[MUSIC] When I was looking for papers for this early Lysogenesis part. I had a difficult choice. There was a paper by Vilan Delberg that looked at the mutual exclusion effect between lamba and. There was a paper by the Lederberg that described lambda, which came actually, came out, was published many years later. And there was this paper by Giuseppe Bertani, and I choose this one for a number of reasons, including the fact that this paper is simple, easy to read and very informative. So the mode of phage liberation by Lysogenic Escherichia coli 1951. Okay, so this paper is also a paper that accidentally became a classic because all the experiments that were published in this paper were done in a bacterial broth that was called LB, LB medium. LB medium is known by every single person who works with recombinant DNA today, because you grow your bacteria in LB. And the LB has, stands for a large number of different names. Some people claim that it is broth. It's true that the LB was invented in Luria's lab. Some people claim that recognition should be given to so they call it the broth medium. And then some people said, wait, wait, wait. But the guy who really made p1 popular is Lennox. So it's probably stands for the Lennox broth. And so, in a memoir paper that wrote 55 years after this one. He said that he just wanted to make things clear that LB stands for Lysogeny broth because he was working on Lysogeny, and he was using a broth and not a synthetic meat. So that's why he called it LB. Okay, so he used the body and string called which was discovered in the mid 20s, early 20s and published for the first time by in 1928. And he uses strain of Shigella. Both given by Josh Lederberg. Okay, so what did he do? He wanted first to establish an adequate, easy, and reproducible way of counting phages away from the bacteria. Bertani wanted to have a good, simple, quantitative way of measuring phage in a culture of Lysogenic bacteria. So, he actually tested two methods, one method was the simplest. It involves centrifugation, spinning the bacteria doesn't get, as a palette. And counting the number of phages in supernatant. That's a centrifugation technique. The other technique he used was to kill the Lysogenic bacteria with streptomycin and to plate the culture on a loan of Shigella made resistant to streptomycin. So that the Shigella could grow but the original easbun strain could not grow. And you can see he did this four times in quadruplicates, and so he measured the amount of free phage, and basically with the two techniques he gets about the same numbers, 5.5 times 10 to the 5, or 4.6 times 10 to the 5. Those are negligible differences. He also measured the amount of living bacteria before he killed them. Or before subjected them to centrifugation. And he compared the amount of phage and the amount of bacteria. So in a culture you have somewhere between 500 and 6,000 bacteria per phage. Most of the organism in the culture are bacteria and there are very few phages. If you do this with lambda You find that the level is about one in ten to the five. Ten to the Five bacteria and one phage, free phage. So he had this quantity. So then he measured the amount of phage in bacteria as the bacteria grow. And found that as long as they grow exponentially, the ratio is constant. If they stop growing, or if you start with a cell takes time to start, they are in their life phase, the ratio changes. What are? What is the phage in this culture. Well in fact Bertanni was both luck and unlucky he was lucky because this particular lisbon strain in fact carries three prophage. Each of this phage can be liberated one is P1, the small plaque. One is P2, which was studied extensively because it has also the capacity to transduce and be, it's a very interesting phage, we won't go into it. And the third one was P3. And P3 was not studies. P3 makes Heterogeneous plaque, some are small, some are large. P3 is a very difficult phase to work with because [INAUDIBLE]. There is an amusing story about P3. For some reasons a scientist named Nancy Kleckner was in charge of organizing a course for students with phage. And that was in 1976, and she was working at Harvard in the city of Cambridge, Massachusetts. And so she resurrected P3, Gloria was at MIT. So they have the strength, they resurrected P3 and she wanted to use page for the work of the students. And then came a big drama, because somebody found out that Nancy Kleckner was trying to use this phage. And this phage is called P3. Now the city council of Cambridge Massachusetts had voted a law prohibiting scientists to work with dangerous organisms in the town, unless these organisms were contained in a safety containment called a P3 lab. And its still called a P3 lab. So there was a big misunderstanding because had no idea that this was not a dangerous organism, this was not a pathogenic virus. This was just a simple e.coli virus. Whose name happened to be p3. And so it was absolutely forbidden. And the authorities at Harvard said, there is no way you can use this phage for a class with students. We're going to be in trouble. So P3 was resurrected for a few days, and went back into oblivion. Okay. Okay. So, this seems to be a complicated experiment, but in fact it's quite easy. What Bertani did was to do basically a luria-delbruck experiment. A fluctuation analysis. He inoculated 70-plus tubes with a small number of bacteria. So the tubes, and he did the experiment four times, and in these four times he had about the same number of tubes, and total was 300 cultures. 300 small tubes, and in each of these tubes he added a small amount of bacteria. This is the average number of bacteria that was inoculated, 300 bacteria. And then he waited at 37 degree for a couple of hours. What happens during these hours, two hours. during these hours the bacterium will replicate and in this condition the bacterium will replicate and undergo about three generation, one two four, eight. Eight. So you will have eight times more bacteria, roughly. So this is three generation, 80 tubes per experiment. And then he asks, in these tubes, do I observe the conversion from the prophage, latent stage to the lytic stage. Do I see one bacterium lysing and liberating phage? That's what he asks. Of course he doesn't let the cells grow too much. He just let them grow for a few hours. And then he measures in this 300 tubes, how many of the tubes have phage in them on the Shigella strain. And he finds that the experiment is very reproducible. Under this condition. He has about 10% of the tubes have a burst. So with 10% of the tubes having a burst, the mean is roughly 0.1. From the mean, remember, you can calculate. The P0 value from a Poisson distribution and the P0 value is roughly 0.889. Roughly 90% of the tube have no burst. He can also calculate P2, this time the poisson P2 not the phage P2. And he can will have two bursts, and he find that the number is very small. So most of the bursts he observed are single burst. One bacterium, lysing. He counts the number of phage, and the number varies between six and 129. This seems a big variation. In fact, it's not such a big variation. This tube, the 129, probably had two bursts. Most likely. So the average burst is about 30 to 50 or 60. So he calculated the burst and he can calculate the burst per generation and this is an important value. He gets one out of 45,000 bursts per cell per generation. So the probability of becoming, of going into the lytic cycle is low, one in 45,000 per generation. He was very surprised because the single burst where all either p1 or p2 or p3 where he never had a burst, a mixed burst. The burst were homogenous. So, at this stage, there was also some discussion about lysogeny and in fact lysogeny he specifically states that lysogeny is not observed if you break open the cells. By grinding, my lysozyme eating, the wall, you don't see it. So the prophage is not a virus ready to go it's not the plane or the car in the garage covered with something and ready to go. In fact, the prophage has the instructions to make the virus, but no parts of the virus. Except the DNA that is in this case integrated. Now, this poses a problem into how you will distribute the prophage from one cell to the other daughter cell. And the way lambda choose was to integrate the prophage into the host chromosome. A phage like P1 use another system called a partition system that is essentially a counting device that will put one plasmid on one side and the other [INAUDIBLE] on the other side, but this is an important problem where you think about the prophage state.
