Lesson 10
Molecular Biology
In module 1 we have begun to develop a foundational knowledge of biochemical concepts and in module 2 we focused on the structure and function of proteins. Now that we have made it to module 3, we will be discussing techniques we use in the lab to design, grow, purify, and analyze proteins. This lesson will focus on the molecular biology used to designing and making the DNA constructs that encode the proteins we are interested in.
Obtaining DNA
In order to obtain (express and purify) the proteins we are interested in (the topic of next week's lesson), we need to have the DNA construct which codes for that protein because we will use bacteria to produce the protein from the DNA we design. (Remember that DNA codes for mRNA in transcription which codes for amino acids in translation - called the central dogma). One way to obtain DNA from something you are interested in is to extract it from that thing itself. In fact, you can even extract the DNA from a strawberry in your own kitchen! However, a lot of times in the lab practically, we just order the DNA sequence for our protein that is made synthetically.
DNA Plasmid
Once we have the DNA for our protein of interest, we need to insert it into a expression vector which we will eventually use to produce the protein in bacterial cells. This vector is a plasmid - a circular DNA molecule found in bacteria or yeast cells. Watch this video from Addgene describing plasmids and how they are useful in the laboratory applications.
As the video described, we insert the DNA for our protein of interest into the plasmid using restriction enzymes (proteins which cut the plasmid within a specific amino acid sequence). One or two different restriction enzymes can be used (when two are used it is called a double digest). In order to determine if the plasmid has been proper digested (cleaved or cut by the enzyme), we can run a DNA gel which will show bands at the corresponding molecular weight of the DNA fragment. Watch this video describing DNA digestion and DNA gel analysis.
Q1: A common DNA plasmid we use in the lab is named pET28b+. Here is a map of this vector showing all of the restriction enzyme cut sites (the base pair of the cleavage site is shown in parenthesis next to each restriction enzyme.) Draw what a DNA gel would look like for each of the following (like in the video):
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Uncut
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Cut by BamHI
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Cut by EcoRV
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Cut by HindIII
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Cut by BamHI and EcoRV
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Cut by BamHI and HindIII
Q2: The plasmid map shows KanR signaling kanamycin (an antibiotic) resistance. Why is this important (this was touched on in the video)?
New England Biolabs (where we purchase most restriction enzymes and cloning supplies from) has great resources for developing protocols. Using the Double Digest Finder, develop a protocol for performing a double digest on pET28b+ using BamHI and EcoRV.
Q3: What temperature should this reaction be performed at? What buffer should be used for this reaction?
A double digest needs to be completed on both the plasmid and the DNA insert (containing the DNA for our protein of interest) so that they have compatible ends (where the cleavage took place). The vector and the insert can then be ligated together using a ligase enzyme which joins the compatible ends together.
DNA Mutation
In certain cases we may want to mutate certain amino acids in our protein of interest. For example, we may mutate an amino acid we think is important for the function of the protein and see what happens to the activity or function of the protein. We might also want to mutant amino acids in order to perform some type of labeling (to detect properties of the protein). For example, in electron paramagnetic resonance (EPR) we covalently attach the spin label required for the experiment onto cysteine residues. Thus, we need to remove any native cysteines (if there are any), and then add in cysteines at the locations for which we want to investigate.
There are several techniques which can be used to introduce mutations into the DNA sequence, but all use primers (short DNA sequences of ~10 - 50 bases) which contain overlapping DNA bases with the plasmid and mismatched DNA bases that encode the desired mutation (insertions and deletions can also be made in this way). PCR (polymerase chain reaction) is then used with the primers (and other components) to amplify (make lots and lots of copies) the desired DNA. Watch this video describing one such technique using a specialized kit from New England Biolabs.
Q4: In lesson 6 we found that the DNA 5'- ATGCCGGATGTG -3' coded for the amino acid sequence - MPDV. Remember also that LspA is an aspartyl protease and requires two aspartic acid amino acids (D) to cleave its lipoprotein substrate. If we wanted to test if the D amino acid in the MPDV sequence above was required for LspA function, what might we mutate the DNA sequence to? Hint: for functional studies, amino acids of interest are often mutated to alanine, one of the smallest amino acids.
Transfomation
After we produce the plasmid containing the DNA for our protein of interest, we must perform a transformation in order to get the DNA plasmid into bacterial cells which we will use to grow our protein (we will talk about protein growth and purification in the next lesson). These cells are called competent cells because they easily take up extracellular DNA. We generally use E.coli cells, but there are many different strains that can be used for different applications. You can watch this video to see a transformation being performed.