This MCQ module is based on: Cloning Vectors Competent Host
TOPIC 42 OF 64
Cloning Vectors Competent Host
🎓 Class 12
Biology
CBSE
Theory
Ch 9 – Biotechnology: Principles and Processes
⏱ ~14 min
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Cloning Vectors and the Competent Host
A gene cut cleanly out of a genome is still a fragment with no future. It needs a vehicle that can replicate inside a host, a way of telling the cells that took it up from the millions that did not, and a host willing to swallow DNA in the first place. This part deals with all three.
What a cloning vector is
You know that plasmids and bacteriophages have the ability to replicate within bacterial cells independent of the control of chromosomal DNA. That independence is exactly what makes them useful.
Bacteriophages, because of their high number per cell, have very high copy numbers of their genome within bacterial cells. Some plasmids may have only one or two copies per cell, whereas others may have 15–100 copies per cell, and their numbers can go even higher. If we are able to link an alien piece of DNA with bacteriophage or plasmid DNA, we can multiply its numbers equal to the copy number of that plasmid or bacteriophage.
Vectors used at present are engineered, not merely borrowed. A modern cloning vector is built so that it helps easy linking of foreign DNA, and easy selection of recombinants from non-recombinants.
The four features a vector must have
(i) Origin of replication (ori)
This is a sequence from where replication starts, and any piece of DNA when linked to this sequence can be made to replicate within the host cells. It is also responsible for controlling the copy number of the linked DNA. So if one wants to recover many copies of the target DNA, it should be cloned in a vector whose origin supports a high copy number.
(ii) Selectable marker
In addition to ori, the vector requires a selectable marker, which helps in identifying and eliminating non-transformants and selectively permitting the growth of the transformants. Transformation is the procedure through which a piece of DNA is introduced into a host bacterium.
Normally the genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, tetracycline or kanamycin are considered useful selectable markers for E. coli. This works for a simple reason: normal E. coli cells do not carry resistance against any of these antibiotics, so there is no background of survivors to confuse the result.
(iii) Cloning sites
The cloning sites of a vector are the restriction-enzyme recognition sites at which the alien DNA is actually ligated in. In order to link the alien DNA, the vector needs to have very few — preferably single — recognition sites for the commonly used restriction enzymes. The presence of more than one recognition site within the vector will generate several fragments, which will complicate the gene cloning.
The ligation of alien DNA is carried out at a restriction site present in one of the two antibiotic resistance genes. For example, you can ligate a foreign DNA at the BamH I site of the tetracycline resistance gene in the vector pBR322. The recombinant plasmids will then lose tetracycline resistance due to the insertion of foreign DNA, but can still be selected out from non-recombinant ones by plating the transformants on ampicillin containing medium.
The procedure runs like this. Transformants growing on ampicillin containing medium are then transferred onto a medium containing tetracycline. The recombinants will grow in the ampicillin containing medium but not on the one containing tetracycline, while non-recombinants will grow on the medium containing both antibiotics. In this case, one antibiotic resistance gene helps in selecting the transformants, whereas the other gets inactivated due to insertion of alien DNA and helps in the selection of recombinants.
A better way: insertional inactivation of a colour reaction
Selection of recombinants by the inactivation of antibiotic resistance is a cumbersome procedure, because it requires simultaneous plating on two plates having different antibiotics. Alternative selectable markers have therefore been developed which distinguish recombinants from non-recombinants on the basis of their ability to produce colour in the presence of a chromogenic substrate.
Here, a recombinant DNA is inserted within the coding sequence of the enzyme β-galactosidase. This results in the inactivation of the gene for the synthesis of this enzyme, which is referred to as insertional inactivation. In the presence of a chromogenic substrate, bacteria whose plasmid does not carry an insert give blue coloured colonies; where an insert is present, the β-galactosidase gene is inactivated and the colonies produce no colour. Those colourless colonies are identified as the recombinant ones.
One plate instead of two. The antibiotic method needs two media, two platings and a comparison between them. The colour method needs one plate and one look. Both rely on exactly the same idea — that inserting DNA into a gene destroys it — but the second turns that destruction into something the eye can read directly.
