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7.12G: Plasmids as Cloning Vectors - Biology

7.12G: Plasmids as Cloning Vectors - Biology



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Plasmids can be used as cloning vectors, allowing the insertion of exogenous DNA into a bacterial target.

LEARNING OBJECTIVES

Illustrate how plasmids can be used as cloning vectors

Key Points

  • All engineered vectors have an origin of replication, a multi- cloning site, and a selectable marker.
  • Expression vectors (expression constructs) express the transgene in the target cell, and they have a promoter sequence that drives expression of the transgene.
  • Transcription is needed for a plasmid to function, without the proper sequences to transcribe parts of a plasmid it will not be expressed or even maintained in host cells.
  • Vectors can have many additional sequences that can be used for downstream applications—purification of proteins encoded by the plasmid and expressing proteins targeted to be exported or to a certain compartment of the cell.

Key Terms

  • Kozak sequence: a sequence which occurs on eukaryotic mRNA and has the consensus (gcc)gccRccAUGG. The Kozak consensus sequence plays a major role in the initiation of the translation process. The sequence was named after the person who brought it to prominence, Marilyn Kozak.
  • transcription: The synthesis of RNA under the direction of DNA.
  • polyadenylation: The formation of a polyadenylate, especially that of a nucleic acid

Vectors

In molecular biology, a vector is a DNA molecule used as a vehicle to transfer foreign genetic material into another cell. The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. All engineered vectors have an origin of replication, a multi-cloning site, and a selectable marker. The vector itself is generally a DNA sequence that consists of an insert (transgene) and a larger sequence, which serves as the “backbone” of the vector. The purpose of a vector that transfers genetic information to another cell is typically to isolate, multiply, or express the insert in the target cell. Vectors called expression vectors (expression constructs) express the transgene in the target cell, and they generally have a promoter sequence that drives expression of the transgene.

Plasmids

Plasmids are double-stranded, generally circular DNA sequences capable of automatically replicating in a host cell. Plasmid vectors minimally consist of the transgene insert and an origin of replication, which allows for semi-independent replication of the plasmid in the host. Modern plasmids generally have many more features, notably a “multiple cloning site”—with nucleotide overhangs for insertion of an insert—and multiple restriction enzyme consensus sites on either side of the insert. Plasmids may be conjugative/transmissible or non-conjugative. Conjugative plasmids mediate DNA transfer through conjugation and therefore spread rapidly among the bacterial cells of a population. Nonconjugative plasmids do not mediate DNA through conjugation.

Transcription

Transcription is a necessary component in all vectors. The purpose of a vector is to multiply the insert, although expression vectors also drive the translation of the multiplied insert. Even stable expression is determined by stable transcription, which depends on promoters in the vector. However, expression vectors have a two expression patterns: constitutive (consistent expression) or inducible (expression only under certain conditions or chemicals). Expression is based on different promoter activities, not post-transcriptional activities, meaning these two different types of expression vectors depend on different types of promoters. Expression vectors require translation of the vector’s insert, thus requiring more components than simpler transcription-only vectors.

Expression vectors require sequences that encode for:

  • A polyadenylation tail at the end of the transcribed pre-mRNA: This protects the mRNA from exonucleases and ensures transcriptional and translational termination and stabilizes mRNA production.
  • Minimal UTR length: UTRs contain specific characteristics that may impede transcription or translation, so the shortest UTRs are encoded for in optimal expression vectors.
  • Kozak sequence: a vector should encode for a Kozak sequence in the mRNA, which assembles the ribosome for translation of the mRNA.

The above conditions are necessary for expression vectors in eukaryotes, not prokaryotes.

Modern vectors may encompass additional features besides the transgene insert and a backbone:

  • Promoter: a necessary component for all vectors, used to drive transcription of the vector’s transgene.
  • Genetic markers: Genetic markers for viral vectors allow for confirmation that the vector has integrated with the host genomic DNA.
  • Antibiotic resistance: Vectors with antibiotic-resistance allow for survival of cells that have taken up the vector in growth media containing antibiotics through antibiotic selection.
  • Epitope: A vector containing a sequence for a specific epitope that is incorporated into the expressed protein. Allows for antibody identification of cells expressing the target protein.
  • Reporter genes: Some vectors may contain a reporter gene that allow for identification of plasmid that contains inserted DNA sequence. An example is lacZ-α which codes for the N-terminus fragment of β-galactosidase, an enzyme that digests galactose.
  • Targeting sequence: Expression vectors may include encoding for a targeting sequence in the finished protein that directs the expressed protein to a specific organelle in the cell or specific location such as the periplasmic space of bacteria.
  • Protein purification tags: Some expression vectors include proteins or peptide sequences that allows for easier purification of the expressed protein. Examples include polyhistidine-tag, glutathione-S-transferase, and maltose binding protein. Some of these tags may also allow for increased solubility of the target protein. The target protein is fused to the protein tag, but a protease cleavage site positioned in the polypeptide linker region between the protein and the tag allows the tag to be removed later.

Novel Expression Vectors Based on the pIGDM1 Plasmid

Escherichia coli is one of the most widely used hosts for the production of heterologous proteins. Within this host, the choice of cloning vector constitutes a key factor for a satisfactory amplified expression of a target gene. We aimed to develop novel, unpatented expression vectors that enable the stable maintenance and efficient overproduction of proteins in E. coli. A series of expression vectors based on the ColE1-like pIGDM1 plasmid were constructed. The vectors named pIGDMCT7RS, pIGDM4RS and pIGDMKAN carry various antibiotic resistance genes: chloramphenicol, ampicillin or kanamycin, respectively. Two derivatives contain the inducible T7 promoter while the third one bears the constitutive pms promoter from a clinical strain of Klebsiella pneumoniae. The pIGDM1-derivatives are compatible with other ColE1-like plasmids commonly used in molecular cloning. The pIGDMCT7RS and pIGDM4RS vectors contain genes encoding AGA and AGG tRNAs, which supplement the shortage of these tRNAs, increasing the efficiency of synthesis of heterologous proteins. In conclusion, pIGDMCT7RS, pIGDM4RS and pIGDMKAN vectors, with significantly improved features, including compatibility with vast majority of other plasmids, were designed and constructed. They enable a high-level expression of a desired recombinant gene and therefore constitute a potential, valuable tool for pharmaceutical companies and research laboratories for their own research or for the production of recombinant biopharmaceuticals.

Keywords: Bacterial expression system E. coli Expression vectors Recombinant protein production.


Cloning Vectors/Plasmids

Download the video from iTunes U or the Internet Archive.

Good morning. So, I see we have a lot of parents here.

How many parents have we got here?

Welcome to the parents. How many of the parents have done the reading for today? [LAUGHTER] Good, because we'll call the parents too, right? We'll see what happens.

All right, so, where are we? We've talked about this diagram that I keep coming back to. If you want to study biological function the two traditional ways to do that were to look at genetics or to look at biochemistry: genetics, the study of an organism with one broken component, those components being genes biochemistry: the study of the purification of individual components from an organism away from the organism, particularly the most important such components being proteins.

What do they have to do to each other?

The unification in molecular biology that occurred in the middle of the century from the 1950s into the ‘60s and really up to 1970 or so, we came to a conceptual understand that genes encode proteins, and therefore these two different ways of looking at the organism: organism minus a component, components minus an organism were complementary points of view, and in theory, you could go from a gene sequence to a protein Sequence, a protein sequence back to a gene sequence, to go for a gene sequence to its function, its function to a protein, except for one to a tiny detail. This was all just conceptual. Conceptually we understood by about 1970 that the DNA made the RNA made the protein. The protein carried out the function but as of then, you couldn't individually work with or purify the DNA corresponding to any particular gene. All of the inferences had been indirect inferences: indirect inferences from bacterial genetics, bacterial regulation or Meselson-Stahl experiments, and all sorts of interesting indirect ways working out the genetic code, but it didn't let you read anything.

This was a problem. Some people in the late 1960s said, great, molecular biology is over.

We understand in principle how life works. Now let's go understand how the brain works. And there was an exodus of some people from molecular biology into neurobiology to now go nail the brain, figured that would be worth another ten years or so. But in fact, remarkably, people began to focus on how you could get to work with individual specific genes. Now, what's so hard about that? I mean, it's not very hard to crack open a red blood cell and purify different proteins. You can purify hemoglobin.

