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1. A group of researchers at the U.S> Department ofAgriculture has isolated a new Gram-positive bacterium related toListeria ivanovii from a recent outbreak of f...

Question

1. A group of researchers at the U.S> Department ofAgriculture has isolated a new Gram-positive bacterium related toListeria ivanovii from a recent outbreak of food poisoningdue to contaminated cheese. They have determined that thewildtype bacterium can invade epithelial cells and spread from cellto cell. The researchers find that for intestinal epithelialcells, binding and uptake of L. ivanovii could be blockedin coincubation with galactose and lactose, as well as a mixture ofcrude ganglio

1. A group of researchers at the U.S> Department of Agriculture has isolated a new Gram-positive bacterium related to Listeria ivanovii from a recent outbreak of food poisoning due to contaminated cheese. They have determined that the wildtype bacterium can invade epithelial cells and spread from cell to cell. The researchers find that for intestinal epithelial cells, binding and uptake of L. ivanovii could be blocked in coincubation with galactose and lactose, as well as a mixture of crude gangliocides. Describe how that might go about experimentally identifying potential host cell surface receptors (glycolipids) involved in mediating the cellular uptake of the bacteria. 2. You expect that the new isolate Lkb has virulence properties that allow it to survive in the host to cause disease. You decide to conduct comparative genome sequence analysis of the new koala isolate Lkb strain ACM 39xx with that of the already-sequenced avirulent L. koalarum (Lk) strain ACM 3666, but in order to do so, you first need to sequence the Lkb genome. a. What sequencing technology would you choose to perform this? Be sure to provide your rationale for choice in terms of cost, efficiency, and outcome. b. Provide an overview of the experimental steps you would take to sequence the Lkb genome and identify genes that are unique to Lkb versus Lk.



Answers

One of the first organisms that was genetically modified using recombinant DNA technology was a bacterium that normally lives on the surface of strawberry plants. This bacterium makes a protein, called ice-protein, that causes the efficient formation of ice crystals around it when the temperature drops to just below freezing. Thus, strawberries harboring this bacterium are particularly susceptible to frost damage because their cells are destroyed by the ice crystals. Consequently, strawberry farmers have a considerable interest in preventing ice crystallization.
A genetically engineered version of this bacterium was constructed in which the ice-protein gene was knocked out. The mutant bacteria were then introduced in large numbers into strawberry fields, where they displaced the normal bacteria by competition for their ecological niche. This approach has been successful: strawberries bearing the mutant bacteria show a much reduced susceptibility to frost damage. At the time they were first carried out, the initial open-field trials triggered an intense debate because they represented the first release into the environment of an organism that had been genetically engineered using recombinant DNA technology. Indeed, all preliminary experiments were carried out with extreme caution and in strict containment (Figure $010-16)$
Do you think that bacteria lacking the ice-protein could be isolated without the use of modern DNA technology? Is it likely that such mutations have already occurred in nature? Would the use of a mutant bacterial strain isolated from nature be of lesser concern? Should we be concerned about the risks posed by the application of recombinant DNA techniques in agriculture and medicine? Explain your answers.