(iv) Vectors for cloning genes in plants and animals
We learnt the lesson of transferring genes into plants and animals from bacteria and viruses, which have known for ages how to deliver genes into eukaryotic cells and force them to do what the bacterium or virus wants.
Agrobacterium tumifaciens, a pathogen of several dicot plants, is able to deliver a piece of DNA known as T-DNA to transform normal plant cells into a tumour, and to direct these tumour cells to produce the chemicals the pathogen requires. Similarly, retroviruses in animals have the ability to transform normal cells into cancerous cells.
A better understanding of this art has let biologists convert the tools of pathogens into useful vectors. The tumour inducing Ti plasmid of Agrobacterium tumifaciens has now been modified into a cloning vector which is no more pathogenic to plants, but is still able to use those mechanisms to deliver genes of our interest into a variety of plants. Retroviruses have likewise been disarmed, and are now used to deliver desirable genes into animal cells.
So, once a gene or a DNA fragment has been ligated into a suitable vector, it is transferred into a bacterial, plant or animal host, where it multiplies.
| Feature | What it does | The problem it solves |
|---|---|---|
| Origin of replication (ori) | Starts replication; controls copy number of the linked DNA | A loose fragment cannot be copied, and you may need many copies |
| Selectable marker | Permits growth of transformants, eliminates non-transformants | Only a few cells in millions take up the vector |
| Cloning sites | Very few, preferably single, sites for common restriction enzymes | Several sites would generate several fragments and complicate cloning |
| Insertional inactivation | Second marker broken by the insert — antibiotic or colour based | Transformed is not the same as recombinant; this separates the two |
| Eukaryote-capable vectors (Ti plasmid, disarmed retroviruses) | Deliver genes into plant and animal cells | Plasmids alone cannot transform eukaryotic cells |
The competent host
Since DNA is a hydrophilic molecule, it cannot pass through cell membranes. Why not? Because a membrane's interior is a hydrophobic layer of lipid tails, and a large, highly charged, water-loving polymer has no way through it. So in order to force bacteria to take up a plasmid, the bacterial cells must first be made competent to take up DNA.
This is done by treating them with a specific concentration of a divalent cation such as calcium, which increases the efficiency with which DNA enters the bacterium through pores in its cell wall. Recombinant DNA can then be forced into such cells by:
- incubating the cells with recombinant DNA on ice;
- placing them briefly at 42°C — the heat shock;
- then putting them back on ice.
This sequence enables the bacteria to take up the recombinant DNA.
Three other ways in
Chemical competence is not the only route.
- Micro-injection: recombinant DNA is injected directly into the nucleus of an animal cell.
- Biolistics, or the gene gun: suitable for plants, in which cells are bombarded with high velocity micro-particles of gold or tungsten coated with DNA.
- Disarmed pathogen vectors: which, when allowed to infect the cell, transfer the recombinant DNA into the host.
📐 Activity: two plates, four colonies
You have ligated a gene into the BamH I site of the tetracycline resistance gene of pBR322 and transformed E. coli. You plate the cells on ampicillin medium, then transfer the surviving colonies onto tetracycline medium. Four colonies behave as follows.
- Colony A grows on ampicillin, grows on tetracycline.
- Colony B grows on ampicillin, dies on tetracycline.
- Colony C does not appear on the ampicillin plate at all.
- Colony D grows on ampicillin, and grows on tetracycline, but PCR shows your gene is present.
Predict: which colony do you keep, and what has happened in each of the other three?
Colony B is the one you keep. It is a recombinant. The insert has broken the tetracycline resistance gene, so the cell is resistant to ampicillin but not to tetracycline — exactly the pattern that identifies a recombinant plasmid.
Colony A is a non-recombinant transformant. It took up a plasmid, but one that re-closed without an insert. Both resistance genes are intact, so it grows on the medium containing either antibiotic.
