BiologyISC Class 12 · Biotechnology - Principles and Processes

Cloning Vectors and pBR322: ISC Class 12 Biology

Updated 15 October 2026 · Fact-checked

A cloning vector, usually a plasmid or phage, carries foreign DNA into a host and replicates it. A good vector has an origin of replication (ori), a selectable marker such as an antibiotic-resistance gene, and single recognition sites for common restriction enzymes. pBR322 has ori, rop, amp^R and tet^R genes and sites such as EcoRI and BamHI.

Notice that ori and the two resistance genes are on one circle, and each cloning site (like BamHI or PstI) sits inside tetR or ampR.

Illustration drawn for QuestionBuddy.

Understand Cloning Vectors and pBR322

Why vectors. A foreign piece of DNA cannot multiply in a host unless it is part of a DNA that has an origin of replication. Plasmids (autonomously replicating, circular, extra-chromosomal DNA) and bacteriophages replicate in bacteria independently of the chromosome, so linking a gene to them makes it multiply. Copy numbers: some plasmids have only 1-2 copies per cell, others 15-100 or more; phages have very high copy numbers. The linked gene multiplies to the copy number of the vector.

Features of a good cloning vector.
1. Origin of replication (ori): the sequence where replication starts; any DNA linked to it replicates in the host. It also controls the copy number of the linked DNA, so for many copies choose a vector whose ori supports a high copy number.
2. Selectable marker: helps identify and eliminate non-transformants and selectively permit growth of transformants. Genes for resistance to ampicillin, chloramphenicol, tetracycline or kanamycin are used for E. coli; normal E. coli carries no resistance to these.
3. Cloning sites: very few, preferably single, recognition sites for commonly used restriction enzymes. More than one site would cut the vector into several fragments and complicate cloning.
4. Small size, easy isolation and purification (helpful practical features).

pBR322 (draw-and-label). An engineered E. coli plasmid; p = plasmid, B = Bolivar, R = Rodriguez (its makers). Labels on a circle:

ori: origin of replication;
rop: codes for proteins involved in replication of the plasmid;
amp^R: ampicillin-resistance gene, with the PstI site inside it;
tet^R: tetracycline-resistance gene, with the BamHI and SalI sites inside it;
unique restriction sites: HindIII, EcoRI, BamHI, SalI, PvuII, PstI, ClaI (NCERT's figure).

Place HindIII and EcoRI near the start of tet^R and ClaI close by; PvuII between tet^R and ori; keep each site written once.

Selection of recombinants by antibiotic resistance (insertional inactivation).
1. Ligate the foreign DNA at the BamHI site in tet^R: recombinant plasmids lose tetracycline resistance but keep amp^R.
2. Plate transformed cells on ampicillin: all transformants (recombinant and non-recombinant) grow; non-transformants die.
3. Replica-plate onto tetracycline: non-recombinants grow, recombinants do not. Colonies that grew on ampicillin but are missing on tetracycline are the recombinants.
This needs two plates, so it is cumbersome.

Blue-white selection (chromogenic substrate). The vector carries the gene for β-galactosidase (lacZ). Foreign DNA is inserted within this coding sequence, inactivating it (insertional inactivation). On a medium with a chromogenic substrate (X-gal):

Colony	Plasmid	β-galactosidase	Colour
non-recombinant	no insert	made; splits X-gal	blue
recombinant	insert in lacZ	not made	white (colourless)

Only one plate is needed.

Vectors for plants and animals.

Agrobacterium tumefaciens: a pathogen of many dicots that delivers T-DNA into plant cells, turning them into tumours that make chemicals for the pathogen. Its Ti (tumour-inducing) plasmid has been disarmed: no longer pathogenic but still delivers genes of interest into plants.
Retroviruses: can turn normal animal cells cancerous; disarmed retroviruses deliver desirable genes into animal cells (used in gene therapy).
BAC and YAC (bacterial and yeast artificial chromosomes): carry very large DNA inserts; used in sequencing large genomes.

Copy-number arithmetic. Copies of an inserted gene = number of host cells × copy number per cell; cells after g generations from one cell = 2^g.

Cloning Vectors and pBR322: key facts

Three essentials: Origin of replication (ori) starts replication; controls copy number
Selectable marker antibiotic-resistance gene; picks out transformants
Cloning site single site for a restriction enzyme; vector opens once; Write the role of each, not just the name.
pBR322 parts: ori origin of replication
rop proteins for plasmid replication
Ampicillin-resistance gene contains the PstI site
Tetracycline-resistance gene contains the BamHI and SalI sites
Other single sites HindIII, EcoRI, PvuII, ClaI; BamHI and SalI lie in tet^R; PstI in amp^R.
Antibiotic selection: Insert at BamHI tetracycline-resistance gene inactivated
Recombinant grows on ampicillin, not on tetracycline
Non-recombinant transformant grows on both
Untransformed cell grows on neither; Non-recombinants grow on both.
Gene copies: $copies = N_{0} \times 2^{g} \times copy number$; g = generations.