You can purify different enzymes. Biochemistry allows you to purify different components from each other. I want to purify an enzyme: let's crack open a yeast cell, separate the proteins over some column that separates them based on their size or their charge, and I'll get purer and purer fractions. I'll assay each fraction to see which one has the enzymatic activity. But basically I use the physical chemical properties of the proteins to separate them into different buckets. Why not do that with, say, the human DNA and purify out the gene for beta globin, that encodes the beta component of hemoglobin?

What would be the problem of just using physical chemical purification to purify one human gene from another? Well, I mean, it's one very big molecule. Well, I could sheer it up.

Maybe I'll just break it up. Now, let's purify the beta globin containing part. It all looks the same. It's just DNA. It's one chemical polymer with pretty boring properties, and they're not very different.

Any particular DNA sequence in any other DNA sequence basically about the same molecular weight, same charges, there's nothing to separate them by. How are you going to purify beta globin? That was the problem. That's where recombinant DNA came in was recombinant DNA was a remarkable and totally different way of purifying individual components. And the basis of it was this notion of cloning.

If I want to purify out from the human genome, how big is the human genome? The human genome is about three billion bases long. If I want to purify a particular gene, let's say beta globin or some other gene, typical gene, might be on the order of 30,000 letters long.

This is one part in 105 purification I've got to achieve.

Any given gene is only one part in 105 of the human genome. And then, what about a typical mutation? Maybe the mutation that causes sickle cell anemia by changing a single nucleotide and beta globe, well, that's one base pair. So that means I'm trying to identify something that's on the order of one part in 109 actually, a little less than one part in 109 of the whole genome.

Carrying out purifications like: really kind of hard to imagine. But the way it was done was by the invention of cloning.

Let me briefly overview of the idea of cloning, and then we'll dive into the details. The idea of cloning was, the way to purify individual molecules would just be to take the molecules and just dilute them so that there was only one of each model. That's very pure, isn't it? The problem is it's not very much, so you need a way to take a single copy of a molecule, and then make many copies of it. So purification's not hard. You just dilute it down so you work with single molecules but then you need to copy it back again and again and again, and no biochemical technique involves, say, fractionating a cell and replicating some enzyme, you know, copying some enzyme.

You can't copy enzymes, but you can copy DNA, and that was the basis of it. So here's the way it goes. The basic overview we'll look at is take your DNA and cut your DNA of interest, maybe the human genome, into pieces at defined sites Then, paste your DNA, which is more technically ligate, the word we use.

Paste your DNA to some other DNA called a vector. So, cut your DNA and paste your DNA. Each piece of your, say, human DNA gets stuck to some piece of vector.

Insert this DNA into vectors that can replicate in bacteria.

So, I'm going to actually take my piece of human DNA and not just ligate it to any piece of DNA.

I'm going to take my human DNA, and I'm going to ligate it to a vector that has all of the machinery, all of the ability to be copied in a bacteria. Then what I'm going to do is I'm going to transform my DNA into a host cell, a host bacterial cell. Transform means introduce. When we talk about transforming DNA, we're not talking about changing it. It's the word that's used for taking my DNA, stuck into a vector, and introducing it into bacterial cells. Ideally, each bacterial cell would carry one such DNA molecule, and then what I want to do is I want to plate my cells, and select those that carry human DNA, my DNA DNA I've put on it. So, I'm going to put them on a Petri plate and I want only the bacteria that happen to have picked an individual piece of human DNA to grow. So, that's the trick. It's a very simple trick. Take total human DNA, cut it up into pieces, glue it to a vector that's able to be copied so that it's able to be replicated in bacteria, put the vectors into bacterial cells every bacterial cell picks up no more than one vector.

You plate it out, and you simply arrange so that the only cells that grow are those that picked up the piece of human DNA.

And then, every one of these colonies is the descendent of a single bacterial cell that picked up a single human molecule, but is obligingly copying that molecule for you again and again and again and again.

And thus, you have what we refer to this whole collection here is called a library of clones.

This is called a recombinant library because every piece of the human genome is somewhere in here.

You know, this one here probably is active, and maybe this one here maybe is collagen-11 and that one there might, ah, there's beta globin. OK, actually when you look at the plate there's no way to tell but in principle they're all there. So, there will be this question of, how do we look at a library and pull out what the right one is? But somewhere in there should be a bacterial colony that has pure beta globin gene, the DNA for beta globin.

The next lecture will be about how you actually find it.

But today let's just build this library. So our goal is to be able to build a library like this.

So, we have to figure out how to cut DNA, paste, DNA, vectors, etc., etc. So that's what our subject will be today. Let's dive in. First, cutting DNA, how do you cut DNA? Restriction enzymes, etc. It turns out that the way you could cut DNA at particular places is as follows. Let me take a piece of DNA.

Here's a double-stranded piece of DNA. We'll go A, G, C, T, A, G, A, A, T, T, C, T, T, A, C, C, hydroxyl there, three primad. Let's go back on the other strand. What do we have? G, G, T, A, A, G, A, A, T, T, C, T, A, G, C, T, hydroxyl there, three prime. There's my double stranded piece of DNA. It turns out that there exists an enzyme that recognizes that exact sequence: G, A, A, T, T, C. The enzyme goes by the name Eco R1.

This protein, this enzyme, scans along the the DNA, and it finds this sequence: G, A, A, T, T, C.

Actually it's on this strand. What about on the other strand does it say? Same thing. But it's a reverse palindrome.

It's symmetric. That's very good.

And it turns out most restriction enzymes do that. OK, so what it does when it finds that, with the benefit of colored chalk that has just shown up here is it cleaves the DNA fragment like that.

And what it gives you then is a broken double strand with an overhang, T, T, A, A, five prime, three prime, three prime, five prime. This has a hydroxyl here this. This has a phosphate there. And then this other fragment here is A, A, T, T, C, T, T, A, C, C, G, G, T, A. So, what happens is, and this has a hydroxyl five prime, three prime, three prime, five prime I get into two fragments of DNA that have been broken there and have it over. The overhang is complementary.

Those two sequences match each other. There's what's called a five prime overhang and they're complementary So, we have complementary, that is matching, five prime over X. This is called Eco R1 because it's purified, this particular enzyme, from E coli strain R and it's the number one such enzyme that was purified from it. So, it is very simple nomenclature here. Now, here's a question. Why do bacteria have an enzyme like this? There are some people who feel that the reason is that this enzyme is here precisely to allow molecular biologists to cut and paste DNA, and this represents impressions likely, me among them.

How did anybody find this stuff? Well, shaggy dog story, I have to tell you the following shaggy dog story.

So, this is a fun shaggy dog story, and it's an MIT shaggy dog story because it comes from the work of Salvador Luria, who is a very famous biologist who worked here at MIT. So, Salvador Luria was studying bacteriophage. Remember, bacteriophage are the viruses that infect bacteria. So, he was studying bacteriophage, and he took his bacteriophage and used it to infect a strain of bacteria, strain A, and he also used it to infect a strain of bacteria, strain B. So when he did that, what you do is you plate a lawn of bacterial cells.

You kind of have a slush of bacterial cells that you plate here with virus mixed in, and wherever there's a virus, the virus grows, replicates, and either kills or slows down the growth of the cells so that bacterial cells grow everywhere else, but where a viral particle landed there's an absence of bacterial cells and that hole in the lawn, this whole thing is called a lawn of bacteria, and the holes in the lawn are called plaques.

So, when he did this, he found that when he did it on strain A he got a bunch of plaques and when he did it on strain B, he didn't, no plaques. So what what's the simplest explanation for this?

Strain B is different somehow. It's resistant to the virus. I don't know, the virus has to come in and do various things, and strain b isn't compatible with the virus or something like that. No big deal. So it's a resistant strain. But, occasionally you'd get a plaque.

Very occasionally, you'd have an occasional plaque.

So now, how would this be? I said the strain was resistant. How could there be an occasional plaque?

Mutation in, could it be imitation in the bacteria?

Sorry. Well, if it was a mutation in the bacteria there would be one bacteria that had the mutation. It was now susceptible, and it would die. But, the lawn would kind of grow because the cells around it wouldn't have a mutation.

So it's probably not a mutation in the bacteria but what could be? Maybe a mutation of the virus: what if it was a mutation in the virus that was able to overcome the resistance? Ah, so that's OK.

So, what this must be is the existence of a resistant virus that is a virus that can overcome the resistance of the bacteria. So far: perfectly normal, no problem. Now, let's do the following experiment. Let's take this resistant virus, and grow it, again, on strain A and grow it on strain b. What do you think is going to happen when I grow it on strain A? It'll grow lots of plaques. It still grows on strain A, and now what's going to happen when I grow it on strain B? If this was really a mutation that made it able to grow on strain b then it gets lots of plaques because it's now gained the ability to grow on strain B, and sure enough, that's what happens. So, there's nothing funky yet. But now, suppose I take one of these resistant viruses that I isolated here on strain B, I grow it again here on strain A. It grows. I grow it on strain B.