Colony C never took up a plasmid at all. Normal E. coli cells do not carry resistance against ampicillin, so an untransformed cell simply dies on the first plate. This is precisely the job of the selectable marker: identifying and eliminating non-transformants.
Colony D is the interesting one. Your gene is present but tetracycline resistance survived — so the insert did not go into the BamH I site of the tetⁿ gene. It may have integrated elsewhere, or the cell may carry two plasmids, one with the insert and one without. The lesson is that a selection scheme reports on the marker, not on your gene; confirmation by PCR or sequencing is what finally proves what you have.
🎯 Interactive: Choose the Right Vector Feature
Each situation below fails for want of one specific vector feature or method. Pick the situation and see which one, and why.
🎯 Competency-Based Questions
Q1. A vector carries a single antibiotic resistance gene and an ori. A student inserts a gene into it and transforms E. coli, plating on the antibiotic. Hundreds of colonies grow, but only a few turn out to carry the insert. Explain why the selection failed to distinguish them, and redesign the experiment.
Why the selection could not work. A selectable marker separates transformants from non-transformants — it identifies and eliminates cells that never took up a plasmid, and selectively permits the growth of those that did. It says nothing about whether the plasmid taken up carries an insert. A vector that re-closed on itself during ligation still carries a perfectly good resistance gene, so it survives the plate exactly like a recombinant.
The redesign, version one. Use a vector with two antibiotic resistance genes, such as pBR322, and ligate the foreign DNA at a restriction site inside one of them — for example the BamH I site of the tetracycline resistance gene. Plate the transformants on ampicillin, then transfer them to tetracycline. Recombinants grow on ampicillin but not on tetracycline, because the insert has inactivated the tet gene; non-recombinants grow on both.
The redesign, version two, and why it is better. Selection by inactivation of antibiotic resistance is cumbersome, since it requires simultaneous plating on two plates with different antibiotics. Instead, insert the gene within the coding sequence of β-galactosidase and supply a chromogenic substrate. Colonies without an insert turn blue; colonies with an insert suffer insertional inactivation of the gene and stay colourless. One plate, and the answer is visible at a glance.
Q2. Why must a cloning vector have very few — preferably single — recognition sites for the commonly used restriction enzymes, when having many sites would seem to offer more choices?
Because the enzyme cuts every site it finds. A restriction endonuclease does not choose one site out of several; it cuts wherever its recognition sequence occurs. The presence of more than one recognition site within the vector will therefore generate several fragments, which complicates gene cloning.
What goes wrong in practice. With two sites, digesting the vector cuts it into two pieces, and the essential features may end up on different pieces — the ori on one, the selectable marker on the other. Re-ligation then produces a mixture of products, most of them useless, and the recombinant you want becomes a small minority that is hard to recover.
The design answer. Modern vectors concentrate single sites for many different enzymes into one short stretch, so you have a wide choice of enzymes while each individual enzyme still cuts the vector only once. Choice comes from having many different single sites, not from having many copies of the same site.
Q3. Pure plasmid DNA added to an ordinary culture of E. coli does not transform it. Explain the obstacle in terms of molecular structure, and describe the full treatment that overcomes it.
The obstacle. DNA is a hydrophilic molecule and therefore cannot pass through cell membranes. The membrane's interior is a hydrophobic region of lipid tails, and DNA is a large polymer carrying a dense negative charge on its phosphate backbone. It is both too water-loving and too charged to cross a lipid barrier.
Making the cell competent. The bacterial cells must first be made competent to take up DNA. This is done by treating them with a specific concentration of a divalent cation such as calcium, which increases the efficiency with which DNA enters the bacterium through pores in its cell wall.
The transfer itself. Recombinant DNA is then forced into such cells by incubating the cells with the recombinant DNA on ice, followed by placing them briefly at 42°C — the heat shock — and then putting them back on ice. This sequence enables the bacteria to take up the recombinant DNA.
Alternatives when this will not do. Micro-injection delivers recombinant DNA directly into the nucleus of an animal cell; biolistics, or the gene gun, bombards plant cells with high velocity micro-particles of gold or tungsten coated with DNA; and disarmed pathogen vectors carry the DNA in by infecting the cell.
