Recombinant DNA
Step 1: Isolation
A restriction enzyme (endonuclease) is a DNA-cutting enzyme that recognises a specific target sequence and cuts DNA into two pieces at or near that site (recognition site).
Most restriction enzymes produce cut ends with short, single-stranded overhangs called sticky ends. If two molecules have matching overhangs, they can base-pair and stick together. However, some produce blunt ends.
Step 2: Prepare the plasmid
Use the same restriction enzyme that you used to remove the DNA fragment in step 1 to make a single cut in a plasmid. This is going to be the plasmid vector that is inserted back into a bacterium.
Step 3: Glue in the DNA
DNA ligase seals gaps in DNA backbone. Therefore, when it’s used in recombinant DNA, it will link the plasmid and the DNA fragment to make a recombinant plasmid containing the gene.
DNA ligase links the phosphate group of one DNA strand to the hydroxyl group of the other DNA strand to form a single sugar-phosphate backbone/combined piece of DNA.
Step 4. Bacterial Transformation
Plasmids can be introduced into bacteria in a process called transformation.
Specially prepared bacterial cells are given a heat shock that encourages them to take up foreign DNA.
Plasmids normally also contain an antibiotic-resistance gene, so this is an easy way to see which bacteria took it up. You can grow bacteria on agar plates that have been applied with an antibiotic. If they survive, you have a winner.
You could also use fluorescence markers or enzyme marker.
You would need to collect samples from many colonies to see if they have taken up the DNA fragments correctly. Surprisingly, only 1% of bacteria take up plasmids, and even then, may not even contain the DNA fragment. PCR can help to check this (more on this later).
Step 5. Produce your protein
When a bacterial colony with the right plasmid has been found, then it can be grown in a culture.
The bacteria start to transcribe the gene and will produce the coded for functional protein.
The bacteria are then split open to release the product and the end product is then purified.