Origin of replication (ori)	starts replication; controls copy number
Selectable marker	antibiotic-resistance gene; picks out transformants
Cloning site	single site for a restriction enzyme; vector opens once

ori	origin of replication
rop	proteins for plasmid replication
Ampicillin-resistance gene	contains the PstI site
Tetracycline-resistance gene	contains the BamHI and SalI sites
Other single sites	HindIII, EcoRI, PvuII, ClaI

Insert at BamHI	tetracycline-resistance gene inactivated
Recombinant	grows on ampicillin, not on tetracycline
Non-recombinant transformant	grows on both
Untransformed cell	grows on neither

How to answer Cloning Vectors and pBR322 questions

ISC sets 'choose the right vector' items from diagrams, the features of a good vector with roles, labelled pBR322, antibiotic and blue-white selection, and Ti plasmid or retrovirus vectors.

1For a vector choice, check each option for ori, a selectable marker and single (not repeated) sites; pick the one with all three and give two reasons that match it.
2Give the role with each feature: ori starts replication and sets copy number; marker selects transformants; single site lets the vector open once.
3For antibiotic selection, state where the insert goes, what is lost, and which plate recombinants fail on.
4For blue-white, link colour to the enzyme: insert → no β-galactosidase → X-gal not split → white.
5Draw pBR322 as a circle with ori, rop, amp^R, tet^R and at least five sites.
6For plants, name the disarmed Ti plasmid of Agrobacterium; for animals, disarmed retroviruses.

Cloning Vectors and pBR322 in real life

The idea, met in an everyday situation. Answer the question in your own words, then compare with the model answer. The one-line hook is what to remember.

Choosing a high-copy vector

Suppose a lab needs a large amount of a gene's DNA for sequencing and has two plasmids that differ only in their ori: one keeps about 2 copies per cell, the other about 50.

Which should they use, and how many more copies would they get from one transformed cell after 10 generations?

Try it first, then show the solutionSolution

Copy number is controlled by ori, so the 50-copy plasmid gives more DNA per cell.
After 10 generations one cell gives 2¹⁰ = 1024 cells.
Low-copy: 1024 × 2 = 2048 copies; high-copy: 1024 × 50 = 51 200 copies.
So the high-copy vector gives 51 200/2048 = 25 times as much DNA.

Answer: Use the high-copy plasmid: 51 200 versus 2048 copies, 25 times more.

Memory hook: Ori decides how many.

Tricks to remember Cloning Vectors and pBR322

Short memory aids. Say each one aloud once, then use it on a question the same day.

O-M-S
Origin, Marker, Site: the three things a vector must have.
Use it when: Features of a good vector.
Insert kills colour
Insert inside lacZ → no enzyme → no blue → white = winner (recombinant).
Use it when: Blue-white logic.
Bolivar and Rodriguez's plasmid
p-B-R: plasmid, Bolivar, Rodriguez.
Use it when: Expanding pBR322.

Cloning Vectors and pBR322: solved examples

Ordered from board level to HOTS. Try each one before you open the solution.

Example 1: Features with roles · 3 marks · Short answerBoard level

List three features of a good cloning vector and give the role of each.

Try it first, then show the solutionSolution

Origin of replication: where replication begins; controls the copy number of linked DNA (1 mark).
Selectable marker (e.g. ampicillin-resistance gene): identifies and eliminates non-transformants, allowing only transformants to grow (1 mark).
Single recognition sites for common restriction enzymes: the vector opens at one point to receive the insert without breaking into fragments (1 mark).

Answer: ori, selectable marker, single cloning sites, with the roles above.

Example 2: Label pBR322 · 3 marks · Short answerBoard level

Draw a labelled diagram of the E. coli cloning vector pBR322. What does rop code for?

Try it first, then show the solutionSolution

Circle with ori, rop, amp^R and tet^R labelled (1 mark).
At least five restriction sites: HindIII, EcoRI, BamHI, SalI, PvuII, PstI, ClaI, with BamHI and SalI shown in tet^R and PstI in amp^R (1 mark).
rop codes for proteins involved in the replication of the plasmid (1 mark).

Answer: Labelled circular map; rop = replication proteins.

Example 3: Selecting with two antibiotics · 3 marks · Short answerHarder board level

A researcher ligates her fragment into pBR322 after opening the plasmid with BamHI, then transforms E. coli. Describe how antibiotic plates reveal which colonies are recombinant, and why this method is called cumbersome.