For this question. We're trying to understand how C T cells were to present antigen from different strains of, say, a flu virus. So in this experiment, they're using two different flu virus strains. They have a 1968 flu virus and a 1934 flu virus. And what they are doing is they are making proteins from the genetic sequences of each of these, and they're finding out that the 1968 gives a response by the T cells, whereas the 1934 strain has no T cell response or very little. And this is measured by the number of cells that are being Liszt when they contain the proteins from each of these flu strains, so they give us a graph. To represent this, they have the number of cells, or the percent of cells, life on the Y axis. And they're presenting us with multiple bars measuring the number of cells being laced so they have a bar for the 1968 virus, a bar for the 1934 virus, and you can see there's a significant difference between the number of cells BIST for each of these strains from there they are measuring the number of cells that lies when they contain a certain amino acid sequence. So the first section is for cells containing amino acids from the 1968 stream. But as the second sequence of bars are amino acid sequences from the 1934 strain. So as you can see in the 1934 strain, you have two different sequences. You have a 3 65 to 3 80 and you, of course, have a 3 69 to 3 82. And both of these have very little cell isis among all the cells, whereas in the 1968 we have three different bars, the most significant ones being the one showing many cells being Liszt, which is the 3 65 23 80 and the amino acid numbering sequence. And you also have 3 69 23 82. So between these two amino acid sequences, the difference between each of these is causing this significant change in the number of cells being recognized by your T cells and being Liszt. So the main difference in amino acid sequence between these two it's going to be those first few starting amino acids. So here, for the first part of question a the amino acid sequence responsible for this T cell recognition are going to be the amino acids 365 to 369. We know that 369 to 380 is not causing this response. Or else we would see the bar in this second part for the 1968. Also induce that large number of style license. So here for part two, a question A. We're also being asked why these other viral proteins do not cause this significant sell like Isis. That's because of the recognition of the anti Jinbei your T cells. T cells are not all powerful, and they're not going to recognize and induce apoptosis in all kinds of cells with viral antigens. It just so happens that they can recognize some sequences, and by chance, they happen to recognize the 365 2, 369 amino acid sequence. If they recognized all amino acid sequences, they risk the chance of endangering our own cells, which, of course, have sequences of amino acids. And this limited recognition kind of helps that specific response of T cells and prevent them from overreacting. But I say we have part B. It's asking us about how the MHC type one molecule works and how it's applied to this experiment. So if you remember how MHC molecule works specifically MHC one, this occurs in all living cells. And when they have, say, a viral strand inside their genomic structure, they're going to create viral proteins to have these be viral proteins. The cell is going to recognize that these viral proteins are not their own. They're going to connect them to an MHC molecule. And when it's complex with the viral protein, it's going to move it to the surface of the cell, where it can present this auntie gin to your professional antigen, presenting cells such as your B cells or macrophages, and that will allow for your cell mediated response and the like isis of the cells. So, in order for this to work for this experiment, they're measuring the number and percent of cells being laced. So the best way to go about this is there going to use a 96 well plate, which is basically just 96 specimen holders where they're going to have the infected cell on the inside containing these viral proteins. They're going to line these 96 well plates with the cells they're infecting, and they're going to infect them with different amino acid sequences. So this would be like an amino acid strands of 3 65 23 80. Or they might do the 3 69 to 3 82. And they're going to do this for each the 1968 strain as well as the 1934. And to make sure they can measure the number of cells Liszt. They're going to need a probe or some sort of marker that they can check inside of the well. So what they're going to do is to each of these amino acids, the viral protein or the viral genetic sequence they're going to infect each of these cells with is going to include what we call a probe or some sort of marker. So this could be a marker that has some sort of fluorescence. It could be some sort of radioactive marker, something they can use to detect and measure the amount in the super needn't or the remaining fluid in the 96 well plate. Basically, it's going to be included inside the cell, and if the cell lice is, it's going to be released into the fluid of the 96 well, plate, whereas if it does not light, it will still remain inside the cell, where it won't be measured in the fluid. So what they're going to do is there going to centrifuge each other? 96 well, plates, and they will get a button of live cells on the bottom, and they will get a number of the probe for the marker for the concentration inside of the fluid or the super name. So they're they're able to compare the number of cells they initially put in and compare that two. The amount of probe in the Super Natan, where this would be the inverse of it, will be the probe divided by the cells, and that would give them the percentage of cells that were laced. So here, for part B, they're going to introduce the virus and some sort of probe into the cells, and from there they're going to compare the amount of probe and the Super Matan to the number of cells they started with, and that's going to give them the percentage of cells that were Liszt in the solution. This way, they'll be able to reliably compare the number of live cells and not include that in the number of life cells they originally started out with.

