 In our previous videos we learned about gene cloning, we saw how a gene of interest gets inside of a vector and through transformation gets inside of a host cell, where all this takes place inside a test tube and by the end of the process we expect that in the test tube we will get cells of this three possible genetic makeup. So we will have cells that has the recombinant DNA, we will also have cells that has the empty plasmid and we will also have cells that has not taken up anything at all. Now we do not want these two types of cells to grow any further because we are interested in the gene that we care about and that is present in this first type of cell that has the recombinant DNA. Now the question is how do we select this type of cells from millions and billions of cells that are present in the test tube? Well there comes the saviour that is selectable, selectable marker, well this selectable marker are genes in the plasmid and in this video we will see how they help us to isolate the type of cells we want. Now before we even jump into the process of selecting the type of cells we want we need to let the bacterial cells grow in a petri dish. We will have to pour a little bit of content from the test tube into a petri dish having nutrient medium and what will happen after that? If you leave it overnight you will see that the next morning your petri dish is full of bacterial colonies. Now these bacterial colonies are formed by division of bacterial cells and millions of bacterial cells pile one above the other to give the appearance of this circular patches you see here. Now for the ease of understanding let's say we just have five colonies. Now the interesting thing about bacterial colonies is that every single cell in a colony is an identical copy of each other, it is an exact replica of one another. As a bacterial cells do not undergo sexual reproduction there is no variation and therefore every single cell in a colony will be the same. Let's say if we extract one cell from this colony and it has only an empty plasmid inside of it that means every single cell of that colony will have only the empty plasmid. Well having said that let us assume let's just consider that the recombinant DNA is present in cells of this particular colony. Say let me number them let's say this is colony one, two, three, four and five. So the first and second colony has cells with recombinant DNA so let me number these cells as well and let's say the third and fourth colony has cells which is just the empty plasmid. So third and fourth colony has this type of cells and the fifth colony has cells that has nothing inside of it. Let's say this is the type of cell present in the fifth colony. Well now our goal should be to extract the first and the second colony but how would we know that in a lab? How would we differentiate? Well what about we find out a way to stop the growth of the other three colonies the third, fourth and fifth one or maybe how about find a way to just kill them? Well we know about a chemical substance that kills or stops the growth of bacterial cells and that is antibiotics. How about we go ahead and put some antibiotics into the medium? Well wait won't that kill the first and the second colonies as well because those are bacterial colonies too and we do not want those colonies to die because it has the recombinant DNA. So what should we do next? How about while designing the plasmid we put in a gene that would give resistance to antibiotic. To be specific let's say the antibiotic we are using here is ampicillin a fancy name given to an antibiotic. Well how about while designing the vector we put in an ampicillin resistance gene inside of it. That way when when we pour in ampicillin in the medium any cell that has the vector inside of it will be able to grow but cells that has not taken up the vector will not be able to grow in the medium so that way we can get rid of this type of cell the cell that has not taken up the vector which means that we can get rid of this fifth colony but wait the problem is not solved yet we still have colonies with two different type of cells and if you can see both these cells has the vector inside of it and as we have inserted the ampicillin resistance gene they have the ampicillin resistance gene inside those vectors so put this vector has the ampicillin resistance gene inside of it and now the question is how do we differentiate between the two because both the cells have the vector inside of it and making any further change in the vector would create the change in both the cells this is when scientists came up with an idea of inserting another antibiotic resistance gene into the vector say the antibiotic resistance gene here is for tetracycline cycline this is another fensiname for another antibiotic and this is this gene here is resistant to the tetracycline antibiotic but now you may think how is that even helping us both our cells now are resistant to another antibiotic which is tetracycline but how is that even helping us differentiate between the two well here comes the master stroke of the scientist what they did they inserted the multiple cloning sites inside this tetracycline resistance gene now if you can recall from our previous video we discussed about multiple cloning sites those were sites in which we put in the gene of interest now when the multiple cloning side is situated inside of another gene here it is the tetracycline resistance gene it inactivates the gene that was previously there well to tell you in simple words that is similar to inserting a new word inside another word for example