pGLO Bacterial Transformation Lab

hi everyone doc romba are here to explain some of the principles involved in bio-rad's pee glow bacterial transformation lab as well as to review the basic protocol and what to expect when you're doing this lab so essentially what we're doing is taking a genetically engineered plasmid and transforming bacteria with it through a heat shock protocol we can then use the you know the principles of the scientific method use our scientific reasoning to show how a bacterium can take up a plasmid and express the genes in that plasmid and exhibit the traits created by the proteins generated so it's really an important fun lab to reinforce or protect perhaps teach gene expression to your students so basically what we're seeing here is the act of transformation taking a plasmid putting it into a subset of bacteria growing up that bacteria and then making those bacteria Express the gene make a protein eventually show this trait and in this case it's glowing so it's a fun lab the pico a plasmid shown here so plasmids you know as a review are these extra chromosomal loops of DNA and they have genes on them so you have your large chromosomal loop and your small plasmids so basically you see to the right here a map of bio-rad's pee glow plasmid and there are some key features here so as you might recall from lessons on DNA replication basically DNA replication starts at an origin of replication and so that's what we have here this ensures that the bacterial plasmid is copied into each clone during binary fission as the bacteria multiply so you get copy after copy after copy of the plasmid in DNA for further expression the BLA gene shown here on the plasmid shown in red encodes the enzyme beta lactamase which is an effective very effective antibiotic this enzyme basically works by hydrolyzing or breaking down any well not any a certain class of antibiotics including ampicillin which is what we use in this lab so it basically breaks that antibiotic down so essentially what you're doing is by giving bacteria this gene you're conferring the trait of antibiotic resistance to any bacterium that possesses that plasmid the next facet of this map I want to direct you to is the RSC gene okay here shown in balloon blue basically this this is the arabinose operon regulator so you're gonna see here in a moment what bio-rad has done to utilize the arabinose operon but basically what you have here is the gene that encodes the regulator that can control gene expression okay so basically the regulator blocks downstream expression of a gene in a certain conformation and that conformation can be changed when you add an inducer which we'll get to here in a moment last but not least the star of the show I want to guide your attention to GFP so it encodes the jellyfish green fluorescent protein so GFP in this case is downstream of the arabinose promoter so it's under the control of that promoter which can be blocked by the RC protein the regulator so we're gonna be able to induce expression of this downstream GFP gene with the sugar arabinose next what we have here is a little depiction of certain elements of the arabinose operon and what bio-rad has done to utilize this operon okay so the molecular biologists at bio-rad have put this Rabbitohs operon to use to regulate gene expression the reason I love this lab so much is it really reinforces that regulation that prokaryotic regulation that the operon affords and operon itself is a concept that has a lot of different facets to it so I really like it in fact molecular biology is is founded on the fact that DNA and genes are modular in nature that is to say that they can be transferred between organisms and they behave the same way in each organism so the genes can be expressed equally the same in both organisms and for ap biology students out there that's a really key part of big idea one really really integral idea for evolution so basically what we're seeing in this picture in this first line GFP the GFP gene downstream of the arabinose operon so where we're regulating its so RSC that regulator protein in this particular conformation blocks the promoter so you can't even see the promoter in this because it's blocked by that regulatory protein however if you add a RAB nose which I'm going to depict as this if you add a ravenous you structurally change to the confirmation of ro changes such that now you can actually see the operon promoter there so the promoter for those BA D genes and and basically now what can happen here in line 3 is that RNA polymerase can come to the Rabbitohs promoter and start expression right so basically you you open up the promoter so that RNA primer is combined there and start transcription and that's basically what we're seeing here in line 4 is that the RNA polymerase has proceeded and it has transcribed this GFP gene here into messenger RNA which will eventually be translated next into that GFP protein so a really interesting lab here okay so what we'll talk about next here is some of the AP Biology surrounding ways that bacteria diversify their genomes there are other screencasts that that talk about transduction and conjugation as ways for bacteria diversify their genomes I'm gonna focus here on transformation okay so transformation is an important one we're using it in fact for the lab and what transformation is is organisms have the ability to be transformed see here my eraser so when this bacterium here I can erase this little segment right here for example if this if this bacterium were to open say the bacterium died and this cell