clots in patients with heart diseases and thrombotic disorders.

Plant tissue culture
Most methods of plant transformation applied to genetically modified crops require that a whole plant is regenerated from isolated plant cells or tissues that have been genetically transformed. This regeneration is conducted in vitro so that the environment and growth medium can be manipulated to ensure a high frequency of regeneration. In addition to this, the regenerable cells must be accessible to gene transfer by whatever technique is chosen. The primary aim is therefore to produce, as easily and as quickly as possible, a large number of regenerable cells that are accessible to gene transfer. The subsequent regeneration
step is often the most difficult step in plant transformation studies

Plasticity and totipotency
Two concepts, plasticity and totipotency, are central to understanding plant cell culture and regeneration. Plants, due to their sessile nature and long life span, have developed a greater ability to endure extreme conditions and predation than have animals. Many of the processes involved  in plant growth and development adapt to environmental conditions. This plasticity allows plants to alter their metabolism, growth, and development to best suit their environment. Particularly important aspects of this adaptation, as far as plant tissue culture and regeneration are concerned, are the abilities to initiate cell division from almost any tissue of the plant and to regenerate lost organs or undergo different developmental pathways in response to particular stimuli.
When plant cells and tissues  are cultured  in vitro  they generally exhibit a very high degree of plasticity, which allows one type of tissue or organ to be initiated from another type. In this way, whole plants can be subsequently regenerated. This regeneration of whole organisms depends upon the concept that all plant cells can, given the correct stimuli, express the total genetic potential of the parent plant. This maintenance of
genetic potential is called  totipotency. Plant cell culture and regeneration do, in fact, provide the most  compelling evidence for totipotency. In practical terms though, identifying the culture conditions and stimuli required to manifest this totipotency can be extremely difficult and it is still a largely empirical process.
The culture environment When cultured in vitro, all the needs of the plant cells, both chemical and physical, have to met by the culture vessel, the growth medium, and the external environment (light, temperature, etc.). The growth medium has to supply all the essential mineral ions required for growth and development. In many cases (as the biosynthetic capability of cells cultured in vitro may not replicate that of the parent plant),
it must also supply additional organic supplements such as amino acids and vitamins.
Many plant cell cultures, as they are not photosynthetic, also require the addition of a fixed carbon source in the form of a sugar (most often sucrose). One other vital component that must also be supplied is water, the principal biological solvent. Physical factors, such as temperature, pH, the gaseous environment, light(quality and duration),and osmotic pressure, also have to be maintained within acceptable limits.

Plant cell culture media
Culture media used for the cultivation of plant cells in vitro are composed of three basic components:
1 essential elements, or mineral ions, supplied as a complex mixture of salts;
2 an organic supplement supplying vitamins and/or amino acids; and
3 a source of fixed carbon; usually supplied as the sugar sucrose.
For practical purposes, the essential elements are further divided into the following categories:
1 macroelements (or macronutrients);
2 microelements (or micronutrients); and
3 an iron source.
Complete plant cell culture medium is usually made by combining several  different components,
Media components
It is useful to briefly consider some of the individual components of the stock solutions.

Macroelements
As is implied by the name, the stock solution supplies macroelements required in large amounts for plant growth  and development. Nitrogen, phosphorus, potassium, magnesium, calcium, and sulphur (and carbon, which is added separately) are usually regarded as macroelements. These elements usually comprise at least 0.1% of the dry weight of plants.

Nitrogen is most commonly supplied as a mixture of nitrate ions (from KNO3) and ammonium ions (from NH4NO3). Theoretically, there is an advantage in supplying nitrogen in the form of ammonium ions, as nitrogen must be in the reduced form to be incorporated into macromolecules. Nitrate ions therefore need to be reduced before incorporation. However, at high concentrations, ammonium ions can be toxic to plant cell cultures and uptake of ammonium ions from the medium causes acidification of the medium. For ammonium ions to be used as the sole nitrogen source, the medium needs to be buffered. High concentrations of ammonium ions can also cause culture problems by increasing the frequency of verification (the culture appears pale and ‗glassy‘ and is usually unsuitable for further culture). Using a mixture of nitrate and ammonium ions has the advantage of weakly buffering the medium as the  uptake of nitrate ions causes
OH− ions to be excreted. Phosphorus is usually supplied as the phosphate ion of ammonium, sodium, or potassium salts. High concentrations of phosphate can lead to the precipitation of medium elements as insoluble phosphates.

