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.
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.
- 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.
- 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.
colonies on the master plate contain plasmids?
- 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!)
colonies on the master plate contain recombinant plasmids?
have now identified the bacteria containing the plasmids with
the gene you require. These bacteria are said to be transformed.
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.
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.