Gene transfer techniques- Plasmids and Balistic

1. Gene transfer techniques 
In the laboratory, specific enzymes may be used to cut and splice DNA:
Restriction enzymes break DNA at specific parts of the molecule (nucleotide base sequences) - usually leaving so called "sticky ends".
This can be done to both DNA from which genes are being taken, and to DNA in which genes are being inserted. Then, DNA ligase enzymes may be used to rejoin such sections into the other DNA.
The DNA containing the selected gene for the desired characteristic may then be inserted into cells of the target organism by means of vectors. There are 2 main types of vectors:plasmids and viruses (see previous notes on micro-organisms).

1.1 Gene transfer using plasmids 
Agrobacterium tumefaciens is a bacterium that contains a section of DNA called a plasmid in addition to its usual component of DNA. This tumour inducing plasmid has the ability to incorporate its DNA into the cells of the plant host, thus acting as a medium to allow the insertion of other genes into crop plants during genetic engineering.

1.2 Gene transfer using viruses
lambda (λ) phage - a bacteriophage which can modify bacteria, stays in the DNA of the host, and replicate the host’s DNA. It does not harm the host during the replication process, but it evolves with the host DNA. The microphage can remain inside the host indefinitely without having any harmful effect. These bacteriophages can be modified using restriction enzymes and foreign DNA. When the bacteriophages are opened, gene modification can be carried out by inserting the desired viral DNA to integrate with the host cells's "chromosome".

2. Others

2.1 Ballistic techniques
Minute tungsten particles are coated with the DNA to be inserted, then shot into the target cells with an explosive charge.

2.2 Electroporation
In this technique, a brief pulse of electric current is passed through the cell, temporarily increasing surface permeability so that DNA is taken up from the surrounding liquid. This has been especially useful with pollen tubes and has resulted in the genetic transformation of seeds.

author: Monica  

Health hazards

Health hazards of GM foods

There are various health hazards that can be contributed by GM food. There are a wide range of health hazards which are ranged from rashes to cancer. There are studies which prove that there is an increase in the risks for cancer, birth defects and other health effects which are linked to the use of super weed-killers. Glyphosate which is a broad spectrum herbicide which is commonly used in herbicide resistant crops are known to be possible hazards to health which is proven in an article from the year 1999.  It is proven via laboratory studies that adverse health reactions can be caused by glyphosate even at low doses. In an experiment done on animals, results show that glyphosate causes diarrhea, salivary gland lesions, liver damage and cataracts. There is also an increase in thyroid pancreas and liver tumors after the animals consumed products with doses on glyphosates in it.

Even though glyphosate in our food poses danger to human health, there is still a legal limit recommendation of dosage of 20mg per kilo of glyphosate residue in GM product which is given by the WHO (world health organisation).

 Bt which is known as bacillus thuringenesis is a natural insecticide which is used to spray on crops to control crop eating insects. Bt have poissibilities to cause intesitinal problems which can also lead to severe damage. This fact is not negligible as rats have been used during experiments to test for the health effects of Bt residues from the plantations. However the there are high possibilities that anyone who consumes an unacceptable dosage of bt will most likely suffer from diarrhea.

~liwen helped to post

Widening of social gap

Widening of social gap from the rich and poor

Qualities of GM seeds have been modified and their detriments may only occur throughout the years. Farmers are prone to pick the most efficient seeds which causes the planting of the same crops in all areas. By doing so, resistance to diseases will be reduced when crops were attacked. The same type of crops will be affected by the same disease and this may eliminate out all food supply. Other than that, the genetically modified seeds may widen the gap between the rich and the poor farmers. The poorer farmers will not be able to afford the more expensive genetically modified seeds thus causing them to stick to their old methods. The richer farmers therefore will use the new seeds and increase their yield and wealth thus increasing the disparity gap between the rich and the poor, genetically modified seeds will lead to an increase in the unemployment rate. Many will be jobless, further increasing the disparity gap. (Ngeography, 2007)