Try it first, then show the solutionSolution

The BamHI site lies in tet^R; the insert inactivates it, so recombinants are ampicillin-resistant but tetracycline-sensitive (1 mark).
Plating on ampicillin keeps only transformants; replica plating on tetracycline shows that non-recombinants grow while recombinants do not, so recombinants are the colonies missing from the tetracycline plate (1 mark).
It needs simultaneous plating on two plates with different antibiotics, so it is cumbersome (1 mark).

Answer: Recombinants: grow on ampicillin, not on tetracycline; two plates make it cumbersome.

Example 4: Blue and white colonies · 2 marks · Short answerHarder board level

Explain how blue and white colonies help in identifying transformed cells with recombinant DNA.

Try it first, then show the solutionSolution

The foreign gene is inserted within the β-galactosidase gene of the vector, inactivating it (insertional inactivation); recombinant cells cannot make the enzyme, cannot act on the chromogenic substrate and form white colonies (1 mark).
Non-recombinant cells have an intact gene, make β-galactosidase, convert the substrate to a blue product and form blue colonies (1 mark).

Answer: White = recombinant (gene interrupted); blue = non-recombinant.

Example 5: Counting recombinants · 3 marks · Short answerHarder board level

After transformation with pBR322 carrying an insert in tet^R, 200 colonies grow on an ampicillin plate. On replica plating, 150 of them grow on tetracycline. (a) How many colonies are recombinant? (b) What fraction of transformants is recombinant? (c) Why did no colonies of untransformed cells appear?

Try it first, then show the solutionSolution

(a) Those that grew on ampicillin but not on tetracycline: 200 − 150 = 50 (1 mark).
(b) 50/200 = 0.25, i.e. 25% (1 mark).
(c) Untransformed E. coli have no ampicillin resistance and die on the ampicillin plate (1 mark).

Answer: (a) 50. (b) 25%. (c) They lack amp^R.

Example 6: Pick the vector · 4 marks · Short answerHOTS / competency

Suppose three plasmids are offered for cloning a BamHI fragment: P has amp^R and a single BamHI site but no ori; Q has ori, amp^R and tet^R, with one BamHI site inside tet^R; R has ori and amp^R, but its only BamHI site lies inside the ori sequence. (a) Which plasmid should be chosen? Give two reasons. (b) Explain why each rejected plasmid fails. (c) How would recombinants be detected with the chosen plasmid?

Try it first, then show the solutionSolution

(a) Q: its ori lets the insert replicate in the host, amp^R is a selectable marker, and its single BamHI site matches the fragment (1½ marks).
(b) P cannot replicate in the host because it has no ori; in R, inserting DNA at the BamHI site would disrupt the ori, so the recombinant plasmid could not replicate (1 mark).
(c) Insertion at BamHI inactivates tet^R: recombinants grow on ampicillin but not on tetracycline (1½ marks).

Answer: Q; P lacks ori and R would lose its ori on insertion; recombinants are ampicillin-resistant and tetracycline-sensitive.

Where students lose marks in Cloning Vectors and pBR322

The slips examiners see most often, and what to write instead.

Mistake: Reasons not matching the chosen vector
In 2024 examiners reported that the vector chosen did not match the reasons given, and that candidates could not tell Vector B from Vector C.
Do this: Check each option for ori, marker and a single site before choosing; quote the features of your choice.
Usually costs: Up to 2 marks.
Mistake: Not knowing the role of each feature
Examiners (2024) noted that roles of ori, selectable marker and restriction site were not understood.
Do this: Learn one line of role per feature.
Usually costs: 1 mark each.
Mistake: Swapping blue and white
In 2025 examiners reported colours interchanged, and 'blue colony' and 'white colony' repeated without insertional inactivation.
Do this: Write the chain: insert → lacZ inactivated → no β-galactosidase → white.
Usually costs: 1 to 2 marks.
Mistake: Plating recombinants on the wrong antibiotic
Saying recombinants grow on tetracycline when the insert is in tet^R is a common slip.
Do this: Insert in a gene = that resistance is lost.
Usually costs: 1 mark.

Exam tips

Write amp^R and tet^R with superscript R.
In diagrams, place BamHI inside tet^R; it is the site used for insertional inactivation in NCERT.
Use 'transformant' for any cell that took up a plasmid and 'recombinant' for one whose plasmid carries the insert.
Say 'chromogenic substrate' in blue-white answers.
Mention 'disarmed' for Ti plasmid and retroviruses.

Practice questions on Cloning Vectors and pBR322

Write each answer on paper first. Open the hint only if you are stuck, and the answer only when you have finished.