Hi, everyone. My name is Eric. And let's review problem 47. So in this question, they're asking us to describe the process of molecular cloning. Now, molecular cloning is the process where you take your DNA of interest, you insert it in a plasmid, and then you're going to place it in a host to replicate and produce mawr amounts of your DNA of interest. And the process to do that is actually what we're looking for here. So we're looking at four different choices A, B, C and D, and I'll be walking us through what is the correct answer and why. The other answers are not correct. So the first answer is letter A. So let's start with a So they're saying that the foreign DNA and the plasma are cut with the same restriction enzyme, and the DNA is inserted into a gene. So let's start there. So our plasma is a circular piece of DNA, and it's mostly common found inthe e bacteria. So with the plasmid, we're going to cut at specific sites with the restriction enzyme, and that's going to allow us to insert our foreign DNA into the same spot and in the problem. They described it as cutting into the lack Z gene. So by cutting into the lack see Jean, we're actually going to be disrupting this gene. And the legacy gene is responsible for metabolizing lactose. So lactose is a molecule, and Black sea is the gene that allows the host to break down and utilize lactose. So that would be the first part where we're going to be inserting our DNA of interest inside the laxity gene. All right, and then did said that the DNA and the vector are a kneeling, so then that would allow us to make a new plasmid. But instead our DNA of interest will be bonded in the spot where the Lexie should be, all right. And then this plasma is going to be transferred into a bacterial host, and it's going to be selected for using ampicillin, right? So somewhere on this plasmid will be an amp resistant jean, so that allows the bacteria to survive on episode. And so that's how we know if the plasma has been taken up into the bacteria. The ones that survive on AMP Resistance is what we're looking for, and once we have it, into a bacterial host. We're going to be able to produce more plasmids. I mean, with our DNA of interest. So it's going to replicate and produce our plasma with our DNA of interest and how we can tell that our DNA has been taken up into the correct spot is that if we introduce what is called X gal X gal is similar to lactose, so X gal is actually the artificial version of black toas. So by introducing X gal, if the bacteria cannot metabolized X gal, then that means that our bacteria have successfully taken up before and DNA and therefore our molecular cloning is successful. So part a following those steps is true. The answer would be part egg. But to further explain, let's look at B, c and D. So and be already the first step is the nature ring using high heat, so that is not correct. We don't de nature. We have to restriction enzyme cut first, so be is not an answer. Let's look at sea, so see they're going to cut. Okay, so that's a good part. They're starting with cutting, and they inserted into the plasmid. Then they allowed to kneel. But then the thing is here, is that they They said at the end here that it shows an inability to synthesize X gal. We're not synthesizing X gal. We're introducing X gal into the environment. We're giving it X gal so that we can see if it metabolizes it or not. So because we're not synthesizing X gal, see is not the correct answer either. And finally, let's look at D. So in part D, they're saying that they want to introduce it into a viral vector. And that is not part of molecular cloning. Because typically, we're going to be focusing it in a bacterial host, right? And additionally, we're not synthesizing X gal. So that is also another reason why we're not picking answer D. So the process of molecular cloning the answer is a thanks for watching. And I'll see you next time. Bye bye

When the UV radiation damages the Cl protein, it stopped repressing the C. R. O. Gene. The C. R. O. Gene is the gene that switches the bacteria Feige into the lyric lifecycle. Ah The crow gene also further represses the D. L. G. Despite the fact that you could reverse or turn off the using lamp, this process has already started and the subsequent increase of crow protein within the host cell will result in the production of new virus particles and the ultimate license and release of these particles.

Hello, everyone. Today I will be helping you with the 70th problem of the chapter 14 problems. So you have a that is asking you Thio, as described in 14.3 transformation or H G T was reported by Griffith in 1928 in an experiment in which which of the following occurred. So which want to know is that you have the heat pathogen with the stuff in it was he treated and then you have a non pathogenic thing. Basically, what happened is that the stuff went into the pathogen and killed the mouse. So if you look at all of them one says he treated pathogenic bacteria recovered their pathogen Jenna City, when incubated with non pathogenic bacteria. Then you have two presidents were transferred to non pathogenic bacteria would just fall. So too, is not it. Your three non pathogenic bacteria acquired pathogenesis ity when incubate Oh, in a broth containing heat treated pathogenic bacteria. Then you have four. Police said No, it's not two or four. And then what happened is that it is three. Because one says that the pathogen a factory recovered their pathogenesis ity, which is false. So then you have B which is asking you Ah, Griffis. So basically what happened in the Griffiths experiment and that basically it's asking you, um, what was observed in the chase and Hershey experiment? So then you know that the bacteria fage culture, um as in four grown in a nutrient containing medium and radioactive labeled using sulphur isotope do not transfer to the bacteria incubated in an unlabeled nutrient containing so that is true. So it is for and then you have ah c R c, which is transformation and transaction increased variation within populations of bacteria um, and R k bacteria by which the following. So, um, it's basically figured it out. See if a transferring dina among different species, which is fault, which is true. That is it actually it and then you have B. This is transferring Freedia across the cell memory, which is false. You have see that says transferring DNA between different strains of the same species false. They have de facto test site toeses of bacteria phages. So then you have, um d that says identify a factor that might affect transformation or hte GT than design a plan to evaluate the dependence of how it will happen. So for me, I would do Ph and then I would see, um if the D nature ring of the protein, um, made it so that the information cannot transfer to a non pathogenic. So it's very similar. Do this experiment. Except this one would be treated with a very low pH. And then it would be mixed with, ah, non pathogenic one. And see if the DNA were that the pathogens transferred to the non pathogenic would. So if you found the cell phone, I hope you have a good


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