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What are recombinant plasmids?
A plasmid is a circular piece of DNA found in some bacteria. Recombinant plasmids are man made with new DNA sequences in them. They can be used theraputically is some medical conditions.
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Answer The plasmid have a "reporter gene" inside it, generally resistance to specific antibiotic. the plasmid is transformed into bacteria that don't have …resistance to that specific antibiotic drug, and than the cultured on a petri-dish that contain the antibiotic drug. Only bacteria that had receive the plasmid will have resistance and grow, all the other will die.
Recombinant plasmids are engineered plasmid molecules that contain a DNA sequence inserted into it from a foreign source.
it's used to insert genes into a plant.
There are two main methods: using chemicals and using electricity. A combination of certain solutions, such as calcium chloride, and exposure to particular temperature…s, makes bacteria more competent (i.e. "receptive", if you like). Another way to allow plasmids to enter bacteria is to apply a pulse of electricity; this method is known as electroporation. It is more efficient and consistent, but more expensive. These methods are the result of trial-and-error (officially called empiricism), and just why they work nobody knows.
The gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics and a multiple cloning site (MCS, or polyli…nker), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this location. Next, the plasmids are inserted into bacteria by a process called transformation. Then, the bacteria are exposed to the particular antibiotics. Only bacteria that take up copies of the plasmid survive, since the plasmid makes them resistant. In particular, the protecting genes are expressed (used to make a protein) and the expressed protein breaks down the antibiotics.
Orginal Plasmids are extra chromosomal genetic material present in eukaryotes and some prokaryotes. Recombinant plasmids contain a gene of intrest ie,individual gene carrying… a specific function can be inserted in to a specific site on original plasmid in cell culture via transformation.So the recombinant plasmid contain both gene of intrest and native genes.
functions as a vector
When the original function of the gene in the plasmid is altered or another gene is inserted in the non- coding region of the plasmid is called the recombinant plasmid.
The plasmid must include a genetic marker that allows for selection of recombinant cells. This marker is usually antibiotic resistance, normally ampicillin in bacteria and Gen…eticin in mammalian cell lines. After treating the cells with the appropriate antibiotic, only the cells that have the vector will have survived. In short, they aren't identified, they're selected for.
Isolation of a plasmid from a bacterium
Researchers employ electricity to weaken the cell wall and membrane or simply use heat-shock to stimulate the competent cell to absorb foreign DNA since some bacteria natu…rally do so.
The recombinant DNA can be inserted into a bacterial cell by a method called transformation/calcium chloride treatment. It is the free uptake of DNA by the competent cell. In …this method the recombinant DNA is added to the broth culture containing bateria, to this CaCl2 is added and incubated for 12 hrs. The recombinant DNA gets precipitated on the host cell due to CaCl2. This is subjected to heat shock treatment ( 42 degree celsius ) for 2 minutes during which the pores are created on the host cell wall and the recombinant DNA gets integrated with the host cell DNA.
plasmids are very useful in recombinant DNA technology because ; 1. it has own origin of replication. 2.it has some selective markers like ; Amp^r, ter^r etc. 3. it has uniqu…e recognition sequence . 4.it has specific restriction sites . 5. it is small in size because in RDT foreign DNA is inserted , if the vector is large sized then after insertion it will creat problem . but due to the small size plasmids donot creat any problem.
One of the most common ways these days is from cDNA. RNA is extracted from human cells, purified, and then treated with an enzyme called reverse transcriptase which is able to… make DNA from RNA templates (this DNA made from RNA is called cDNA). The advantage of using cDNA is that in the genome human genes are typically distributed across multiple exons spread over tens or even hundreds of thousands of basepairs of DNA. Such a massive segment of DNA is extremely hard to manipulate and far too large to insert into a plasmid. However, in cDNA, all the introns have been spliced out (because cDNA is made from mature mRNA). To isolate a particular gene from cDNA, PCR is often used to selectively amplify one gene's cDNA using specific primers. To insert the amplified cDNA into a plasmid, the traditional approach was to use restriction enzymes - enzymes that cut precise DNA sequences. The great thing about many restriction enzymes is that they cut DNA but leave behind "sticky ends". Thus if you cut both your cDNA and a plasmid with a particular restriction enzyme, the resulting sticky ends will allow the human cDNA to be taken up by the plasmid (the sticky ends will mesh). The sticky ends will have to be sealed by an enzyme called DNA ligase. However, there are other ways these days - often involving recombination to insert the PCR product directly into a plasmid without resorting to restriction enzymes and ligations.