Q1 · 1 mark · MCQBoard level
In pBR322, the BamHI site lies within the gene for resistance to:
(a) ampicillin (b) tetracycline (c) kanamycin (d) chloramphenicol
Hint
Insert here for tet selection.
Answer
(b) tetracycline.
Q2 · 2 marks · Short answerBoard level
What is meant by copy number? Which part of a vector controls it?
Hint
Copies per cell.
Answer
Copy number is the number of copies of a plasmid (and any DNA linked to it) present in one cell. It is controlled by the origin of replication (ori).
Q3 · 1 mark · Assertion-reasonHarder board level
Assertion (A): A vector with several recognition sites for the same restriction enzyme is preferred for cloning.
Reason (R): Several sites produce several fragments of the vector.
(a) Both A and R are true and R is the correct explanation of A.
(b) Both A and R are true but R is not the correct explanation of A.
(c) A is false but R is true.
(d) Both A and R are false.
Hint
Fragments complicate cloning.
Answer
(c). A single site is preferred; several sites break the vector into fragments.
Q4 · 2 marks · Short answerHarder board level
How has the Ti plasmid of Agrobacterium tumefaciens been made into a useful vector?
Hint
Disarmed.
Answer
Agrobacterium naturally transfers T-DNA from its Ti plasmid into dicot cells, causing tumours. The Ti plasmid has been modified (disarmed) so it no longer causes disease but still delivers the gene of interest into plant cells.
Q5 · 2 marks · Short answerHarder board level
In a blue-white screening, 300 colonies grow, of which 240 are blue. How many recombinant colonies are there, and what percentage is this?
Hint
White = recombinant.
Answer
Recombinant (white) = 300 − 240 = 60, i.e. 60/300 × 100 = 20%.
Q6 · 2 marks · Short answerHarder board level
Why is blue-white selection preferred to selection by antibiotic resistance?
Hint
Plates.
Answer
Antibiotic-based selection needs simultaneous plating on two plates with different antibiotics, which is cumbersome; blue-white selection distinguishes recombinants from non-recombinants by colour on a single plate.
Q7 · 3 marks · Short answerHOTS / competency
Suppose a student inserts a gene at the PstI site (inside amp^R) of pBR322 and then plates the cells only on ampicillin. Predict what she will see and correct her plan.
Hint
Which resistance is lost?
Answer
Recombinants lose ampicillin resistance, so on ampicillin only non-recombinant transformants grow; she will miss her recombinants. She should first plate on tetracycline (selects all transformants, since tet^R is intact) and then replica-plate on ampicillin: colonies that fail on ampicillin are the recombinants.
Q8 · 3 marks · Case-basedHOTS / competency
Read and answer.
Suppose one transformed E. coli cell carrying a plasmid with copy number 20 divides every 30 minutes in a fresh medium.
(a) How many cells are there after 3 hours? (1)
(b) How many copies of the cloned gene do they carry in all? (1)
(c) Which vector feature would you change to raise the yield? (1)
Hint
6 generations.
Answer
(a) 3 h = 6 generations: 2⁶ = 64 cells. (b) 64 × 20 = 1280 copies. (c) Use a vector whose origin of replication supports a higher copy number.

Test your recall

Answer in your head or on paper first, then open the card. Pulling an answer out of memory makes it stick far better than re-reading.

1.What does rop code for in pBR322?Show answer
Proteins involved in the replication of the plasmid.
2.Which enzyme gene is used for blue-white screening?Show answer
β-galactosidase (lacZ).
3.What colour are recombinant colonies?Show answer
White (colourless).
4.Name a vector used to deliver genes into plants.Show answer
Disarmed Ti plasmid of Agrobacterium tumefaciens.
5.What controls copy number?Show answer
The origin of replication (ori).
6.Name two antibiotic-resistance genes in pBR322.Show answer
amp^R and tet^R.

Cloning Vectors and pBR322: frequently asked questions

What is a cloning vector?

A DNA molecule, such as a plasmid or bacteriophage, that can carry foreign DNA into a host cell and replicate it there.

What are the features of pBR322?

An origin of replication, the rop gene, ampicillin- and tetracycline-resistance genes, and single sites for enzymes such as HindIII, EcoRI, BamHI, SalI, PvuII, PstI and ClaI.

What is insertional inactivation?

Loss of a gene's function because foreign DNA is inserted into it; used to tell recombinants from non-recombinants.

Why are recombinant colonies white in blue-white screening?

The insert disrupts the β-galactosidase gene, so the substrate is not converted to a blue product.

Which vectors carry very large DNA pieces?

Bacterial artificial chromosomes (BAC) and yeast artificial chromosomes (YAC).