Microelements
Microelements are required in trace amounts for plant growth and development, and have many and diverse roles. Manganese, iodine, copper, cobalt, boron, molybdenum, iron, and zinc usually comprise the microelements, although other elements such as nickel and aluminium are found frequently in some formulations. Iron is usually added as iron sulphate, although iron citrate can also be used. Ethylene diamine tetra-acetic acid (EDTA) is usually used in conjunction with iron sulphate. The EDTA complexes with the iron to allow the slow and continuous release of iron into the medium. Uncomplexed iron can precipitate out of the medium as ferric oxide.
Organic supplements
Only two vitamins, thiamine (vitamin B1) and myoinositol (considered a B vitamin),are considered essential for the culture of plant cells  in vitro. However, other vitamins are often added to plant cell culture media for historical reasons. Amino acids are also commonly included in the organic supplement. The most frequently used is glycine (arginine, asparagine, aspartic acid, alanine, glutamic acid,glutamine, and proline are also used), but in many cases its inclusion is not essential. Amino acids provide a source of reduced nitrogen and, like ammonium ions, uptake causes acidification of the medium. Casein hydrolysate can be used as a relatively cheap source of a mix of amino acids.

Carbon source
Sucrose is cheap, easily available, readily assimilated, and relatively stable, and is therefore the most commonly used carbon source. Other carbohydrates (such as glucose, maltose, galactose, and sorbitol) can also be used and in specialized circumstances may prove superior to sucrose.
Gelling agents
Media for plant cell culture  in vitro  can be used in either liquid or ‗solid‘ forms, depending on the type of culture being grown. For any culture types that require the plant cells or tissues to be grown on the surface of the medium, it must be solidified(more correctly termed  gelled). Agar, produced from seaweed, is the most
common type of gelling agent, and is ideal for routine applications. However, because it is a natural product, the agar quality can vary from supplier to supplier and from batch to batch. For more demanding applications, a range of purer (and in some cases, considerably more expensive) gelling agents are available. Purified agar or agarose can be used, as can a variety of gellan gums.

Plant growth regulators

Plant growth  regulators are the critical media components in determining the developmental pathway of the plant cells. The plant growth regulators used most commonly are plant hormones or their synthetic analogues.
Classes of plant growth regulator There are five main classes of plant growth regulator used in plant cell culture, namely:
(1) auxins;
(2) cytokinins;
(3) gibberellins;
(4) abscisic acid;
(5) ethylene.
Each class of plant growth regulator will be looked at briefly below.
Auxins
Auxins promote both cell division and cell growth. The most important naturally occurring auxin is indole-3-acetic acid (IAA), but its use in plant cell culture media is limited because it is unstable to both heat and light. Occasionally, amino acid conjugates of IAA (such as indole-acetyl-l-alanine and indole-acetyl-l-glycine), which are more stable, are used to partially alleviate the problems associated with the use of IAA.
It is more common, though, to use stable chemical analogues of IAA as a source of auxin in plant cell culture media. 2,4-Dichlorophenoxyacetic acid (2,4-D) is the most commonly used auxin and is extremely effective in most circumstances. Other auxins are available and some may be more effective or ‗potent‘ than2,4-D in some instances.
Cytokinins
Cytokinins promote  cell division. Naturally occurring cytokinins are a large group of structurally related purine derivatives. Of the naturally occurring cytokinins, two have some use in plant tissue culture media zeatin andN6-(2-isopentyl)adenine (2iP). Their use is not widespread as they are expensive(particularly zeatin) and relatively unstable. The synthetic analogues kinetin and6-benzylaminopurine (BAP) are therefore used more frequently. Non-purine-based chemicals, such as substituted phenylureas, are also used as cytokinins in plant cellculture media. These substituted phenylureas can also substitute for auxin in someculture systems.
Gibberellins
There are numerous, naturally occurring, structurally related compounds termed gibberellins. They are involved in regulating  cell elongation, and are agronomically important in determining plant height and fruit-set. Only a few of the gibberellins are used in plant tissue culture media, GA3 being the most common.
Abscisic acid
Abscisic acid (ABA) inhibits cell division. It is most commonly used in plant tissue culture to promote distinct developmental pathways such as somatic embryogenesis
Ethylene
Ethylene is a gaseous, naturally occurring, plant growth regulator most commonly associated with controlling fruit ripening in climacteric fruits, and its use in plant tissue culture is not widespread. It does, though, present a particular problem for plant tissue culture. Some plant cell cultures produce ethylene, which, if it builds up sufficiently, can inhibit the growth and development of the culture. The type of culture vessel used and its means of closure affect the gaseous exchange between the culture vessel and the outside atmosphere and thus the levels of ethylene present in the culture.

