A very common method of cloning uses restriction enzymes. Restriction enzymes were first discovered by Ronald W. Davis from Stanford. The basic mechanism is shown in the image below.
We use a version of sequence and ligation independent cloning (SLIC).
We call this LICKI…(for you Pokemon fans, this is named after Lickitung)
(This protocol was created and developed by Thaddaus Huber in the Snow Lab)
With the plasmid completed with the gene of interest, we now transform the plasmid into E. coli via Heatshock. See the protocol below.
After cloning, you should make A LOT of your plasmid for future transformations and to check that the sequence in the plasmid is correct. We use 5-alpha competent E. coli. This cell line loves to make lots of the DNA.
After growing the cultures to an optical density of absorbance of 1, they are ready to die… in other words, we can break the cells open to retrieve the hundreds to thousands of plasmids they replicated for us. We call this breaking of cells “mini-prep”. See the protocol below on how to mini-prep.
Once we have verified that our plasmid sequence is correct, we are ready for expression of the protein. There are many ways to over-express your protein but we love the strong T7 promoter. The schematic below is a simplified version of the induction process.
Factors to consider when setting up an expression experiment:
- Prevent a leaky T7 promoter with 1% glucose in the starter media (don’t want to express too soon if the protein is toxic to the cells)
- RPM to shake
- Time for outgrowth
- When to induce (what optical density)
- What concentration of IPTG (or inducing agent)
All of these factors come into play to brew up the best stew of your protein of interest.
How do we know if we got over-expression?
We can check our results with SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis).
Let’s start with the bad news. This is a gel of an unsuccessful overexpression…
Lane 1 is the control while lanes 2-4 are the expression condition trials. This may look like a success, but the same protein is in the control.
Lane 4 is the control and lanes 6-7 are the trials.
Lane 8 is the control and lanes 9-11 are the trials.
Now the good news! This is a gel of a successful over-expression. The first lane is the control with the lanes following the expression trials. All four expressed the protein!
Once it is confirmed that you can over-express the protein with your expression protocol, you can grow large batches (the Liter scale) of the protein for purification.
Next step, we want to get our protein away from all the other proteins that E. coli makes on a regular basis.
First, we have to kill the cells. Ok, we just break them up with super strong sound waves in a process called sonication.
Then, we can apply the supernatant, with the soluble protein floating around in solution, to multiple rounds of purification.
A Nickel Column is our first choice. This is a quick way to separate your protein if the protein was expressed with a histidine tag.
As you can see, in the last tube, there are still some impurities. So, we use a secondary method. The next step seperates by size: Size Exclusion Column
Finally, we have pure enough protein to combine with the DNA for crystallization.