Hi, in this video we'll talk about restriction enzyme and restriction sites. So restriction enzymes are considered as molecular seizures because using restriction enzyme you can cut DNA in specific location. So the restriction enzymes are mostly isolated from bacterias but have you ever wondered that why restriction enzymes are required for bacteria?
I mean like why does bacteria produce restriction enzymes? So this is a confusing question, right? And why does bacteria need to produce restriction enzyme? It turns out that the enemy of the bacteria, that means the bacteriophage, which can which kind of kill bacteria and survive on that, they invade bacterial bacteria and put their genome inside.
And the bacteriophage can be prevented from attacking E.coli if the bacteria is producing a restriction enzyme. So restriction enzymes work like a defense system for bacteria. So bacteria normally put their DNA inside. That DNA can integrate into the bacterial genome in a lysogenic cycle or bacteria might also undergo a lytic cycle. So these enzymes, restriction enzymes, ensures that the virus, the fudge virus, might not survive and this restriction enzyme kind of cleaves the DNA of the fudge virus and thereby protects the bacteria against the fudge virus infection.
Now that is why the restriction enzymes are a defense mechanism or immune mechanism for bacteria we can think of. Now these restriction enzymes don't cleave their own DNA of the bacteria. Why is that so? Because soon after the DNA is replicated in the bacteria, it is methylated and these restriction enzymes do not recognize DNA with a methylated at cytosine. So they don't cleave their own DNA, but they can cleave the foreign DNA that comes from the fudge virus.
And that is how restriction enzymes are useful for bacteria itself. But how that is important for biologists? And how do scientists even figure out this thing that bacteria has this kind of mechanism and we can use that mechanism in purpose of biology? So first of all, we should talk about the work of Brittany.
and Luria. By the way, the LB broth or LB agar that we use today to grow bacteria is named after them. So what they were doing is they were like you working on lambda phage and they seen that lambda phage can easily grow on E.coli plate and they form plaques in the plate and uh but so when they change this strain to a different E.coli strain known as E.coli strain K, they saw that there are few plaques produced.
I mean not compared to like E.coli strain C. They thought that there is something that is preventing the fudge to infecting the bacteria and thereby there are like less kind of bacterial viral plaque and that might be strain specific. So they wondered what are the difference between the strain K and strain C. Later they call them restriction factor because it restricts the virus to grow.
in presence of that strain. So there is some kind of factor, magical factor, that is preventing the viral growth. Later on, 10 years down the line, Arbor and Meselcent figured out that these protective factors or the restriction factors are nothing but enzymes which can chop off DNA and cleave the viral DNA.
So that was a big discovery in the field. problem is they thought that can we try to use that restriction enzyme and try to manipulate the DNA or manipulate the genome of an organism. The only problem that they faced is like the restriction enzyme that they found was not specific. Now we know the restriction enzyme they found was a type 3 restriction enzyme which cut in a random location. But eventually 10 years down the line people found out a type 2 restriction enzyme.
type 2 restriction enzyme has specific recognition site and they only cut in specific recognition site not anywhere else so they are very specific so thomas kelly and his co-workers found that specific restriction enzyme subtypes which are which they isolated from hemophilus influenza bacteria hind was the name of the first restriction enzyme and they saw it cut into a specific sequence and specific location and and there are like specific length of fragments were produced from this cut. And that was a game changer in this field. All the field knew that they can use this strategy to manipulate genome and cut a specific length of DNA.
And that is how the restriction enzyme came into the picture and molecular cloning start. Now, there are restriction enzymes which could produce sticky ends and which could produce blunt ends. It depends upon the choice of the restriction sites.
For example, this is an example of a sticky end which is protruding out in terms of cloning or... ligation this kind of sticky ends like get better than the blunt end now these are some examples of like popular restriction enzymes that are isolated from specific bacterial strain and there are a variety of bacteria which produce different different type of restriction enzymes now nowadays we use this restriction strategy and put these restriction sites into a cloning vector for example here it is plasmid and in its multiple cloning site we put the restriction recognition sites such that we can easily use these molecular scissors which is this restriction enzymes and cut that according to our own choice and we can put our dn of interest in that as well so here is the workflow so here is your uh plasmid which has the restriction recognition site in the green zone we can see and we can cut that with restriction enzyme and our gene of interest we can also cleave it with the same restriction enzyme. Later on we can ligate the two and thereby our gene of interest is now inside in the plasmid of interest and it is directed to the specific location where we want it because there are multiple different restriction enzyme of our own choice so we can use whichever combination we like to and after that we transfer that into the back bacteria and as the bacteria grows our gene of interest is also getting increased in number in in format of a plasmid and that is a very important strategy and that is how molecular cloning started so restriction enzyme was a breakthrough in the process of molecular cloning so i hope you enjoyed this video if you like this video give it a quick thumbs up but this is an introductory video of restriction enzyme we'll talk about more details about it mechanism of action and many other things it's star activity non-specific activities etc so if you like this video give it a quick thumbs up don't forget to like share and subscribe thank you