Transcript for:
Phage Biology and Applications

[Music] [Applause] [Music] [Applause] okay welcome to microbiology lecture eight which is about phage phage biology and some of the really cool applications that come out of phage so we're going to talk about today like what we're talking about what are phage which are basically bacterial viruses we're going to talk about the to life modes that phage can undergo where they can either lyse bacteria or integrate into their genome and we're going to talk about then how sometimes these integrated bacteriophages give different abilities to microbes or if I were one example with a current outbreak of e.coli o157 h7 but we're also going to talk about how scientists have used phage as a way to do genetic engineering and also used phage as a way to treat disease so a lot of really interesting things about phage and one of them is illustrated in the case which is shown in point two and this is about a current outbreak that's happening right now in romaine lettuce so right now in romaine lettuce 98 people have become sick and 22 in 22 states from eating romaine lettuce that is contaminated with a type of e.coli which is called oh one 57 h7 that's the strain 46 of these people have been hospitalized including ten evade kidney failure and basically so far people epidemiologist have been able to track the contaminated ìletís back to a particular farm in Yuma Arizona but they know that actually the lettuce didn't pick up the you cola at that farm it picked up the e.coli in the food chain like wow the lettuce was being processed and so we know that we work on you cola in the lab and without getting sick so why is this eco lie so bad and basically the short answer is that this type of e.coli the o157 h7 has numerous genes that have been added to its genome and one of them some of them in code toxins and one of them has come from a phase that is integrated into the genome so talk about how that happens and we'll talk more specifically about 157 h7 so as I said before bacteria our phage are viruses that infect bacteria or archaea and they basically if you remember what a virus is back from like very first lecture we talked about a virus the definition of that is that they're a cellular obligate intracellular parasites that basically do not stay intact throughout their lifecycle throughout life and they are composed of proteins and nucleic acids and so there are two basically types of life cycles that bacteria phage and viruses can undergo one is the ability to lysis cells which is called lytic on host cell or target cell is lysed or this mode which is called lysogenic in which the phage integrates into the genome and doesn't Lysa cell i know unfortunately these have names that are not exactly because these cells are lysed and this is called lysogenic some that's a little confusing I know so it's just kind of a term you're gonna have to memorize so first we're going to talk about an example of a little is called T 4 and then we'll talk about an example of a lysogenic virus called lambda and so if you look at point 4 on the NEX on this page you'll see that the a picture of the t4 phage and they look kind of like I drew them here they have a couple of key parts one is the head and this contains the nucleic acid and a few proteins that are needed for early action and then the tail here is the part that recognizes the host cell okay and so one of the the very first step that's going to happen when bacteriophage interacts with a with a bacterium is that basically so we got our phage the bacterial interaction here we're talk about T for the first step is going to be attachment that makes sense the phage has to attach to the bacteria and so basically they're it's a specific interaction meaning that there's a protein on the phage that interacts with a molecule on the bacterial cell in the case of phage t4 in interacts with LPS on the bacterial cell specifically the core portion of LPS and so basically if you remember what LPS is it's in the outer membrane of gram-negative cells it's got its lipid a it's got its core polysaccharide ur that it's got an O an engine and so basically t4 is able to interact with the core portion of the of the LPS on the outer membrane and because this is a it's kind of a its name core because it's it's a central part of the LPS and it's found in many types of bacteria so LPS is able to it I sorry t4 is able to interact with e.coli and other bacteria that have a similar core part of LPS so Salmonella Shigella these all have a very similar core portion of LP so t4 can target all these bacteria so then after attachment there's a penetration step we're basically the phage injects the nucleic acid in and so this is both a mechanical process carried out by the phage itself and is aided by one of the early proteins carried in the inside the phage head here which is lysozyme you remember lysozyme because it's the enzyme that is able to cut peptidoglycan and that makes sense because now the phage basically wants to shoot it's a DNA through here basically through the outer membrane through the peptidoglycan and then through the cell membrane so the DNA has to move all the way through all those layers to get inside the cytoplasm of the bacterial cell so then after penetrate after this step when the new the nucleic acid is brought inside then the basically the first thing is there's early transcription if you look at the figure that's at the bottom of that page figure x2 you can see sort of a diagram of a timeline showing how transcription occurs it basically at time zero is when these first steps happen attachment and penetration and then right away as soon as the the phage genome gets inside the cytoplasm here now the first transcription is going to happen and this is going to be on phage genes that have promoters that are exact matches