Saturday, April 28, 2007

TAXOL PRODUCTION AND PLANT BIOTECHNOLOGY


Sh. Dehshahri , G. Asghari

Pharmaceutical Research Center, Faculty of Pharmacy, Isfahan University of Medical Sciences, Isfahan, I.R. Iran

In recent years have seen great achievements in the production of taxoids using cell cultures.
for example paclitaxel production yield improvement: from about 1mg/l to more than 100mg/l.

in 1994, Phyton and ESCA genetic conducted taxus suspension cultures in 75,000-l and 2500-l bioreactors respectively. In 2001, the commercial production of paclitaxel using plant cell suspension cultures was achieved by Samyang Genex.

This successful industrial application of a plant cell culture will trigger future research on the production of plant-derived beneficial compounds.


What is taxol?
paclitaxel (TaxolTM ) is a diterpenoid alkaloid originally isolated from the bark of pacific yew, Taxus brevifolia .
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Sunday, April 22, 2007

Factors affecting volatile terpene and non-terpene biotransformation products in plant cell cultures

W. Zhu, G. Asghari, G.B. LockwoodU
School of Pharmacy & Pharmaceutical Scienes, Uni¨ersity of Manchester, Manchester M13 9PL,
UK
Received 7 January 2000; accepted in revised form 16 February 2000

Introduction
The production and accumulation of volatile essential oil constituents in plant cell cultures has been reviewed 1,2 . Although plant cells cultured in vitro are considered to be totipotent, i.e. contain all necessary genetic material to carry out any or all of the functions shown by the intact plant, in practice many either fail to produce essential oil constituents or produce a few in only very low levels. It is often accepted that as undifferentiated cultures contain no structures such as trichomes or vittae for storage of these constituents 3 none will accumulate. In many systems particular enzyme systems needed for a biosynthetic step have been shown to be present , but inoperative, and this inhibits production of the end product(s). A number of workers have attempted to improve production and accumulation of these compounds by feeding precursors 1,2 , but levels are still well below those of intact plants. We decided to use a range of techniques to investigate if levels of biotransformation products could be increased. Biotransformation of geraniol acetate to geraniol by plant cell cultures has not previously been reported, although there is one report of the reverse reaction occurring, in a study of the biogenesis of monoterpenes using cultures derived from Muscat grapes 4.
However, a few reports have described the biotransformation of other terpene acetates into their parent alcohols by plant cell cultures 5,6, but in both instances the parent alcohol was only the major product, not the sole product. In an attempt to show variation in levels of biotransformation of volatile terpenoids and non-terpenoids, we chose suspension cultures of Peganum harmala L._Zygophyllaceae. for the feeding experiments.

Keywords: Peganum harmala; Biotransformation; Essential oil , Suspension cultures


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Tuesday, April 10, 2007

Generating Genetically Modified Plants

HAMID M. SADEGHI
Pharmaceutical Research Center, Faculty of Pharmacy, Isfahan University of Medical Sciences, Isfahan, I.R. Iran

Transformation

Two mostly used transformation pathways for making transgenic plants are:
•Bacterial carriers
Biolistics

Bacterial Carriers
Agrobacterium tumefaciens contains a plasmid, a small circular piece of DNA that has its own origin of replication and is replicated independently of other nuclear material, that is key to its use in genetically modifying plants. This plasmid, called a tumor-inducing (Ti) plasmid, interacts with compounds released by fractured plant cells. When a wounded plant is exposed to A. tumefaciens, it integrates a stretch of its DNA, called transferred DNA (T-DNA), to the plant's genome.Normally, the bacterium transfers its own T-DNA, but if the T-DNA is removed and replaced with another gene, A. tumefaciens can be used to introduce that gene into the plant genome, thereby providing a vector for scientists to engineer beneficial genes into plants. Studies have shown that native T-DNA genes are not necessary for this process. Inserted genes get transferred to plants as long as two repeated border sequences of 25 base pairs flanking the genes are present in the vector.


Biolistics
The gene gun is part of the gene transfer method called the biolistic (also known as biobalistic or particle bombardment) method. In this method, DNA or RNA adhere to biological inert particles (such as gold or tungsten). By this method, DNA-particle complex is put on the top location of target tissue in a vacuum condition and accelerated by powerful shot to the tissue, then DNA will be effectively introduce into the target cells. Uncoated metal particles could also be shot through a solution containing DNA surrounding the cell thus picking up the genetic material and proceeding into the living cells. The efficiency of the gene gun transfer could be depended on the following factors: cell type, cell growth condition, culture medium, gene gun ammunition type, gene gun settings and the experimental experiences, etc.


