Lecture 02. Common Tools in Chemical Biology.

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Lecture 02. Common Tools in Chemical Biology.
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Lec 02. Introduction to Chemical Biology --Common Tools in Chemical Biology
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Weiss, Gregory Alan
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UCI Chem 128 Introduction to Chemical Biology (Winter 2013) Instructor: Gregory Weiss, Ph.D. Description: Introduction to the basic principles of chemical biology: structures and reactivity; chemical mechanisms of enzyme catalysis; chemistry of signaling, biosynthesis, and metabolic pathways. Index of Topics: 0:03:11 Our Story Thus Far: Principles to Organize Biology 0:18:39 Modular Architecture Allows Combinatorial Synthesis 0:30:40 Common Tools in Chemical Biology 0:47:04 Fluorophores Allow Visualization of Molecules Inside the Cell 0:52:24 Assays to Detect Molecules in Solution and Cells 0:58:41 Viruses for Gene Delivery 1:02:18 Phage-Displayed Protein Libraries 1:04:55 Vast Libraries of DNA and RNA 1:07:09 Small Molecules Provide Control over Cell Processes 1:10:26 MOdel Organisms for Biology and Chemistry 1:13:41 Bacteria Used to Define DNA as Responsible for Transferring Heredity 1:15:32 Fruit Fly (Drosophilia)
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OK welcome back the a quick Sanchez everything OK great thank you and welcome back today were going to be finishing up the topic that we were talking about last time last time we were talking about ,comma tutorial approaches in chemistry and then we'll talk a little bit about more about our common editorial approaches in biology and I'll show you a couple of examples of this OK but not sufficient OK so OK
so again we're here we just completed our survey of biomolecules I'm going to complete the topic of making common combinations of biomolecules and then I will talk about tools for chemical biology and this is really important because these are the tools that you're going to be using when you write your proposals so I'm glad you're all here today because of you actually need to hear this to be allowed to write a good chemical biology proposal which recall last time I told you I was going to substitute for the final exam this class there is no final exam in this class will not have a final instead on the very last day of class you will hear me a 10 page or so proposal written proposal with figures and I will be original idea something that no 1 on the planet has thought before you will be the 1st and it can be really fun because it's really great to come up with creative ideas and that's really the ultimate goal of science and science is really a creative enterprise articles are to invent new concepts to tell people New Visions of the universe and to do this we have to to understand somehow event these new of experiments to do OK somebody talking to you today about the tools in your toolkit that you're going to be using to do this assignment
I know Kerry talked about these announcements and skipping some stuff so I office hours I had office hours yesterday that got derailed by a student emergency and I know a least 1 you send me an e-mail about that I apologize and I will have office hours today and In addition of I send an e-mail back to that students so I apologize if he came by yesterday there is a student health emergency that absolutely needed my attention so I had to close my door to deal with that OK so apologies there and other office hours tomorrow so Miriam will have a office hour on Friday and hoping critical will be back next week and electricity to Argillite of office hours next Tuesday because so any questions about any of announcements things like that things that we talked about last time questions about the cost structure so I got e-mail from someone and I apologize for not replying to the e-mail was when he went to post online the on the slides that I'm flipping through and the answer is I'm going to try to get to that today and then my plan is to basically posts all of my slides from Creek from the previous year and said I wouldn't we still have a guideline for what the slides will look like chances are all heavily modify these slightly modified his depending on how much time I have before each lecture I mean literally 5 minutes before lecturers making changes to the slides Summers a possible the stop me from doing that I just love this still much fun so because that all the posting of that kind of a guideline for what the slides will look like in advance and then I'll come back with something that's more definitive a so the ended stays lecture than office all the weak ones lies in a definitive way but I'm also going to post last year's week-to-week three-week for etc. OK sounded had a questions about OK great I'm OK so when the