if we take the word tetracycline and we divide it into two and we insert a new word say insulin here now the word that was previously there loses its meaning I mean Tet and racycline individually makes no sense right similarly when we insert a new gene inside of another gene the gene that was previously there becomes inactive and that inactivation was caused by the insertion of new gene right so we call it insertion inactivation and this insertion in activation will take place in any cell that has the insert inside of it which means the tetracycline resistance are here stands for resistance so the resistance power of tetracycline will be lost by any plasmid that has the gene of interest inserted inside of it so the recombinant cell that has the insert inside of it will not have tetracycline resistance now can you think what might happen if we allow both these cells to grow in the presence of tetracycline can you pause for a while and think about it now in the presence of tetracycline any cell that do not have the resistance power would die now as we have inactivated the resistance power in the recombinant DNA the cells having the recombinant gene would die which means the first and the second colony will die or we may say they would stop growing but we do even want them to stop growing because at these where the two colonies we ultimately focused on extracting of this patriarch right these colonies had the recombinant DNA but now what we have done is stop the growth of this recombinant cells well you need not freak out because we take care of it in the lab let me show you how it is done so what happens is we keep this plate untouched we keep it as a master plate let me write it down this is a master plate just like we make photocopies of important documents we create replica plates now you need not worry about how do we create them because that's not very important what's important is in both this petri dishes along with nutrient medium we now have antibiotics in here in the first petri dish we have ampicillin and in the second petri dish we have tetra cycle now how about you pause for a while and think about the fate of these colonies in both the petri dish all right let's start with the fifth colony the colony that has not taken up the vector since it has got no vector it has no gene for ampicillin resistance and no gene for tetra cycle in resistance therefore it won't grow in both the petri dishes so the fifth colony won't grow in both the petri dishes all right what about the third and the fourth colony this colonies have cells with empty plasmid that means it has an ampicillin resistance gene intact that means it would grow in the presence of ampicillin and it has an intake tetra cycle in resistance gene which means it will be able to grow in the presence of tetra cycle all right what about the first and the second colony the cells that has the recombinant DNA inside of it what will it do in the presence of ampicillin since it has an ampicillin resistance gene intact inside of it it will be able to grow in the presence of ampicillin but then due to insertional inactivation we have inactivated the tetra cyclin resistance gene right therefore it won't grow in the presence of tetra cyclin all right while working in a lab we will not have the information of which cells are present in which of these colonies let me get rid of these numbers first okay so the only information we will have is what vectors we are using what genes are present in our vector and where is the multiple cloning side where the insertional inactivation would occur well just with those information we need to tell which of these colonies has the recombinant DNA just by looking at these two petro dishes all right let's try to do that when we look at the first petro dish we'll see that the first second third and fourth colony are growing fine so that means it has resistance to ampicillin and that is possible only if they have taken in the vector but the fifth colony is not growing in the presence of ampicillin that means they have cells that did not ingest the vector all right now let's look at the second petro dish in here the fifth colony is not growing that means it has not taken in the vector no resistance against tetra cyclin as well now the third and the fourth colony seems to be doing just fine even here it has a tetra cyclin resistance gene intact but the difference is in the first and the second colony it's growing in the presence of ampicillin but not growing in the presence of tetra cyclin mm-hmm interesting right so what must have happened the insertional inactivation must have happened that has inactivated the tetra cyclin resistance gene this also means that it has the gene of interest inside of it so these are the two colonies that we would want to extract now that we know the first and the second colony has the cells with recombinant DNA inside of it we can go ahead and extract those colonies from the master plate and carry on the experiment with it all right now if you look back and see what actually helped you to select the first and second colonies it was actually the antibiotic resistance gene in the vector the ampicillin resistance gene and the tetra cyclin resistance gene so we call this genes as selectable markers all right this was all about antibiotic acting as selectable markers and now after watching this video you should now be able to tell what is a selectable marker how antibiotic resistance gene act as selectable markers and what is insertional inactivation now if you're not able to answer any of these you can always go back and watch the video