Agrobacterium versus biolistics (particle bombardment)
In general Agrobacterium is considered the method of choice for transformation. Advantages include:
•Low copy number of the transgene
•Higher proportion of stable transformants
•Large DNA segments can be transferred
•More time efficient


Other techniques that can be used:

Calcium phosphate precipitation
Electroporation
Gene silencing
Gene splicing
Lipofection
Microinjection
Viral carriers


Protein Based Testing Methods

•Transgenes encode for novel proteins
•Immunoassay techniques using antibodies
•Analyte must be known
•Interference from non-specific interactions of proteins, surfactants (saponins), phenolics, fatty acids and phosphatases
•Polyclonal antibodies
•Monoclonal antibodies

Key words: Western Blot ,ELISA Microwell Plate , Antibody-coated Tube ,ELISA Considerations , DNA-Based Methods , Southern Blot , PCR Fragment Confirmation

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Cloning and Mutagenesis

HAMID M. SADEGHI

Pharmaceutical Research Center, Faculty of Pharmacy, Isfahan University of Medical Sciences, Isfahan, I.R. Iran

Mutagenesis

Some Necessary Tools of Genetic Engineeirng
Restriction enzymes
Plasmids


Restriction endonucleases cleave only double stranded DNA by recognition of specific sequences within the DNA molecule.

•Protruding Ends (also referred to as "sticky" ends or "staggered" ends).
A. Protruding 5' (PO4) ends: occurs when the enzyme cuts to the left of the central axis of symmetry
5'......G*AATTC......3'
3'......CTTAA*G......5'
5'......GOH PO4AATTC......3'
3'......CTTAAPO4 OHG......5'


Ligase

The reaction requires the expenditure of two high energy phosphate bonds. Notice that the phosphate on the 5' end of the nick is used to make the phosphodiester bond. ATP provides only energy, not a phosphate.

Plasmids


Plasmids are molecules of DNA that are found in bacteria separate from the bacterial chromosome. They:
•are small (a few thousand base pairs)
•usually carry only one or a few genes
•are circular
•have a single origin of replication
Plasmids are replicated by the same machinery that replicates the bacterial chromosome. Some plasmids are copied at about the same rate as the chromosome, so a single cell is apt to have only a single copy of the plasmid. Other plasmids are copied at a high rate and a single cell may have 50 or more of them.

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INTRODUCTION TO GENETIC ENGINEERING


HAMID M. SADEGHI
Pharmaceutical Research Center, Faculty of Pharmacy, Isfahan University of Medical Sciences, Isfahan, I.R. Iran



The most commonly engineered crops are canola, chicory, corn, cotton, squash, tomatoes, and soy. Currently in the US, 35% percent of all corn is genetically engineered as well as 55% of all soy and cotton. Although cotton is most commonly thought of as a material, cottonseed oil is used in foods. Soy is more widely consumed than many would think, as 60% of processed foods contain soy. Consumer Reports of September 1999 shows that products such as Boca Burgers, Jiffy Corn Mix, Ovaltine Malt, Bravo Tortilla Chips, various infant formulas, and Bac-Os Bacon Flavor Bits contain genetically engineered ingredients
Nutritional effects

No tests have been conducted on the effects of genetically engineered food on the human body. At this time, we could not do longitudinal studies on people who are consuming foods containing genetically engineered ingredients because foods are not labeled. Thus, it would be impossible to develop a control group.
One significant problem with genetically engineered foods is the risk to people with specific food allergies. For example, Brazil nut genes were inserted into soybeans to increase their protein content. The product was ready to go to market when tests showed that the soybeans caused allergic reactions in people with allergies to Brazil nuts. In addition, new allergens may be introduced into the food system through genetic engineering.
Why are crops genetically engineered?

Currently, 85% of genetically engineered crops are engineered for herbicide or pest resistance. The other 15% are engineered to be viral resistant or have more "desirable traits" such as increased oil content or shelf life. These desired traits are not necessarily preferred by or beneficial to the consumer, but they fit within the framework of our industrialized, concentrated agriculture system, which entails large-scale production and long distance transport.


Key words: Genetic , Transgenic organism , Genetic Engineering
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