to the Sigma 70 consensus and so basically the phage nucleic acid comes in its DNA so it has on it it has sequences that are minus 35 s minus 10 s minus minus 10 basically and these are in front of genes that need to be made very early and basically these are exact matches and so they bind very tightly to the host cell Sigma factor Sigma 70 and then RNA polymerase and so they basically sucked all the Sigma 70 away from the host cell promoters and drag it over here to the fates promoters and start producing lots of phage there early fade genes and some of these early fades genes are genes for a variety of things but one of them one set of genes are some nucleases called endo 2 and endo 4 and these basically nucleases cut up the bacterial genome and the reason they so basically now in the cell there's two genomes really there's I should correct this picture over here there's the bacterial genome well at this stage I through there's a bacterial genome and there's a phage genome and so the the first genes that are transcribed by the fades make proteins that cut up the bacterial genome only and the reason the these protists can tell it's the bacterial genome is because these enzymes cut up DNA that has a normal sea as the sea bass and the phage DNA all has a modified sea and this C is modified it's a D oxy hydroxymethyl sited een so it has a couple of modifications on it and this makes it so that these endonucleases basically can't see can't detect that anymore they can only check the normal bacterial genome with a normal see and then they cut up the bacterial genome in two pieces and now there's basically no more bacterial genome to be used and so this has two purposes one is that it basically prevents the bacterial genome from being transcribed and the second thing is it basically provides nucleotides for the fade so dntps nucleotides for the phage okay so the early transcription here fates promoters are initially transcribed because they find segment 70 faster better because they're exact matches to consensus but then as time goes on now the bacterial genome gets totally destroyed so there's no more bacterial promoters so now all the resources of the cell can go towards basically making phage and then as we move through the rest of the stages you the next steps are middle transcription and late transcription which are basically making the next kind of products that a phage needs so basically making lots of copies of the genome and making the heads and the tail pieces basically to build phage and then these are all packaged together kind of shown is in this diagram and then the host cell lysis and releases the phage and from t4 basically we'll get about a hundred phage per bacteria so there's a massive amplification we started with one phage entering and then when the bacteria lyse 100 fates come out so there's a lot of amplification of the phage in any phage infection this fact that bacteria lyse cells can be used as one I started that phage light cells is one way is the one we can use that as a way to monitor the number of phages in the sample and so this is talked about in point D where we talk about the plaque assay put down here and there's a couple of figures figure X 3 and X 4 which show you the plaque assay so basically how these are done is that we mix together some bacteria some agar and some phage and we pour these in a petri dish and basically what we've got here the key to this is that we have a lot of bacteria and there's so many in there that they don't be the colonies there's not really single colonies they're all run together into what's called a lawn and so if we supposedly look from the side view we're going to pour our solution into the petri dish here it's going to fill it up and there's gonna be so many bacteria in there that basically it's going to become opaque and so this is what it would look like if there is no phage but if there's phage they're gonna lie some of the cells and so when we pour them in there's going to be areas where the bacteria grow but then there's an area where there's basically lysis and it's going to look like a little clear zone and so you can see that in the images which show it better than I'm doing it maybe from this view if you look from the top view you'll see basically solid growth and then areas where there's a clearing and those are the plaques and those plaques came about because the phage infected bacteria and then lice the bacteria and now there's basically no bacteria there anymore and so it's kind of like the opposite of a colony so a colony is a mound of back Karia in this case the plaque is like a area of dead bacteria and all around it our our confluent bacteria and so you can use you can use this kind of essay to ask are there phage in the sample in this sample and so and you would have to answer yes or no at least if their phage that can do the lytic part of the lifecycle they have to be able to lyse the bacteria in order to form these plaques and so the other form of the life cycle though is called lysogenic so let's talk about that and this is a case where again so the word is lysogenic this is a case where infection does not always destroy the host cell instead and sometimes the phage DNA actually integrates into the bacterial genome and then as such it's basically can be passed to the offspring when the bacteria divide and so an example of this is the lambda phage which people write owes the group Greek letter lambda and so basically there's some ways in which the life cycle between lambda and phage t4 are similar one of these is that there's very specific attachment lambda uses a pouran it's a kind of a special porn it's porn for maltose so it's a porn that has some specificity in addition to this size and it's specific for maltose so there's specific attachment there's highly controlled gene expression meaning