~liwen helped to post

Religious concerns and beliefs, ethical concerns

Religious concerns and beliefs

cows given more potent GM growth hormones could suffer from health problems related to growth or metabolism and lastly, new GM organisms could be patented so that 'life' itself could become commercial property through patenting. Usually, ministers, vegetarians and others will be found on either both sides of the debate regarding foods that were modified contain genes from animals or species that are prescribed by various religions. (Parker, 2003)

Ethical concerns

Crops are modified to provide immunization against common types of diseases, and crops are able to withstand particular growing conditions such as drought and soil acidity. Surpassing the benefits of GM crops could be counterproductive from an environmental associated with conventional agriculture.  GM crops can limit the need of chemical inputs such as pesticides, herbicide and fertilizers and can prevents further soil erosion and loss of moisture in vulnerable areas by reducing the need for ploughing (parker, 2003)

~liwen helped to post

consumer rights- outcrossing

defination:
Outcrossing

Outcrossing typically occurs accidentally, but poses a real health risk to consumers of genetically-modified crops. Outcrossing occurs when genetically-modified seed used to grow genetically-modified crops is mixed up with a conventional crop with the similar type of gene is transferred to the conventional crop. This becomes a problem since some crops are not safe for human consumption and are grown strictly for such things as animal feed. (Corp, 2011)

~liwen has helped in posting

The risk of Gene modification (at a glance)

Genetic engineering is capable of introducing dangerous allergens and toxins into foods (Mohan, 2005). As a gene is a building block for a protein, the introduction of a foreign gene that is not originally produced by the organism, will has the possibility of evoking an allergy that is not found in the conventional food crop (Wesseler, 2005).

This is because the cellular metabolisms are altered and may result in the production of dangerous by-products such as toxins and allergens. Furthermore, the newly-introduced protein may be enzymes that may interupt with the growing process of the food-producing organism, where the vitamins and mineral production may be short-changed (Mohan, 2005).

go to here to see the beenfits of gene modification

author: monica

Polymerase chain reaction (PCR)

The polymerase chain reaction (PCR) is a scientific technique in molecular biology to magnify and increase a single or a few copies of a piece of DNA across various extent, generating thousands to millions of copies of a particular DNA sequence (White, 1993). One of the major concept in PCR is thermal cycling, a process where the sample is heated and cooled alternately into a defined series of temperature steps.

During thermal cycling, two strands of the DNA double helix gets separated under high temperature. After heating, the sample is then cooled, where DNA polymerase uses each strand as a template to select the target DNA (Narayanasamy, 2010). The DNA polymerase used are also heat stable. An example will be Taq polymerase, an enzyme originally isolated from the bacterium Thermus aquaticus (Maier, 2009).

This DNA polymerase, through enzymatic action, assembles a new DNA strand from nucleotides (Mullis, 1994). This process is done by using single-stranded DNA as a template and DNA oligonucleotides, which are required for initiation of DNA synthesis (White, 1993). PCR is used in functional analysis of genes, the diagnosis of hereditary diseases and infectious diseases. PCR’s ability in targeting selected DNA also made this process widely used in genetic manipulation (Maier, 2009).

author: monica

Thermogravimetric Analysis (TGA)/Differential Thermal Analysis (DTA)