Plant growth regulators and tissue culture
Generalizations about plant growth regulators and their use in plant cell culture media have been developed from initial observations made in the 1950s. There is, , some considerable difficulty in predicting the effects of plant growth regulators: this is because of the great differences in culture response among species, cultivars, and even plants of the same cultivar grown under different conditions., some principles do hold true and
have become the paradigm on which most plant tissue culture regimes are based.

Auxins and cytokinins are the most widely used plant growth regulators in plant tissue culture and are usually used together, the ratio of the auxin to the cytokinin determining the type of culture established or regenerated A high auxin to cytokinin ratio generally favours root formation, whereas a high cytokinin to auxin ratio favours shoot formation. An intermediate ratio favours callus production.

Culture types
Cultures are generally initiated from sterile pieces of a whole plant. These pieces are termed  explants, and may consist of pieces of organs, such as leaves or roots, or maybe specific cell types, such as pollen or endosperm. Many features of the explant are known to affect the efficiency of culture initiation. Generally, younger, more rapidly growing tissue (or tissue at an early stage of development) is most effective. Several
different culture types most commonly used in plant transformation studies are as follows.

Callus
Explants, when cultured on the appropriate medium, usually with  both an auxin and a cytokinin, can give rise to an unorganized, growing, and dividing mass of cells. It is thought that any plant tissue can be used as an explant, if the correct conditions are found. In culture, this proliferation can be maintained more or less indefinitely, provided that the callus is sub cultured on to fresh medium periodically. During callus formation,
there is some degree of dedifferentiation (i.e. the changes that occur during development and specialization are, to some extent, reversed), both in morphology (a callus is usually composed of unspecialized parenchyma cells) and metabolism. One major consequence of this dedifferentiation is that most plant cultures lose the ability to photosynthesize. This has important consequences for the culture of callus tissue, as the metabolic profile will probably not match that of the donor plant. This necessitates the addition of other components-such as vitamins and, most importantly, a carbon source-to the culture medium, in addition to the usual mineral nutrients. Callus culture is often performed in the dark (the lack of photosynthetic capability being no drawback) as light can encourage differentiation of the callus. During long term culture, the culture may lose the requirement for auxin and/or cytokinin. This process, known as habituation, is common in callus cultures from some plant species(such as sugar beet).
Callus cultures are extremely important in plant biotechnology. Manipulation of the auxin to cytokinin ratio in the medium can lead to the development of shoots, roots, or somatic embryos from which whole plants can subsequently be produced. Callus cultures can also be used to initiate cell suspensions, which are used in a variety of ways in plant transformation studies.

Protoplasts
Protoplasts are plant cells with the cell wall removed. Protoplasts are most commonly isolated from either leaf mesophyll cells or cell suspensions, although other sources can be used to advantage. Two general approaches to removing the cell wall (a difficult task without damaging the protoplast) can be taken: mechanical or enzymatic isolation. Mechanical isolation, although possible, often results in low yields, poor quality, and poor performance in culture due to substances released from damaged cells. Enzymatic isolation is usually carried out in a simple salt solution with a high osmoticum, plus the cell-wall-degrading enzymes. It is usual to use a mix of both cellulase and pectinase enzymes, which must be of high quality and purity.
Protoplasts are fragile  and damaged easily, and therefore must be cultured carefully.Liquid medium is not agitated and a high osmotic potential is maintained, at least in the initial stages. The liquid medium must be shallow enough to allow aeration in the absence of agitation.  Protoplasts can be plated out on to solid medium and callus produced. Whole plants can be regenerated by organogenesis or somatic embryogenesis from this callus. Protoplasts are ideal targets for transformation by a variety of means.
Root cultures
Root cultures can be established in vitro from explants of the root tip of either primary or lateral roots and can be cultured on fairly simple media. The growth of roots  in vitro  is potentially unlimited, as roots are indeterminate organs. Although the establishment of root cultures was one of the first achievements of modern plant tissue culture, they are not widely used in plant transformation studies.
Shoot tip and meristem culture The tips of shoots (which contain the shoot apical meristem) can be cultured  in  vitro, producing clumps of shoots from either axillary or adventitious buds. This method can be used for clonal propagation. Shoot meristem cultures are potential alternatives to the more commonly used methods for cereal regeneration as they are less genotype dependent and more efficient (seedlings can be used as donor material).
Embryo culture
Embryos can be used as explants to generate callus cultures or somatic embryos. Both immature and mature embryos can be used as explants. Immature, embryo-derived embryogenic callus is the most popular method of monocotyledon plant regeneration.