that there's late middle and early late early late a middle gene expression but one way and way to lambda phage is different is that there is this decision point where the set lambda can either adopt a lysogenic lifestyle or oolitic lifestyle can do either one so it can do lysis or lace ogyny so license is kind of a another word for lytic lysis or lysogeny and this is considered a decision because basically for phage to be able to undergo this kind of a life cycle the cell needs to have a lot of resources because from one cell we're going to make a hundred phage and so this requires a lot of transcription and translation and DNA replication in order to produce all these phage so if there's a phage that can make a decision typically the decision is based on whether it'll go the lytic route if the bacteria are dividing and this means that our growing have to be actually divided but they have to be growing and feeds this means that the bacterium is healthy there's maybe a lot of food around probably other bacteria around and so typically in this case the phage will go will lysis el and go the lytic route the other option though is to go the lysogeny route and it will do this the phage will go this route if the bacteria are not dividing and the ideas again that maybe they don't have enough of precursors like nucleotides and amino acids to be able to support the phage to form all these phage particles and so if if that's not the case then the fades decides to basically the best option is just to integrate in the genome and wait it out and so we'll go with the lysogenic option in that way and that's shown in Figure 18.5 where you can see again basically a version of this diagram and so when when the bacteria integrated into the genome basically they integrate into a specific site and you can see that in the figure 18.6 and so lambda integrates at a specific site on the chromosome it's called at to be and you can see that in that diagram and basically on the phage there's a homologous site so using homologous recombination with the phage at B at P sorry so it's attachment site in the phage and attachment site in the bacteria these two are homologous to each other they line up and then by homologous recombination the phage will integrate at a specific site in the chromosome and so then the phage will stay with the bacterium as shown here until conditions get good so maybe many generations here plus a division and so f not sorry I said it wrong it's not what the conditions get good after many generations the phage will hop out exercises and then we basically go back to this plan here where we go the phage can become lytic again and the conditions usually the drive this are actually the known conditions are when actually the cell undergoes damage so it's kind of the opposite of good conditions the cell is damaged and then the phage says that oh man this is not working for me I'm going to hop out and so when the phage hops out though it was in the genome if we do our genome here we do our phage in there so this is our phage so normally when it excites that excises very precisely right at these two points hops out and basically you get kind of an exact version of what you started with and you end up with phage so you end up with that pieces but every now and then the excision isn't very precise and so instead of exactly hopping out like this maybe there's a little bit of there's this site and a little bit of an error there so we end up with a little bit of a deletion on the genome and a phage that's a little bit longer it's got some extra stuff on it and so now when this page goes to the next cell it's going to carry some of these genes with it and this is a way in which phage can be a mechanism for horizontal gene transfer they bring a little bit of the chromosomal genes with them when they move to the next host post and so this is thought to be the way that phage kind of can expand what they carry with them to pick up some new genes and so when you think about this if we had a phage that was like lambda do you think that lambda would form plaques in this plaque as they would land with lambda phage form plaques and your options are yes/no or sometimes right probably sometimes because I said that sometimes lambda will go this route lytic which is the way plaques will be formed and this would be if the bacteria were growing really happily there there the bacteria they infect but if the bacteria they affected were growing kind of poorly then you might think no they would just integrate and in this form they would not form plaques okay so one case where a lysogenic phage has been really important is in the e.coli o157 h7 and if you look at table 26.2 you can see that there are lots of bacteria that carry hawks and jeans on phages and so toxin genes are particularly harmful code for toxin so toxins are proteins that damage us damage a host cell and so having a toxin gene encoded on the phage is really interesting because I just said that fades are a really good way to move genes around and so if some of the genes that the phage are moving around our toxin genes this is a way that microbes can evolve to be more harmful to us and if you look at that table you can see that there are lots of toxin genes that are carried on phage so diphtheria which is one we're all vaccinated against the the toxin is coated on a phage cholera which causes diarrhea the toxin is coated on the phage several forms of Staphylococcus and then the example we're going to talk about the e.coli o157 h7 which carries shigga toxin on the phage okay so let's talk more about what is a coli o157 h7 so I said that it carries a extra gene that codes for a toxin called sugar toxin so let's first talk about the disease so the way you get what the way you gave you coli o157 h7 which is also called a hack and I'll we'll get to why