It grows. If I take it again from strain B and I repeat this, it'll still grow on strain A and still grow on strain B. Let's take one, though, from strain A. It's the resistant one which we have just now happened to have grown on strain A. And now, let's grow it again on strain A versus on strain B. And sure enough, it continues to grow on strain A, no problem.

And we grow it now on strain B. And, what shall we get? Well, it should grow on strain B, right, because it was a mutant virus, and it gained the ability to grow on either.

We passage it through B, it grows. We passage it through A.

But the answer was nothing, no growth. How can that be?

We had a virus. We agreed that was a mutant virus that had picked up the ability to grow on strain B, and we demonstrated it has now on either A or B.

We then reached in, and grabbed a copy of it here from strain A, having grown on strain A, and we try it again and it now won't grow on strain B. If this was a mutation, I mean, maybe the mutation reverted, right?

It was a reversion of the mutation. It mutated back. Is that plausible?

No, come on. The chance that all of the copies there would mutate back, come on. I mean, you could repeat this several times and this is always what happens.

What does that tell you about this mutation in the virus?

It can't be a mutation of the virus because if it was a mutation, it would be transmitted through.

But, passing through strain A makes it lose its ability to grow on strain B. But as long as you keep passing it through strain B, it can grow on strain B. This is not your typical genetics. So, Salvador Luria loved this.

And, he really worked out what was going on. And somehow, well, so anyway, they referred to this as strain B having the ability to restrict the growth of the virus. Strain B can restrict the growth of the virus.

That's where this word restriction enzyme comes from. What's really, truly going on here underneath the shaggy dog story? It took a long time before the shaggy dog story that Salvador Luria was the one to really demonstrate is fully worked out. But, what turns out to be the case is that strain B has a restriction enzyme.

That's how it restricts the growth. It has one of these enzymes that can cut DNA at a specific place.

When the virus comes into strain B, it injects its DNA, and the enzyme comes along and cuts the virus's DNA, protecting the bacteria. It's got its own little defense mechanism: pretty cool, pretty cool. So, any DNA that's introduced, if it has the sequence here, it'll take G, A, A, T, T, C, the bacteria cuts it. Wait a second, the bacteria has its own DNA. Why doesn't it chop up its own chromosome? Well, I mean, so one simple possibility would be that if this thing is looking for the sequence, G, A, A, T, T, C in the genome, maybe it's the case that the bacteria has arranged that its own DNA never has a G, A, A, T, T, C. That would be a simple solution, right? But is it a plausible solution? Why not? But just statistically, how often do I expect to encounter a G, A, A, T, T, C? What's the frequency of any given six letter word in a four letter alphabet? It's about one in 46. So, about one in 46 positions will be a G, A, A, T, T, C, and that's about 4,000 letters. So, every 4, 00 letters, I expect to encounter a G, A, A, T, T, C. How big is the E coli genome?

4 million letters. So, how many G, A, A, T, T, Cs will there be? About 1,000 of them. It's just not plausible to imagine that it doesn't have the sites. So, your idea is that if it has these sites, it's got to arrange to protect its own sites. So, how is it going to protect its own sites?

Covers it or something. You could imagine something covers it or something, but you want to alter your own, so it turns out you're exactly right. What happens is there is an enzyme that comes along, and at this position, attaches a methyl group. It modifies the DNA by attaching a methyl group. It turns out that that methyl group is enough to prevent the restriction enzyme from binding.

So, this blocks the restriction enzyme. So, that way the bacteria is able to distinguish between its own DNA, which is methylated, and the viral DNA. So, wait a second, how does that explain my virus that manage to grow? How did my virus manage to grow? It would need to have gotten itself modified also to be protected.

Could that happen by chance? What if the methylation enzyme, the methylase, which is floating around in the cell, “accidentally” methylated the virus's DNA? What would happen then? The virus would become immune.

So, suppose the bacteria was pretty clever, and had a lot more restriction enzyme, and only a little bit of methylase?

Well, you'd imagine that most of the time the restriction enzyme would cut up the viral DNA first.

But every once in a while, the methylase would get there first and protect the virus's DNA.

That becomes an immune virus because it can't be cut by the enzyme anymore. And, if I take that, and I grow it again on strain B, it'll now produce lots of plaques because it was methylated. And, if I grow it again on strain B, it remains methylated because once it's methylated and comes into the cell, it's not cut. And so, its descendants will get methylated.

But, what happens if I ever grow that methylated virus on strain A? Strain A doesn't have the restriction enzyme, and it doesn't have the methylase. So, the progeny phage that grew up on strain A aren't methylated.

They're no longer protected. The protection that the virus has is the protection that comes from this methylation enzyme.

It's not the sequence of the DNA. It's the attachment to these methyl groups. And so, it turns out that if you ever pass this virus through strain A, passage through strain A, the resulting DNA loses is unmethylated. And now, it can be cut.

And it can be cut. Well, this explained the weird results of Luria, that somehow bacteria had a complex defense mechanism of a restriction enzyme and a cognate methylase. The restriction enzyme would cut the sequence. The chromosome would be protected by methylating that site, and usually it would work fine.

Occasionally the bacterial virus would get methylated.

It would be protected as long as it continues to go through strains that have this restricted methylation system. That was it. Now, this shaggy dog story took a couple of decades to work out, and eventually led to Nobel prizes for the discovery of restriction enzymes. They're extremely important because although bacteria do this to protect themselves, they have also given us the perfect tool to now cut DNA where we want to cut DNA. Now, what if you wanted to cut at a G, A, A, T, T, C? You've got Eco R1.

But what if you wanted to cut it cut it in another sequence?

Well, it turns out that if you want to cut it at G, G, A, T, C, C there's an enzyme called Bam H1. If you want to cut it at A, A, G, C, T, T or A, A, G, C, T, T, there's an enzyme called Hmd 3. If you want to cut it at just G, A, T, C like this, C, T, A, G, an enzyme called Mbo 1. And, there are enzymes that cut it this way, enzymes that cut it this way, enzymes that cut it this way, enzymes that recognize four bases, six bases. There are even enzymes that recognize eight bases.

It turns out that bacteria have elaborated zillions of different restriction enzymes that recognize different sequences. This perfect for molecular biologists. Bacteria, of course, are much smarter than we are, having been out this much longer, have developed all of these tools for engineering.

All we have to do is borrow them. So how do you get Eco R1?

We grow out that strain of E coli you purify Wco R1.

And how do you get Hmd 3? You grow up strain of haemophilus influenza. You purify the enzyme. At least, that's how primitive molecular biologists did it. If you wanted to work with a restriction enzyme, you'd grow up the bacteria.

You'd purify the enzyme yourself, and you would just use it in your laboratory. Of course today what does a modern molecular biologist do if he or she should want Hmd 3?

It's in the catalog. So the catalog has 200 restriction enzymes. Yup, PSI-1 is new, on sale, 500 units for $400.

Let's see what Eco R1 is going for.

Eco R1: look at this, 50,000 units $200. That's a good price for Eco R1 because it's a very famous enzyme here. So all you have to do is you give them your credit card number and you have it tomorrow by FedEx. So that's how restriction enzymes are obtained today. So, next up, we can cut DNA any place we want to. We now need to glue DNA together.

Suppose I cut DNA, human DNA, and I'm going to cut it. I'll just take human DNA, your DNA, which I've purified, and I'm going to cut it at all its Eco R1 sites. I can take any other DNA I want.

I don't know, I could take zebra DNA. I could take anything and I could also cut it at Eco R1 sites. I could mix them together, and after mixing them together the fragments will float around and remember this down here has T, T, A, A. This fragment over here from some other piece T, T, A, A, this could be human DNA. This could be zebra DNA if you want to. It doesn't matter. It could be bacterial DNA.

These fragments overlap. They'll hydrogen bond a little bit, but that of course won't introduce a covalent bond here.

I'd really like to make a covalent bond. I would like to attach the piece of DNA from one source to the piece of DNA from the other source by doing the opposite of the restriction enzyme. The restriction enzyme cut at these locations. I would now like to catalyze the rejoining of the sugar phosphate backbone here.

