Putting the plasmids into Bacteria to make Clones

Firstly the bacteria cells are treated with chemicals or shockwaves to make their walls permeable and increase their chance of taking up a plasmid. These bacteria are then mixed and incubated with the recombinant plasmids.

The antibiotic resistance genes on the plasmids are used as gene markers to see which bacteria have taken up plasmids. The plasmid below has a red gene for tetracycline resistance and a green gene for Ampicillin resistance. The other points marked on the plasmid are where various different restriction enzymes will cut the plasmid up.

1 - The bacteria are spread onto plates of agar so they can grow into individual colonies. Each bacterium within a colony is genetically identical due to asexual reproduction.

2 - The colonies are then transferred/ by blotting onto new plates with agar containing Tetracycline. (The colonies are blotted onto the new plates in exactly the same positions). Only those colonies of bacteria that have taken up plasmids will be able to survive. Without the plasmid they will not be resistant to Tetracycline and will die.

Which colonies on the master plate contain plasmids?

3 - However, they must now be checked to see if those colonies that survived the Tetracycline actually contain recombinant plasmids. The Tetracycline plates are blotted onto Ampicillin agar once more and this time only the bacteria containing plasmids without the gene in will be able to grow (that is, these plasmids just glued themselves back together and did not take up the gene we want!)

Which colonies on the master plate contain recombinant plasmids?

You have now identified the bacteria containing the plasmids with the gene you require. These bacteria are said to be transformed.

The transformed bacteria can now be grown or fermented on a large scale. Each time the bacteria cells divide the plasmid inside will also divide and replicate a copy of the gene.

If the engineering has been a success, the bacteria will use the code on the inserted gene to produce the protein you want. This can then be extracted and purified for use.

However, these days scientists are far more interested in Inserting genes into multicellular living organisms and altering the way they perform or function forever.