it's called that a heck so you you get it by eating it it's on contaminated food and water and one thing that's really it so you eat it you ingest it and one thing that's really bad about e.coli o157 h7 is that the dose that you need then you need to get an infection is about ten organisms ten bacteria is extremely low and meaning that it's a lot of work to clean up food that much to not have ten bacteria so keep that in mind so after you eat them the bacteria you ingest them and then bacteria travel through the stomach and end up in the intestine and there they multiply and adhere to your cells and if you look at that little figure at the bottom of the page figure x5 this kind of shows some of the differences between different eco lies so the middle e.coli is your kind of normal commence Li coli which doesn't cause disease and nobody you can see is that there's been a variety of new genes that have come in from either plasmids or phage which have given the different e-coli different abilities and then all the resulting eco lies shown at the bottom are the so-called pathogenic e.coli so they the commensal one doesn't cause disease and all the other ones do cause disease and so what one set of genes that ecoli acquired is called Li which is shown on that figure and that is a set of genes that allows E coli to adhere to your cells and then once it's adhering so it's kind of like a really tight association with your cells it can deliver toxic products and so the e coli o157 in addition to having the genes for the lis also have the genes for the shigga toxin so they make a protein called sugar toxin and this gets released it can be delivered to the cells and shigga toxin there's two things about it we need to know one is that it binds to a receptor a host cell receptor so again it's a protein maybe the bacteria it binds to a host cell receptor to get inside cells to get inside your cells that's a human actually human cells and this receptor is only found on intestinal cells and kidney cells and the second thing about the sugar toxin is that the way it works is that it Cleaves mammalian ribosomal RNA and stops translation mammalian translation okay so okay so e so E coli o157 produces has the lead genes which allows it to adhere and then it has the sugar toxin genes which are carried by a phage and they the sugar toxin genes code for sugar toxin and this produces a toxin that enters mammalian cells but only intestinal kidney cells and Cleaves ribosomal RNA and stops translation I should say one more thing is that the toxin is released in the intestine but actually gets into the bloodstream so this is the sugar toxin and travels to the kidney well travels all over the body but travels and then in the kidney it can get it can intoxicate this kidney cells okay so basically we've got our hack coming in and seeing a testin producing these products these products intoxicate the intestinal cells kind of locally but then in addition the toxin gets into the blood goes all over the place and ends up getting into the kindy cells because the receptor for Shikha toxin the sugar toxin receptor is found on the kidney cells as well and this gives Ecola l 157 h 7 its other name which is an Toro hemorrhagic ecoli it causes bloody diarrhea in the intestine so let's see intro hemorrhagic hemorrhagic means bleeding and then it causes also hemolytic uremic hemolytic uremic syndrome which is kid'n a form of kidney failure and so basically you know a bloody diarrhea you have this kidney failure and this is why enter a hemorrhagic a heck you coli are particularly bad and so basically as I said before the genes for the sugar toxin are carried on a integrated bacteriophage so I use the word temperate but what I should say what I would say instead is that shigga toxin so I don't know more words to know here he's carried on a lysogenic phage that's and that's a phage that's integrated it's kind of like the fate the sugar toxin phage is called the STX phage it's kind of like lambda so it's a lambda relative one way it's different from lambda is it integrates in six sites on the genome and so there's not just one copy of the xes phage but there's six copies per genome and so ehekk it's really bad because of this integrated sugar toxin phage which is releasing sugar toxin and causes a really bad disease epidemiologically this is a real challenge because basically a hack or a 157 I'm gonna write you hack cuz it's shorter is basically found in the intestines of many animals mammals I think also even reptiles a lot of things have a heck finally many animals intestine the big animal that seems to have a lot of it is cattle and what's kind of interesting is these are all carried as commensals it's carried as a commensal meaning it doesn't cause disease the ehekk doesn't cause disease and these animals mostly because they don't have the host cell receptor for the sugar toxin so they don't have this STX receptor so these animals lack the X s TX receptor so they have a coli and it's basically weak and everything and may before but they are like we don't care we don't get the toxin can't get into ourselves so it doesn't harm us and what's also interesting is in studying like where in the intestine there was some really nice work analyzing cow intestine to see where is he hacked located and they scientists analyze the whole intestine you know chopping it up in pieces and saying yeah here you hack here like looking looking and then they didn't find it and they realized they had skipped up first a part of the intestine the part that's really near the recto anal junction and it turns out be heck lives right there right at the recto anal junction spill then and junction and basically it's essentially right where the feces exit the intestine as the feces come out they squeeze past that that junction point there and then that's