So I would like to rejoin the sugar phosphate backbone. I have a hydroxyl here. I have a phosphate here, and I would like to ligate them together. So how I manage to ligate? What kind of fancy chemistry do I do to ligate these pieces of DNA together? I don't do any fancy chemistry. I again sit at the feet of bacteria who have solved all these problems before. And I ask bacteria, how do you do this? And they say, well, we have an enzyme called ligase. So, you purify ligase from bacteria, you add that, and ligase ligates the fragments together. Why do bacteria have an enzyme ligase?

For a pair of their own DNA. Things go wrong this is part of the DNA maintenance scheme of bacteria. They have an enzyme ligase to appear their own breaks in DNA and, obligingly, you can purify DNA ligase. So you add ligase, today, of course, if you need a ligase, how do you get it? It's in the catalog, absolutely. So, you can glue together any of those things you want. All right, next up, what DNA do I want to stick together? I mean, here I made a silly example.

I'm going to stick some human DNA to some zebra DNA. Why do that? I mean, just to show you that I can doing it, right? I'm just demonstrating that I could stick any DNA to any DNA. Remember, once I've got a piece of DNA it doesn't know whether it came from a human or a zebra. It's just the molecule. You can stick the molecules together, right? But what do I really want to attach my human DNA to?

I want to attach it to attach it to some other DNA that has the ability to grow on its own within bacteria. Vectors: I need to make, here's what I would really like. I would like to have a piece of DNA that has some sequences that contain the recognition sites for replication. I'd like to have some replication initiation sites here. So, a piece of DNA that, remember, because the bacterial chromosome itself, here's my bacteria, bacteria'chromosome replicates itself, and it has the ability to start DNA replication at multiple sites called origins of replication. But, what I would really like is to be able to construct in the laboratory a synthetic piece of DNA that also would function as an origin of replication because then what I could do is in vitro take my piece of DNA, attach it to this vector, and it would now have the ability to grow the bacteria. How am I going to make a piece of DNA?

What kind of engineering tricks can we do to create a small piece of DNA that has all the machinery needed to be able to be copied and replicated just like bacterial chromosomes? That's a pretty fancy feat of engineering.

How are you going to do that? Sorry? OK, so who are you going to ask? If you wanted to do this, you're going to ask the experts. Who are the experts? Viruses or bacteria, or basically, if you want to do anything, the place to ask is the folks who have the most experience. And, the folks who have the most experience are almost always prokaryotic organisms because they are by far the most evolved things on this planet. Anything that can replicate itself and grow every 20 minutes or something like that has had a lot more generations of evolution than you have. And therefore, they are much more optimized than we are. And so you go ask and say, has any bacteria worked out how to do this? Turns out bacteria have worked out how to do this just fine. In fact, most bacteria, at least many bacteria, contain within them, in addition to their own chromosome, small circles of DNA. These are called episomes.

This is the chromosome. Epi means on top of or in addition to. So in addition to the chromosome, there's an episome. The episome is in fact an autonomously replicating piece of DNA that has an origin. And it replicates. Why do bacteria have episomes? It turns out episomes often contain genes. One fo the genes they contain, or some of the types of genes they contain, are resistance genes. There might be, for example, a penicillin resistance gene contained on an episome, or a streptomycin resistance gene.

It turns out the bacteria have these episomes containing resistance genes, and they're not in the chromosome.

They're separate. Now, why would they do that?

It turns out when a bacterium dies and a cell cracks open, the DNA spills out. The next door neibhored bacteria has mechanismis to suck up DNA from the environment. You never know. It might find something interesting out there.

So, it turns out that bacteria are rather promiscuously exchanging pieces of DNA all the time. And so, a bacteria that has an episome that has a penicillin resistance gene can spread it to other bacteria, and it's very nice. It's compact.

It's on its own little episome, autonomously replicating piece of DNA. This is great for bacteria wanting to spread drug resistance. It's not good for human populations, for example, because this is how drug resistance spread through populations. This is why we have spreads of penicillin resistance. Now, of course, wait a second, this whole mechanism of spreading drug resistance, we've only had antibiotics since the 1940s. How did bacteria devise this so quickly? Sorry? Many generations since 1945?

That would be very impressive.

Yeah, but, I mean, why do they have this episome mechanism, the ability to spread DNA and all that? That's an awful lot to evolve in 50 years? Yeah? Something natural like penicillin. It turns out, we didn't think of penicillin. Who thought of penicillin?

Fungi. Right, again, we learn from the lower organisms. Penicillin comes from fungi. Bacteria have been fighting off penicillin for millions and tens of millions of years. So, we may be very proud of our penicillin and all that.

But, they've been at this for a very long time.

This is about war between bacteria and fungi.

That's what this is, OK? So, that's why these things are here. They're here so that bacteria can have these resistance genes against fungi and things like that that make antibiotics. Antibiotics are natural. We've made a few new ones, but most of the antibiotics have been made by nature.

And so, if I wanted to replicate DNA, if I wanted to attach my human DNA to a piece of DNA that's capable of autonomous replication, autonomously replicating circles of DNA, these autonomously replicating circles of DNA are also called plasmids. And that's the word we'll mostly use for them, plasmids. All I need to do is purify a plasmid from a bacteria. So, I find a bacteria that has plasmids.

I purify the plasmid, and then I can cut open the plasmid at the Eco R1 site, OK? So, this plasmid will have an ORI, an origin of replication. I'll cut it open at the Eco R1 sight. I'll take human DNA fragments that I've cut with Eco R1. I'll mix them with plasmid DNA that has been opened up, has an origin. Ligase will come along, join this up, and now I have a circle of DNA that has all the machinery to autonomously replicate, plus my human DNA. Now, if I wanted to get a vector, or an honest to goodness plasmid, I can go to a bacteria, grow it up, purify the plasmid, and cut it. Or alternatively, if I needed the plasmid, say, tomorrow, it's in the catalog. The next section of the catalog has a long list of plasmids here. There's a plasmid there, right? It's a nice plasmid.

Oh yes, let's see, puck is a very good plasmid.

PBR 322 is a good plasmid. The whole section, all this purple stuff are the plasmids. So, you can get the plasmids too.

You place one order, you get the restriction enzymes, you get the ligases, you get the plasmids, no problem. So, I can then take total human DNA, cut up, cut up, cut up, cut up, add in plasmid, and I'm going to ligate together. And then, having ligated my human DNA to my plasmids, I'm going to mix with bacteria. I take some bacterial cells. I add my mixture of these plasmids containing human DNA. And now all I have to do is persuade the bacteria to suck up my plasmids containin human DNA.

How do I teach bacteria to suck up DNA? They do that for a living. That's what they do. They're always spreading material.

They have that ability. All we're doing is we're using their ability. So you get the sense that the kind of engineering that really works in biology is engineering that exploits what nature has been doing for a very long time.

Rather than butting your head up against the problem, usually somebody has solved it, and it's almost always bacteria. So, you've transformed the bacteria.

Now, there are a few tricks you can use to make them a little more transformable.

You can add calcium phosphate, and blah, blah, blah, but you can sort of persuade them to take up the DNA. And then all you have to do is plate them out on a plate.

Plate them out fairly dilutely so there are a lot of single bacterial cells that land on the plate, and wait for them to grow up. Each one of these had a single plasmid, a different plasmid than the next guy over. Wait a second, each one?

How do I guarantee that every bacteria in my test tube took up a plasma? Is that plausible? I mean, I can't guarantee that every bacteria is going to take up a plasmid. Maybe I'll add so much plasmid that every bacteria will take one up. Oh, but that's a bad idea because why? Because then a lot of them will take up more than one.

You don't want to do that. You really only want to have at most one. So, if you were going to arrange so that at random you only have about one, you've got to have a lot that are zero. So, this is a problem. I mean, it's a real waste. My library is going to have large numbers of bacteria that don't have any plasmid. In fact, this transformation process is not so efficient. It's not so efficient. So, we have a little bit of a problem here is that some of these guys will have human DNA.

But, most of them won't. So, what can I do to arrange that any bacteria that did not pick up a plasmid was incapable of growing? Add a resistance gene to the plasmid. Suppose I were so clever as to add to that plasmid, penicillin resistance. So, not just an origin of replication, but suppose I also had a resistance gene here, say, for penicillin resistance or streptomycin resistance, or ampicillin tends to be a very big favorite, ampicillin resistance. Then, my plasmid would have ampicillin resistance gene encoded on it, an enzyme that can, say, break down ampicillin, so on and one way to to my perch you plate I just said ampa cell and now i'll even though most of the bacteria have not picked up a plasmid, only those bacteria that have picked up a plasmid have the ampicillin resistance gene and can grow on an ampicillin containing plate.

Now, how do I get a plasmid with an ampicillin resistance gene? It's in the catalog. It's all there, right? In fact, these occur naturally.

