Simulation · Biology · Class 12
Heat shock: getting DNA into E. coli
From the lesson Plant and animal vectors, and competent hosts in Biotechnology: Principles and Processes. Change the values and watch what happens.
Heat shock: getting DNA into E. coliBiology · Class 12
The idea behind it
NCERT §9.2.2 (iv), §9.2.3
- Methods of moving genes into plants and animals were learnt from bacteria and viruses. These pathogens deliver genes into eukaryotic cells and make the cells serve them.
- Agrobacterium tumefaciens, a pathogen of several dicots, delivers a piece of DNA called T-DNA. The T-DNA turns normal plant cells into a tumour that makes chemicals the pathogen needs. Retroviruses turn normal animal cells cancerous.
- The tumour-inducing (Ti) plasmid of Agrobacterium has been modified into a cloning vector. It no longer causes disease but still delivers chosen genes into many plants. Disarmed retroviruses deliver desirable genes into animal cells.
- DNA is hydrophilic and cannot pass through cell membranes, so bacteria must first be made competent to take it up.
- Cells are treated with a set concentration of a divalent cation, calcium for example, which helps DNA get in through pores in the cell wall more efficiently.
- The cells are incubated with recombinant DNA on ice, placed briefly at 42°C (heat shock), and returned to ice. This lets them take up the DNA.
- Micro-injection: recombinant DNA is injected directly into the nucleus of an animal cell.
- Biolistics (the gene gun), used for plants: tiny particles of gold or tungsten, coated with DNA, are fired into the cells at high velocity.
- Disarmed pathogen vectors transfer recombinant DNA into the cells they are allowed to infect.