where all the heck is in the heck just gloms onto the feces so right at the feces exit point I guess you can see just like we're ready to go ready to come on to the feces and as such it is shed in the feces and so cows you know when they're living like in their cow house they kind of rub up against each other and so feces tends to end up in a variety of places like on the hair and so this makes it really challenging that the see heck is coming out in the feces cow feces ends up all over the place and so to deal with a hack there's a couple of recommendations and the other thing actually is that it's in the feces and people use cow feces for fertilizer like it's a really common practice to use manure as a feet as a fertilizer and so this could be a problem though if your manure has a heck in it and then you throw it onto your lettuce for example then you can imagine that now the yuck can get onto the onto the lettuce and so what they recommend is that if you're going to use your manure you store it for a few days think about for greater than 30 days and that can that decreases the amount of e.coli o157 h7 it can't survive that long the other thing is when you're slaughtering cattle is to watch out for the feces so if people have to be really careful about slaughtering and there's been a lot of really good practices in the slaughtering industry to make this better so basically to remove fur outside the slaughtering facility and then clean off the carcass and be really careful because fur hair whatever cattle have watch out for feces and then of course if you are using meat they recommend to cook it this doesn't work so well for lettuce and so initially a heck is the famous pathogen that was in the jack-in-the-box hamburgers and the reason it was it was ground up from the cattle meat which had some feces in it and then people didn't cook the burgers at well and then they ate the burgers and then they got the e.coli but now actually most cases of e.coli outbreaks are found on things that you eat raw like lettuce and so people don't really cook their romaine lettuce and so now the goal is to really decrease get that you call it a 157 h7 out of the food chain and I put a link to an article that came out into forbes about how complex our food chain really is now and in this case they know the lettuce was grown in Arizona but then that wasn't where the oh one 57 h7 was the heck it wasn't on that farm it was sometime in the processing of the lettuce as it moved to the place location where is washed and then the location where was chopped up and then where it was bagged somewhere along that really complex food chain the o157 h7 got in there and what's also challenging is this fact that really only needs 10 bacteria to cause disease so this means that people had to be really unbelievably anal ha no pun intended I'm getting the e.coli o157 h7 out of the whole system like when people shed feces in human feces there's about 10 to the 14th bacteria per gram and we call it cow feces are huge they weigh a lot so there are each cow feces has just unbelievable numbers of bacteria in it and the idea that you got to get rid of all of them except 10 is really a very interesting challenge for microbiologists and for food food people who work with food science to be able to do this we just talked about a hack you call it 157 h7 and how it's been modified by a phage and we talked about how phages can do they can when they infect a cell they can either integrate in the genome or they can undergo replication and lysis cells so in either of those cases they those outcomes are not that good for the bacterial cell particularly when the bacterial cell lysis so you can imagine that bacteria would benefit from in ways to fight against phage and indeed they do and so we're going to talk about two ways that bacteria have learned or are capable of fighting against phages so the first one and this is in point seven of the notes the first one is using restriction enzyme so I think pretty much all bacteria encodes and restriction enzymes ecoli encodes one called eco r1 and restriction enzymes are proteins that cut specific sequences of DNA so for example ecoli encodes one an enzyme which is called eco r1 and then it cuts a sequence g-a-a-t-t-c so enzymes are usually named for the organism they come from so eco tells you it actually comes from eco lie and so basically you coli makes an enzyme this enzyme cuts this sequence g-a-a-t-t-c and this sequence is found all throughout the e coli genome so eco light does not want to cut its own genome that would be lethal and so basically the way e.coli protects against this cutting is to modify this sequence so e.coli also encodes an enzyme called eco r1 methylase and what eco r1 methylase does methylase eco Roman methylates puts a methyl group which is ch3 on one of the A's in that sequence and so basically now when this sequence is methylated so basically we have a situation here where we have g-a-a-t-t-c it's unmethylated and basically now this can be cut cut by eco r1 but if the sequence is methylated now we have our methyl group stuck on there now it can no longer be cut not cut and so what ecoli does is make sure that all the sequences that are g-a-a-t-t-c are methylated by the EQ on methylase and one way it does this is by encoding them in an operon together and so basically if we looked at the genes for the eco r1 methylase and eco r1 they're encoded by one promoter and basically they produce a polycistronic mrna because for two genes and this makes sure that any time when d Collies making eco r1 which would kill it it's always making eco r1 methylase which will methylate the sequence and protect the coli from the action of eco r1 and so this is how the e.coli genome looks e coli genome is all methylated on every g-a-a-t-t-c but the phage genome is not so when a