You can, with restriction enzymes, move the ampicillin resistance gene to your favorite plasmid. If you don't like that, you can put in kanamycin resistance, etc., etc., etc. So, that's how you do it. So, we've got the big picture here.

We have now gotten a library, the Library of Human Fragments contained in E coli. The library is a big Petri plate or many Petri plates, each one of which is a colony.

Each colony has a single vector with an origin, a resistance marker, and a distinct piece of human DNA. In this library lives somewhere the gene for Huntington's disease. Over here is a gene for cystic fibrosis, over here a gene for Duchenne muscular dystrophy, over here a gene for diastrophic dysplasia, over here a gene for etc. etc. The only detail, now, you've got a library. You've managed to purify each piece of human DNA away from every other piece of human DNA.

The only question now is how do you use the library?

How do you go to the library and withdraw the correct volume from the shelf? How do you find the one you're looking for? So, we have converted the problem of purification, which in every other form of biochemistry starts by saying, I'm going to purify something based on its distinctive properties, to I'm going to randomly purify everything.

Everything would be purified in its own bacteria, and I've now converted to the problem of finding the one that I want in my library. Next time, we'll talk about how you go to the library and find what you want.


Plasmids: Definition, Types and Replication | Microbiology

In this article we will discuss:- 1. Definition of Plasmids 2. Physical Nature and Copy Number of Plasmids 3. Properties 4. Incompatibility 5. Types 6. Replication 7. Plasmid Curing 8. Use of Plasmids as Coning Vectors.

Definition of Plasmids:

In addition to bacterial chromosome (nucleoid), bacterial cells normally contain genetic elements in their cytoplasm. These genetic elements exist and replicate separately from the chromosome and are called plasmids. The very existence of plasmids in bacterial cytoplasm was revealed by Lederberg in 1952 while working on conjugation process in bacteria.

Lederberg coined the term ‘plasmid’ to refer to the transmissible genetic elements that were transferred from one bacterial cell to another and determined the maleness in bacteria.

Literally, thousands of plasmids are now known over 300 different naturally occurring plasmids have been isolated from strains of Escherichia coli alone. Besides naturally occurring plasmids, many artificially modified plasmids have been developed and used as vectors in the process of gene cloning (genetic engineering).

Physical Nature and Copy Number of Plasmids:

The physical nature of plasmids is quite simple. They are small double-stranded DNA molecules. Majority of the plasmids are circular, but many linear plasmids are also known.

Naturally occurring plasmids vary in size from approximately 1 kilobase to more than 1 megabase, and a typical plasmid DNA is considered to be less than 5% the size of the bacterial chromosome. Most of the plasmid DNA isolated from bacterial cells exist in the supercoil configuration, which is the most compact form for DNA to exist within the cell.

The copy number refers to the fact that different plasmids occur in cells in different numbers. Some plasmids are present in the cell in only 1-3 copies, whereas others may be present in over 100 copies. Copy number is controlled by genes on the plasmid and by interactions between the host and the plasmid.

Properties of Plasmids:

(i) They are specific to one or a few particular bacteria.

(ii) They replicate independently of the bacterial chromosome.

(iii) They code for their own transfer.

(iv) They act as episomes and reversibly integrate into bacterial chromosome.

(v) They may pick-up and transfer certain genes of bacterial chromosome,

(vi) They may affect certain characteristics of the bacterial cell,

(vii) Plasmids differ from viruses in following two ways.

(viii) They do not cause damage to cells and generally are beneficial.

(ix) They do not have extracellular forms and exist inside cells simply as free and typically circular DNA.

Incompatibility of Plasmids:

In some cases, a single bacterial cell contains several different types of plasmids. Borrelia burgdorferi that causes Lyme disease, for convenience, possesses 17 different circular and linear plasmids.

In a condition when a plasmid is transferred to a new bacterial cell that already possesses another plasmid, it is commonly observed that the second (transferred) plasmid is not accommodated and is lost during subsequent replication.

This condition is called plasmid incompatibility and the two plasmids are said to be incompatible. A number of incompatibility groups of plasmids have been recognised in bacteria. The plasmids of one incompatibility group exclude each other from replicating in the cell but generally coexist with plasmids from other groups.

Plasmids of an incompatibility group share a common mechanism of regulating their replication and are thus related’ to one another. Therefore, although a bacterial cell may possess various types of plasmids, each is genetically distinct.

Types of Plasmids:

Various types of plasmids naturally occur in bacterial cells, and the most favoured classification of such plasmids is based on their main functions encoded by their own genes.

Following are the main type of plasmids recognised on the basis of above mentioned characteristic feature:

1. F-plasmid (or F-factor):

F-plasmid or F-factor (“F” stands for fertility) is the very well characterised plasmid. It plays a major role in conjugation in bacteria E. coli and was the first to be described. It is this plasmid that confers ‘maleness’ on the bacterial cells the term ‘sex-factor’ is also used to refer to F-plasmid because of its this property. F-plasmid is a circular dsDNA molecule of 99,159 base pairs.

The genetic map of the F-plasmid is shown in Fig. 5.31. One region of the plasmid contains genes involved in regulation of the DNA replication (rep genes), the other region contains transposable elements (IS3, Tn 1000, IS3 and IS2 genes) involved in its ability to function as an episome, and the third large region, the tra region, consists of tra genes and possesses ability to promote transfer of plasmids during conjugation. Example F-plasmid of E. coli.

R-plasmids are the most widespread and well-studied group of plasmids conferring resistance (hence called resistant plasmids) to antibiotics and various other growth inhibitors.

R- plasmids typically have genes that code for enzymes able to destroy and modify antibiotics. They are not usually integrated into the host chromosome. Some R-plasmids possess only a single resistant gene whereas others can have as many as eight.

Plasmid R 100, for example, is a 94.3 kilobase-pair plasmid (Fig. 5.32) that carries resistant genes for sulfonamides, streptomycin and spectinomycin, chloramphenicol, tetracyclin etc. It also carries genes conferring resistance to mercury.

Many R-plasmids are conjugative and possess drug- resistant genes as transposable elements, they play an important role in medical microbiology as their spread through natural populations can have profound consequences in the treatment of bacterial infections.

Virulence-plasmids confer pathogenesity on the host bacterium. They make the bacterium more pathogenic as the bacterium is better able to resist host defence or to produce toxins.

For example, Ti-plasmids of Agrobacterium tumefaciens induce crown gall disease of angiospermic plants entertoxigenic strains of E. coli cause traveller’s diarrhoea because of a plasmid that codes for an enterotoxin which induces extensive secretion of water and salts into the bowel.

Col-plasmids carry genes that confer ability to the host bacterium to kill other bacteria by secreting bacteriocins, a type of proteins. Bacteriocins often kill cells by creating channels in the plasma membrane thus increasing its permeability. They also may degrade DNA or RNA or attack peptidoglycan and weaken the cell-wall.

Bacteriocins act only against closely related strains. Col E1 plasmid of E. coli code for the synthesis of bacterioein called colicins which kill other susceptible strains of E. coli. Col plasmids of some E.coli code for the synthesis of bacteriocin, namely cloacins that kill Enterobacter species.

Lactic acid bacteria produce bacteriocin NisinA which strongly inhibits the growth of a wide variety of gram-positive bacteria and is used as a preservative in the food industry.

5. Metabolic plasmids:

Metabolic plasmids (also called degradative plasmids) possess genes to code enzymes that degrade unusual substances such as toluene (aromatic compounds), pesticides (2, 4-dichloro- phenoxyacetic acid), and sugars (lactose).

TOL (= pWWO) plasmid of Pseudomonas putida is an example. However, some metabolic plasmids occurring in certain strains of Rhizobium induce nodule formation in legumes and carry out fixation of atmospheric nitrogen.

A brief summary of important types of bacterial plasmids, their hosts, and properties is given in Table 5.2.

Replication of Plasmids:

Plasmids replicate autonomously because they have their own replication origins. The enzymes involved in plasmid replication are normal cell enzymes particularly in case of small plasmids. But, some large plasmids carry genes that code for enzymes that are specific for plasmid replication.

Plasmids possess relatively few genes, generally less than 30, and the genes are concerned primarily with control of the replication initiation process and with apportionment of the replicated plasmids between daughter cells the genetic information carried in plasmid genes is not essential to the host because the bacteria that lack them usually function normally.

Since the plasmid DNA is of small size, the whole process of its replication takes place very quickly, perhaps in 1/10 or less of the total time of cell division cycle.

