 Imagine, you have discovered a gene out of a mouse genome and this gene codes for a protein, protein that can cure cancer. Great, right? Now the next thing you would want to do is to create more and more of this protein and for large scale production of anything we need factories, right? And the factories we will use here are host cells. Mostly bacterial host cells are used inside which our gene gets expressed and we extract the proteins out of it. But for the factory to get started, we'll have to introduce the gene into it, right? And that is done by a vector. These vectors take in our gene of interest and gets inside of a host cell through a process called transformation. Now, bacteriophage and plasmids are the two things that are commonly used as vectors and the most widely used plasmid vector is PBR322. PBR322 is one of the first and most widely used plasmid vectors. Now in this video, we will try to understand the significance of each part of this vector and also how do we incorporate the gene of interest inside of this vector. Alright, before we go directly into the parts of PBR322, let me answer a question that keeps coming about vectors. Like why do we need a vector? Can't we directly put our gene of interest into a host cell? Well the answer is no. We can't because in DNA replication, we have seen that a gene alone is not self-sufficient to replicate and express itself. It needs other parts and the vector provides just that. It not only carries the gene that we care about but it also provides other parts which is necessary for this gene to get expressed inside any cell. So we just cannot do without a vector. Alright, having said that, let's come to our vector PBR322. Here P comes from the word plasmid and BR comes from the word bolivar and rodrix. These are the two brilliant minds, the scientists that created this vector and 322 is the number given by the lab to this particular vector. Well there is no data that tells us why this number came up but we can assume that maybe it was sample number 322 that ultimately turned out to be the perfect vector. Alright, so PBR322 is a vector that was designed from bacterial plasmids. These plasmids naturally occur within bacterial cells. They are circular double-stranded and codes for few genes. These plasmids are then extracted genetically modified and made into perfect vectors. Now as PBR322 is made of plasmids, it also looks like one, circular and double-stranded. Now the first thing you'd want your plasmid to do as soon as it enters a host cell is to replicate, right? Now there should be a point in this closed plasmid from where the replication would start. Let's say this is the point from where replication starts and we call this point as the ORI, O-reside which is origin of replication initiation. The name pretty much speaks for itself. This is the place from where initiation of replication takes place. So this place opens up like a bubble like this and in that bubble two complementary strands in the opposite direction starts forming. Now this bubble opens up even more as this strands proceeds further until we get two separate plasmids. So as you can see we got two different plasmids from one because of replication and this would not have even started if we didn't have this origin of replication. So to be a vector the first and foremost thing, the very necessary thing is that it should have an ORI. Alright, now that we know from where the replication would start let's talk about how many times do we want our vector or the plasmid to replicate inside of a host cell. Now a very obvious answer comes to our mind that the more the number of replication the better it will be because our gene will be expressed more, will extract more proteins out of it. Well there is another side to it. Imagine the protein that we want to extract which is curing human cancer may not be a healthy protein for our host cell. In fact it can be a toxic to our host cell. So in such a situation we would prefer that our plasmid make fewer copies inside of this host cell. Obviously because we don't want our host cells to die right? Now the thing is who controls the copy number of the plasmids? Well there is a gene for it. In case of PBR322 there is a protein just near the ORI which is called ROP. Let me write it down. ROP which is repressor of the promoter gene. We have discussed about ROPs in DNA replication. Now to tell you in simple words this ROP senses the number of replication this plasmids have undergone and when it feels like it should stop it codes for a protein that goes and binds to the ORI site and stops its further replication. That way it can keep a check on the copy number. So while we insert a gene we should be mindful about what impact it will have on a host cell and accordingly we will select our vector which will have either high copy number or low copy number. Now to speak about PBR322 this is a vector which has low copy number. It gives around 10-20 copies per host cell. Alright now that we have a origin of replication a gene to keep a check on the number of replication this seems to be a good time we put our gene of interest into this vector but as you can see this is a closed circular loop and we have to cut open this plasmid somewhere to insert our gene right? So just like in the real world we use caesars we use molecular caesars in gene cloning and the molecular caesar here is called restriction enzyme. We have a whole different video on restriction enzymes and if you have gone through that you will know that unlike normal caesars restriction enzymes cannot randomly cut anywhere it wants. There are specific sequences that each restriction enzyme recognises and can make a cut only at that particular sequence. Say the restriction enzyme we will use is ECO ECO R1 and the specific sequence it can recognise say is G A A T T C let's put 5 prime as this is a DNA we will have to put the 5 prime and 3 prime ends say this ECO R1 restriction enzyme recognises only this particular sequence now it will go in search of the sequence all around the plasmid and wherever it finds this particular sequence it will make a cut there say that particular site is this one now let me clear the board a little bit alright this site we call it as restriction site restriction site now just imagine a condition of having the restriction site for ECO R1 at multiple places say this is one this is another one this is the third one this is the fourth one this here is the fifth one and we have six restriction sites for ECO R1 can you pause for a while and think of what can happen if we have so many sites that can be recognised by a single restriction enzyme well it will break down our plasmid into multiple fragments and that will be useless to us therefore it is preferred that one restriction enzyme should have or a maximum of two cloning sites and not more than that but different restriction enzymes will have their own recognition sites in a vector and we call them multiple cloning sites you may not worry about that now we will make a complete different video on it so after we have made the cleave the next thing we will do is insert our gene of interest into it our gene goes and binds to the sticky ends of the vector and the DNA ligase ligates it now when we allow this vector to go and get inside of host cells and we culture the host cells there are possibilities that we may get cells that do not have anything inside of it or that may contain just the plasmid part without the gene of interest or if we are lucky enough we may get the entire recombinant DNA inside of it now to differentiate between those type of cells scientists have incorporated another gene into the vector which we call selectable markers these are mostly antibiotic resistance gene and when we allow all type of cells to grow in the presence of a particular antibiotic say this is an ampicillin ampicillin resistance gene alright and we allow all different type of cells to grow in the presence of ampicillin only those cells will survive that has the vector and this resistant gene inside of it that way we are able to select the cells we want and the other thing about selectable marker is that vectors have not just one but two selectable markers when it comes to PBR322 it has tetracycline resistance gene as its second selectable marker and if you can see we have the gene of interest somewhere in between the tetracycline resistance gene let's not complicate things with all this right now we will have a future video that talks about selectable markers and why we have gene in between them and also don't get carried away by the fancy names of these antibiotic resistance genes all we need to know is that selectable markers helps us in distinguishing our cells from the one that do not have the recombinant gene inside of it alright now let's quickly summarize the different parts we learned in the vector the first is the ORI from where the replication would start the second is a gene called ROP that takes care of the number of replications the third thing we learn is about restriction sites and that one restriction enzyme should not have more than two restriction sites or cloning sites and in those cloning sites we will insert our gene of interest and the fourth and the last important point is that it should have selectable markers that will help us distinguish the cells we want alright now that we have everything we need inside this vector this vector is ready to be cloned inside of a host cell you can culture those cells, extract proteins out of it and help the world fight cancer