DNA cloning and recombinant DNA
Introduction to DNA cloning.
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- If bacteria already have plasmids, when they take up the genetically altered plasmid is the bacterias native plasmid kicked out or do they become neighbors?(27 votes)
- Vector plasmids are generally small enough that it's uptake is negligible to the overall size of DNA in the host. In cases where the host nucleus is smaller, bacteriaphage viruses with individual strands are used as vectors, and only inject small amounts of genetic material(17 votes)
- how to isolate a gene which is separated by restriction enzyme from DNA strand?(9 votes)
- Restriction enzymes recognize where to cut by recognizing certain 6 base pair recognition sequences (for example, GAATTC).
“Restriction enzymes cut double-stranded DNA at specific locations based the pattern of bases found at those locations. These enzymes predictably cut both strands because the sequences they recognize are palindromic”
DNA ligase enzymes can be used to stick the cut DNA pieces back to each other or to a new DNA sequence
If cut by the same type of restriction enzyme, they will have the same type of sticky ends (because the restriction enzyme recognizes only a certain type of recognition sequence). That way, it is easy to pair up the different DNA in the vector molecule with the DNA sequence because they have the same sticky ends
“Recombinant vector molecule cannot be created unless the vector and source DNA is cut with the same restriction enzyme”
The process of separating and isolating DNA fragments after the sequence was cut off:
DNA fragments can be separated by gel electrophoresis.
Since DNA fragments are negatively charged molecules (because the phosphate group has a negative charge), they can be separated by forcing them to move towards the anode (positively charged electrode, attracting the negative charge) under an electric field through a medium.
The medium is Agarose gel, so DNA fragments [the big leftover DNA and its cut off sequence] separate according to their size though the sieving effect produced by the Agarose gel.
The smaller DNA fragments will move further down because of the complex sugar structure of Agarose gel letting smaller DNA pieces to pass more easily. Then, the separated bands of DNA are cut out from the Agarose gel and then extracted from the gel piece using elution (process of extracting one material from another by washing with a solvent)
- Around8:14Sal mentions that you can add an antibiotic resistance gene to the plasmid to select for the bacteria that took up the plasmid. But going back to the "paste" step - how do you know that the plasmid took up both the antibiotic resistance gene AND the target gene? I assume that the percentage of plasmids that take up the gene is not 100% so it stands to reason that there would be a portion of plasmids that take up only one of the genes.(8 votes)
- Great question! Basically, you would know after two steps:
From my understanding, an antibiotic resistance gene would be inserted into the plasmid first. By growing those bacteria in an environment that contains the antibiotic, you can select for the ones that took up the antibiotic resistance gene now contained in the plasmid.
After isolating that plasmid (that now has the antibiotic resistance gene), you can add the target gene through the "paste" step. When grown on antibiotic-containing media, you will select for bacteria that took up the plasmid containing both the antibiotic resistance gene and your insert.(2 votes)
- How do recombinant plasmids transform into E.Coli using Ca ion?
Comparing two transformational methods is electric impulse and using Ca ions?(4 votes)
- calcium chloride makes E. coli competent (able to take up extracellular DNA from the environment in order to perform transformation [have a genetic alteration due to the extracellular DNA] ).
"The addition of calcium chloride to a cell suspension (state in which the particles of a substance are dispersed but not totally dissolved in a fluid) promotes the binding of plasmid DNA to lipopolysaccharides. Positively charged calcium ions attract both the negatively charged DNA backbone and the negatively charged groups in the lipopolysaccharides inner core. The plasmid DNA can then pass into the cell upon heat shock"
E.coli is a Gram-negative bacteria so has lipopolysaccharides, therefore it is required to use the calcium chloride to allow the plasmid with DNA to pass through and be accepted.
“E. coli cells stain Gram negative”
Why lipopolysaccharides are in the way for Gram negative bacteria:
Gram-negative bacteria have 2 plasma membranes (unlike Gram-positive that have 1).
so its structure is [from inside to outside]: plasma membrane, thinner call wall than Gram-Positive, another plasma membrane, capsule.