Most plasmids in gram-negative bacteria replicate in a manner similar to the replication of bacterial chromosome involving initiation at the replication origin site and bidirectional replication around the DNA circle giving a theta (Ө) intermediate.

However, some plasmids of gram-negative bacteria replicate by unidirectional method. Most plasmids of gram-positive bacteria replicate by a rolling circle mechanism similar to that used by phage φx174. Most linear plasmids replicate by means of a mechanism that involves a protein bound to the 5′-end of each DNA strand that is used in priming DNA synthesis.

Plasmid Curing:

Plasmids can be eliminated from bacterial cells, and this process is called curing. Curing may take place spontaneously or it may be induced by various treatments, which inhibit plasmid replication but do not affect bacterial chromosome replication and cell reproduction. The inhibited plasmids are slowly diluted out of the growing bacterial population.

Some commonly used curing treatment agents are acridine dyes, ultraviolet (UV) and ionizing radiation, thymine starvation and growth above optimal temperatures. These curing treatment agents interfere with plasmid replication than with bacterial chromosome replication.

Use of Plasmids as Cloning Vectors:

Significance of plasmids dramatically increased with the advent of recombinant DNA technology as they became the first cloning vectors, and even today they are the most widely used cloning vectors especially in gene cloning in bacteria.

They enjoy this status because they have very useful properties as cloning vectors that include:

(i) Small size, which makes the plasmid easy to isolate and manipulate

(ii) Independent origin of replication, which allows plasmid replication in the cell to proceed independently from direct chromosomal control

(iii) Multiple copy number, which makes them to be present in the cell in several copies so that amplification of the plasmid DNA becomes easy and

(iv) Presence of selectable markers such as antibiotic resistance genes, which make detection and selection of plasmid-containing clones easier.

The plasmid vector is isolated from the bacterial cell and at one site by restriction enzyme. The cleavage converts the circular plasmid DNA into a linear DNA molecule.

Now the two open ends of linear plasmid are joined to the ends of the foreign DNA to be inserted with the help of enzyme DNA ligase. This regenerates a circular hybrid or chimeric plasmid, which is transferred to a bacterium wherein it replicates and perpetuates indefinitely.

One of the most widely used plasmids in gene cloning in bacteria is pBR322, which has both resistance genes for ampicillin and tetracycline and many restriction sites. When a foreign DNA is inserted into the ampicillin resistance gene of pBR322, the plasmid is no longer able to confer resistance to ampicillin.


Cloning Vectors: Types & Characteristics

A vector is a DNA molecule which is used for transporting exogenous DNA into the host cell. A vector is capable of self-replication and stable integration inside the host cell.

The molecular analysis of DNA has been made possible only after the discovery of vectors. The whole process of molecular cloning involves the following steps:

  1. Digestion of DNA fragments of the target segment and the vector DNA with the help of restriction enzymes,
  2. Ligation of the target segment with the vector DNA with the help of DNA ligases, and
  3. Introduction of the ligated segment into the host cell for propagation.

General characteristics of a vector:

  • It should have an Origin of Replication, known as ori, so that the vector is capable of autonomous replication inside the host organism.
  • It must possess a compatible restriction site for insertion of DNA molecule.
  • A vector should always harbour a selectable marker to screen the recombinant organism. This selectable marker can be an antibiotic resistance gene.
  • For easy incorporation into the host machinery, a vector should itself be small in size and be able to integrate large size of the insert.

CLONING VECTOR

A cloning vector is also a fragment of DNA which is capable of self-replication and stable maintenance inside the host organism. It can be extracted from a virus, plasmid or cells of a higher organism. Most of the cloning vectors are genetically engineered. It is selected based upon the size and the kind of DNA segment to be cloned.

The cloning vectors must possess the following general characteristics:

  • It should small in size.
  • It must have an origin of replication.
  • It must also be compatible with the host organism.
  • It must possess a restriction site.
  • The introduction of donor fragment must not intervene with the self-replicating property of the cloning vector.
  • A selectable marker, possibly an antibiotic resistance gene, must be present to screen the recombinant cells.
  • It should be capable of working under the prokaryotic as well as the eukaryotic system.
  • Multiple cloning sites should be present.

Importance of Cloning Vectors

Cloning Vectors are used as the vehicle for transporting foreign genetic material into another cell. This foreign segment of DNA is replicated and expressed using the machinery of the host organism.

A cloning vector facilitates amplification of a single copy DNA molecule into many copies. Molecular gene cloning is difficult without the use of the cloning vectors.

History of Cloning Vectors

Herbert Boyer, Keiichi Itakura, and Arthur Riggs were three scientists working in the Boyer’s lab, University of California, where they recognized a general cloning vector. This cloning vector had restriction sites for cloning foreign DNA and also, the expression of antibiotic resistance genes for the screening of recombinant/ transformed cells. The first vector used for cloning purposes was pBR322, a plasmid. It was small in size, nearly 4kB, and had two selectable markers.

Features of Cloning Vectors

1. Origin of Replication (ori)

  • A specific set/ sequence of nucleotides where replication initiates.
  • For autonomous replication inside the host cell.
  • Foreign DNA attached to ori also begins to replicate.

2. Cloning Site

  • Point of entry or analysis for genetic engineering.
  • Vector DNA at this site is digested and foreign DNA is inserted with the aid of restriction enzymes.
  • Recent works have discovered plasmids with multiple cloning sites (MCS) which harbour up to 20 restriction sites.

3. Selectable Marker

  • Gene that confers resistance to particular antibiotics or selective agent which, under normal conditions, is fatal for the host organism.
  • Confers the host cell the property to survive and propagate in culture medium containing the particular antibiotics.

4. Marker or Reporter Gene

  • Permits the screening of successful clones or recombinant cells.
  • Utilised extensively in blue-white selection.

5. Inability to Transfer via Conjugation

  • Vectors must not enable recombinant DNA to escape to the natural population of bacterial cells.

Types of Cloning Vectors

E. Bacterial Artificial Chromosome (BAC)

F. Yeast Artificial Chromosome (YAC)

G. Human Artificial Chromosome (HAC)

A. Plasmids

  • Plasmids were the first vectors to be used in gene cloning.
  • They are naturally occurring and autonomously replicating extra-chromosomal double-stranded circular DNA molecules. However, not all plasmids are circular in origin.

  • They are present in bacteria, archaea, and eukaryotes.
  • The size of plasmids ranges from 1.0 kb to 250 kb.
  • DNA insert of up to 10 kb can be cloned in the plasmids.
  • The plasmids have high copy number which is useful for production of greater yield of recombinant plasmid for subsequent experiments.
  • The low copy number plasmids are exploited under certain conditions like the cloned gene produces the protein which is toxic to the cells.
  • Plasmids only encode those proteins which are essential for their own replication. These protein-encoding genes are located near the ori.

Examples: pBR322, pUC18, F plasmid, Col plasmid.

Nomenclature of plasmid cloning vector: pBR322 cloning vector has the following elements:


Plasmid Cloning Permits Isolation of DNA Fragments from Complex Mixtures

A DNA fragment of a few base pairs up to � kb can be inserted into a plasmid vector. When such a recombinant plasmid transforms an E. coli cell, all the antibiotic-resistant progeny cells that arise from the initial transformed cell will contain plasmids with the same inserted sequence of DNA (Figure 7-3). The inserted DNA is replicated along with the rest of the plasmid DNA and segregates to daughter cells as the colony grows. In this way, the initial fragment of DNA is replicated in the colony of cells into a large number of identical copies. Since all the cells in a colony arise from a single transformed parental cell, they constitute a clone of cells. The initial fragment of DNA inserted into the parental plasmid is referred to as cloned DNA, since it can be isolated from the clone of cells.

Figure 7-3

General procedure for cloning a DNA fragment in a plasmid vector. Although not indicated by color, the plasmid contains a replication origin and ampicillin-resistance gene. Uptake of plasmids by E. coli cells is stimulated by high concentrations of CaCl (more. )

DNA cloning allows fragments of DNA with a particular nucleotide sequence to be isolated from a complex mixture of fragments with many different sequences. As a simple example, assume you have a solution containing four different types of DNA fragments, each with a unique sequence (Figure 7-4). Each fragment type is individually inserted into a plasmid vector. The resulting mixture of recombinant plasmids is incubated with E. coli cells under conditions that facilitate transformation the cells then are cultured on antibiotic selective plates. Since each colony that develops arose from a single cell that took up a single plasmid, all the cells in a colony harbor the identical type of plasmid characterized by the DNA fragment inserted into it. As a result, copies of the DNA fragments in the initial mixture are isolated from one another in the separate bacterial colonies. DNA cloning thus is a powerful, yet simple method for purifying a particular DNA fragment from a complex mixture of fragments and producing large numbers of the fragment of interest.

