 In our previous video, we discussed how we can use two antibiotic-resistant gene as selectable markers to select different bacterial colonies. But there is a process which is considered less time-consuming and easier than the process that we already know. And there, instead of using two antibiotic-resistant genes, we use an enzyme-producing gene. And that process is called blue-white screening. So in this video, let's discuss what a blue-white screening is, how an enzyme-producing gene can be used as a selectable marker, and in which way is this method easier and quicker than the one we already discussed. Let's begin by discussing the need to screen different bacterial colonies. What is the need to do that in the first place? Well, during gene cloning, by the end of transformation, we end up getting cells of different genetic makeup. We get cells that has taken in the plasmid along with the insert. We also get cells that has taken in just the empty plasmid without the insert. And we get cells that has not taken up anything at all. Now we want the cells that has the insert to grow and not the other two. So there comes the need to screen different bacterial cells or to be precise to screen different bacterial colonies. Alright, let's begin screening them. And we begin by getting rid of cells that do not have the plasmid inside of it. And we do it just the way we discussed in our previous video. We insert an antibiotic-resistant gene into the plasmid. And what happens then? All the cells that has the plasmid will have this antibiotic-resistant gene except for this poor fellow here who could not even ingest the plasmid. Now the antibiotic-resistant gene we have used is resistant to the antibiotic ampicillin. Now, when we allow the cells to grow in a petri dish that has ampicillin, this poor fellow won't even grow in that medium. So we will get rid of cells that do not have the plasmid inside of it. But the problem is not solved yet. We are still left with two different type of cells. One has the insert and one has the empty plasmid. And we want the cells to grow that has the insert, right? Now to differentiate between these two, this time we do not use another antibiotic-resistant gene but an enzyme-producing gene called LEG-Z. And this gene codes for an enzyme called beta-galactosidase. Well, a fancy name given to an enzyme, beta-galactosidase. And what this enzyme does? It digests something that's found in the milk you drink, lactose. So any cell that has the plasmid inside of it will have this LEG-Z gene. It will produce an enzyme and digest lactose. Now as you can see, both our cells has the plasmid inside. That means both will be able to digest lactose, right? But that's not the case. When allowed to digest lactose, the cell that has the empty plasmid does digest lactose but not the one that has an insert. So can you think of a possible reason why is the cell that has an insert cannot digest lactose? Alright, let's find the answer from what we learned in the previous video. We learned about insertional inactivation, which means inserting of the new gene, the gene that we care about inside another gene. And this time it is inside LEG-Z gene. And when we do that, we inactivate the gene that was previously there. So whenever this LEG-Z gene is inactivated by the insertion of an insert, it stops producing enzyme and therefore it cannot digest lactose. So any cell that has the insert inside of it won't be able to digest lactose. Alright, now you must be wondering how is digesting lactose or not digesting lactose helping us differentiate between these two cells? Because in a Petri dish when we will have so many colonies, how would we know which of these colonies are digesting and which of these colonies are not digesting lactose? Imagine how wonderful it would be if say digesting lactose would have produced some kind of color. So we could easily tell that the colored colonies are the ones that has an intake LEG-Z gene. That means it do not have the insert inside of it. So we could easily distinguish between two different type of cells. But that's not happening here. Lactose is not producing any color. So that is when scientists came up with the idea of using a chromogenic substrate instead of lactose and that chromogenic substrate is called X-Gal. X-Gal is artificially prepared in the lab which is a chromogenic substrate and on being broken down or digested it produces blue precipitates. And that way the cells or the colonies that can digest X-Gal appears blue in color. So we see blue colonies in our Petri dish. Well when I first learned about it I was really curious to know that an enzyme which is naturally produced to digest lactose how come it can now digest X-Gal? Well X-Gal is a modified form of galactose but how come an enzyme that can digest lactose is now digesting X-Gal? So what I found out was that X-Gal is made in such a way that its structure resembles the natural substrate of beta-galactosidase that is lactose. So X-Gal has a similar structure to that of lactose and that is why beta-galactosidase is able to digest or break down X-Gal. An X-Gal being a chromogenic substrate produces blue precipitates on being broken down. And what is a chromogenic substrate you ask? Well a chromogenic substrate is artificially designed in such a way that it resembles a natural substrate as we discussed already. Here it resembles X-Gal resembles lactose and on being broken down by an enzyme it produces color. So here X-Gal on being broken down by beta-galactosidase produces blue precipitates and that's how we are able to recognize colonies that has an intake-lexi gene that can produce an enzyme, digest X-Gal and appear to be blueish in color. And when millions of cells grow together in a colony all producing blue precipitates the blue color becomes very prominent it becomes visible to the naked eyes. Now what about the cells that has an insert? The cells that says, we don't have an active-lexi gene we don't produce an enzyme so we can't digest X-Gal. So what color do you think they appear in the patriotish? Well from the name blue-white screening you can guess that they might appear white in color. Well you're right but why do they appear white? Because most of the time we use equalize as host cells and most varieties of equalize are naturally white in color. So when millions of equalize grow together their colonies appear to be white in our patriotish. So finally we have it a patriotish of blue colonies and white colonies and now we know that the white colonies are the ones that has the insert inside of it. So we will allow the white colonies to grow further and we'll take out the blue colonies from the patriotish and stop their growth. And this method of distinguishing bacterial cells is called blue-white screening. Now the question is when we have already learned to distinguish between bacterial colonies in our previous video using two different antibiotic resistant gene as selectable markers why are we learning a different method to screen bacterial colonies? Well this is because the method of blue-white screening is considered easy and gives you quick results. So let's compare the previous method that we learned using two antibiotic resistant gene with blue-white screening. Well as you can see in the previous method we had too many patriotishes. We had a master plate and two replica plates. Now why do we create two replica plates? In the first plate we allow the cells to grow in the presence of the antibiotic ampicillin. So just like we got rid of the first cell here any cell that didn't have the plasmid inside would not grow in the presence of ampicillin. Now we will be left with two different type of cells one that has the insert and the other that has the empty plasmid but both of them will have a tetracycline resistance gene now and any cell that has the insert will have the insertional inactivation of this tetracycline resistance gene therefore any cell that has the insert won't have resistance to tetracycline so therefore they won't grow in the presence of tetracycline. So we saw that the cells without plasmid won't grow in the presence of ampicillin they won't grow even in the presence of tetracycline and the cells with an insert won't grow in the presence of tetracycline. So we have to monitor both the patriotishes to finally identify the colonies that has our insert. Well I'll recommend you to go and watch the previous video for better understanding but the gist is that in the previous video we were using a number of different patriotishes a number of different antibiotics and it was a multi-step process and was also very time consuming compared to blue-white screening where in a single patriotish we use a chromogenic substrate and an antibiotic and then we see distinguishing colors between different bacterial colonies it is almost like a single step process therefore scientists prefer blue-white screening over the other process because it is less time consuming and is considered comparatively easy. Alright now let's quickly summarize what we learned in this video we have learned that instead of an antibiotic resistant gene we can use an enzyme producing gene as selectable marker here we discussed about lexigene that produces beta-galactosidase enzyme and that digest the chromogenic substrate eggs-gel and on being digested it produces blue precipitates and that is how we find blue colonies in our patriotish and the cells that has an insert that do not digest eggs-gel appears to be white in the patriotish that is how we see blue and white colonies in our patriotish and we call it blue-white screening and this eggs-gel was something new that we learned it is a chromogenic substrate which while being broken down produces blue color and we also learned that blue-white screening is a preferred method of screening because it takes place in fewer steps and is comparatively easy so this is all about blue-white screening