review what we talked about last time if there no questions about any announcements things like that really goes straight into the material look again and so what we talked about last time was the definition of chemical biology Chemical Biology uses techniques and chemistry offer new techniques from chemistry often techniques that had been invented specifically answer problems biology but not always and then these techniques from chemistry argues to address understanding biological systems at the level of Adams and bonds that's the goal of chemical biology to really understand how organisms are living probably do the things they do at the level of Adams bonds OK so I'm really fascinated to know I'm about that hydroxide functional groups that she donates a key hydrogen bond or provides a key um uh Bronstad acid to and some mechanism in an enzyme active site that's the part that makes me right to work this sort of the details of this I basically want to use the pushing that you learned in soft organic chemistry to explain biology and that's the goal of this class and that's a definition of chemical biology so on last time we learned about 2 key principles that organize biology the 1st of these is a central dogma which provides the roadmap for all biosynthesis taking place inside the cell Everything that this cell has to synthesize will flow through the central dogma This is the flow of information for biosynthesis by the cell so everything that yourselves will synthesize is going to be encoded in some way by the DNA inside yourself 0 and can I ask you an empty seat next to you to move over to the right just to open up some set some seats on the edges of some people I know are coming in from other classes so other classes there anything about about what our classes started so if you have an empty seat underwriting dispute over and leave seats on the heads that would be really appreciated behind OK thank you OK so the 2nd key on principle that we discussed was evolution evolution provides a principle that helps us organize vast amounts of knowledge and makes and really in and simplifies biology enormous later on and it's actually a principle that all of you are going to be applying when you design your chemical biology experiments because I will tell you what events that I will not accept any proposals that appalled experiments on humans OK so experimenting on humans has its own special topics that I can actually teach a whole quarter on came requires ethical considerations it requires tremendous design considerations it's nontrivial to sample for example a diverse population of humans and ensure that you're getting diversity so all of those considerations are beyond the realm of this class so instead what I'm going to ask you to do is experiments on a nonhuman organisms you might for example choose cells from humans or you might choose model organisms and by choosing those model organisms you're applying a key principle from evolution which is that that model organism descended from some common ancestor that we share and in doing so I acquired the same mechanisms that govern its chemistry and its chemical biology and so that means that we learn something about this model organism we can then apply that knowledge to understanding how humans work no naturally there's limits to this great if you're model organisms is a salamander and you're interested in understanding how the salamander regenerates its arms when you cut them off which incidentally would be an absolutely fascinating topic for a proposal right but there's a limit to how much analogy to do back to humans right we humans don't have that same mechanism obviously and it would be absolutely fascinating for me to learn from you how it is that you plan to apply the biochemistry major learning about stem cell growth too develops a limb regeneration in humans I would love to learn that a appeared on a so evolution is important to us because it provided it tells us that fundamental processes are more or less the same for every organism on the planet and I'll be show you a few examples in the next few weeks that illustrate this universality of chemical mechanisms In addition we also saw that evolution is is really a tool by which we can evolve molecules to do powerful stuff for us inside the laboratory and I wanna pick that topic up for us today focus on start there and any questions about anything that we saw on Tuesday OK I also got some really fascinating e-mails of front of some virologists an audience to point out there's actually a decline the corner virus protein that is known to start them on a template and then replicate or name that's actually fascinating I wasn't aware of that so there are exceptions to what teaching Your Honor trying to teach you sorta most general thing and yes the will there will be exceptions don't hesitate to point them out to me I'm fascinated by this exceptions to OK so let's pick up where OK before we do 1 last thought about this proposal Simon
but to do the proposal successfully which you have to do is you have to come up with a novel idea OK I will not accept any proposals that don't have something new in the case and all actually asked the TAC do Google searches and and literature searches of Pub that and other sources to verify that would you're proposing to do has not been done before thanks to you whether come up with the creative new idea sounds daunting but let me I'm provides some guidelines on how to do this so the 1st thing that you need but they are a series of experimental tools and their knowledge of the problem peso experimental tools I'm going to provide you today I'm going to give you a toolkit by which you can go out and start to address problems a chemical biology the 2nd portion knowledge of the problems you need to know that actually you know there's a key step in limb regeneration that's not so well understood that 2nd step comes from reading the literature pay in the 1st assignment in this class the juror or journal article book for the Journal article report is designed to help you addressed this 2nd day knowledge of problems OK so in doing the assignments that are required for the class these 2 things are going to come together a paid today we're going to address number 1 and then item number 2 you're