How to synthetically make a protein.

How to synthetically make a protein:

Should probably know this for the final exam on Tuesday.

Step 1: Isolate the gene for the protein by using PCR. With PCR you can use primers to isolate the gene and amplify the gene sequence. (A bunch of copies are made.)
Now you have the gene of interest.

Step 2: Restriction digestion.
-use restriction enzymes to cut plasmid and gene of interest.
-insert the gene of interest into our plasmid @ a specific location
-ligation. (or "litigation" is how I keep saying it on accident) this connects the discontinuous DNA strand and the gene of interest

Side note: Why do we use a plasmid? A plasmid is a circular nonessential piece of DNA that is exchanged btwn bacteria. Use plasmids because they can survive better in different environment: like how they have antibiotic resistance.

Step 3: Transformation
-Heat shock the bacteria forcing it to take up the plasmid. The H.S. is designed to make small holes in the cell wall of that bacteria so plasmids can hopefully enter the bacteria cell.

Step 4: Plate on differential media.
Sometimes can use 3 or more kind but generally only use 2 different plates- like we used LB (Luria Broth agar) and LB+amp in the first lab. The first plate, LB, makes sure the heat shock didn't actually kill off the bacteria. So the LB plate should usually be covered. The other plate used selects for bacteria that took up the plasmids. If even a minimum of a few colonies are present of the second plate then we know the gene of interest is being expressed.

Step 5: Now have to figure out if the plasmid that is present actually contains the gene of interest. For example: in the pGLo lab, GFP would be produced if arabinose is in the media (we put the GFP gene behind arab. metabolism gene) so we could check this expression by using the black light

—> If you notice any mistakes or would like to make an addition, feel free! -Alicia

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