Figure 7-4

Isolation of DNA fragments from a mixture by cloning in a plasmid vector. Four distinct DNA fragments, depicted in different colors, are inserted into plasmid cloning vectors, yielding a mixture of recombinant plasmids each containing a single DNA fragment. (more. )


DNA CLONING VECTORS

Plasmids are the most commonly used cloning vectors. Plasmids are popular because they allow cloning and manipulation of small pieces of DNA thereby being helpful in molecular biology techniques. One of the first widely used plasmid DNA vectors, called pBR322 was designed to have genes for ampicillin and tetracycline resistance and several useful restriction sites.

FEATURES OF DNA CLONING VECTORS

Modern plasmid DNA cloning vectors usually include the features.

  1. SIZE – They should be small enough to be easily separated from chromosomal DNA of host bacteria.
  2. ORIGIN OF REPLICATION(ori) – The site for DNA replication that allows plasmids to replicate separately from the host cell’s chromosome. The number of plasmids in the cell is called the copy number. Most of the desirable cloning plasmids are known as high copy number plasmids because they can replicate to create hundreds or thousands of plasmid copies.
  3. MULTIPLE CLONING SITE – The MCS , also called a poly linker is a stretch of DNA with recognition sequences for many different restriction enzymes. A MCS provides a lot of opportunities for possible DNA inserts to be added.
  4. SELECTABLE MARKER GENES – These genes allow the selection and identification of bacteria that have been transformed with a recombinant plasmid compared to non transformed cells. Example, ampicillin resistance and tetracycline resistance and lacZ gene used for blue – white selection.
  5. RNA POLYMERASE PROMOTER SEQUENCE – These sequences are used for transcription of RNA in vivo and in vitro by RNA polymerase.
  6. DNA SEQUENCING PRIMER SEQUENCE – These sequences permit nucleotide sequencing of cloned DNA fragments that have been inserted into the plasmid.
TYPES OF VECTORS

Each vectors has its significant role in molecular biology techniques. Therefore, there are different cloning vectors.

DNA from bacteriophage lambda one of the first phage vectors used in cloning. The lambda chromosome is a linear 49 kb size structure. Cloned DNA is inserted into restriction sites in the centre of the lambda chromosome Recombinant chromosomes are hen packaged into viral particles in vitro, and these phages are used to infect E.coli growing as a lawn. At each end of the lambda chromosome are 12 nucleotide sequences called cohesive sites (COS) that can base pair with each other . When lambda infects E.coli as a host, the Lambda chromosome uses these COS sites to circularize and then replicate. A primary advantage of using these vectors is that they allow cloning of larger DNA fragments than plasmids.

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2. COSMID VECTORS

Cosmid vectors contain COS ends of lambda DNA, a plasmid origin of replication, and genes have been removed. DNA is cloned into a restriction site, and the cosmid is packaged into viral particle, a s is done with bacteriophage vectors. Bacterial colonies are formed on a plate, and recombinants are screened by antibiotic selection. A primary advantage of cosmids is that they allow for the cloning of DNA fragments in the 20- 45 kb range.

3. EXPRESSION VECTORS

Protein expression vectors allow high level expression of eukaryotic proteins with in the bacterial cells because they contain a prokaryotic promoter sequence adjacent to the site where DNA is inserted into the plasmid. Bacterial RNA polymerase can bind to the promoter and synthesize large amounts of RNA which is then translated into protein. Protein can then be isolated using biochemical techniques.

4. BACTERIAL ARTIFICIAL CHROMOSOMES

Bacterial artificial chromosome (BACs) are large low copy number plasmids, present as one or two copies in bacterial cells that contain genes encoding the F- factor. BACs can accept DNA inserts in the 100 – 300 kb range. BACs were widely used in the Human Genome Project to clone and sequence large pieces of human chromosomes.

5. YEAST ARTIFICIAL CHROMOSOMES

Yeast artificial chromosomes (YACs) are small plasmids grown in E.coli and introduced into yeast cells. A YAC is a miniature version of a eukaryotic chromosome. YACs contain an origin of replication, selectable markers, two telomeres, and a centromere that allows for replication of the YAC and segregation into daughter cells during cell division. Foreign DNA fragments are cloned into the restriction site into the center of the YAC. YACs are useful for cloning large fragments of DNA from 200 kb to approx. 2 megabases (mb= 1 million bases) in size. YACs have also been used in Human Genome Project.

6. Ti VECTORS

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These are naturally occurring plasmids isolated from the bacterium Agrobacterium tumefaciens. A soil borne Gram negative pathogen that infects dicot plants by causing crown gall disease. On entering a cell the A. tumefaciens inserts a apart of its DNA(T-DNA) from the Ti plasmid into the host chromosome. Therefore the plant geneticists exploited this property if the bacteria and used it to insert their gene of interest using A. tumefaciens as a vector into the host cell.

image credit


Vector Cloning

Our custom vector cloning workflow typically consists of three parts: QC of customer-supplied materials (if applicable), sourcing of required vector components (if applicable), and the actual vector cloning step. A brief overview of the price and turnaround breakdown is shown in Table 1 below:

Table 1. Overview of price and turnaround for custom cloning.

a. For shRNA, gRNA and short fragment expression (e.g. enhancer, promoter): starting from $99

b. For protein expression: starting from $159

* Vectors built from VectorBuilder&rsquos standard backbones and components have flat price and turnaround based on Table 2 below.

** The cloning turnaround refers to the time from production initiation to completion. It does not include transit time and QC of customer-supplied materials, and transit time for shipping final deliverable to the customer.

Table 2. Price and turnaround for simple vector cloning.

Vector Type Price Turnaround
shRNA vector $99 7-12 days
gRNA vector (single-gRNA) $99 6-11 days
gRNA vector (dual-gRNA) $299 10-17 days
Expression vector $159 7-13 days

Table 3. Price and turnaround for de novo gene synthesis.

Fragment length Price (USD)* Turnaround
<1.5 kb .12/bp 6-10 days
1.5-3 kb .14/bp 10-14 days
3-5 kb .16/bp 10-16 days
5-7 kb .18/bp 16-20 days

*The de novo gene synthesis fee may be higher when 1) the fragment contains regions that are difficult to synthesize such as high GC content, simple repeats or segmental repeats 2) fewer than three DNA fragments are synthesized.

Deliverables

The default deliverable format is E. coli glycerol stock. Upon request, small amounts of plasmid DNA can be provided if available without additional charge. Multiple scales of high-quality plasmid DNA can be purchased additionally: Miniprep (> 10 ug), Midiprep (>100 ug), Maxiprep (> 300 ug), Megaprep (>1 mg) and Gigaprep (>10 mg). For viral vectors, virus packaging offered at various scales is available as a downstream service.

Quality control

All vectors cloned by VectorBuilder come with a 100% sequence guarantee. Our vectors undergo stringent quality control to ensure that they contain the correct sequence exactly as designed. Common QC includes DNA quantification, A260/280 measurement, electrophoresis of undigested and restriction enzyme digested plasmid DNA, Sanger sequencing, recovering bacteria from glycerol stocks, and re-transformation.

Customers-supplied materials

If customer-supplied materials such as backbones or gene templates are needed, please submit your materials information on "Support" > "Materials Submission Form". Please strictly follow our guidelines to set up shipment to avoid any delay or damage of the materials. All customer-supplied materials undergo mandatory QC by VectorBuilder which may incur an additional QC charge starting from $120 per item, depending on the type and usage of item. Please note that production cannot be initiated until customer-supplied materials pass QC.

How to Order

At VectorBuilder, a combination of various molecular biology techniques is utilized to generate custom vectors based on customer needs. While expression vectors are typically constructed using Gateway cloning or Gibson assembly, shRNA and gRNA vectors are cloned using a ligation-based approach to provide you with your desired vectors at the lowest prices and fastest turnaround times utilizing our highly optimized high-throughput cloning platform. Additionally, we routinely use a variety of other methods including de novo gene synthesis and Golden Gate cloning as needed, on a project-by-project basis.

All vectors at VectorBuilder are cloned using our proprietary host strain VB UltraStable &trade , which is designed to achieve high transformation efficiency (>1x10 9 cfu/ug) and for propagating DNA plasmids with unstable elements such as repeated sequences. Therefore, we highly recommend that you use VB UltraStable &trade chemically competent cells for propagating your plasmids obtained from VectorBuilder. However, our vectors are compatible with and can be propagated in other commonly used bacterial hosts strains such as DH5&alpha and Stbl3.

