ଓଡ଼ିଆ ରେ ଏହି ଲେଖା କୁ ପଢ଼ିବା ପାଁଇ ଏଠାରେ କ୍ଲିକ୍ କରନ୍ତୁ ।
PLASTIC:
Plastic contains organic polymer. The vast majority of these polymers are formed from chains of carbon or with addition of oxygen, nitrogen and Sulphur.
POLYETHYLENE TEREPHTHALATE(PET):
It is the most common thermoplastic polymer resin of the polyester family. PET in its natural state is colorless, semi crystalline resin. It is very light weight so we use it in the manufacturing of bottles, containers etc.
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| Plastic Pollution |
PLASTIC POLLUTION:
In particular, polyesters containing a high ratio of aromatic compounds, such as PET, are chemically inert, resulting in resistance to microbial degradation. These products are remarkably resistant in the environment because of low activity or absence of catabolic enzymes that can break down their plastic constituents.
Majority of the world’s PET production is for synthetic fibers (in excess of 60%) with bottle production accounting for about 30% global demand. Each year 56 million tons of PET are produced. Worldwide 480 billion plastic drinking bottles were made in 2016 (and less than half were recycled).
ENVIRONMENTAL ISSUES:
Large quantities of PET have been introduced into the environment through its production and disposal, resulting in the accumulation of PET in ecosystems across the globe. PET is commonly used in commercially sold water bottles and other bottles. Its harmful effects can prove to be carcinogenic or promote endocrine disruption.
PATH OF DISCOVERY:
Ideonella sakaiensis bacteria was accidentally discovered by scientists in 2016. It is a bacterium that can use the plastic as it’s primary carbon and energy source, found in PET.
It is the world’s first PET – eating bacterium that is gram negative, aerobic, non-spore forming and rod shaped. Contain PET hydrolase (PETase) and mono (2- hydroxyethyl) terephthalic acid hydrolase (MHETase) that can degrade PET by hydrolyzing it. Forms translucent circular colonies after incubation at 30°C in NITE BIOLOGICAL RESEARCH CENTRE no. 802 culture medium. The growth will be retarded above 45 degree Celsius and concentration of NaCl exceeds 3%.
First we have to destroy the cell wall of bacteria by lysozyme. We have to identify the DNA in the genome of the bacteria. Which has the gene coded for 2 enzymes i.e., PETase and MHETase. We have to cut the DNA segment by restriction of endonuclease. If we insert the DNA fragment directly into the host it might get degraded. We need a vector to place the target gene to the host.
The vector is self-replicating. The restriction endonuclease is the same for both vector and target DNA to generate compatible ends to bind. The restriction endonuclease cuts down target DNA and vectors. When the vector is bound to the target DNA it is known as recombinant DNA and after that we will place it in the host.
When the host is bacteria and division occurs then the recombinant DNA is also divided. The vector is plasmid (extra chromosomal DNA i.e., self-replicating). It has a specific origin where we can insert the target DNA and the site is known as multiple cloning sites. Since multiple restriction enzymes have multiple recognition sites. It increases chances of insertion of target DNA in multiple sites.
Selectable marker is a gene sequence, i.e., artificial, introduced inside a vector which will help to develop some kind of phenotypic expression that we can artificially measure.
After recombinant DNA enters into the host and makes different copies of gene the big question is where does the recombinant DNA exist in a host. To answer this in simple words we would say that, there is a genetic library in which the copies of DNA exist.
In a Petri dish containing many different colonies of bacteria it also contains the whole genome of species known as genomic library. But we don’t require the whole genome, we only need the gene which is coded for the enzyme.
Genomic library is important because we have to put all the recombinant DNA into one colony. It takes more time firstly to collect all the required genes to the one colony but after the collection it would be easier for us to identify the required gene for further research.
If for some other reason the gene which is coded for 2 enzymes is infected so the genomic library is not required because the segment of DNA is infected. So, after the transcription the mRNA is produced and after the reverse transcription of mRNA the cDNA is produced.
We need a fragment of cDNA to insert in the vector then recombinant cDNA is placed in the host. Vector is self-replicating so the cDNA is also simultaneously replicated. After the recombinant DNA is transferred in the host cell via the cloning vector, the job of expression of the vector is started. Expression of the desired gene occurs with the help of the expression vector.
Recombinant DNA – mRNA – protein occurs
So, the criteria that must be fulfilled for protein expression are:
- A strong promoter (inducible)- can be a promoter which can be induced by a certain kind of chemical like IPTG; so as to increase the expression of certain proteins.
- Translation and initiation sequence (ribosomal binding site and start codon and a transcription termination sequence).
- The elements for expression that are appropriate for the chosen host (shine dalgarno sequence for prokaryotes and kozak consensus sequence for eukaryotes).
- Purification tag i.e., takeout the required protein. Fusion protein issues (glutathione s-transferase or maltose binding protein for better solubility).
We can take out a gene from the host if the host has a certain gene which is destroying or affecting our gene of interest and we can insert another gene for better performance and create a suitable environment for our gene of interest.
KNOCKOUT- gene completely excluded
KNOCKDOWN- omit expression of desired gene by adding the RNA interference.
Also, if it is possible, we can change the sequence of amino acid and the protein i.e., at the desired site directed by mutagenesis. In this process with change in a single amino acid, we can modify the protein.
We need:
DNA molecule that contains the gene that encodes for the protein. To know the nucleotide sequence on the gene around the site to be altered. We have created a short DNA primer that is complementary to the site to be altered. Even though one of the bases are mismatched, the DNA primer will still appear on the DNA template because all other bases are paired correctly.