wall started to collapse these plasmids could come out and what would happen is the genes that are on these plasmids if they're able to be transformed that is to come into this second bacterium here the genes on that plasmid will be expressed the same as any plasmid that's already there and also the same as the large chromosomal loop so we're going to take advantage of this ability for bacteria to be transformed in our bio-rad p glow transformation lab today okay so let's introduce you to the actual start of the p glow transformation experiment here you could use this as a protocol of course your teacher will give you a protocol to follow but here's basically what's going to happen you're going to designate two different tubes one as minus P glow a tube that will not receive the P glow plasmid and one is positive P glow that will receive the plasmid now both plates both tubes excuse me are going to get colonies of bacteria from that starter plate okay so you will have grown up or be given a starter plate of bacteria the plate contain individual colonies which you'll use for your research you're gonna add these colonies to the transformation solution using the kit the calcium chloride and that's how we start so you're gonna have bacteria in both tubes now one of those tubes the plus P glow tube is also going to receive P glow plasmid so you'll also have a tube with that to add to the plus P glow experiment okay next we're gonna take those bacteria half of which have been exposed to plasmid the other tube is not exposed to plasmid and we're gonna put them both through a heat shock protocol so a heat shock protocol is a form of transformation so to really shock the bacteria you go from ice to heat for a brief time and then you put them back on ice and it's really important to stick to this timeline of 50 seconds in the 42 degrees Celsius water bath and that is because the bacteria are being shocked many of the bacteria will die in this process certainly more than you want if you extend the protocol past 50 seconds okay the thing is though is that many of those bacteria will also live an even smaller subset of those bacteria will have been transformed that is to say they will have taken up the plasmid provided to them okay so basically you're going to do 10 minutes into on ice so you'll have your tubes here you'll transfer them for 50 seconds to a water bath 42 degrees Celsius and then back on to ice okay next the two tubes will receive what is called LB essentially food nutrients for the bacteria so after your incubation you will add lb so each tube will get some lb and essentially what you're doing there is you're saving the survivors okay you want them to start gene expression and basically started enabling themselves to to divide and express genes to make proteins okay so let's get into basically how you're setting up your experiment okay we're gonna put our various - P glow samples and and plus P glow samples on two different plates okay so you're going to have an lb plate basically that's a gel matrix with food to grow on you're going to have 2 LB ampicillin plates these plates are a gel matrix with food but it also has the antibiotic ampicillin so keep in mind that antibiotics kill bacteria so you'll get two of those plates and last but not least you'll get a plate that has all the above but also a ravenous so you'll get an lb at the cylinder buttocks but you'll also have that inducible arabinose sugar okay so you're going to have a plate that can induce expression of that downstream GFP gene okay so let's walk through this a little further and see what we can get out of the experiment so essentially what you'll do is you'll add a sample of - pee glow to your lb plate and this can be seen as a essentially a positive control both both of these are going to serve as controls and keep in mind a control of something you compare your experiment to for for various reasons but essentially you're gonna you're gonna compare your controls to your experimental samples to validate your experimental results okay so essentially adding Heat shocked bacteria to an lb plate which contains food it's going to serve as a positive control for us okay so the they have not received plasmid they have received a heat shock and we do this control for several reasons it lets us know if bacteria can survive a heat shock okay so that might be a question that can be solved by this by this negative control so if this plate doesn't have bacteria then one would predict that none of the other three plates would have bacteria so this is basically telling us if we have bacteria that can survive heat shock and that they were alive and viable to start okay the other control the LB ampicillin plate that receives bacteria that were heat shocked but not in the presence of plasmid will service this negative control okay this is one that you would predict the death of all the bacteria given the fact that this plate contains antibiotic but the bacteria that were plated on it that were heat shocked in the absence of pee glow so these bacteria had never really had a chance to be transformed and obtain that ampicillin resistance gene the BLA gene okay now when you get your results for this particular plate you're gonna ask yourself well they should all be dead if they are alive did you have bacteria that were naturally resistant to ampicillin that would be a problem for this experiment or you could call into question if your ampicillin is actually working so do you have functioning antibiotic that can kill bacteria if they're growing maybe not okay now let's