BiologyCBSE Class 12 · Biotechnology: Principles and Processes

Cloning Vectors and pBR322

Updated 15 October 2026 · Fact-checked

A cloning vector is a DNA molecule, such as a plasmid or bacteriophage, that carries foreign DNA into a host and multiplies it. It needs an origin of replication (ori), a selectable marker such as an antibiotic-resistance gene, and single recognition sites for restriction enzymes. In pBR322, inserting DNA into the tetracycline gene inactivates it, helping identify recombinants.

Foreign DNA cut into BamHI inside tetR breaks that gene, so recombinants resist ampicillin but not tetracycline.

Illustration drawn for QuestionBuddy.

Understand Cloning Vectors and pBR322

Why vectors. Plasmids and bacteriophages replicate inside bacteria independently of the chromosomal DNA. Bacteriophages have very high copy numbers per cell; some plasmids have only 1-2 copies per cell, others 15-100. Linking a piece of foreign DNA to such a vector multiplies it to the vector's copy number.

Features of a good cloning vector.
1. Origin of replication (ori): the sequence where replication starts. Any DNA linked to it can replicate in the host. The ori also controls the copy number of the linked DNA, so to get many copies, use a vector whose ori supports a high copy number.
2. Selectable marker: helps identify and eliminate non-transformants and lets only transformants grow. (Transformation = introducing a piece of DNA into a host bacterium.) Genes for resistance to ampicillin, chloramphenicol, tetracycline or kanamycin are useful markers for *E. coli*, since normal *E. coli* is not resistant to them.
3. Cloning sites: the vector should have 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. Vectors for plants and animals: see below.

pBR322 and insertional inactivation by antibiotics. pBR322 carries ori, rop (codes for proteins involved in plasmid replication), and two resistance genes, ** $am p^{R}$ (ampicillin) and $t e t^{R}$ (tetracycline), with restriction sites such as EcoRI, ClaI, HindIII, BamHI, SalI, PvuII, PstI and PvuI**. BamHI and SalI lie in $t e t^{R}$ ; PstI and PvuI lie in $am p^{R}$ .

Insert foreign DNA at the BamHI site in the tetracycline-resistance gene.
Recombinant plasmids lose tetracycline resistance (the gene is broken) but keep ampicillin resistance.
Plate transformed cells on ampicillin medium: only transformants grow (both recombinant and non-recombinant).
Transfer (replica-plate) these colonies to tetracycline medium: recombinants do not grow; non-recombinants grow on both.

One resistance gene selects transformants; the other, inactivated by the insert, identifies recombinants.

Blue-white selection (easier). Two-plate selection is cumbersome, so a colour-based marker is used. Foreign DNA is inserted within the coding sequence of the enzyme β-galactosidase, inactivating it: insertional inactivation. On a medium with a chromogenic substrate:

No insert → β-galactosidase works → blue colonies (non-recombinant).
Insert present → enzyme inactivated → colourless (white) colonies (recombinant).

Vectors for plants and animals.

*Agrobacterium tumefaciens*, a pathogen of many dicot plants, delivers a piece of DNA, T-DNA, that transforms plant cells into tumours producing chemicals the pathogen needs. Its tumour-inducing (Ti) plasmid has been modified into a vector that is no longer pathogenic but still delivers genes of interest into many plants.
Retroviruses can turn normal animal cells cancerous; disarmed retroviruses are used to deliver desirable genes into animal cells.

Competent host. DNA is hydrophilic and cannot pass through cell membranes, so bacteria are made competent by treating them with a divalent cation such as calcium, then incubated with recombinant DNA on ice, given a brief heat shock at 42 °C, and returned to ice. Other methods: micro-injection (into the nucleus of animal cells), biolistics or gene gun (plant cells bombarded with gold or tungsten particles coated with DNA), and disarmed pathogen vectors.

Flow chart (selection in pBR322): ligation mix → transform E. coli → ampicillin plate (kills non-transformants) → replica to tetracycline plate → colonies that fail on tetracycline = recombinants.