Thermogravimetric analysis (TGA) is a thermal analysis technique which measures the weight change in a material as a function of temperature and time, in a controlled environment. This can be very useful to investigate the thermal stability of a material, or to investigate its behavior in different atmospheres (e.g. inert or oxidizing). It is suitable for use with all types of solid materials, including organic or inorganic materials.
Differential thermal analysis (DTA) is a calorimetric technique, recording the temperature and heat flow associated with thermal transitions in a material. This enables phase transitions to be determined (e.g. melting point, glass transition temperature, crystallization etc.).
Ideal Uses for TGA/DTA Relevant Industries for TGA/DTA
• Thermal stability/degradation investigation of organic or inorganic materials, e.g. polymers, composites, glasses, metals, minerals etc.
• Thermal stability/degradation investigations in inert or oxidative atmospheres, or in vacuum
• Determination of organic/inorganic content of mixtures
• Curing kinetics (e.g, adhesives, polymers)
• Chemical composition measurements (using appropriate reference standards, accurate quantification of sample composition can be determined
• Phase transition measurement (e.g. glass transition, clustering, crystallinity, melting point)
• Quantum - size effect investigation for nanomaterials
• Reaction kinetics with reactive gases (e.g., oxidation, hydrogenation, chlorination, adsorption/desorption)
• Pyrolysis kinetics (e.g., carbonization, sintering) • Semiconductor
• Energy
• Polymers/biomass
• Pharmaceutical
• Biomedical
• Metallurgy
• Ceramics
• Chemicals
• Construction materials
• Optical
• Solar
• Batteries
 
Strengths of TGA/DTA Limitations of TGA/DTA
• Any type of solid can be analyzed, with minimal sample preparation (e.g. powders, pellets, chunks, flakes etc)
• Minimum sample size ( at least 0.1mg)
• Qualitative or quantitative analysis • Solid (or initially solid) samples only
• Data interpretation not always straightforward. Analysis in combination with other techniques is often helpful.

Evans Analytical Group LLC , . (2011). Thermogravimetric analysis (tga)/differential thermal analysis (dta). Retrieved from http://www.eaglabs.com/techniques/analytical_techniques/tga_dta.php


Thermogravimetric Analysis
 
Thermogravimetry thermal analysis (TGA) testing.
Thermogravimetric (TGA) analysis is used for determination of endotherms, exotherms, weight loss on heating or cooling, and more. Materials analyzed by TGA include polymers, plastics, composites, laminates, adhesives, food, coatings, pharmaceuticals, organic materials, rubber, petroleum, chemicals, explosives and biological samples.
TGA materials analysis:
Thermogravimetric analysis uses heat to force reactions and physical changes in materials. TGA provides quantitative measurement of mass change in materials associated with transition and thermal degradation. TGA records change in mass from dehydration, decomposition, and oxidation of a sample with time and temperature. Characteristic thermogravimetric curves are given for specific materials and chemical compounds due to unique sequence from physicochemical reactions occuring over specific temperature ranges and heating rates. These unique characteristics are related to the molecular structure of the sample. When used in combination with FTIR, TGA/FTIR is capable of detailed FTIR analysis of evolved gases produced from the TGA.

TGA thermogravimetric capabilities:
• Compositional analysis of materials
• Decomposition temperatures
• Rate of degradation
• Product lifetimes
• Oxidative stability
• Evaluation of polymer flammabilities
• Thermal stabilities
• Determination of rancidity of edible oils
• Fingerprinting unknown polymers
• Moisture Content
• Volatiles content, VOC analysis
• Analysis of evolved gases using TGA/FTIR
• Competitive product evaluation
• Measurement of oil extender content in elastomers
• Effects of reactive atmospheres on materials
• Determination of inert filler or ash contents
• ASTM D6375 Noack Method
Thermal analysis:
• Thermal Analysis and Testing
• Thermo-Mechanical Analysis
• Thermal Properties Analysis
Intertek Group plc, . (n.d.). Thermogravimetric analysis. Retrieved from http://www.intertek.com/analysis/thermogravimetric/

What is DSC?
 