Reference:
Food Biotechnology  by K.V.Anand Raj & M. Raveendra Reddy

Saturday, August 25, 2012

Biogas (Methane) Production, Biogas Plant, Advantages

Biogas

When methane is produced by the fermentation of animal dung the gaseous products are usually referred as biogas and the installations are called biogas plant or bioreactor. Biogas is a flammable mixture of 50-80% methane, 15-45% CO2, 5% water and some other trace gases.Biogas is produced by biomethenation and is self regulating symbiotic microbial process operating under anaerobic conditions and functions at temperature around 30oC.

Organisms involved are all found naturally in ruminant manures. In such system the animal dung is mixed with water and allowed to ferment in near anaerobic conditions, under ideal condition 10Kg of dry organic matter can produce 3m3 of biogas, which will provide 3hrs of cooking, 3hrs of lighting and 24hrs of refrigeration with suitable equipment.

Biogas Production Overview
biogas plant, biogas technology


Methane as an energy source may have economic value at local small scale production, but there is considerable doubt about the future of commercial large scale process for methane production.
  • An abundance of methane occurs in nature, particularly in natural gas fields and oil field overlays.
  • Methane production by gasification of coal is commercially more attractive.
  • Microbial production of methane is more expensive than natural gas.
  • Costs of storage, transport and distribution of gaseous fuels is not economical.
  • Methane cant be used in automobiles.
Production of Biogas


Production of Biogas

  • Biogas is a mixture of gases produced from the anaerobic digestion of waste materials such as animal & plant waste.
  • A biogas plant which uses only cow dung is called as the"gobar gas plant".
  • The gas is used as fuel for cooking or lighting.
  • Microorganisms involved in biogas production are a group of different Sps. which forms a consortium.
  • Bacteria involved in the initial stages are not strict anaerobes.
Anaerobic Digestion is accomplished by three stages
  1. Solubilization
  2. Acidogenesis
  3. Methanogenesis.
stages of biogas production process

Advantages
  1. Anaerobic digestion of municipal industrial and agricultural wastes can have positive environmental values, since it can combine waste removal, stabilization and net yield of biogas formation.
  2. Solid or liquid residues can be used as fertilizer, soil conditioner or animal feed. hence biomass production continues to have high priority in alternative energy research

Tuesday, August 14, 2012

Strain Improvement - Methods and Applications

STRAIN IMPROVEMENT
Several options are open to an industrial microbiology organization seeking to maximize its profits in the face of its competitors‘ race for the same market. The organization may undertake more aggressive marketing tactics, including more attractive packaging while leaving its technical procedures unchanged. It may use its human resources more efficiently and hence reduce costs, or it may adopt a more efficient extraction system for obtaining the material from the fermentation broth.

The operations in the fermentor may also be improved by its use of a more productive medium, better environmental conditions, better engineering control of the fermentor processes, or it may genetically improve the productivity of the microbial strain it is using. Of all the above options, strain improvement appears to be the one single factor with the greatest potential for contributing to  greater profitability. While realizing the
importance of strain improvement, it must be borne in mind that an improved strain could bring with it previously non-existent problems.

For example, amore highly yielding strain may require greater aeration or need more intensive foam control; the products may pose new extraction challenges, or may even require an entirely new fermentation medium. The use of a more productive strain must therefore be weighed against possible increased costs resulting from higher investments in extraction, richer media, more expensive fermentor operations and other hitherto non existent problems. This possibility not withstanding, strain improvement is usually part of the program of an industrial microbiology organization. To appreciate the basis of strain improvement it is important to remember that the ability of any organism to make any particular product is predicated on its capability for the secretion of a particular set of enzymes. The production of the enzymes, themselves  depends ultimately on the genetic make-up of the organisms. Improvement of strains can therefore be put down in simple term as follows:

  1. Regulating the activity of the enzymes secreted by the organisms.
  2. In the case of metabolites secreted extra-cellularly, increasing the permeability of the organism so that the microbial products can find these way more easily outside the cell.
  3. Selecting suitable producing strains from a natural population.
  4. Manipulation of the existing genetic apparatus in a producing organism.
  5. Introducing new genetic properties into the organism by recombinant DNA technology or genetic engineering. 
MUTATIONS

