 Okay, the materials you will need for this assignment is a roll of packing tape, two paper clamps, small ones are better, two beads, and these beads can be glass beads, wooden beads, plastic beads, as long as they have a hole that you can thread string through, it doesn't matter. A single rubber band, a single marble, a pair of scissors, some string, and this string can be variable thickness, it doesn't matter, this is a waxed coated string, and as long as it will fit into whatever bead you decide, as long as you can thread the bead onto it, it doesn't matter, and the bead should be able to move freely on the string. You will also need the instructions for this assignment including the piece of paper that has the two operons on it shown here and here. So the first step is to take your operon on your sheet of paper and to cut out these operons. In order to cut these out, if you'll notice there are arrows depicted on each of the operons, you don't need to include the arrows in the cutout, cut inside the arrows as close to the frame of each operon as you possibly can. When you're done cutting out the operon, you should have two operons that look like this. We're then going to take the operon and we're going to laminate it, and we're going to laminate it using the packing tape. So take your first operon on a clean surface, I have a glass cutting board that I'm using. Try not to do this on a table that has a good finish on it, or you will find that the tape will take the finish off the table. So you're going to measure the thickness of your packing tape, and you're going to measure it to the operon, and you're going to cut it. And then when the operon is laying on your flat surface, you're going to lay the piece of packing tape over the operon and press down. That gives you a nice, smooth, protective cover on the one side of the operon. You can then peel the operon off the glass surface, or whatever surface you taped it to, and then flip it over so that the sticky end is pointing up. You'll then take your packing tape, measure out a second piece of packing tape that is the same length as the operon, or a little bit longer is probably best, and you're going to lay it down onto the other side of the operon. It doesn't have to be exact because we're going to trim the edges, but this just provides a barrier to protect and strengthen your operon, as well as it gives us a clean, shiny surface to write on. The next step is to trim the excess tape that is surrounding the operon. So you can do this by cutting as close to the edge of the piece of paper or the frame of the operon as possible. You might want to leave a little bit of tape to protect the edge of the operon, but you don't have to leave very much. And there you have your protected, laminated operon. You're going to repeat the steps on your second operon, laminating it as well. Okay, once you have the two laminated operons, you should then go back to your cutout sheet, and you'll notice that on that sheet, I have a second copy of it here, there is an arrow in the PLAC region and an arrow at the end of the A, or the end of the operon. These arrows indicate where we're going to attach the string. So you want to take your piece of paper that has the arrows on it, and you want to take a piece of string, and you want to measure, and measure a piece of string that goes about a half an inch past the arrows, both arrows, and then you're going to trim that string. So I have a piece of string that stands just past both arrows, which you can see here. And then you're going to cut a second string the same length as the first string. So you can either measure again on the arrows, or you can just measure it to the string you've already cut and cut that as well. Okay, so once you have your two pieces of string cut to the appropriate length, you're going to take a piece of packing tape that's about half an inch in width, so I just cut off a piece using my scissors, and it's about half an inch in width. And you're going to take your first operon shown here, and your first piece of string, and you're going to line the string up across the top edge of your operon. And then you're going to take the piece of tape, and you're going to lay it down, lining it up so that the tape doesn't pass the transcription start site. And you're going to stick the tape down so that it traps the string on the top edge of the operon, and then you're going to fold the tape over onto that operon. So as you can see here, the string is now attached to the top edge of my operon. And you're going to do that to both of the operons with both pieces of string. Okay, so you should now have two operons, both that have string taped to the promoter end, and then a free end of the string of the other strings, of both strings. Then you want to take your bead, and this is really important to feed the bead onto the string prior to taping the other end of the string. And you're going to do this one bead on each operon. So now we have both operons that have the bead on the strings. And once the beads are on the strings, then you're going to take another piece of tape that's about a half inch width, and I just take a big piece and cut it as I need it. And you're going to lay the string down along the edge of the operon, and you want to make sure it's fairly tight so that it lays flat along the string with the bead sitting on the top of the operon. So you don't want it to be so tight that it's going to fold the operon. You don't want it to lift the operon off, so make sure it's laying nice and flat. And then at the other end, the opposite end where the A is, you're going to do what you did at the other end and take the piece of tape and lay it over both part of the operon and the string. And then you can peel it off your surface, flip it over, and flip the tape over the end of the operon. Now what you may end up with is an end that has an overhang of both the string and the tape, and we can just easily trim that off. So now you should have an operon shown here that runs along the top edge with the bead that freely slides along the operon. And we'll do the same thing with the other operon. So the next step is to take your clamp and your rubber band. So the rubber band serves a purpose of holding the marble in place on the clamp shown here because otherwise the marble will fall off. So we're going to take the rubber band and we're going to create a trap for the marble to go. So you're going to loop the rubber band over the two metal prongs of the clamp fold it over a second time and that should produce a bottom base that looks like this. So your rubber band is still fairly loose, so you're going to cross it again. And now this time, when you bring it over, only bring it over the first loop then cross it in the center and bring it over the second loop. And that creates an X shape in the center of your rubber band or between the two clips. Now, if your rubber band is still too loose, because yours might be bigger than mine, you can do that a second time to create a double loop, a double X loop in the center. So that's going to be a little tight for me. So I'm going to take the one loop off, but it completely depends on how big your rubber band is. So now I have this area in between the two metal handles of the clamp that I can use to fit the marble into shown here. And you have a diagram of what this should look like that is