Gene Manipulation / Transformation

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Hi.
If we want cell to accept vector dna in transformation, we treated with calcium chloride or chilled on ice etc..
But i have a trouble with one issue about that. Books say that ; We must choose the vector that specific according to the cell that will do transformation. But we will treat with calcium chloride and ice, why we choose specific vector?, this foreign vector can be enforced the cell for accepting. I know cell might accept the vector as a homolog sequence and might do cross with this vector, might take homolog genes into own dna, but it doesnt have to be. Vector might stay in the cytoplasm and i think it dont require the homolog sequences with cell's own DNA
Transformation should occur spontaneously due to bacteria nature, already
 
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  • #2
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Vector might stay in the cytoplasm and i think it dont require the homolog sequences with cell's own DNA
A forcing vector, in order to be part of the already existing DNA double sequence, need to be the homolog, because the DNA sequences are complementary, so also the vector have to be.

Transformation should occur spontaneously due to bacteria nature, already
Why a cell should accept a vector spontaneously?
It can't, you must cut the DNA sequence that correspond to that specific vector, and this one will replace the part you've cut away.
Indeed you need a specify enzyme.
 
  • #3
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A forcing vector, in order to be part of the already existing DNA double sequence, need to be the homolog, because the DNA sequences are complementary, so also the vector have to be.


Why a cell should accept a vector spontaneously?
It can't, you must cut the DNA sequence that correspond to that specific vector, and this one will replace the part you've cut away.
Indeed you need a specify enzyme.
But, gene of interested dont have to goes in nuclear dna i think, it might stay also in cytoplasm instead of making crossover with nuclear dna. Just stay like a plasmid dna with no homolog sequence with nuclear Dna
But, Transformation event can occur without forcing in nature. So why we need extra effort for transferring vector to specific bacterium?
 
  • #4
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But, gene of interested dont have to goes in nuclear dna i think, it might stay also in cytoplasm instead of making crossover with nuclear dna. Just stay like a plasmid dna with no homolog sequence with nuclear Dna
But, Transformation event can occur without forcing in nature. So why we need extra effort for transferring vector to specific bacterium?
In order to answer your question you should take in consideration the fact that not all the bacteria do the transformation spontaneously, some of them does, others, don't.
So, how you can make a bacterium that don't usually do the transformation spontaneously, accept the vector?
You can do that only treating them with chemical substances like calcium chloride.
This will make the bacterium to accept the vector.

Obviously this second option is artificial and possible only in the labs.
There are food and clothes industries that create this artificial condition in order the make transformation to happen.
 
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  • #5
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Plasmids contain an element known as the origin of replication (ORI) that tells the bacterium to replicate the plasmid. Some of these origins work only in specific hosts, so you need to choose a plasmid containing an ORI compatible with your host. If you transform a bacterium with a plasmid lacking an ORI or with an ORI not recognized by the host, the plasmid will enter the bacterium just fine, but it will not replicate itself and will be lost from the bacteria after a few generations. Plasmids with similar ORI sequences will also compete with each other, so you also need to take ORI compatibility into account when transforming bacteria with multiple plasmids.

For more information on the topic see: http://blog.addgene.org/plasmid-101-origin-of-replication
 
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