going to get by Valentine's Day February 14th you'll have a journal article report and then in doing this assignment you'll be looking at the literature you'll start identified problems in the field that interest you because he'll choose a journal article that's relevant your interests I don't what for solace 1 of the dermatologist because maybe you'll find a chemical biology report that I uses skin cells and looks at saved melanoma development in skin cells and looks out at the level of Adams of bonds but I would love to hear more about that and then by doing this assignment you'll start to know 1 of the big unknowns in our skin cell tumor development 1 of the things that people are fascinated by their mother and designing experiments to address and you'll have the tools from this lecture that will allow you to address those problems have somebody OK and so on how to find the problem the 1st thing I need to ask you to do is start reading either science or nature of peso and I assume many of you science majors if you're not a science major reasons OK you're a fascinating case I'd like to talk to you later so come to my office hours introduced solids everyone else I said you're going to get to get a degree in science I'd like you to read the science nature pretty much for the rest your life pick 1 you don't have to read about and furthermore you don't have to readable I carefully just skimmed through them by doing that you will be informed citizens OK you will know more about science than 99 . 9 9 per cent of the people on this planet and furthermore you'll learn something about what's really cutting edge they only have to spend 10 or 15 minutes flipping through science and nature just looking at the headlines of seeing will be discovered the class of quasars out in unison our galaxy just doing that is enough to the help you will certainly have much better be incorrect cocktail parties let's say because and to the that's enough showcases his party eradication effect so and start reading science nature simply flip through them that helps you are identified problems the 2nd ways to look at pub or MetLife which are the same things and I'll be talking some more about pub Madden in a in a future election a so hopefully your you already know what pub that is hopefully you already know how to apply it all be showing you had applied to chemical biology proper problems at a future lecture but these are the 2 ways have to sift through literature defined stuff that's interesting and that grabs your attention because in the end you want your proposal to be about something that really interest you pay your spend a lot of time on this occasion many many hours and if it's not something that totally interest you know that's not somehow related to the bigger picture of your career aspirations it's not could be as much fun and in the end if it's fun you'll do a better job of all get a better proposal backed out of it and that's the part that interest me OK now I was reading I cheered the admissions committee in Department of Chemistry at UC Irvine and I was reading the application essays from all the wonderful applicants who applied to use your mind this year and I came across this wonderful "quotation mark appear at the more you know the more questions you can ask and said so those questions that you could ask those are the questions that you will be in the dressing with your proposals so our goal is to get your knowledge up to the point we can start asking those questions defense and I know this is all very this seems 3 abstract but it's not news obstructed a moment had some good questions so far are dumping too daunted by the assignment and it will all come together but when you're ready OK last
announcements next week's planned next week we're going to be starting on chapter to please him chapter to an advanced take a look through Chapter 2 even before I get to it Chapter Two is a review of their oppression Chapter One was a review of the biology you need to go and next week will be talking about Europe pushing and mechanistic organic chemistry that you need to know To I do Chemical Biology a peso next week wouldn't have to lectures on mechanistic era pushing now here's the deal albeit of town on Tuesday but I've prerecorded Tuesday's lecture and say trying a little experiment this year and I understand that the the video from Tuesday's lecture the last Tuesday's lecture is already available and it's going to be up shortly posted online OK so I will send you the link to last year Tuesday's lecture and at the same time I'll send you the link to the next Tuesday's lecture pay and so that next Tuesday's lecture than you could watch in your pajamas in the comfort of your your dorm room OK and so on we're going to try that for for Tuesday's lecture I think that's actually I think that will work on but I'll know very quickly if it doesn't work said and then Thursday I'll be back said Tuesday albeit Cal State only giving a seminar Thursday they'll all be back 7 but no arrests so that the next week's planned we're going to be reviewing important stuff from organic chemistry is mainly focuses on structural reactivity of Kerviel's if your weekend 51 C please read read this chapter on carbon reactivity structure things like that there might be 2 or 3 chapters for you to read on mechanisms involving Kerviel's especially the algal reaction 90 per cent of carbon-carbon bonds in chemical biology or made using an algal reaction you need to know what an Alltel reaction it's OK if this word Al balls totally unfamiliar to you go there and on