Draw and label: pBR322. Checklist: a circle (plasmid); arcs marking $am p^{R}$ and $t e t^{R}$ genes, ori and rop; small ticks for restriction sites placed correctly (BamHI and SalI on $t e t^{R}$ ; PstI and PvuI on $am p^{R}$ ; EcoRI, ClaI, HindIII between them; PvuII near rop). Labels: ori, rop, $am p^{R}$ , $t e t^{R}$ , EcoRI, ClaI, HindIII, BamHI, SalI, PvuII, PstI, PvuI, 'pBR322 (E. coli cloning vector)'.

Cloning Vectors and pBR322: key facts

Vector essentials: $ori + selectable marker + single cloning sites$; ori controls copy number.
pBR322 selection: $recombinant: am p^{R} (grows on ampicillin), t e t^{S} (fails on tetracycline)$; Insert at BamHI (or SalI) in $t e t^{R}$ ; non-recombinants grow on both.
Blue-white: $blue = no insert; white (colourless) = recombinant$; Insert inactivates β-galactosidase (insertional inactivation).
Copy number: $copies of insert = cells \times plasmid copies per cell$; Plasmids: 1-2 to 15-100 copies per cell.
Making cells competent: $C a^{2 +} \to ice \to 42^{\circ} C heat shock \to ice$; Lets hydrophilic DNA enter bacteria.

How to answer Cloning Vectors and pBR322 questions

Vector questions are about features, pBR322 selection, or blue-white screening. Track what each gene does before and after an insert.

1List the three features of a vector with one line of purpose each (ori: replication and copy number; marker: select transformants; cloning site: single site to insert DNA).
2For pBR322 cases, find where the insert went (which gene) and mark that gene as inactivated.
3Predict growth: the intact resistance gene still works; the interrupted one does not. Recombinants grow on the antibiotic of the intact gene only.
4For blue-white, remember: insert inside β-galactosidase → no enzyme → no colour → recombinant.
5For 'why single site', explain that multiple sites cut the vector into several pieces.
6For plant or animal vectors, name the disarmed pathogen (Ti plasmid of Agrobacterium, retroviruses) and say it keeps delivery ability but not disease.

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.

Selecting recombinants with pBR322

Suppose a student inserts a gene into the BamHI site of pBR322, transforms E. coli, and plates them on ampicillin. She gets 200 colonies. She replica-plates them onto tetracycline, where 140 colonies grow.

How many colonies carry recombinant plasmids, and what happened to the bacteria that did not take up any plasmid? What fraction of transformants are recombinants?

Try it first, then show the solutionSolution

Bacteria without plasmid are not resistant to ampicillin, so they did not form colonies on the ampicillin plate.
All 200 colonies are transformants (have pBR322, recombinant or not).
Recombinants have the insert in $t e t^{R}$ , so they cannot grow on tetracycline: 200 − 140 = 60 recombinant colonies.
Fraction recombinant = 60/200 = 0.3 (30%); the 140 that grew on both antibiotics are non-recombinants.

Answer: 60 recombinant colonies (30% of transformants); non-transformants died on ampicillin; 140 are non-recombinants.

Memory hook: Ampicillin finds the transformed; tetracycline unmasks the recombinant.

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-S-C
Ori, Selectable marker, Cloning site: the three must-haves.
Use it when: Features of a vector.
White is right
In blue-white screening, white (colourless) colonies have the insert.
Use it when: Picking recombinants.
Bam in Tet
BamHI (and SalI) sit inside $t e t^{R}$ ; PstI (and PvuI) inside $am p^{R}$ .
Use it when: Predicting which gene an insert inactivates.

Cloning Vectors and pBR322: solved examples

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

Example 1: Role of ori · 1 mark · Very short answerBoard level

What is the role of the origin of replication in a cloning vector?

Try it first, then show the solutionSolution

Replication starts at ori, so any DNA linked to it replicates in the host; ori also controls the copy number of the linked DNA.

Answer: Starts replication of the linked DNA and controls its copy number.

Example 2: Features of a vector · 3 marks · Short answerBoard level

Explain the importance of (a) selectable marker and (b) cloning sites in a cloning vector.

Try it first, then show the solutionSolution

(a) A selectable marker (e.g. an antibiotic-resistance gene) helps identify and eliminate non-transformants and allows only transformants to grow (1½ marks).
(b) Cloning sites are recognition sites for restriction enzymes where foreign DNA is ligated; there should be very few, preferably single, sites, because several sites would cut the vector into many fragments and complicate cloning (1½ marks).

