Botany · Ch 8 — Biotechnology: Principles and Processes
Competent Host (For Transformation with Recombinant DNA)
Competent Host (For Transformation with Recombinant DNA)
Why DNA Can't Enter Cells on Its Own
DNA is a hydrophilic (water-loving) molecule. Cell membranes, on the other hand, are made of lipids (fats) and are hydrophobic in their interior. A hydrophilic molecule like DNA simply cannot slip through this fatty barrier — it gets repelled. So if you just mix plasmid DNA with bacteria in a test tube, nothing happens. The bacteria will not take it up.
To make transformation happen, you must first make the bacterial cells competent — that is, capable of taking up foreign DNA.
Making Cells Competent: The Calcium Chloride Method
The standard lab technique to induce competence uses a divalent cation — most commonly calcium ions (Ca²⁺) . The bacterial cells are treated with a specific concentration of calcium chloride solution.
Why does calcium work? The positive calcium ions neutralise the negative charges on both the DNA molecule and the bacterial cell surface. This reduces repulsion. At the same time, calcium ions cause changes in the bacterial cell wall, creating temporary pores through which DNA can pass. This treatment increases the efficiency with which DNA enters the bacterium.
The Heat Shock Step
Once the cells are treated with calcium, the actual entry of recombinant DNA is forced by a temperature cycle:
- Incubate on ice — The mixture of competent cells and recombinant DNA is kept in ice-cold conditions (0°C). This slows down the cell's metabolism and makes the membrane more fluid and receptive.
- Brief heat shock at 42°C — The cells are briefly placed at 42°C (the book gives no exact duration) before being returned to ice. This heat shock, following the calcium chloride treatment, increases the efficiency with which DNA enters the bacterium through pores in its cell wall.
- Return to ice — The cells are put back on ice to stabilise them.
This entire procedure — calcium treatment followed by the ice-heat-ice cycle — is the classic method to introduce recombinant DNA into bacterial hosts like E. coli.
Alternative Methods for Introducing DNA
The calcium chloride method works well for bacteria, but not for all cell types. The textbook describes three other important techniques:
| Method | How it works | Used for |
|---|---|---|
| Micro-injection | Recombinant DNA is directly injected into the nucleus using a fine glass needle under a microscope. | Animal cells |
| Biolistics / Gene gun | Cells are bombarded with high-velocity micro-particles of gold or tungsten that have been coated with DNA. The particles penetrate the cell wall and membrane, delivering the DNA inside. | Plant cells (which have tough cell walls) |
| Disarmed pathogen vectors | A pathogen (virus or bacterium) is genetically modified so it cannot cause disease — it is 'disarmed'. This harmless vector is allowed to infect the host cell, and as it does so, it carries the recombinant DNA into the cell. | Both plant and animal cells |
The term 'biolistics' is a blend of biology and ballistics — because it literally shoots DNA-coated particles into cells.