transition into our experimental samples okay essentially what you're gonna do is you're gonna take samples from that plus P glow and plate them onto the other lb ampicillin plate so again there's food here but there's also antibiotics on this plate they will receive heat shock in the presence of P glow plasmid so they have the opportunity for transformation and essentially what we're doing on this plate is we're checking for those transforming bacteria using antibiotics it's a means of selection the biological reasoning here is that the only way that you can live on a plate containing ampicillin is if you have that bla gene okay were you transformed do you have the bla gene we're asking these questions for bacteria and if they're living on this plate most likely they do have that gene and they have obtained this trait of resistance so we're checking for transformation via resistance to ampicillin okay the last plate here is the fun plate it is the L will be alpha cylinder a bad knows plate and essentially yeah we're confirming again resistance to ampicillin but we're also checking for expression of GFP that's the whole purpose of this experiment to see if we can take that jellyfish protein and express it in bacteria okay so remember arabinose is the inducer for the arabinose operon and it basically opens up the promoter for expression of downstream genes so on this plate we're checking to see if we have induced expression of GFP with arabinose so before we advance to the next slide I want you to pause the video and hypothesize what you would see on each plate given the experimental setup this is sort of explaining the controls the experiments where everything goes but I want you to make sure that you understand the lab and are able to predict what you'll see experimentally when you've done that go ahead and press play and you can rejoin us on next for the data discussion part of this particular video so these are the results that that one would receive in this experiment if everything goes well so let's take a look at this lb plate first this plate received Heat shocked bacteria you would see a lot of bacteria just because this is a growth plate there's no antibiotics sure some of the bacteria died in the heat shock process but keep in mind that colonies contain millions of bacteria there are so many bacteria in fact that you would see what we call a lawn here and a lawn is essentially you know you can see from the shading but you're not gonna see colonies you're just gonna see loads of bacteria it's gonna be basically covering the entire plate next on the LB ampicillin plate you shouldn't see anything on this plate there should be no growth whatsoever you plated bacteria that were not exposed to plasmid during heat shock so yes they were heat shock some of them died for that reason but all of them died all those sort of all the survivors died because they were exposed to antibiotic they did not have the plasmid so they could not be transformed they could not take up the BLA gene and express an antibiotic resistance protein so they were not able to obtain the trait of resistance everything is dead there it should be a clean plate the second lb amp plate here well it received bacteria that was exposed to plasmid during heat shock so it should have some bacteria that were transformed and received the BLA gene thus you would see the trade of antibiotic resistance in the form of colonies each colony represents a bacterium that was transformed you do not see a lawn however in this case because many bacteria were not transformed and consequently died because of the exposure to the antibiotic ampicillin but each of these colonies here was a individual bacterium that was transformed and thus can survive in the presence of antibiotics so you have some colonies here last but not least let's take a look at our lb amp arabinose plate this plate received bacteria that was heat shocked in the presence of plasmid like the previous one so you have antibiotic resistance transformants you would also have a rabid nose sugar on this plate remember arabinose is the inducer so it takes the regulator protein and changes its conformation such that RNA polymerase can come to the promoter and start expression so that downstream GFP gene is transcribed and translated resulting in colonies expressing gfp colonies that grow this plate over here although they lived no glowing they are not inducing expression of gfp so if you shine a UV light on this one there's no glowing if you shine a UV light on this one you do get glowing okay so that's really the the the basics of this bio red pee glow GFP expression kit I think it's fascinating I think it's a really fun lab it reinforces a lot of concepts and ap biology it's a great lab and this is what you would see at the very end using a UV pen light on the Elbe amp arabinose plate these are essentially bacteria that are expressing a jellyfish protein called GFP that brings into focus a lot of important ideas certainly a lot of from big idea one and big idea 3 and ap biology anyway I hope this ties it in nicely I hope it gives you a really nice mental picture of the lab and some of the expectations for it and some of the results that you'll get ok let me know how it went see you next time

Channel: Ron Kinser

Views: 17,194

Rating: 4.9012346 out of 5

Keywords: AP Biology, AP Bio, Investigation 8, pGLO, Big Idea 3, plasmid, transformation, bacteria, E. coli, GFP, gene expression, operon, arabinose, Bio Rad, Doc Ron Bio

Id: 4zXIoZBIBtY

Length: 21min 20sec (1280 seconds)

Published: Fri Mar 02 2018