Genes are chemically the segments of DNA molecules except in some viruses, as some viruses are found to  contain RNA as genetic material.  They are normally transmitted with great exactness. But sometimes variations may be caused by physicalor chemical agents resulting in altered phenotype.  The heritable changes in the  genome of a cell are called mutations. Those mutations which occur in the somatic cells are called somatic mutations. These are not transmitted to next generation.
Mutations occurring in the germ cells are called germinal mutations. These mutations influence the gametes and are passed to next generation, generating new variability and contributing to the process of evolution.

Mutations have both advantages as well as disadvantages.Increasing the microbial mutation rates bring out the genetic changes which have been put to many important uses in the laboratory and industries. For example mutations in some plants like tulips producing colourful flowers. Mostly mutations effect the normal existence of cells.  For example in bacteria, auxotrophs are developed from wild type because of mutations.  In humans mutations leads to physiological abnormalities like sickle cell anemia.

TYPES OF MUTATIONS

Mutations are classified in different ways on the basis of one or the other criterion. They may be depending on their origin, depending on  the type of change in base composition, on the basis of type of the cell, on the basis of the nature of their effect,etc. Among all these classification criteria the significant one‘s are

(A) Depending on their origin. 
(B) Depending on the type of change in base composition.

(A) Depending on their origin : Mutations are of two types
  1. Spontaneous mutations
  2. Induced mutations.
Spontatneous mutations: Mutations that occur naturally are called spontaneous mutations. Their origin is indeterminate and unknown.They are generally assumed to be random changes in the nucleotide sequences of genes. Spontatneous mutations are linked to normal chemical processes in the organism that alter the structure or the sequences of genes. For example all the four common bases of DNA have unusual tautomeric forms. Which are,however, rare.Tautomers are the mutually interconvertable structural isomeric forms. Normally nitrogenous bases in DNA present in the keto form. As a result of tautomeric rearrangement they can be transformed into the enol form.

The tautomeric rearrangement changes the hydrogen bonding characteristics of bases. Normally AU and GT base pairs. The tautomeric changes during replication substitutes nitrogenous bases with others. If a purine for purine and pyrimidine for pyrimidine are substituted the type of mutation is called transition mutation. If a purine for pyrimidine and pyrimidine for purine substituted the mutation is called transversion. The transtition and transversion mutations are also termed point mutations. Spontaneous mutations also occurs by frame shifts of DNA.

Once an error is present in the genetic code, it may be reflected in the amino acid composition of the specified protein.  If the changed amino acid is present in a part of the molecule, determining the structure or biochemical activity, functional alteration can occur. Many spontaneous mutations are reported.  For example albinism and hares lip in man; a tobacco mutant producing seventy leaves all of a sudden in a normal progeny
producing an average of twenty leaves.

Induced mutations:  The mutations resulting from the influence of any artificial factor are considered to be induced mutations.  Muller subjected drosophila to powerful x-rays and obtained a number of mutations.  The chemicals or any other means that induce mu8tations are called as mutagens or mutagenic agents.The mutagens acts in different ways like incorporation of base analogs, specific mispairing and intercalation.

Base analogs are structurally similar to nitrogenous bases of DNA, and can be incorporated into the growing polynucleotide chain during replication. Specific mispairing is caused when a mutagen changes a bases structure, by that alters its base pairing characteristics.

The different types of mutations changing the nucleotide number or order of DNA are
  1. Frameshift mutations
  2. Chromosomal mutations
Frameshift mutations:  As pointed out in the third unit the genetic information in DNA is expressed first into mRNA by the transcription.  mRNA is translated to proteins on reading triplet code from a fixed starting codon.  If a single nucleotide is deleted or inserted in the normal sequence then the reading frame changes.
The mutations leading to the change in reading frame are called frame shift
mutation.  These are two types.
  1. Deletion mutations
  2. Insertion mutations
Deletion mutations:  The reading frame of mRNA does not have any punctuation. So if nucleotides deleted it changes the amino acid sequence of protein expressed by it.