Lipopolysaccharides are molecules on the outer leaflet of the second plasma membrane.
Lipopolysaccharides are only present in Gram-negative bacteria like E.coli
- At5:50sal said there are different ways to shock the bacteria but he only mentions the heat shock, what other ways can you shock the bacteria into accepting the plasmid?(4 votes)
- The other major method of transforming bacteria is known as electroporation.
In this method the solution containing bacteria is "shocked" (exposed to a short duration pulse of high voltage). This is believed to create temporary holes in the bacterial membrane that allow DNA to get inside.(6 votes)
- How can we collect the plasmid DNA in the first place??(3 votes)
- Typically when you are performing lab work, you will buy the plasmid from another source. You should examine the plasmid DNA sequence (typically these sequences are well documented online) before you buy it to make sure that it meets certain criteria. When using a cloning vector, it is critical that the cloning vector and the desired gene both have the same restriction enzyme site. This allows for the creation of the same "sticky" DNA ends as shown in the video to allow for the gene to stick to the plasmid. It's also important that you know where this restriction enzyme is cleaving. If the restriction enzyme cleaves in the middle of the antibacterial resistant gene, this gene will no longer work! Remember that it is critical that at least one antibacterial resistant gene is working in the plasmid to allow for selection of bacterial colonies that taken in the plasmid.
Hope this helps!(3 votes)
- Given that the bacteria can resist antibiotics, and it is e-coli, how would you kill it if it starts spreading and infecting people? Will it be dangerous?(3 votes)
- Such a thing can be dangerous and fatal and is, unfortunately, happening right now. Some species of bacteria are becoming resistant to all known antibiotics, known as superbugs. While it will certainly take more testing to ensure that these methods work, some proposed solutions to this problem include rotating antibiotics, creating new antibiotics, and undergoing phage therapy, where viruses known as bacteriophages are used to fight the infection.
Did this help?(4 votes)
- Hi guys! I am thinking with a Chemistry perspective and I suddenly remembered that enzymes are biological catalysts. And catalysts are supposed to catalyse both forward and backward reactions to the same extent without altering the end point of the reaction. So seeing what restriction enzymes do, I presume it catalyses the breaking down of DNA molecules, but breaks only some specific bonds. If so, the end point should be a completely broken down molecule, which luckily takes a super long time because its structure is quite stable. I think I've wandered away from the main point a little, but I just wanted to give more info and be clearer. Anyway, my point is, the forward reaction is cutting a section of DNA molecules and the backward reaction is sealing the sticky end. In this case, why do there seem to be two end points (is the environment changed or something?) and why are two different enzymes/catalysts (restriction enzyme & ligase) needed for forward and backward reactions?(4 votes)
- So, there is an antibiotic resistance gene to separate the bacteria containing the plasmid from the bacteria not containing it. But isn't there any other antibiotic resistance gene to make the difference between the plasmids which correctly inserted the gene and the plasmids which didn't?(1 vote)
- When I was a biology TA in college, we did bacterial transformation not only with an antibiotic resistance gene, but also a gene that coded for proteins that fluoresced. Therefore, the bacteria that took up the plasmid not only survived the antibiotic, but also fluoresced under UV light. That made it a lot easier to see the bacteria that took up the plasmid successfully. The only way that the bacteria would glow (and would have the resistance) is if the bacteria was transcribing the DNA and creating the proper proteins for the resistance and the glow, so if the plasmid wasn't picked up and properly transcribed and translated, it wouldn't work.(4 votes)
- i ask if there is a restriction enzymes that can be used for cutting a specific gene from a virus(3 votes)
a virus is a single or double strand of RNA or DNA.
there are animal, plant, and bacteria viruses, which are all just a packets of DNA/RNA that gets mixed with the hosts DNA/RNA to perform a deviant function.
specific restriction enzymes can cut DNA and RNA.
they were actually discovered studying a virus (Lambda phage). so of course there are some that cut genes from a virus.