attached to your assignment. But at the end of the day, ta-da, we have a marble trapped in the clamp being held by the X shape that we have made with our rubber band. OK, so now you should have both of your operons that have the bead on the string on the top edge. You should have a clamp that is free to open and close like so. And you should have a clamp that has a marble trapped inside it. So in this case, you can't open the clamp because the marble is preventing it from opening. All right, so in order to visualize this activity appropriately, you will also now need your dry erase marker and keep your eraser handy just in case. So your operon consists of a promoter region with a transcriptional start site. This is where transcription will start in creation of the messenger RNA. We also have an operator site as depicted by an O and the three structural genes that are related to the lac operon. They are Z, Y, and A, Z codes for beta galactosidase, Y codes for permease, and A codes for transacetylase. So because this operon is laminated, we can freely draw on it. So if we have a mutation or a broken gene, we can cross it out. We can draw on here if we have some sort of mutation. And then in order to create our scenarios, and then we can freely erase as shown here. The free clamp serves as our repressor molecule. So in the normal case, the lac operon is in the off position, meaning that transcription is not occurring. So weak, and that is because the repressor is bound to the operator region. So if we think about the bead on a string as our RNA polymerase, RNA polymerase will create RNA by starting at the transcriptional start site and moving along the operon in order to create the structural genes. Okay. When the repressor molecule as depicted by the clamp is bound to the operator, and we bind it to the operator by clamping over the string, RNA polymerase is unable to transcribe the gene. See that? So if RNA polymerase is unable to transcribe the gene because you cannot slide the bead, which is RNA polymerase along the string, then we do not get transcription of Z, Y, and A. And we do not get creation of beta-galactosidase permease or transicillase. So this is the off position of the lac operon. The second scenario is when an inducer molecule is present. So the marble depicts the inducer molecule. When the marble is bound to the inhibitor, which is the clamp, the inhibitor is unable to clamp onto the operator. See? So I can't open the clamp because the inducer molecule is bound. So if I can't open the clamp, the clamp is not going to bind to the operator. If the clamp doesn't bind to the operator, then RNA polymerase is free to move along and transcribe the genes Z, Y, and A. So this is a scenario. In the presence of the inducer molecule, in the case of the lac operon, the inducer molecule as depicted by the marble is lactose, well, allolactose, but in the presence of lactose. Lactose binds as the inducer molecule to the repressor or the inhibitor. The inhibitor is unable to bind to the operator and we get transcription of the three structural genes. All right, so in your assignment, I have provided you with a couple of different scenarios. The first scenario is an IS mutant. So an IS mutant means that the repressor protein is unable to be in this confirmation. It's unable to bind lactose. So an IS mutant, if it's unable to bind lactose, it looks like this, the free molecule. So no matter whether or not lactose is present, it is unable to bind to the repressor protein. So you'll never see it in this confirmation. So we can take this and put it aside. An IS mutant, if it never binds lactose, is always free as we can depict by using the clamp to bind to the operator. So if the IS mutant is present, it will always bind to the operator whether or not lactose is present. And if it always binds to the operator, then we will never get transcription of the genes Z, Y, and A. So this is an IS mutant shown here in both the presence and absence of lactose. The second scenario is an OC mutant. So here's where your little marker comes in handy because we can write on here OC. OC means the C stands for constitutive expression of the operator. So this means that no matter whether or not there is a free repressor, meaning not bound to lactose, it can never bind the operator. So if this is present, but we have an OC mutant, this will never bind to the operator. So we can take this molecule, this repressor, and put it aside because it's not going to be able to bind OC. And if it cannot bind OC, then RNA polymerase is free to move along the string and therefore transcribe genes Z, Y, and A shown here. So whether or not lactose is present, an OC mutant is always able to transcribe genes Z, Y, and A. The third scenario is an I-mutant. I- means it's a repressor molecule that looks like this. So it is unable to ever bind O, the operator, whether that operator is an OC mutant or a regular O mutant. So whether or not lactose is present, the inhibitor is unable to bind either the OC or the O mutant. So you're always going to get transcription of genes Z, Y, and A. And then the final can be a combination of mutations of your structural genes. So let's look at two different scenarios. So here we have in a scenario where we have an OC mutant and a Z- mutant, but Y and A are still functional. And then we have a second operon where the operator is not mutated, but Y is mutated. So we depict structural gene mutations with a minus superscript beside the gene. You can also, if you'd like, to just cross out the gene. I sometimes do that to remind myself that this gene Z on this operon will not be transcribed and this gene Y on this operon will not be transcribed. So here we have a regular inducer molecule. So let's look at the first scenario and that's in the absence of lactose. So in the absence of lactose we have an inhibitor molecule that is not bound to lactose. So it's able to bind the operator. Now because this operon is an OC mutant, this inhibitor molecule, repressor molecule, is unable to bind the operator. So on this operon, whether or not lactose is present or not, we always get transcription of the structural genes. But because structural gene Z is mutated off of this operon, we will only get functional permease and transestibilase being made. Whether or not lactose is present. On this operon where we have a functional operator, there are two different scenarios that will occur. The first is that in the absence of lactose, the repressor molecule looks like this. It's a regular clamp that is able to bind the operator to prevent transcription from occurring. So transcription of Z, Y and A will never occur in the absence of lactose. So you will never see functional beta-glactosidase permease or transestibilase. Now in the presence of lactose, this is what the inhibitor molecule looks like. Lactose inhibits, sorry, acts as an inducer molecule by inhibiting the repressor from being able to bind the operator. So in that case, we get transcription of the structural genes. Now because permease, the Y gene, is mutated, in this case, we will only get functional protein for beta-galactosidase and transestibilase. Permease will never be transcribed from this operon because the gene itself that codes for permease protein is mutated and broken. But in the presence of lactose, transcription will occur, but translation of permease will not occur.