Okay so in this video we're gonna be talking about bacterial fage genetics. So bacteriophages, if you have ever heard this term before, you may know what they mean. But essentially they're viruses that infect bacteria bacteria page. And so there are often times used to study bacterial genetics and also bacterial fage genetics. Um and so I want to talk a little bit about how you you know how you actually work with these in lab. So one of the main essays that you used to work with viruses in the laboratory is called a plaque essay. And so what how you do this is you infect a bacterial culture with a bacterial page. Then you plate that bacterial culture onto a Petri dish and grow the bacteria. So now we're growing colonies of bacteria then you can count the number of plaques. And so what is a plaque? It's sort of this like dot on the plate that's formed through the license. So the breaking open of the infected bacteria because the bacteria were infected. So that virus gets in it makes a bunch of copies of itself and then it slices it so that that virus can get out. So when it lice is it it forms this hole on the plate and that place where that hole is called a plaque. So what this looks like here, you can see that there is a bacterial plate here and all throughout here anywhere where it's sort of that like kind of white gray yellow color that's covering the most part that's bacteria. And all of these are plaques. This is where a page has actually infected the bacteria um colony essentially at this point and life this bacteria so it forms these plaques on the plate. And so a viral plaque can be used to study lots of different things including calculating how much virus you have or how infected the viruses or different types of mutations. And so they're super these are super important essays. Now there are three types of pages. There's the profile page and this is a virus that has integrated its genetic material into the bacterial genome. So this is a virus that has its own genetic material right? And when it in fact sometimes that genetic material can actually be integrated into the bacterial genome. So now you have this hybrid of virus and bacterial chromosome um in the bacterial chromosome. And this portion of that that's the viral D. N. A. Is called a profile page And it can lie there for a really long time and then later become active. Um So these are super important. Then you have the virulent pages and these are ones that immediately lice and kill kill the host cell. So the virus gets inside the cell it reproduces so quickly it sort of starts swelling the cell with how fast it's been reproducing how many virus offspring it's made. And eventually that's gonna you know pop the cell it's gonna lice the cell and release that virus into the environment. Then you have the temperate pages and these are actually viruses that remain inside the host for a period of time. It can be a few minutes, it can be a few years, it could be a few decades. It just sort of sits there and it's just like I'm just gonna chill here for a while, I'm not in any hurry, I'm just gonna enjoy the environment before I start having kids. And so um it does that for a period of time and then something triggers it and it's like okay I'm ready to produce a bunch of offspring now. And so it does and so it will eventually lice and kill the cell. But for a very long time it can actually just stay inside the cell. And that's called a temporary page. So those are the three types that you do need to know those vocab words, definitely. So with that let's turn the page.
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Okay. So now let's talk about bacteriophages and mapping. So bacteriophages can actually be used to map the bacterial fage um jeans or genome. So how you do this is the exact same way that you do this with humans essentially or other types of plants or anything use recombination frequencies. So how you do this is you take a bacterial culture right? They infect bacteria. So you take bacteria and you perform a mixed infection which means that you have two strains of bacterial pages which have different phenotype is essentially and um you mix them together and you infect this one bacterial culture. So in this case we're going to say that we have this virus here which has the H. Plus and the R. Plus genes and virus to which has H. Minus and R minus. So if it has H plus it's gonna create purple colonies. And if it has are it's gonna create small colonies and anything else is different. So now we have now we can look for in a typically on the bacterial for the parental and the recombined. So the parental is gonna be purple small and the other parental is gonna be not purple large. And so we can look at the parental versus the recombined which would be purple large or non purple small. And I have an example of this if you're a little confused but essentially we're looking for these genotype. So we're looking for mixtures of positive and minus minus and positive and the recombination frequency here instead of looking at the plants or the seeds or the animals or whatever the offspring itself you're looking at the effect on the bacteria. So this is gonna be the number of recombined colonies versus the number of total colonies. So here we have an example. So we have these two viruses here. H plus R. Plus and H minus R minus. Now the H plus R. Plus is going to give to parental phenotype. Right? So when the H. Plus R plus infects the bacteria it's gonna be purple small. Or I guess in this case it's gonna be black large depending on the parental that did. But if there's two recombinant phenotype is here. Right? It could be purple large or um black small. And the only way that these are gonna form is there some kind of recombination. And so jeans that are closer together right are going to be more um not not genes that are closer together. You can use the recombination frequencies to map the bacterial genes how close they are together. Remember the closer that they are the smaller the recombination frequency is gonna be. So here's the bacteria. We can count the number of recombination. It's so we have recommended here we have a combination here let me actually count them two and we have 3456 6/7 89 10 11 12 12. This is going to be 50%. So these genes are very far apart. Right? Because the recombination frequencies 50%. If they if the recombination frequency was 5% we would say these genes are very close together right there, five map units apart. And so um yeah so that's how you use bacterial phages to map bacteriophage genes. Now bacteriophages actually have this interesting form of recombination called intra genic recombination. And this is recombination occurring inside Of a gene. This doesn't really happen in humans or plants right. The entire gene can recombine but it's not like a portion of a gene. And and you know, you know it's a code on 72 through code on 130 is given that never really happens. But in bacteriophages it can. And so this results in the unique ability to be able to map the position of these mutations inside of a gene. So this was actually studied by a man called Benzer, it's his last name and he studied this gene called the R 11 locus of the T four bacteriophage is just the type of T four bacteriophage. And this are 11 Locusts was very highly mutated. So There was over 20,000 independent R. 11 mutants that he was able to collect. And he actually crossed them together. So he did this cross here. Now he did some shortcuts and things that he didn't actually do. 20,000 With all combinations of other 20,000. He had shortcuts and things that he used and not going to talk about them. So but he got it down to a manageable level of something that a human could actually do. It was still a lot of work but it was still feasible and so he was able to cross them. And so each cross recovered re competence and then he was able to say, okay, well, mutation A mutation B r 50% apart and mutation B and C are 10% and Z is 72 not 72 7.2%. And so all of these different combinations he was actually able to map. So here's our 11 Locusts and this isn't actually where the mutations are. This is just a depiction. But each one of these represents a mutation. So he was able to take those 20,000 mutants and actually, you know, say, okay, well these these mutants are here and these are here, this is how far they are apart from each other. So it's a ton of work. But it allowed him to be able to identify specific, you know, actual mutations within a gene. Which was really the first time this was done. We can do this much easier today just by sequencing it. But at the time this was really novel and really important to be able to understand using recombination frequencies to map bacterial page genetics. So with that plus how we've done
A plaque assay studies viruses through what measurement?
The number of bacterial colonies that grow on a plate
The number of bacterial colonies that are lysed upon infection of a bacteriophage
Comparison between the number and size of bacterial colonies
Rate of Infection
A mixed infection of two bacteriophage strains is often used for what purpose?
To sequence bacteriophage genomes
To measure bacteria resistance
To study conjugation
To map bacteriophage genes
A mixed infection of two bacteriophage strains was performed. Infection of bacteriophage strain 1 causes the bacteria to be red and large, while infection of bacteriophage strain 2 causes the bacteria colony to be black and small. The following results were obtained. Using this data, determine the distance between the color and size genes.