it was noticed that certain strains of E.Coli reduced the bacteriophage infection by cutting the virus's DNA
about restriction enzymes:
"These enzymes are found in bacteria and archaea and provide a defence mechanism against invading viruses. Inside a prokaryote, the restriction enzymes selectively cut up foreign DNA in a process called restriction digestion"
for certain E.Coli strains:
"restriction is caused by an enzymatic cleavage (separation) of the phage DNA, and the enzyme involved was therefore termed a restriction enzyme"
- [Voiceover] Let's talk a little bit about DNA Cloning. Which is all about making identical copies of a piece of DNA. And usually it's a piece of DNA that codes for something we care about, it is a gene that will express itself as a protein that we think is useful in some way. Now you might have also heard the term cloning in terms of the Clone Wars in Star Wars or Dolly the sheep and that is a related idea. If you're cloning an animal or an organism, like a sheep, well then you are creating an animal that has the exact genetic material as the original animal. But when we talk about cloning and DNA cloning we're talking about something a little bit simpler. Although, as we'll see, it's still quite fascinating. It's identical copies of a piece of DNA. So how do we do that? Well let's say that this is a strand of DNA right over here and I'm just drawing it as a long, but this is a double-stranded, and I'll just write it down, this is double stranded. I don't want to have to take the trouble of keep drawing the multiple strands. Actually, let me just draw, let me just try to draw the two strands just so we remind ourselves. So there we go. This is the double-stranded DNA and let's say that this part of this DNA has a gene that we want to clone. We wanna make copies of this right over here. So gene to clone. Gene to clone. Well, the first thing we wanna do is we wanna cut this gene out some how. And the way we do that is using restriction enzymes. And there's a bunch of restriction enzymes, and I personally find it fascinating that we as a civilization have gotten to the point that we can find and identify these enzymes and we know at what points of DNA that they can cut. They recognize specific sequences and then we can figure out well which restriction enzyme should we use to cut out different pieces of DNA, but we have gotten to that point as a civilization. So we use restriction enzymes. We might use one restriction enzyme, Let me use a different color here, that latches on right over here and identifies the genetic sequence right over here and cuts right in the right place. So that might be a restriction enzyme right over there and then you might use another restriction enzyme that identifies with the sequence at the other side that we wanna cut. So let me label these. These, those things right over there those are restriction enzymes. Restriction enzymes. And so now you would have, after you applied the restriction enzymes, you will have just that gene. You might have a little bit left over on either side but essentially you have cut out the gene. You've used the restriction enzymes to cut out your gene and then what you wanna do is you wanna paste it into what we'll call a plasmid. And a plasmid is a piece of genetic material that sits outside of chromosomes but it can reproduce along, or I guess we can say can replicate along with the machinery of the, the genetic machinery of the organism. Or it can even express itself just like the genes of the organism that are in the chromosomes, express themselves. So then so this is where we cut, let me write this, we cut out the gene and then we wanna paste it then we wanna paste it into a plasmid. And plasmids tend to be circular DNA so we will paste it into a plasmid. And in order for them to fit there's oftentimes these overhangs over here. So you might have an overhang over there, you might have an overhang over there. And so the plasmid that we're placing in might have complimentary base pairs over the overhangs, which will allow it easier, it will become easier for them to react with each other if they have these overhangs. So let me, we're pasting it into the plasmid. And this is amazing because obviously DNA, this isn't stuff that we can, you know, manipulate with our hands the way that we would copy and paste things with tape. You're making these solutions and you're applying the restriction enzymes. The restriction enzymes are just in mass cutting these things. They're bumping in just the right way to cause this reaction to happen then you're taking those genes and you're putting them with the plasmids that happen to have the right sequences at their ends so that they match up and then you also put in a bunch of DNA ligase. DNA ligase, to connect the backbones right over here. And we also saw DNA ligase when we studied replication. So that is DNA ligase, which you can think of it as helping to do, helping to do the pasting. And so now we have this plasmid and we want to insert it into an organism that can make the copies for us. And an organism that's typically used, or a type of organism is bacteria and E. coli in particular, and