Thousands of waste material sites surrounding the world contain non-degradable radioactive material. A whole lot of risk and expense of cleaning up such wastes by physicochemical methods and hence the other methods are being pursued for cleanup of those sites. One effective method is to engineer radiation-resistance microbes that degrade or change such wastes into less dangerous compounds. Deinococcus radiodurans, the most radiation-resistance organism ever known and undergo 1, 500 Kilorad/hr gamma or UV radiation and develop in radioactive environment. This feature is useful for remediation of radioactive waste material by bringing out the gene from Pseudomonas putida spp. into Deinococcus radiodurans genome (pressure R1- ATCC BAA-816) which is in charge of degradation. This recombinant stress will degrade the hazardous radioactive material and clean up the sea environment by switching into less hazardous compounds.
Nuclear weapon development in earlier 5 years has generated almost all of the wastes that was directly discharged into the water and now are contaminating a large number of sites. In america, about 1 / 3 of the reported misuse sites are radioactive, with radiation levels up to 10 mCi/L in or near to the contaminating resources (14). These highly dangerous wastes contain inorganic and organic contaminants including radionuclide such as 235Uranium, heavy metals such as mercury, and solvents such as toluene (14).
There is little potential client for cleanup of these misuse sites by physicochemical method together because of extreme charge, danger and strength of labor. The clean-up cost of these throw away sites by physicochemical method was approximated to be $90 billion 1988 and recently to be about $265 billion (15). Unless new costs effective clean up solutions are developed, theses throw away increasing will threaten to the sea life and humans.
Numerous microorganisms (particularly Pseudomonas sp. ) have been detailed that have the ability to degrade, transform and detoxify the organic and natural and inorganic pollutants (16-21). Most of the micro-organisms are hypersensitive to the damaging effect of radiation within the radioactive misuse and are not suited for remediation of radioactive waste. Therefore, radiation-resistant micro-organisms that can degrade radioactive waste material have to be found in nature or manufactured in the laboratory to solve this issue.
The most radiation amount of resistance organism is learned till now could be Deinococcus radiodurans (Figure-1) (22-23). It is pink is color because of carotenoid pigment within it. It contains 4-10 identical copies of an chromosome (2. 65 Mbp), two megaplasmid (412 and 177 kbp) and a plasmid (46 kbp) (4-6). This is a non-pathogenic, spherical formed, Gram Positive aerobic bacteria bacterium that can increase constantly in the occurrence of ґ 1, 500 Kilorad/hr Gamma or UV rays with no influence on either its expansion rate or its capacity to express foreign genes (27).
Deinococcus radiodurans bacterium gets the capability to expand in the radioactive environment. This property is very helpful for bioremediation of radioactive substances by including genes from the Pseudomonas putida sp. The ability of an microorganism to remediate the radioactive waste material is associated with the ability to enhance these materials into less harmful compounds.
Toluene is water-insoluble liquid of benzene derivative. It really is an aromatic hydrocarbon in case inhaled in large amount, it causes neurological harm which may lead to death also. When it is mixed in this particular, it is difficult to degrade toluene. A Deinococcus radiodurans bacterium does not have a gene to degrade toluene. So, when this bacterium is made by adding TOD gene from E. coli sp. , with the ability to degrade toluene (1).
Metals like Hg, in ionic are very poisonous. When Hg (II) is mixed with water it takes lot of energy to degrade in a natural way. So, when the gene accountable for degrading ionic Hg (II) that is Hg(II) amount of resistance gene (merA) from E. coli stress BL308 into Deinococcus radiodurans, it withstand the toxic effect of the steel and enhance those metals to less dangerous and less soluble chemical says(2).
IR resistant bacterias Deinococcus radiodurans have high intracellular awareness of Mn/Fe percentage. When higher level of radiation subjected to the bacterium, its key target of natural action is on protein not on DNA. Mn(II) ions are sent out all over the cell but Fe exists in a region where cell department occurs. Mn(II) ions protects health proteins from oxidative changes by presenting carbonyl group involved with it (9-10).
The most researched of Deinococcus sp. is D. radiodurans. Unlike other varieties of deinobacteria, it is most liable and comfortable to genetic manipulation due to its natural transformability by both high molecular weight chromosomal DNA and plasmid DNA (28-29). The natural capacity of transformation of the bacterium has enlightened the many different approaches for genetic alternation permitting it highly for molecular inspection (29-31).
The goal of this project is to engineer a stress of D. radiodurans that are capable of degrading radioactive waste material. Popular organism those are capable of degrading wastes are not able to endure in these sites because of their awareness to the radiations. Generally, most of the organisms are sensitive to the detrimental ramifications of ionizing radiation and almost all of the bacteria becoming studied aren't acceptable to remediate radioactive misuse. For example, Pseudomonas sp. is very very sensitive to radiation (more very sensitive than E. coli) and not suited to remediate radioactive misuse. Therefore, radiation resistant organisms are needed to be found in the type or built in the laboratory that can remediate radioactive wastes.
The main goals of this job are:
My working hypothesis is that a Deinococcus radiodurans bacterium expands in radioactive environment. This can be used for bioremediation of radioactive substances. It would be making possible by incorporating genes responsible for degradation of waste material from Pseudomonas putida spp.
Goal 1 - D. radiodurans will be cultivated on TYG broth medium and P. putida on Basal Sodium Medium. Chromosomal DNA is isolated and plasmid DNA is isolated from P. putida (36). Restriction digestion of plasmid DNA and Chromosomal DNA will be achieved to cut the specific region of DNA (37). Electrophoresis of restriction digested DNA will be the next step to check their DNA is minimize at the precise site. Plasmid DNA will be extracted by using electro-elution. Centrifuge the eluted DNA with dialysis buffer which will remove polluted agarose debris.
Goal 2 - A clone will be prepared by introducing the gene from P. putida to D. radiodurans by change. pl3 shuttle vector is utilized for change (32).
Horizontal gene transfer can be carried out in various ways like transduction, bacterial conjugation and gene copy real estate agents (35). But change is the dominant method to transfer a gene from one microorganism to another microorganism.
Goal 3 - Once the gene is transferred to D. radiodurans from P. putida, testing will be the next step. It will be done by two methods-Gel Move and Colony PCR. Gel shift (EMSA- Electrophoretic Ability to move Change Assay) method will determine the difference between the altered DNA and non-transformed DNA. Transformed DNA will run slowly and gradually on gel because of its high molecular weight than the non-transformed DNA. By evaluating with the marker DNA, it will be easy to visualize the variations. Colony PCR method determines the add size or orientation in the vector. If the insert exists, the size of the vector increase. This is determined by growing each colony in the liquid medium and plasmid then purified by swift boiling method digestion by limitation enzymes that excise the put, followed by parting on agarose gel electrophoresis.
Goal 4 - When a cloned D. radiodurans is ready, mass media search engine optimization will be crucial for faster growth of the cloned copies. Radioactive materials concentration will be evolved from lower to higher focus. Higher and lower glucose awareness will be altered for greater results. Some other elements will be added if required.
A long term goal of the project is to engineer the cloned copied of D. radiodurans genetically such that it will overexpress. However, to overexpress the cloned copies, first the complete gene series, promoters and regulators must be identified and understood. When the results are not satisfactorily good, use different microorganisms instead of P. putida. Actual field trial of this organism and if it's successful large level production of the organism is the last step.