Differential Scanning Calorimetry (DSC) is unsurpassed for understanding the stability of biological systems. DSC directly measures heat changes that occur in biomolecules during controlled increase or decrease in temperature, making it possible to study materials in their native state
DSC measures the enthalpy (∆H) of unfolding due to heat denaturation. A biomolecule in solution is in equilibrium between the native (folded) conformation and its denatured (unfolded) state. The higher the thermal transition midpoint (Tm), when 50% of the biomolecules are unfolded, the more stable the molecule. DSC is also used to determine the change in heat capacity (ΔCp) of denaturation
Applications include:
• Liquid biopharmaceutical formulations.
• Process development
• Assessment of biocomparability during manufacturing
• Protein stability and folding.
• Assessment of the effects of structural change on a molecule’s stability.
• Characterization of membranes, lipids, nucleic acids and micellar systems
• Antibody domain studies.
• Rank order binding.
MicroCal Inc, . (2008). What is dsc?. Retrieved from http://www.microcal.com/technology/dsc.asp


Another definition
An instrument which measures the rate of heat evolution or absorption of a specimen which is undergoing a programmed temperature change. A recorder prints out the data as a plot of increase in heat per increase in temperature, versus temperature. The instrument has been utilized to study the curing characteristics and related properties of thermosetting resins.

CRC Press LLC, . (1989). Differential scanning calorimeter . Retrieved from http://composite.about.com/library/glossary/d/bldef-d1641.htm

original author: Nat 
help to post: monica

Some Analytical chemistry techniques for used for food testing

Chromatography 
Chromatography is a chemical separation technique that is commonly applied in chemical analysis. This analytical process/method can be applied to in many various experiments. It is known for its efficiency and versatility. Chromatography can divide substances into is most basic components in a single step and at the same time give a rough estimation of the quantity of each part. This technique is applied on samples of liquid, solid or gaseous nature.

HPLC (High Performance Liquid Chromatography) is a column chromatography method used to separate and identify compounds in a given solvent sample. The solvent is forced through high pressure in a column chromatograph. This allows a very small particle size, which gives a greater surface area for phase interactions.
GLC (Gas-liquid Chromatography) is usually used in organic chemistry works, in separating gaseous and volatile substances. It tests the purity of a substance.

TLC (Thin Layer Chromatography) on the other hand, is a type of liquid chromatography. It separates different structured chemical compounds according to the rate at which these compounds move. In turn, it identifies the different compounds present.
Resolution is used to measure the purity of the substances. A resolution of a chromatography system measures the separation between two peaks on a graph. The better the resolution, the better the purity of the separated components. In relation to the GM rice, chromatography will be able to break down the system to allow easy identification of the different building components that make up the product. From there, it can be determined if the GM food product is safe for consumption with no particularly harmful effects on the human body.

Atomic Absorption spectroscopy (AA spectroscopy)
During production and processing of food, trace elements and their counterparts are most likely incorporated into the system. This is a crucial problem as even traces of metals, for example, can result in a toxic food product. AA spectroscopy is a method used to detect the presence of trace elements such as metal, in a given sample. This process involves the absorption of light by the metals. The sample is first vaporized, and the vapor that results contains free atoms of the particular element. Light is then shined and the atoms present get agitated and absorb the light. As such, the intensity is lowered and hence will compute to a higher or lower absorbance value respective to the experiment conducted. As concentration goes up, so does the absorbance. By applying AA spectroscopy to our product, trace elements of metals can be detected, should they be present. In turn, we will then similarly be able to determine the safety of the product to our consumers.

LD50
In the measurement of toxicity in chemicals, LD50 is used as a form or measurement. LD50 stands for Lethal Dose-50%. This refers to the dose that would essentially kill 50% of the population tested. It is expressed in milligrams of material per kilogram of subject’s body weight.
Generally, the higher the LD50 value, the less toxic the substance is to the body, Vice versa.
For example, with reference to testing on a population of rats, the LD50 value of cane sugar is 29700 mg/kg. This means that it takes 29700mg/kg of cane sugar to kill 50% of the population on rats tested. However, it only takes 192mg/kg of caffeine to have the same effect.
Hence, caffeine has a higher toxicity value in comparison to cane sugar.
Similarly, the LD50 value of our GM rice will determine its toxicity.