Q4. Agrobacterium tumifaciens causes tumours in plants, and retroviruses cause cancer in animals. Explain how biotechnology turned two disease agents into two of its most valuable tools, and what exactly was changed in each.
What the pathogens already knew how to do. We learnt the lesson of transferring genes into plants and animals from bacteria and viruses which have known for ages how to deliver genes to transform eukaryotic cells and force them to do what the pathogen wants. Agrobacterium tumifaciens, a pathogen of several dicot plants, delivers a piece of DNA called T-DNA that transforms normal plant cells into a tumour and directs those cells to produce the chemicals the pathogen requires. Retroviruses in animals have the ability to transform normal cells into cancerous cells.
What was changed. In each case the delivery mechanism was kept and the harmful cargo and consequence removed. The tumour inducing Ti plasmid of Agrobacterium has been modified into a cloning vector that is no more pathogenic to plants but is still able to use those mechanisms to deliver genes of our interest into a variety of plants. Retroviruses have similarly been disarmed, and are now used to deliver desirable genes into animal cells.
Why this was necessary at all. Ordinary bacterial plasmids replicate in bacteria; they have no way of entering a plant or animal cell and being maintained there. Without borrowing a eukaryote-adapted delivery system, genetic engineering would have remained confined to microbes.
Q5. Explain why a vector's origin of replication determines not only whether the cloned DNA is copied but also how much product you can get, and how you would choose a vector for (a) making large quantities of a protein and (b) maintaining a gene stably over many generations.
The dual role of the ori. The origin of replication is a sequence from where replication starts, so any piece of DNA linked to it can be made to replicate within host cells. But it is also responsible for controlling the copy number of the linked DNA. Some plasmids have only one or two copies per cell; others have 15 to 100, and their numbers can go even higher.
Why copy number governs yield. If we are able to link an alien piece of DNA with plasmid DNA, we can multiply its numbers equal to the copy number of the plasmid. More copies of the gene in each cell means more templates for transcription, and therefore more protein per cell — so if one wants to recover many copies of the target DNA, it should be cloned in a vector whose origin supports a high copy number.
(a) For large quantities of protein: choose a high copy number origin, which maximises gene dosage and product per cell.
(b) For stable long-term maintenance: a low copy number vector is the better choice. A high copy number plasmid is a heavy metabolic burden — every copy must be replicated at each division and every gene on it expressed — so cells that lose the plasmid grow faster and take over the culture. A low copy number vector costs the cell little and is therefore retained more faithfully.
🧠 Assertion–Reason Questions
For each pair, decide whether both statements are true and whether the reason correctly explains the assertion.
Assertion (A): Antibiotic resistance genes make good selectable markers for E. coli.
Reason (R): Normal E. coli cells do not carry resistance against ampicillin, chloramphenicol, tetracycline or kanamycin.
Both A and R are true, and R is the correct explanation of A.
A marker is only useful if the untransformed host fails the test. Because normal E. coli is sensitive to all of these antibiotics, every colony that grows on the antibiotic medium must have taken up the vector — so the marker identifies and eliminates non-transformants while selectively permitting the growth of transformants.
Assertion (A): A recombinant pBR322 carrying an insert at the BamH I site cannot grow on tetracycline.
Reason (R): The BamH I site lies within the tetracycline resistance gene, which is inactivated by the insertion.
Both A and R are true, and R is the correct explanation of A.
This is insertional inactivation. The recombinant plasmids lose tetracycline resistance due to the insertion of foreign DNA, but can still be selected out from non-recombinant ones by plating the transformants on ampicillin containing medium — one gene selects the transformants, the other identifies the recombinants.
Assertion (A): Bacterial cells must be treated with calcium ions before transformation.
Reason (R): Calcium ions cut the bacterial cell wall open so that DNA can enter.
A is true but R is false.
