What are the specific scientific techniques and processes involved in the technology?
The recombinant DNA technology in bioremediation allows faster and effective decomposition of contaminants by the microbes. The genetically engineered microorganisms have greater degrading ability since they are inserted with recombinant plasmids facilitated for better metabolism of specific contaminants. When the certain gene that codes for degradation is detected, it is recombined with the engineered plasmids, which are then transferred into the appropriate bacteria via appropriate technologies such as electroporation and particle bombardment.[8]
Plasmids are small, circular, double-stranded DNA molecules that are present in the cytoplasm of bacteria. They are expressed and replicated in the host bacteria cell using the enzymes and ribosomes present in the microorganisms. In the case of bioremediation, plasmids enable the host bacterial cells to break down specific kinds of contaminants and chemicals. Also, the bioremediation process will become much more effective when there is a higher number of plasmids in a microorganism.
One of the basic steps of the recombinant technology in bioremediation includes formation of recombinant DNA. First, artificial plasmids are engineered to contain a single recognition site that can be cut by various restriction enzymes. When an artificial plasmid is cut, the chosen gene for effective degradation that has been excised using the same enzyme will anneal into the plasmid. This occurs naturally since the certain gene possesses the same complementary ends as the linearized plasmid, as they are cut by the same enzyme. DNA ligase is then added to reform the phosphodiester bonds between the fragments through condensation reaction, driving out a molecule of water. This will result in a circular piece of DNA carrying the gene fragment. This plasmid is called the recombinant DNA, a combination of the original plasmid DNA and the foreign DNA segment. This will then be introduced into a bacterial cell, where it replicates to form many copies within the cell.[9]
However, prior to the formation of the recombinant DNA, the certain gene fragments that codes for degradation of specific chemical must be replicated in order to create the recombinant plasmids faster and more efficiently. This replication is done by the process called “polymerase chain reaction“ (PCR). In PCR, DNA is subjected to a temperature in the 94°C–96°C range, where the hydrogen bonds are broken between complementary bases, causing the strands to separate. These strands are then used as templates to build complementary strands. The temperature is then brought down to the 50°C–65°C range for the DNA primers anneal with the template strand. Once the primers have annealed, Taq polymerase starts building complementary strands with free nucleotides in the solution at a temperature of 72°C.[10]
Once the recombinant DNA is made, the contaminants-degrading bacteria can be manipulated to take up the recombinant DNA via electroporation and particle bombardment. Particle bombardment mostly involves a gene gun, which is a device designed to deliver recombinant DNA or plasmids into a bacterial cell. In particle bombardment, particles like heavy metals are used to rupture the cell membrane, allowing the recombinant DNA to enter through the breaks in the cell membrane.
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Moreover, electroporation is another mechanical way to introduce the DNA into a microorganism through the cell membrane. Both the microorganism and the recombinant DNA are suspended in the solution set up by the electroporation apparatus. When the apparatus is turned on, the voltage discharges through the liquid of the cell suspension. In this process, a great electric pulse disturbs the phospholipid bilayer, enabling DNA to pass into the cell. This causes the formation of temporary aqueous pores, and the electric potential across the cell membrane rises so that the charged DNA is driven across the membrane through the pores.