Answer: Marker separates transformants; single cloning sites allow clean insertion.

Example 3: Insertional inactivation in pBR322 · 3 marks · Short answerHarder board level

A foreign DNA is ligated at the BamHI site of pBR322 and E. coli is transformed. How will you identify (a) transformants and (b) recombinants?

Try it first, then show the solutionSolution

BamHI lies in $t e t^{R}$ , so the insert inactivates tetracycline resistance; $am p^{R}$ stays intact (1 mark).
(a) Plate on ampicillin: only cells carrying pBR322 (transformants) grow (1 mark).
(b) Replica-plate onto tetracycline: recombinants fail to grow ( $t e t^{R}$ inactivated); non-recombinants grow on both (1 mark).

Answer: Ampicillin selects transformants; failure on tetracycline identifies recombinants.

Example 4: Blue-white screening · 3 marks · Short answerHarder board level

Explain how recombinant colonies are identified using a chromogenic substrate. Why is this method preferred to antibiotic-based insertional inactivation?

Try it first, then show the solutionSolution

The foreign DNA is inserted within the coding sequence of β-galactosidase, inactivating the gene (insertional inactivation) (1 mark).
On a medium with a chromogenic substrate, colonies without an insert make the enzyme and turn blue; recombinant colonies cannot make it and stay colourless (1 mark).
It is preferred because it needs a single plate, whereas antibiotic inactivation needs simultaneous plating on two plates with different antibiotics (1 mark).

Answer: Insert inactivates β-galactosidase; recombinants are white, non-recombinants blue; single plate is simpler.

Example 5: Insert in the ampicillin gene · 3 marks · Short answerHOTS / competency

Suppose the foreign DNA is ligated at the PstI site of pBR322 instead. Predict the growth of recombinant and non-recombinant transformants on ampicillin and on tetracycline plates, and say which antibiotic must be used first.

Try it first, then show the solutionSolution

PstI lies in $am p^{R}$ , so the insert inactivates ampicillin resistance; $t e t^{R}$ remains intact (1 mark).
Recombinants: grow on tetracycline, fail on ampicillin. Non-recombinants: grow on both (1 mark).
Plate first on tetracycline to select transformants, then replica-plate on ampicillin; colonies that fail on ampicillin are recombinants (1 mark).

Answer: Recombinants are tet-resistant but amp-sensitive; select on tetracycline first, then screen on ampicillin.

Example 6: Disarmed pathogens as vectors · 2 marks · Very short answerHarder board level

How has Agrobacterium tumefaciens been turned into a useful vector for plants? Name the animal counterpart.

Try it first, then show the solutionSolution

Agrobacterium naturally transfers T-DNA from its Ti plasmid into dicot plant cells, causing tumours; the Ti plasmid has been modified so it no longer causes disease but still delivers genes of interest into plants (1 mark).
Animal counterpart: disarmed retroviruses, used to deliver desirable genes into animal cells (1 mark).

Answer: Disarmed Ti plasmid delivers genes without tumours; disarmed retroviruses in animals.

Where students lose marks in Cloning Vectors and pBR322

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

Mistake: Blue colonies taken as recombinants
Colour is linked to 'success'.
Wrong working
Blue colonies have the gene of interest.
Right working
Blue = no insert; white (colourless) = recombinant.
Do this: Insert breaks the colour gene, so no colour.
Usually costs: 1 mark.
Mistake: Wrong plate order
Students plate on the inactivated antibiotic first.
Wrong working
Insert in $t e t^{R}$ : plate first on tetracycline.
Right working
Plate first on ampicillin (intact gene) to get transformants; then check tetracycline.
Do this: Select transformants with the intact gene.
Usually costs: 1-2 marks.
Mistake: Confusing transformant and recombinant
Both terms describe engineered cells.
Wrong working
All colonies on ampicillin are recombinants.
Right working
Colonies on ampicillin are transformants; only those with the insert are recombinants.
Do this: Transformant = has a plasmid; recombinant = plasmid with insert.
Usually costs: 1 mark.
Mistake: Saying ori is a selectable marker
Features are mixed up.
Wrong working
ori helps select transformants.
Right working
ori controls replication and copy number; antibiotic-resistance genes are selectable markers.
Do this: One function per feature.
Usually costs: 1 mark.

