Devise a scheme of cutting out YGOI insert from the donor plasmid and placing it into...

Devise a scheme of cutting out YGOI insert from the donor plasmid and placing it into the recipient plasmid.
How would you separate the transformed cells (those that have picked up the recombinant plasmid) from non-transformed cells?

Expert Solution

YGOI stands for your gene of interest.

This technique is applicable when a gene of interest is used to multiply to recover desired product.

For this process a gene of interest from a specific donor, a cloning vector that is used to to multiply into a host cell after its introduction and a specific host are required.

Let's gets stepwise process

1. The gene of interest is separated from a particular donor or plasmid mentioned here. For the separation of the gene the enzyme restriction endonuclease (RE) is used. This enzyme cuts DNA at a specific site. This enzyme RE belongs to a class of compound called nuclease which is used to cut or separate the genetic material. It may be of endonuclease or exonuclease.

RE belongs to endonucleases as it cuts DNA on interior sites on a restricted location.

2. This gene of interest is used to ligate with the cloning vector. For this purpose the enzyme used is ligase.

Remember, the genetic material of cloning vector is also cut with the same RE.

But, here it is already mentioned plasmid as recipient, so it is also called here as cloning vector if it is used to clone or making multiple copy of GOI.

So, since now the plasmid has an extra chromosomal material after ligation of foreign DNA, it is called as transformed plasmid or cells.

3. After ligation of the gene to the plasmid it is introduced into a specific host for gene multiplication purposes or product manufacturing.

But, here the question arises that how to separate or identify the transformed cells(recombinants) from non-recombinants, a variety of process is applicable for this purposes. Common methods used for this purpose are insertional selection inactivation method and the blue white selection method.

In the blue white selection method a selectable marker is developed which diffentiates recombinants from non-recombinants on the basis of colour production in the presence of chromo genic substrate.

In this method the recombinant DNA is inserted within the coding sequence of enzyme beta galactosidase. This results into insertional inactivation of the enzyme. The presence of chromogenic substrate gives blue coloured colonies if the plasmid in the bacteria does not have an insert in it. Presence of insert results into insertional inactivation of the beta galactosidase and the colony donot produce any colour, these are identified as recombinant colonies.

gladiator answered 11 months ago

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