the need to spend a lot of the time this week and reading about it getting familiar with the again of would assume that you know about a noble reaction when we get to it from now on the other hand in your review of US off organic chemistry don't get worked up about reactions for the said is a Carbondale containing compounds anything that you learned in and out of 51 see about how to make a carbon using PCC and is more or less worthless for this class of hedge against PCC is not found themselves it's doubly toxic and so on I think it is as your skimming through because a reviewing if necessary don't get too worked up about memorizing a bunch of being reactions and stuff like that of her instead focuses on mechanisms to focus on the reactivity understand how carbon work that sort of thing that's what you really need to know going into the next few weeks of this class those along said of announcements but pixel everyone for coming up that area that's get started on the actual the new material and I want to talk to you today about Tibet ,comma tutorial approaches burst and I'm going to pick up on the last slide that I showed you last time and to make sure that it's skim through it so quickly that it didn't make any sense to you and then will go on to the next topic OK case last time
oops I was talking about
Modular Architecture in organic synthesis is a what what I wanted but the give me 1 moment to figure this out I guess will live with this guy and so on modular architecture is a design principle that allows you to synthesize compounds in a way that allows access to ,comma tutorial libraries and last time we talked about this principle of cometary were libraries ,comma Torah libraries are big collections of different molecules and in ,comma tutorial library you have a different set of modules that are shuffled around and recombined in a way that makes a whole series of different molecules OK and we talked about last time about this class of compounds called Denzel dies if he had this name should be the name classic compound in the vaguely familiar to you this is reporting class of compounds that's found almost ubiquitously in medicinal chemistry and the use amongst other things the entire depressants and so you could make a commoner library of the based upon this spends days he'd scaffold by varying the or functionality shown here and you can do this by a very straightforward synthetic plan that involves the recombination of a power of AI could a teetotaler together with the panel and so this is a compound has been key Quito and Adelind functionality together can add some sort of help heal he lied and an acid let's just say acid he lied and mean and so these also not together to give you this benzoate appeared framework and that show you the mechanism for lesson is not so important for discussion skip over but you can imagine having say you know 20 different versions of this key talent base compound with different are ones indifferent or to lose 20 odd threes over here .period 20 compounds that have different our 3 and then say 825 compounds that have different art forms when you put this all together and you would do this in individual reaction flask you'll end up with a large number of different compounds OK so that is due 2020 20 pesos 20 of these 20 these 20 of these funds if we make all possible combinations of those how many compounds will we end up with how many bands of appearance 2010 mostly because 22 the 3rd of which it is it it doesn't thank you OK that's a scary enough OK so 8 thousand compounds can very readily be synthesized by starting with the it's simply 60 different of precursor compounds and that's pretty powerful if you at 8 thousand different bands that I opinions each 1 that is potentially some buyout activity then I'm back collection could have a lot of very powerful a new therapeutic compounds that for example OK and we talked about some other different modular framework 2nd now I want shift years that's the example of using that ,comma tutorial chemistry at the end of this synthetic laboratory this principle of course borrows heavily from biology and it turns out that European system uses a
similar principle to develop diverse molecules called into bodies which are 1 of the 1st lines of defense against foreign invaders OK so if heaven forbid you decided to come take the apple up off the ground over there and started chewing away on it and you would find a lot of foreign bacteria that Apple and so on and likely likely on into bodies would play some role in fighting off those foreign bacteria OK so here's the way this works so in the body's job is to be binding proteins their job is to grab on to non self molecules so I'm going to refer to this classic compounds as professional binding proteins that's what they do for a living in a cave that so their professions and on it's 1 of the new systems 1st lines of defense structurally they look like this I told you about earlier from 1 convention for looking at protein structures looking at using a ribbon to trace out the backbone I did tell you really want these errors mean companies curlicues will get to that later but a different convention Pellecchia protein structures just maps surface onto the outside of the protein structure a case of you were able to have you know special electron microscopy eyes you know eyes had amazing power resolution and vision ability with the at the bodies really will look like is something like this because they have a sort of bumpy exterior now this stuff down there and I've colored this into body to highlight