8 min readPathway sheet1-mark MCQ, and a 2 or 3-mark question on making cells competent or selecting transformants; also a NEET favourite

Competent host and inserting recombinant DNA

DNA cannot cross cell membranes, so bacteria are made competent with calcium ions and a heat shock. Other cells get DNA by micro-injection, gene gun or disarmed pathogens.

Why cells resist DNADNA is hydrophilic (water-loving), so it cannot pass through the cell membrane

Competence treatmentA specific concentration of a divalent cation such as calcium (Ca²⁺)

Heat shockCells with recombinant DNA kept on ice, then briefly at 42°C, then back on ice

Selecting transformantsIf the recombinant DNA carries ampR, only transformed cells grow on an ampicillin plate

Making E. coli competent and transforming it

A **competent** cell is one that can take up DNA from its surroundings.

1
Treat the bacterial cells with a specific concentration of a divalent cation, such as calcium.
This raises the efficiency with which DNA enters through pores in the cell wall
2
Incubate the cells with the recombinant DNA on ice.
3★
Place them briefly at 42°C: the heat shock.
4
Put them back on ice. The bacteria now take up the recombinant DNA.

★ key step

Other ways to get DNA into a host cell

Method	How it works	Used for
Micro-injection	Recombinant DNA is injected directly into the nucleus with a very fine needle	Animal cells
Biolistics (gene gun)	Cells are shot with tiny high-velocity particles of gold or tungsten that carry a coating of DNA	Plant cells
Disarmed pathogen vectors	A pathogen that can no longer cause disease infects the cell and transfers the recombinant DNA	Plants (Agrobacterium) and animals (retroviruses)

Inserting recombinant DNA and picking out transformants

1
Competent E. coli cells take up recombinant DNA that carries an ampicillin resistance gene.
2
Cells that took it up become ampicillin-resistant: they are transformed.
3
Spread all the cells on agar plates containing ampicillin.
4★
Only transformants grow; untransformed cells die. The ampicillin resistance gene is the selectable marker.

★ key step

Transformant vs recombinant

TransformantAny host cell that has taken up a plasmid, with or without the foreign gene. It grows on the antibiotic of its marker.

RecombinantA transformant whose plasmid carries the foreign gene. It is picked out by insertional inactivation of a second marker.

Every recombinant is a transformant, but not every transformant is a recombinant.

In real life

The hepatitis B vaccine given to Indian babies is made by putting the gene for a viral surface protein into yeast cells. Every such product begins with getting recombinant DNA into a host cell.

Exam angle

2 marks: “Why must a bacterial cell be made competent before transformation? How is this done?” DNA is hydrophilic and cannot cross the membrane. Treat cells with a divalent cation such as calcium; incubate with recombinant DNA on ice, give a heat shock at 42°C, then return to ice.

3 marks: “Describe three methods of introducing recombinant DNA into host cells, naming the host each suits.” Micro-injection: directly into the nucleus of an animal cell. Biolistics: plant cells bombarded with gold or tungsten particles coated with DNA. Disarmed pathogens: Agrobacterium (plants) or retroviruses (animals) infect and transfer the DNA.

2 marks: “How does an ampicillin resistance gene help to select transformed E. coli cells?” Normal E. coli is killed by ampicillin. Recombinant DNA carries ampR, so on an ampicillin plate only transformed cells grow. ampR acts as a selectable marker.

Remember it

Make bacteria competent with calcium, cold, hot, cold: Ca²⁺, ice, 42°C, ice.