Normal sequence :
  DNA   AAA  GCT  ACC  TAT  CGG  TTA
 mRNA  UUU  CGA  UGG  AUA  GCC  AAU
 Protein  Phe  Arg  Trp  IIe  Ala  Asn

Addition mutation :
           DNA  AAA  GCT  ACC  ATA  TCG  GTT
        mRNA  UUU  CGA  TGG  TAT  AGC  CAA
        Protein  Phe   Arg  Trp  Tyr  Ser  Gin

Deletion mutation :
  DNA   AAA  GCT  CCT  ATC  GGT
mRNA  UUU  CGA  GGA  UAG
Protein  Phe  Arg  Gly  Stop

Deletion mutations are of variable length ranging in deletion of the number of nucleotides. Deletion of three successive nucleotides will not effect all the protein composition. It is with one amino acid less only, as the codon is triplet code.
    
Dyes like acridines can bring about deletion mutations. In heterozygous diploid eukaryotes, a deletion involving the dominant alleles amy result in the expression of the recessive phenotype.

Insertion mutations:  Inserting nucleotides into a normal gene results in a mRNA,
in which the reading frame is altered.  This type of mutations causing insertion
of nucleotides are called insertion mutations.
 
Chromosomal mutations:
 The mutations effecting the number, size, shape and gene
complements are chromosomal mutations.  These are of different types like a chromosomal segment may be lost by deletion, or it may undergo inversion or it may be translocated to a different site or may be duplicated to tandem repeats.

MUTAGENS :
Mutations inducing agents are called mutagens.  They create mutations in different ways.  Depending on the nature of mutagens they are of two types
  • Physical mutagens
  • Chemical mutagens
Physical mutagens:  Mutations can be naturally or artificially induced by a variety of physical mutagens.  H.J.Muller, founder of genetics, demonstrated in 1927 that mutations can be artificially induced by treating flies with x-rays. Similarly L.J.Stadler in 1928 demonstrated and increase in the rate of mutations due to x-rays in barely and maize.  Besides x-rays gamma rays can also induce mutations.
The physical agents are broadly divided into two types :
  1. Ionizing radiation
  2. Non-Ionionizing radiation
Ionizing radiation: X-rays and gamma (Y) rays are ionizing radiations. They have short wavelength and high penetration power.  They can penetrate into deeper tissues causing ionization of the molecules along their way. When X-rays penetrate into cells, electrons are ejected from the atoms of molecules encountered by the radiation.  As a result the stable molecules and atoms change into free radicals and reactive irons.  The radicals and ions can initiate a variety of chemical reactions, which can affect the genetic material, resulting in point mutations. i.e., affecting only one base pair in a given location.  The rate of mutation increases with the increasing dose of X-rays administered.

Nonionizing radiation:  Ultra Violet (UV) rays are nonionizing radiations. They have long wavelength and low penetration power.  The purines and pyrimidines absorb UV radiation most intensely at about 260 nm.This property has been useful in the detection and analysis of nucleic acids.In 1934 it was discovered that UV radiation is mutagenic. The major effect of UV radiation is formation of pyrimidine dimmers, particularly between two thymines. Cytosine-cytosine and cytosine-thymine dimmers are less prevalent.
The dimers damage the DNA structure and effects normal replication.

Chemical mutagens:  Charlotte Auerbach, author of Science of Genetics‖ was the first to find that mutatins can also be induced due to certain chemicals. Chemical mutagens can remove, replace or modify DNA bases.

Alkylating agents:  Alkylatin of nitrogenous bases by the alkylating agent either removes the base or modifies it.  Guanine residues can be alkylated by the methyl methane sulfonate and ethyl methane sulfonate. These agents alkylates guanine at N7 and weakens the purine-deoxyribose linkage.  This leads to deppurination creating ga at that site.  N-methyl-N1-nitro  –N  –nitrosoguanidine CH3-N(NOC(NH)-NH-NO2  is a powerful mutagen in E.coli.  Some alkylating agents change the GC positin ina nucleotide to AT.

Intercalating agents:  Intercalating agents produces frame shift mutation in bacteriophages like T4.  For example acridines are mutagenic to bacteriophphages but not to bacteria.  As the acridines are unable to enter bacterial cell.

Base analogs:  Base analogs are structurally similar to normal nitrogenous bases and can be incorporated into the growing polynucleotide chain during replication.  These analogs will have base pairing properties direrent from the bases they replace. One of  the first base analog formed to induce mutations in phage T2  is 5 bromouracil (BU) an analog of thymine.  In the normal keto form BU base pairs with adenine.But its tautomeric enol form pairs with guanine like cytosine.

References
Food Biotechnology Course material,by K V Anand Raj