on its structural components OK so into bodies it turns out are and are composed of a total of war chains 2 of these chains are called light chains they're shown up here at the at the top ingredients and then this sort of sigh and color on and this purple color and and then there's too heavy chains have the details not so important that get worked up about memorizing how many chains each protein has here's what's important to take into bodies have evolved a mechanism that allows them to recognize diverse binding partners and they do this and by having a series of flexible loops that can accommodate different shapes that they need to bind to OK so I'm turning out to the very tips that to the top of the antibody body up here which is labeled binding site this is where the body will try to attempt to bind to that foreign invaders let's say you picked up a virus when you bit into the Apple now the viruses floating around your bloodstream so that into bodies .period attempt to bind to the exterior of this virus and if we Izumi in and over here this is the tippy top this is just the 1 this is called FAB region of the of the antibodies to the fabric to land value over here and you could see and then in this theater roles here this is an antibody binding to a small molecule so it's pointing to some target the exact target not so important for us but notice how the target is cradled in these loops table loops are gripping this body very gently but it bit but sided with repeating this antigen gently but the intention is fully buried in these loops so these loops are flexible to accommodate many different potential binding partners that flexibility is critical that means they can recognize you know virus wanna virus to working good Ethiopia pick up some totally different viruses they also think that 1 up to hook you hope and on at the same time these provide enough other types of molecular recognition which will talk about later that allows strong enough binding to muster an immune response and then the antibodies basically sound alarm the redcoats are coming and then get the onion response to go into high gear to start killing off that foreign invaders 10 so very 1st line of defense against foreign invaders now the problem and the big challenge is that some of these antibodies need to recognize stuff that you're so you're nearer human organism you I have never seen in your life OK that means that if you travel to India you travel to Ohno of the palace freezer wherever it is if you travel and you pick up some new organism or some new foreign beta enter body there ,comma a library of antibodies needs to be ready to recognize that and of course of you know this stuff has never been seen before the never trained on that so that about this strategy that union system uses is to have a vast collection of potential binding partners OK so make a big collection of different and bodies each 1 with structural differences to be ready to recognize any particular 1 type of data OK now here's the other thing so the size of the
collection is huge occasion these antibodies are produced by immune cells called T-cells which look like that's so 4 B lymphocytes this collection is a fairly enormous it's estimated to be on the low order of about 10 billion or so different bodies OK but earlier I I told you that the and human gene it is only about 20 4 thousand genes price so obviously there can't be found 10 billion different molecules in the system at each encoded by its own gene so instead the strategy that immune system has evolved is a strategy whereby different gene segments are recombined in a way that then produces a comet or a library of different antibodies OK so let me show you so there 40 of these variable genes the module's 25 diversity model 6 joining models and they're shown here so here's the Devedjian said energy and then bye ,comma tutorial did it ,comma tutorial Jean assembly these are brought together to encode the antibody heavy changing OK so that encodes the heavy chain that I showed on the previous slide similarly the light chains are produced by another type of common at Oriel Jean assembly whereby 1 of these visas picked out and etc. One of the Jesus picked out etc OK so in doing this you can get a theory vast library of different antibodies furthermore the into body diversity pool is further diversified by I'm a series of genetically relations that includes 1 variable Jean joined so when the genes are joined together they're not sort of glued together neatly instead there's little parts that are clipped off or added and then furthermore there's a process called Type mutation that goes through and I am makes tiny little mutations in the coding sequences as well so in it and you end up with around 10 billion or so different and 2 bodies each 1 different structurally and potentially able to recognize whatever foreign invader you happen to counter during your life it makes sense OK so to summarize what we're saying is a strategy for ,comma twirl synthesis that's used the laboratory and also used by your ourselves OK in both cases there are these modules that are shuffled around and then rejoined them in literally random fashion to give us a vast collection of different molecules and then we hope that these different molecules are going to be functional when the time comes that we actually need them it makes sense to take their question over here we used to call there's so many of you know some of them against those of OK yes so there's a separate processors who tracks out things that recognize self as well yeah that's interesting question as well so there expressed wish Joshua because OK