Dose-response curve 
The dose-response curve correlates to exposure of a substance and its induced effects.
With this dose-response curve, we can not only determine lowest amount of material that causes the effect, we can also find out the actual effect the respective chemical has on the subject and also determine the rate at which the effect takes place.
Below is an illustration of LD50 of 20mg/kg and how it comes about in relationship with the dose-response curve.

 (picture will be posted later). 
Dose-response curve Ld50 value of 20mg/kg.
Image from: "Dose-response relationship." In: Encyclopedia of Earth. Eds. Cutler J. Cleveland

author of research: Sutha
help to post: monica

genetic modification (in more details)

Firstly, the donor organism,’s DNA is cut by a restriction enzyme. The cut-up section is called “sticky-ends”, which enables the cut sections to meet up with other DNA strands (Mohan, 2005). Proceeding on, plasmids that induce tumour is treated with the same restriction enzyme, opening out the circle of DNA leaving 2 sticky ends. These plasmids consists of Agrobacterium tumefaciens ‘s DNA (Roller, 1998).

Then, the sticky ends are then mixed with plasmid DNA. Next, DNA ligase enzymes are necessary to rejoin the plasmids. However, these ligase enzymes can only be activated with specific conditions such as temperature. The rejoined plasmids are then reintroduced into the bacterium and the bacterium is cultured under standard microbial methods (Mohan, 2005).

The bacterium is then introduced into the plant, where the plant will grow a gall in reaction to the bacterium. A certain number of cells from the gall might contain the required insecticidal gene (Roller, 1998). These sections of the gall may be encouraged to grow by special plant tissue culture techniques, bulked up in the lab before conditions in the medium are changed to encourage growth of roots and shoots (Mohan, 2005). The resulting small plants are then transferred into the field, along with other crops.

author: monica

various lab methods used for food testing

Differential scanning calorimetry (DSC)
Differential scanning calorimetry is a thermo-analytical technique in which the difference in the amount of heat required to increase the temperature of a sample and reference is measured as a function of temperature (Flammersheim, 2003). During an analysis, heat flows in (endothermic process) or flows out (exothermic process) of the sample as it undergo physical transformation (Dean, 1995). The differential scanning calorimeters are able to measure the difference in heat flow between the sample and reference and the amount of heat absorbed or released during such transitions (Sheehan, 2009). In the study of food science, DSC is used in the correlation measurement of water dynamics with food texture (Dean, 1995).

Loop-mediated isothermal amplification (LAMP)
Loop mediated isothermal amplification (LAMP) is a single tube technique for the amplification of DNA. LAMP is used in nucleic acid amplification which uses single temperature incubation. During the amplification process, two or three sets of primers and a polymerase with high strand displacement activity are directed at the target sequence (Narayanasamy, 2010). The sequence is amplified at a constant temperature of 60 - 65 °C, and at the same time, replication activity is also carried out. At the end of the process, the release of Magnesium pyrophosphate results in turbidity, thus allowing visible results (Tian, 2008). Due to the specific nature of the action of these primers, the amount of DNA produced in LAMP is considerably higher than PCR based amplification (Tian, 2008).

author: monica.

Examples of labelling requirements

GMO type	Hypothetical examples	Labelling required?
GM plant	Chicory	Yes
GM seed	Maize seeds	Yes
GM food	Maize, soybean, tomato	Yes
Food produced from GMOs	Maize flour, highly refined soya oil, glucose syrup from maize starch	Yes
Food from animals fed GM animal feed	Meat, milk, eggs	No
Food produced with help from a GM enzyme	Cheese, bakery products produced with the help of amylase	No
Food additive/flavouring produced from GMOs	Highly filtered lecithin extracted from GM soybeans used in chocolate	Yes
Feed additive produced from a GMO	Vitamin B2 (Riboflavin)	No
GMM used as a food ingredient	Yeast extract	Yes
Alcoholic beverages which contain a GM ingredient	Wine with GM grapes	Yes
Products containing GM enzymes where the enzyme is acting as an additive or performing a technical function		Yes
GM feed	Maize	Yes
Feed produced from a GMO	Corn gluten feed, soybean meal	Yes
Food containing GM ingredients that are sold in catering establishments		Yes (the FSA's legal view is that labelling is required across EU Member States under EC Regulation 1829/2003).  http://www.food.gov.uk/gmfoods/gm/gm_labelling ~liwen~