so what we could do is, we could, let's say that we have a bunch, let's say you have a vial right over here. You have a vial and it has a solution in it with a bunch of E. Coli. A bunch of E. coli. And you actually wouldn't be able to see it visually but there is E. coli in that solution. And then you would put your plasmids, which would be even harder to see, in that solution and somehow we want the E. coli, we want the bacteria to take up the plasmid. And the technique that's typically done is giving some type of a shock to the system that makes the bacteria take up the plasmids. And the typical shock is a heat shock. And this isn't fully understood how the heat shock works but it does and so people have been using this for some time. So if you have a bacteria, you have a bacteria right over here, it has its existing DNA, so this is its existing genetic material right over there, let me label this. This is the bacteria. You put it in the presence of our plasmids so you put it in the presence of our plasmid and you apply the heat shock and some of that bacteria is going to take in the plasmid. It's going to take in the plasmid. And so just like that, it's going to take it, it's going to take it in. And so what you then do is you place the solution that has your bacteria, some of which will have taken up the plasmid, and you put it and then you try to grow the bacteria on a plate. So let me draw that. So let me draw, so here we have a plate to grow our bacteria on, and it has nutrients right over here that bacteria can grow on. It has nutrients. It has nutrients, and so you could say, okay well put this here and then a bunch of bacteria will just grow. So you would see things like this, which would be many, many, many cells of bacteria, there would be colonies of bacteria. You could just let them grow but there's a problem here. Because I mentioned some of the bacteria will take up the plasmids and some won't. And so you won't know, hey when this bacteria, when it keeps replicating it might form one of these, it might form one of these colonies. So this is a colony that you like. So this one is a good colony, put a checkmark there. But maybe this colony is formed by an initial bacteria or a set of bacteria that did not take up the plasmid so it won't contain the actual gene in question. So you don't want that one. So how do you select for the bacteria that actually took up the plasmid? Well, what you do is besides the gene that you care about that you want to make copies of, you also place a gene for antibiotic resistance in your plasmid. So now you have a gene for antibiotic resistance here, and so only the bacteria, and I think it's amazing that we as humanity are able to do these types of things, but now only the bacteria that have taken up the plasmid will have that antibiotic resistance. And so what you do is in your nutrients you grew nutrients plus antibiotics, plus an antibiotic. Antibiotic, and so this one will survive 'cause it has that resistance. It has that gene that allows it to not be susceptible to the antibiotics. But these are not going to survive. They're not even going to happen. They're not even going to grow because there's antibiotics mixed in with those nutrients. And so this is a pretty cool thing. You started with the gene that you cared about, you cut and pasted it into our plasmid. Let me write the labels down, into our plasmid that also contained a gene that gave antibiotic resistance to any bacteria that takes up the plasmid. You put these plasmids in the presence of the bacteria or you provide some type of a shock, maybe a heat shock, so that some of the bacteria takes it up and then the bacteria starts reproducing. And as it reproduces it also is reproducing the plasmids and because it has this antibiotic resistance it is going to grow on this nutrient antibiotic mixture and the other bacteria that did not take up the plasmids are not going to grow. And so just like that you can take this, you can take this colony right over here, and put it into another solution or continue to grow it and you will have multiple copies of that gene that are inside of that bacteria. Now the next question, and I'm over simplifying things fairly dramatically is well you now have a bunch of bacteria that have a bunch of copies of that gene, how do you make use of it? Well, the bacteria themselves, let's say that gene is for something you want to manufacture say insulin for diabetics, well you could actually use that bacteria's machinery, we used its reproductive machinery to keep replicating the genetic information, but you can also use its productive machinery, I guess you could say, it's going to express its existing DNA but it can also express the genes that are on the plasmid. And in fact that's what would give the bacteria its antibiotic resistance but if this gene was say for insulin, well then the bacteria will produce a bunch of insulin, a bunch of insulin molecules, which you might be able to use in some way. And I'm not going to go into all the details of how you will get the insulin out and how you could make use of it, but needless to say, it's pretty cool that we can even get to this point.