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hi friends hope you are fine in this video we will be discussing how antibiotic resistant gene works as a selectable marker in the selection of recombinant colonies within five minutes. Let's begin with the definition of a selectable marker? A selectable marker is a region or gene sequence here it is the antibiotic resistance gene of the vector that helps in the selection of recombinant colonies that contain our gene of interest. Let's take an example pBR 322 vector, here as you see there are two selectable marker regions this is the ampicillin resistance region and this is the tetracycline resistance region. These are actually gene sequences that will make proteins so that that provide antibiotic resistance to make it more clear suppose this is a bacterium, if this bacterium is having this vector with both the selectable markers intact that is ampicillin resistance region and tetracycline resistance region, then this bacterium can grow in both tetracycline and ampicillin containing medium. Hope this much is clear. Now let us see how this helps in selection so this is the pBR 322 vector so this is a ampicillin resistance region and this is the tetracycline resistance region and there are restriction sites within the selectable marker regions. Suppose we are using Bam H1 restriction enzyme, that is within the tetracycline gene sequence to insert our gene of interest, so we are making a cut using Bam h1 then we are inserting our gene of interest, once the gene of interest is inserted into this tetracycline gene sequence then this gene is no more functional. This is called as insertional inactivation, that means inactivation of a gene upon insertion of another gene within that gene. This gene of interest once inserted will disrupt the continuity of this tetracycline resistance gene sequence making it non-functional. This insertional inactivation actually helps in selection of recombinant colonies. Now let us see how after a transformation experiment, we will be getting three types of colonies, the first one is non-transformed, majority will be non-transformed without our vector and second transformed there are two types, one transformed with non-recombinant vector that means the vector remains intact without our gene of interest, the second one is the transformed with recombinant vector, here insertional inactivation has happened the gene of interest is there so we need to select these colonies transformed with recombinant vector. so we'll be transferring these into tetracycline containing medium and ampicillin containing medium. The method we use is replica plating using a velvet, we'll be taking the imprint of these colonies and transferring that into our required medium, so that the spatial organization or the position of the colonies will be the same as that of the master plate so what happens is non-transformed as it doesn't have this antibiotic resistance gene ampicillin resistance gene or tetracycline resistance gene it cannot grow on tetracycline containing medium or ampicillin containing medium, so it can be easily removed. Then these two, the first one transformed with non recombinant vector can grow in both tetracycline containing medium and ampicillin containing medium as it has genes for resistance, here our gene of interest is not inserted so both genes are functional, so transformed with non-recombinant vector can grow in both retrocyclin containing medium and ampicillin containing medium. Third one transformed with recombinant vector cannot grow on tetracycline medium as the gene of insert is inserted within the gene sequence of tetracycline gene making that gene non-functional whereas it can grow in ampicillin containing medium so by comparing the position with the master plate we can easily find out the colonies which are transformed with our gene of interest and that is the purpose of replica plating also here the colonies that can grow in ampicillin containing medium but cannot grow in tetracycline containing medium is a one with our gene of interest and this is how antibiotic resistance gene is used for the selection of transformed recombinant colonies.