GM wheat

GM wheat — fast facts:

• GM wheat is at least seven years away from commercialisation.
• Approved field trials of some GM wheat varieties are now underway to assess the plants’ agronomic performance and characteristics.
• GM wheat will undergo stringent scientific assessment to ensure its safety for human health and the environment as part of the approval process by specialist regulatory authorities.
• The development of GM wheat varieties is a global collaborative effort involving scientists from both public and private sectors using proven technology.
• A recent survey in the USA showed strong support for GM wheat with almost three quarters of respondents indicating they would purchase products made with GM wheat, if the wheat was produced to use less water, land and/or pesticides.

http://www.biofortified.org/2010/12/long-dialogue-between-stakeholders-about-genetically-modified-wheat-starts-in-australia/

~nat~

The two analytical methods used for testing GM food

 

info taken from Asian food Information centre (org)  

How are GM foods labeled?

Previously, i have posted an article on the straits times for the Consumer Association of Penang, Malaysia regarding the labeling of GMOs; especially the agricultural ones. This is part of the government efforts to protect consumers health by having mandatory laws in place. 

 

However, there are still many questions and issues raised if labeling of GM foods becomes mandatory. 

First, are consumers willing to absorb the cost of such an initiative?  
If the food production industry is required to label GM foods, factories will need to construct two separate processing streams and monitor the production lines accordingly. Farmers must be able to keep GM crops and non-GM crops from mixing during planting, harvesting and shipping. It is almost assured that industry will pass along these additional costs to consumers in the form of higher prices. 

Secondly, what are the acceptable limits of GM contamination in non-GM products? 
The EC has determined that 1% is an acceptable limit of cross-contamination, yet many consumer interest groups argue that only 0% is acceptable. Some companies such as Gerber baby foods and Frito-Lay have pledged to avoid use of GM foods in any of their products. 

But who is going to monitor these companies for compliance and what is the penalty if they fail? Once again, the FDA does not have the resources to carry out testing to ensure compliance. 

What is the level of detectability of GM food cross-contamination? 
Scientists agree that current technology is unable to detect minute quantities of contamination, so ensuring 0% contamination using existing methodologies is not guaranteed. Yet researchers disagree on what level of contamination really is detectable, especially in highly processed food products such as vegetable oils or breakfast cereals where the vegetables used to make these products have been pooled from many different sources. A 1% threshold may already be below current levels of detectability. 
 

Finally, who is to be responsible for educating the public about GM food labels and how costly will that education be? 
Food labels must be designed to clearly convey accurate information about the product in simple language that everyone can understand. This may be the greatest challenge faced be a new food labeling policy: how to educate and inform the public without damaging the public trust and causing alarm or fear of GM food products. 

In January 2000, an international trade agreement for labeling GM foods was established. More than 130 countries, including the US, the world's largest producer of GM foods, signed the agreement. The policy states that exporters must be required to label all GM foods and that importing countries have the right to judge for themselves the potential risks and reject GM foods, if they so choose. This new agreement may spur the U.S. government to resolve the domestic food labeling dilemma more rapidly. 

References:

Peterson, G., S. Cunningham, L. Deutsch, J. Erickson, A. Quinlan, E. Raez-Luna, R. Tinch, M. Troell, P. Woodbury, and S. Zen. The risks and benefits of genetically modified crops: a multidisciplinary perspective. Conservation Ecology. (2000)

Retrieved from: http://www.consecol.org/vol4/iss1/art13/

haha, some more interesting pictures i have found about labeling of GM food.


I hope after reading you have a better understanding about GM food and its labeling issues. Also, the concerns raised.

However, Labeling VS Food safety issues is still present now..




~Shirley~