The origin of replication on your plasmid is for low-copy replication

The number of plasmid copies per bacterial cell is determined by the origin of replication on the plasmid. Some origins have inherent low copy number. Check the copy number of the origin of replication on your plasmid. For low-copy plasmids, increase the amount of E. coli culture for plasmid DNA prep in order to obtain satisfying DNA yield.

The volume of bacterial culture is too low for plasmid prep column

Please check the binding capacity of your plasmid prep column and whether your plasmid is high- or low-copy plasmid. For mini preps, we recommend that you harvest 1-5 ml of overnight bacterial culture. For maxi preps, if the plasmid is high-copy plasmid, we recommend using 100-150 ml of overnight bacterial culture if the plasmid is low-copy plasmid, we recommend using 300-500 ml of overnight bacterial culture. Typically, for high-copy plasmid,

5 ug of plasmid DNA can be extracted from every 1 ml of culture in mini prep and

500 ug of plasmid DNA can be obtained from 150 ml of culture for low-copy plasmid (e.g. pET), 1.5-2.5 ug of plasmid DNA can be harvested from every 1 ml of culture in mini prep and 150-200 ug of plasmid DNA can be obtained from 150 ml of culture.

Only a fraction of bacteria in the liquid culture contain plasmids

Some antibiotics, ampicillin in particular, degrade fast in liquid culture. As a result, bacteria that do not contain plasmids can propagate to a significant fraction of the culture, causing poor yield of the plasmid prep. To avoid this, please prepare ampicillin containing growth medium freshly before use and make sure that enough ampicillin is supplied. Also, when culturing ampicillin-resistant bacteria, do not let the liquid culture saturate for too long before harvesting. Besides insufficient antibiotics in the culture, extracting plasmid DNA from very old culture can also result in low yield, since many bacterial cells are dead and plasmid DNA they contain is degraded. Therefore, try to extract plasmid DNA from fresh culture. If plasmid prep cannot be performed immediately, you can spin down the bacteria and store the pellet in -80°C freezer for later plasmid prep.

The liquid culture is directly inoculated from E. coli stab culture

Direct inoculation of a liquid culture from the E. coli liquid stock or stab culture you have received from VectorBuilder can very occasionally result in low yield. We recommend streaking the stock onto an LB agar plate containing the appropriate antibiotic first, and then inoculating a liquid culture with a fresh colony growing from that plate. Detailed user instructions can be found by going to menu item Learning Center > Documentation > Stab Culture.

You have not carefully followed the manual of the plasmid prep kit

If you use a plasmid prep kit, please carefully read the manual before use. Improper operations can often lead to poor performance of the kit.

The mini/maxi prep column is low-quality

Some brands of plasmid DNA prep columns perform poorly or inconsistently for DNA preparation.


Metagenomics

Cloning Vectors and Metagenomic Library Structure

The choice of cloning vector and strategy largely reflects the desired library structure (i.e., insert size and number of clones) and target activities sought. To obtain a function encoded by a single gene, small DNA fragments (<10 kb) can be obtained and cloned in Escherichia coli into standard cloning vectors (e.g., pUC derivatives, pBluescript SK(+), pTOPO-XL, and pCF430). Various enzymes, such as amidases, hydrolases, cellulases, and antibiotic resistance determinants, have been identified in functional screens of metagenomic libraries harboring inserts smaller than 10 kb. Conversely, to obtain targets encoded by multiple genes, large DNA fragments (>20 kb) must be cloned into fosmids, cosmids, or bacterial artificial chromosomes (BACs), all of which can stably maintain large DNA fragments. Two vectors, namely pCC1FOS and pWE15, have been used for cloning large DNA fragments from various microbial communities. The pCC1FOS vector has the advantage that, when in the appropriate host (e.g. E. coli Epi300), its copy number can be controlled by addition of arabinose in the medium to increase DNA yield. Microbial sensing signals, antibiotic resistance determinants, antibiosis, pigment production, and eukaryotic growth modulating factors have been identified from metagenomic libraries constructed with pCC1FOS. Additionally, the presence of considerable flanking DNA on fosmid or BAC clone inserts facilitates phylogenetic inference about the source of the fragment.


Cloning Vectors Questions and Answers

Question 1 : Cosmids lack

  1. genes coding for viral proteins
  2. origin of replication
  3. marker genes coding for replication
  4. cleavage site for the insertion of foreign DNA

Question 2 : Cryptic plasmids

  1. do not exhibit any phenotypic trait
  2. exhibit many phenotypic traits
  3. exhibit one phenotypic traits
  4. exhibit antibiotic resistance

Question 3 : Phagemid consist of

  1. plasmid vector carrying λ phage’s cos site
  2. plasmid vector carrying λ attachment (λ att) site
  3. plasmid vector carrying origin of replication of λ phage only
  4. plasmid vector carrying origin of replication of plasmid only

Question 4 : Maximum size of foreign DNA that can be inserted into an insertion vector is

Question 5 : Plasmids which are maintained as multiple copy number per cell are known as

Question 6 : Cosmid vectors are

  1. plasmids that contain fragment of λ DNA including the cos site
  2. phages that lack cos site
  3. plasmids that have no selection marker
  4. cryptic plasmids

Question 7 : EMBL 3 and EMBL 4 are replacement vectors, which can clone DNA up to

Question 8 : Size of the DNA that can be packaged into a λ phage is

Question 9 : Charon 34 and Charon 35 are the examples of

Question 10 : Cosmid vectors are used for

  1. cloning small fragments of DNA
  2. cloning large fragments of DNA
  3. cloning prokaryotic DNA only
  4. cloning eukaryotic DNA only

Question 11 : Plasmids which are maintained as limited number of copies per cell are known as

Question 12 : Cos site of the cosmids

  1. consists of 12 bases
  2. helps whole genome in circularization and ligation
  3. both (1) and (2)
  4. contains cleavage site

Question 13 : M 13 is an example of

Question 14 : Phagemid vectors are

  1. combination of plasmid and phage λ
  2. combination of phages and cosmid
  3. phages carrying properties of plasmids
  4. all of the above

Question 15 : Single stranded vectors are useful

  1. for sequencing of cloned DNA
  2. for oligonucleotide directed mutagenesis
  3. for probe preparation
  4. all of the above

Question 16 : Inserted DNA in λ gt 11 can be expressed as

  1. β-galactosidase fused protein
  2. free protein in the cytoplasm
  3. free protein that is secreted out
  4. all of the above

Question 17 : Plasmid incompatibility is

  1. inability of a plasmid to grow in the host
  2. inability of two different plasmids to coexist in the same host cell in the absence of selection pressure.
  3. both (1) and (2)
  4. none of the above

Question 18 : P1 cloning vector allow cloning of DNA of the length of

Question 19 : Charon 34 and Charon 35 can clone DNA upto

Question 20 : Charon vectors are different from EMBL vectors because

  1. they have more extensive range of restriction targets with in their polylinkers
  2. physical separation of lambda arm from central fragment is required
  3. both (1) and (2)
  4. physical separation of lambda arm from central fragment is not required

Question 21 : P1 cloning vector is the example of

Question 22 : λ ZAP vector is an example of

Question 23 : λ gt 10 and λ gt 11 vectors can propagate cloned fragments up to

Question 24 : Select the wrong statement about plasmids?

  1. It is extrachromosomal
  2. It is double stranded
  3. Its replication depends upon host cell
  4. It is closed and circular DNA

Question 25 : Stuffer is

  1. the right arm of the vector DNA
  2. the left arm of the vector DNA
  3. central fragment of the vector DNA
  4. none of the above

Question 26 : Conjugative plasmids

  1. exhibit antibiotic resistance
  2. do not exhibit antibiotic resistance
  3. carry transfer genes called the tra genes
  4. do not carry transfer genes

Question 27 : Maximum size of foreign DNA that can be inserted into a replacement vector is

Question 28 : Which of the following is not true about phagemid?

  1. Contain functional origin of replication of the plasmid and λ phage
  2. May be propagated as a plasmid or as phage in appropriate strain
  3. Contain λ att site
  4. Can only be propagated as phage

Question 29 : pBR 322 has/have which of the following selection marker(s)?

Question 30 : A plasmid can be considered as a suitable cloning vector if

  1. it can be readily isolated from the cells
  2. it possesses a single restriction site for one or more restriction enzymes
  3. insertion of foreign DNA does not alter its replication properties
  4. All of the above

Question 31 : Difference between λ gt 10 and λ gt 11 vectors is that