changing gears and so the last topic in Chapter One is a survey of the tools that we need a chemical biology to be able to address problems and address the frontiers of chemical biology so I'm going to have a very quick survey in the next 15 minutes or so I'm going to share with you a series of different tools that you can then use in your proposals a place that they could as you're trying to put together a tool and this is going to be the hammer saw the nail gun whatever OK so these are the things that you need to and but to address to design experiments a chemical biology OK so again this is useful for planning your proposal Siemens 1 but this also provides a toolkit for further experiments were going to be referring to this toolkit quite a bit in this class so later in the quarter albeit that say Oh yeah you member those into bodies that I mentioned earlier and those are now going to be a and this kit is very diverse and vast and it ranges from chemical reagents to the entire model organisms and there's a huge amount of diversity and in the in that in that range of different tools so chemical biology as a field uses all kinds of different techniques it uses techniques for molecular biology at uses techniques from the very latest in nonlinear optics and into image sells them and everything in between OK in addition I also want you to know these tools because I want you to altered design experiments on the fly to determine you know X became a very common mid-term question for me would be How would you design an experiment to address you let in what kind of us signaling chemical signaling is being used by the gut bacteria your gut bacteria to too let's let them let their neighbors that sugar has arrived occur such as a pretty interesting question and want to know whether you you do that on OK in addition I want you to know how to describe negative and positive controls were going to be talking about experiments and all the experiments have both negative and positive controls so when we talk about that topic 1st a pace-setter going to be designed experiments you need to know 1st when a negative control is and what a positive control as it is you need to be altered designees into any experiment that you want to design OK so good experiments have both positive and negative control positive control 1st as a positive control is a set of experimental conditions that provide expected resource response were a positive result OK so in this case you could basically wanna know does the conditions in my class produce in the ghetto prepare produce a amplified DNA or something like that and so what you'll do is I'll start with a on sample that you know should work a certain way and your experiment OK it should give you a predetermined result and it should be completely consistent every time it should be very itchy give you that expected result every time so this tells us that our experimental apparatus is working at a need to know this because I'm oftentimes experimental apparatus in chemical biology labs isn't simply a stir and you know hot plate reading just test hot plate by sticking your fingers on a for a nanosecond of the chemical apparatus might be you know a tiny little migrant migration a fugitive and even you shot and a bunch of different reagents unit tend agents all of which are clear none of which you can really assay all that readily so what you do is you set up a set of conditions where you know the results and then you see that we can result is read capitulated under experimental conditions present this as a positive control and you always want to have 1 of these good experiments have positive controls good experiments also have negative controls so this is where you leave out some experimental conditions in your experiment maybe leave out the test sample OK so earlier I was talking to you about them trying to assay but let's just say I'm some sort of microorganisms found in your stomach that responds to the presence of sugar OK and maybe you want to know how and whether that microorganism releases in all the signal to its neighbors can't just about experiment so you're experiment your experimental apparatus will be measuring the concentration of it all your positive control will be say some bacteria that you know releases and all and that tells you whether it's from his working the negative control can be entirely missing the bacteria because you do the exact same experiment but you will need not the bacteria and no in Dole should result if you see and all resulting that tells you that you have a problem that tells you that's you have a say in contaminant for example this should result in a failed experiment on negative results so it's experimental condition missing a key element say the test sample the thing that you're trying to test case and again it should result in a failed experiment if it does not result in a failed experiment that tells you that in your conditions you have some sort of source a contamination you absolutely need these negative controls OK because all too often a chemical biology we have lots and lots of contaminants and there are lots lots of false positives and we just don't like that kind of thing you want to know that if you're going to tell your friends down the hall that you discovered on a new basis in the the DNA sequence and you wanna know that actually that's the real thing a credit unit that telling your good friend US-Soviet turns out to be totally


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