Exam tips

Draw pBR322 with sites in the right genes; examiners check BamHI/SalI in $t e t^{R}$ and PstI/PvuI in $am p^{R}$ .
Use the phrase 'insertional inactivation' explicitly.
When describing competence, include calcium, ice, 42 °C heat shock, ice.
Mention copy number with ori whenever ori is asked.
Name both disarmed vectors: Ti plasmid (plants) and retroviruses (animals).

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 blue-white screening, recombinant colonies are:
(a) blue (b) colourless/white (c) red (d) green
Hint
β-galactosidase inactivated.
Answer
(b) colourless/white.
Q2 · 1 mark · Assertion-reasonHarder board level
Assertion (A): Bacterial cells are treated with calcium ions before transformation.
Reason (R): DNA is hydrophilic and cannot pass through cell membranes on its own.
(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 true but R is false.
(d) A is false but R is true.
Hint
Competent cells.
Answer
(a).
Q3 · 2 marks · Very short answerBoard level
Why should a cloning vector have only one recognition site for a given restriction enzyme?
Hint
Number of fragments.
Answer
With one site, the enzyme opens the circular vector at a single point to accept the insert. More than one site would cut the vector into several fragments, which would complicate cloning (the pieces may be lost or rejoin wrongly).
Q4 · 2 marks · Very short answerHOTS / competency
Suppose a bacterium carrying a recombinant plasmid with a copy number of 40 per cell divides every 30 minutes. How many copies of the inserted gene are present after 5 hours, starting from one bacterium? Why would a researcher choose a vector with a high-copy ori?
Hint
5 hours = 10 generations.
Answer
10 generations → 2¹⁰ = 1024 cells; copies = 1024 × 40 = 40,960. A high-copy ori gives many copies of the target DNA per cell, so more of the gene (or its product) is recovered.
Q5 · 4 marks · Case-basedHarder board level
Read and answer.
Suppose a diagram of pBR322 shows ori, rop, $am p^{R}$ , $t e t^{R}$ and restriction sites. A student inserts DNA at the SalI site.
(a) Which gene is inactivated? (1)
(b) On which antibiotic plate will the recombinants grow, and on which will they fail? (1)
(c) What is the function of rop, and why must ori never be cut? (2)
Hint
SalI is in $t e t^{R}$ .
Answer
(a) $t e t^{R}$ (tetracycline resistance). (b) Grow on ampicillin; fail on tetracycline. (c) rop codes for proteins involved in the replication of the plasmid. ori is where replication begins and controls copy number; if cut, the plasmid could not replicate in the host.
Q6 · 2 marks · Very short answerHarder board level
Suggest the method best suited to introduce recombinant DNA into (a) a plant cell with a cell wall, and (b) the nucleus of an animal cell. Give the reason for each choice.
Hint
High-velocity particles; direct injection.
Answer
(a) Biolistics (gene gun): tiny gold or tungsten particles coated with the DNA are fired at the plant cells at high velocity and carry the DNA through the wall; a disarmed Agrobacterium (Ti plasmid) vector is an alternative for many plants. (b) Micro-injection: the recombinant DNA is injected directly into the nucleus of the animal cell.

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.Two antibiotic-resistance genes in pBR322?Show answer
$am p^{R}$ and $t e t^{R}$ .
2.What does rop code for?Show answer
Proteins involved in plasmid replication.
3.Which enzyme gene is used for blue-white screening?Show answer
β-galactosidase.
4.Which divalent cation makes cells competent?Show answer
Calcium.
5.At what temperature is the heat shock given?Show answer
42 °C.
6.What does Agrobacterium transfer into plant cells?Show answer
T-DNA (from its Ti plasmid).

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 a good cloning vector?

An origin of replication, a selectable marker, and very few (preferably single) recognition sites for restriction enzymes.

What is insertional inactivation?

Inactivation of a gene when foreign DNA is inserted within it, used to identify recombinants (e.g. loss of tetracycline resistance or of β-galactosidase activity).

How does blue-white screening work?

Inserting DNA into the β-galactosidase gene stops enzyme production, so recombinant colonies stay colourless on a chromogenic substrate, while non-recombinants turn blue.

Why is the Ti plasmid used in plant biotechnology?

It naturally delivers T-DNA into plant cells; once disarmed it no longer causes tumours but can still deliver useful genes.