phage DNA comes in the phage DNA will be cut because it's not methylated and so e-coli is a couple of restriction enzymes of function just like this or basically its own genome is protected by methylation of that restriction enzyme site and the phage DNA will be cut because it's not methylated and so this is a way that bacteria can protect themselves against phage when the phage come in by destroying them so the other way is using the CRISPR system and so basically this is a bacterial system that essentially archives small pieces of DNA and it stands for clustered regularly interspaced short palindromic repeats and this is because of how it looks on the bacterial chromosome so if we draw it basically there's little repeat sequences that are the same and our clustered so these are little DNA sequences that are basically the same and in between them is what's called the spacer so we've got our repeat and then we got in between the repeats we got spacers I'll just draw two and this is all basically encoded on the bacterial genome so on the genome it's pretty easy to pick out because these repeats like this are a little bit unusual to have that and so what happens in this system is that when a phage comes in or when a foreign DNA comes in it's recognized by the cast one and in cast two enzymes which cut it up and basically take some of that DNA and insert it into as a spacer into the CRISPR array and so you can see in the figure in the notes basically figure 10.14 that we start with some foreign DNA coming in its cut cleaved by the caste system into a piece that then is integrated into the spacer region and so this then is basically a little copy of whatever came in so basically this piece of DNA is one portion a little big phage genome let's say it's right here so we have a match there and then what happens is this space this a CRISPR set here is transcribed into a big mRNA and then processed into units that basically have the spacer there's our blue spacer and next to it are the repeats and the repeats pair up and fold into a like a hairpin there so these are the repeats and this is the spacer and then the spacer because it's again it's a match to this um incoming phage if that phage comes in again this spacer can target the repeats to an incoming phage that matches and so one thing about these repeats is that one of their properties is that they bind to an enzyme called cast nine out there which is a nuclease which does a double-stranded cut of whatever it's a mirror and so the fact that this spacer brings the repeats and cast nine let's put cast light on here to over to the incoming phage now the cast iron will cut the incoming phage basically we live we have a three in there somewhere and then for Whoopi cast nine cuts the target that matches the spacer and so this is really a way in which the bacteria can remember what phages they've encountered phage comes in it's processed and stuck into this repeat system as a little spacer and then because this is on the bacterial chromosome on the genome it is going to be passed to the offspring so offs we've never encountered the phage but they have this little piece that's like a record of what phage they encountered and this is being transcribed and processed and so if a phage comes in it's basically ready to go and attack it and so this system is really wonderful as a way for bacteria to remember what phages they encountered and is super been super interesting figuring out like how this works and a lot of interesting studies still being going on today like example very recently people were curious like how does the CRISPR system know that this is foreign DNA we said over in this example the restriction systems knows it's foreign that it's a foreign DNA coming in because it's not methylated like the host genome in this case that actually seems to be related to really fast replication and during the really ramped up replication some of the errors that are made are corrected by removing chunks of DNA and those are the chunks and actually get encoded into the space to put into the spacer so it's because the phage is replicating so fast so but people just discovered that a few years ago and so really at the forefront of figuring out even how this works in the bacterial system but in addition this has become a very powerful tool for genome editing it's the basis so this is it's good for the bacteria but it's also the basis for genome editing and so some of that is shown in images basically the weight why it's a basis for genome editing is because this this spacer sequence really precisely brings the cast nine to any sequence to a sequence in the genome and so we can as a scientist can actually edit the spacer and make it into any sequence you want so basically we can modify the spacer and if you look at figure x8 they're basically when the cast 9 and the spacer are brought to any sequence they're gonna the cast on is going to cut the DNA just like I said and many cells have systems for repairing these double-stranded breaks so it's a double strand break here and so two of them are shown in Figure x8 one of them is non-homologous end joining we're basically just the cut is reese tich together but typically there's some little errors in there it's not precise and so whatever was there will have like an extra nucleotide inserted or maybe one taken out and this could cause a frameshift so that could be one kind of way that we could cause a mutation in any gene we want but the other way that's really powerful is the fact that during another form of repair of double-stranded breaks which is called shown on there was homology directed repair basically if we also include if we add in addition to the spacer sequence that we want we add some new DNA that we want to integrate into the genome after the cut happens the new DNA will be integrated at a