Cells are indeed treated with a specific concentration of a divalent cation such as calcium, but nothing is cut. The treatment increases the efficiency with which DNA enters the bacterium through pores already present in its cell wall — it makes the cell competent. The cell survives the treatment and goes on dividing, which it could not do if its wall had been cut open.
Frequently Asked Questions - Cloning Vectors and the Competent Host
What is a cloning vector?
A cloning vector is a DNA molecule, usually a plasmid or bacteriophage, that carries foreign DNA into a host cell and replicates there. Plasmids and bacteriophages have the ability to replicate within bacterial cells independent of the control of chromosomal DNA, and vectors used at present are engineered so that they help easy linking of foreign DNA and selection of recombinants from non-recombinants.
What are the features required to facilitate cloning into a vector?
There are four. An origin of replication, which is the sequence from where replication starts and which controls the copy number of the linked DNA. A selectable marker, which helps in identifying and eliminating non-transformants and selectively permitting the growth of the transformants. Cloning sites, that is very few and preferably single recognition sites for the commonly used restriction enzymes. And, for eukaryotes, vectors capable of cloning genes in plants and animals.
What is a selectable marker and why do antibiotic resistance genes work as one?
A selectable marker is a gene that helps in identifying and eliminating non-transformants and selectively permitting the growth of the transformants. Genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, tetracycline or kanamycin are useful selectable markers for E. coli because normal E. coli cells do not carry resistance against any of these antibiotics.
What is transformation?
Transformation is the procedure through which a piece of DNA is introduced into a host bacterium.
How are recombinants selected using pBR322?
A foreign DNA is ligated at the BamH I site of the tetracycline resistance gene in pBR322. The recombinant plasmids lose tetracycline resistance due to the insertion of foreign DNA but can still be selected by plating the transformants on ampicillin containing medium, and then transferring them onto a medium containing tetracycline. Recombinants grow on ampicillin but not on tetracycline, whereas non-recombinants grow on both. One antibiotic resistance gene helps in selecting the transformants while the other gets inactivated due to insertion and helps in selecting the recombinants.
What is insertional inactivation and how does the blue colony method work?
Insertional inactivation is the loss of function of a gene because foreign DNA has been inserted within it. Because selection by inactivation of antibiotic resistance is cumbersome and needs plating on two different antibiotics, alternative markers were developed that differentiate recombinants on the basis of their ability to produce colour in the presence of a chromogenic substrate. A recombinant DNA is inserted within the coding sequence of beta-galactosidase, inactivating it. Colonies whose plasmid has no insert appear blue, while colonies with an insert produce no colour and are identified as recombinant.
How are genes cloned in plants and animals?
By using vectors borrowed from pathogens. Agrobacterium tumifaciens, a pathogen of several dicot plants, delivers a piece of DNA known as T-DNA to transform normal plant cells into a tumour. Its tumour inducing Ti plasmid has now been modified into a cloning vector which is no more pathogenic to plants but is still able to deliver genes of our interest into a variety of plants. Similarly, retroviruses, which can transform normal animal cells into cancerous cells, have been disarmed and are now used to deliver desirable genes into animal cells.
Why can DNA not pass through the cell membrane, and how is a host made competent?
DNA is a hydrophilic molecule, so it cannot pass through cell membranes. To force bacteria to take up a plasmid, the cells must first be made competent by treating them with a specific concentration of a divalent cation such as calcium, which increases the efficiency with which DNA enters the bacterium through pores in its cell wall.
Describe the heat shock method of transformation.
Recombinant DNA is forced into competent cells by incubating the cells with the recombinant DNA on ice, then placing them briefly at 42 degrees Celsius, which is the heat shock, and then putting them back on ice. This enables the bacteria to take up the recombinant DNA.
What are micro-injection and biolistics?
They are alternative methods of introducing alien DNA into host cells. In micro-injection, recombinant DNA is directly injected into the nucleus of an animal cell. In biolistics, also called the gene gun and suitable for plants, cells are bombarded with high velocity micro-particles of gold or tungsten coated with DNA. A third alternative uses disarmed pathogen vectors, which transfer the recombinant DNA into the host when allowed to infect the cell.
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