very high frequency and so basically by slightly modifying this CRISPR system that exists normally in bacteria people have been able to make system that can do really amazing genome editing and what was the real breakthrough in the system was that people knew about these double-stranded breaks that double-stranded breaks would be repaired in one of these two ways people knew that was going to be a very powerful way to do genome editing to modify genomes but they had no way to make a break at a very specific sequence with any kind of efficiency and so when this system was discovered and it was realized that AHA the whole way this works is by homology to the spacer bringing this cutting enzyme to specific site that is a way to make a double-standard break anywhere you want and so now we'd you know enter the genome Edit by basically modifying this spacer sequence and that brings a caste 9 and then we also then just insert whatever genes we want as shown figure x8 there and so again this has been a super powerful system but it arose from basically a defense defense system okay so the last thing I want to talk about with regards to phage is that they are also being explored as antibacterials so you remember back from a few drawings ago we basically had a phage infecting an e a cell and essentially after a few steps we got basically to a step where the cells are lysed and the light cell is dead so basically we can use phage if we if you have an infection and you treat yourself or treat the infected person with phage the phage will cause the cells to lyse and so it's kind of a kin to an antibiotic that essentially it's a it's a tool that we can use to stop a bacterial infection hasn't been it's it's being developed now more and more and I gave two links in the notes one is to a company called amplify bio that is basically developing phage technology and it has some interesting stuff on their website I encourage you to look at that the other is actually a really amazing story of a person at UCC a person who is a professor at UC San Diego who got a super bad infection and the super bad infection was from an organism called Acinetobacter and the super bad infection was resistant to every antibiotic there was nothing that would work against it so it's a super bad and basically he was not doing well at all obviously because he had this infection and his wife had taken microbiology and remembered about phages and she looked into the possibility of using phage as a way to treat her husband who was very very ill and tracked down some phages and it was incredible it was totally experimental but they found some phage that worked against Acinetobacter and they were able to inject them into the guy and he actually was cured so it's a very interesting story so there links there is from the UC San Diego news release about how this is being used I think it's personal I think it's a really powerful technique it probably will work well in conjunction with antibiotics the UC San Diego guy professor was treated with antibiotics during his in addition to the phages one of the region's reasons is that when phage infect cells here they have a very specific interaction between a receptor in the phage and a very easy way that bacteria become resistant to phages is just to mutate the receptor and so the the cells can become resistant pretty easily to phage but nonetheless I think there's a lot there's a lot to explore there and especially as we need new ways to treat infection I think that phage probably will play an important role in there's obviously a lot of companies that think so too and so it'll be very interesting to see how some of these phage based products develop there are a few that are commercially available they're typically topicals let you put on outside not that you eat we're not that get injected but the the possibility is that they will develop into more useful drugs so basically today we talked about fades biology covering how phage can do lysis they can lay cells and this is a convenient way that maybe we can use pages of therapy by their ability to lice cells we talked about how we measure that using the plaque assay which are those little holes in the lawn of bacteria and then we talked about how another mode for phage is how they can integrate into the genome and when they integrated into the genome they kind of like big basically hide out and get passed from one generation to the next and in some cases they seem to carry other genes with them we looked at the case with a heck a coli o157 h7 which has acquired a phage an integrative phage that confer that codes for the shigga toxin gene and shigga toxin which is a very important part of its how it causes disease and then we ended talking about two systems that bacteria have developed to become resistant to our fight against phage one of them is using restriction enzymes to basically chop up foreign DNA that comes in and the other is using the CRISPR system which is kind of a more sophisticated way to chop up DNA it's based on specific recognitions and not just differences between the types of DNA and that this has led really to a revolution in biology that now we can use a system this understanding of a really basic biology of how bacteria fight against phage we scientists have been able to use this as a way to develop genome editing in all sorts of cells including human cells and this has really been really remarkable unbelievably fast-moving field the first papers on it were it was just discovered really in bacteria in the late 90s early 2000s really understood in after 2005 and then adapted in 2012 so it's really been moving really rapidly it's remarkable but there's a lot still understand we don't really understand all of how it even works in bacteria so I think be really interesting to see where this develops in the next few years