Methods in biochemistry and protein engineering Flashcards
(37 cards)
There are many components in a biochemist or molecular biologist toolbox, name four and what their used for in short.
- Type II restriction endonucleases: cleave DNA at specific sequences and produce defined ends, can be useful in cloning. Modifications like methylation can hinder or enable cuts depending on restriction enzyme.
- DNA ligase: covalently couples DNA ends together.
- DNA polymerases: DNA synthesis, nick translation.
- Reverse transcriptase: Generates cDNA from RNA template, extremely useful to clone eukaryotic genes as you get rid of all non coding parts of the gene.
- Exonucleases: used to shorten down DNA (not used so much anymore tho)
- Alkaline phosphatase: removing phosphoryl groups in end of DNA, very useful to ensure that DNA don’t self ligate.
Type II restriction endonucleases produce defined ends. What two types of ends can they produce, when is what useful?
Restriction endonucleases can produce staggered ends (with overhang) or blunt ends (no overhang). Staggered ends can only pair with the opposite overhang, which provides specificity but less effective ligation. These can be cut again if needed, as palindromic seq is still there. Blunt ends can pair to any other end, less specificity but more effective ligation. Can’t be cut again tho.
Some families of restriction enzymes produce the same ends, the ends can be ligated together but the palindromic recognition seq is lost, so they cant be cut again. Can be useful.
Why do you often use two different restriction enzymes when cutting the donor DNA?
You use two different restriction enzymes 1. to be able to predict the direction of the fragment once its inserted, 2. to ensure that the ends don’t re-ligate.
Studies of genes and gene products often require molecular cloning, to get more material to work with. What is molecular cloning and what do you need to do it?
Molecular cloning is producing many identical copies of DNA fragments, e.g. like a gene.
To perform cloning, you need:
- a vector, something that can take up the DNA fragment and multiply it.
- a host cell, that can supply energy for survival and replication. E. coli most common.
Give two examples of vectors used in molecular cloning.
- Plasmids: small, circular dsDNA originating from bacteria, that can stably carry foreign DNA. The fragment can be up to 3000bp.
- Phages: a bacterial virus where you have removed the major part of the viral genome and only kept the genes for packaging and delivery. The fragment can be up to 10 kbp and the most commonly used one is called lambda.
- BAC (bacterial artificial chromosome) for very large fragments and used in genome studies.
When you have created a vector containing the donor DNA, how do you get it into the host cells?
By transformation: you treat the membrane with ions, and then use heat shock or electroporation to make the cell membrane less rigid and more permeable.
Explain the term “propagation” in cloning.
Propagation means letting the transformed cells divide to amplify the number of clones. Then we can lyse the cells and isolate the plasmids. The cells own genome is so big so it will precipitate.
In short, explain how you would go about cloning a donor DNA fragment using E. Coli as host.
- cleave the cloning vector with two different restriction endonucleases.
- cleaving the donor DNA with the same restriction enzymes to make sure the ends are compatible with the cleaved vector.
- Use DNA ligase to insert the donor DNA into the vector.
- Transform the plasmids into E. Coli by heat shock.
- Propagate the cells to produce many plasmid copies.
- lyse the cells
- isolate the plasmids and BAM you’re done.
Alternatively, after step 5, you could induce expression of the gene, incubate and then move on to step 6 to isolate for example a protein.
What is required of a vector for it to be used?
- For plasmids, they need to include the unique restriction sites and have a marker, most common is genes for antibiotic resistance, so that you can select for only the bacteria containing the plasmid.
- For viral vectors like lambda, they also need to include the restriction sites and they need contain the genes needed for packaging and the resulting genetic material needs to be long enough to package.
Plasmids usually contain resistance for multiple antibiotics, why is this useful?
Because if you choose restriction sites that disrupt one of these markers you can re-streak colonies confirmed to contain the plasmid on plates with a mix antibiotics to see which don’t make it on the plate containing the antibiotic you disrupted. These colonies contain the plasmid with the desired insert.
How do you go about packaging recombinant DNA in viral vectors?
You simply mix the the DNA with empty phage heads and tails, and they will assemble by themselves bc the reaction has a negative delta G (thermodynamically favorable).
Explain the blue white selection system in short.
The blue white selection system is based on the lac operon, which encodes for lactose metabolism genes. A synthetic version of lactose; X-gal is added, that when broken down leaves a blue product behind. If you use restriction enzymes that disrupts the lac operon and add X-gal, the colonies containing the insert will be white while the colonies not containing the insert will be blue.
To confirm that you have isolated the DNA fragment of interest, how do you proceed?
Use the same restriction enzymes that you used to cut it out of the donor genome, run in gel and analyse the fragment pattern. If you see a band of the predicted length, you can cut the band out and BAM you have isolated the fragment.
Explain the basics of sanger sequencing in short.
Sanger sequencing, aka the chain termination method which involves making many copies of a target DNA region using the same principle as PCR but with some tweaks:
- You use the same “ingredients” as in a PCR but you have a mix of normal bases and dideoxybases (ddNTPs) which lack the 3’OH group and therefore blocks further elongation once added. You also only use one primer (forward or reverse) to only get the same fragment sequenced, otherwise you’d get conflicting signals.
- Run the reaction, several cycles basically guarantee that a ddNTP will have incorporated at every position
- outcome: Many fragments of different lengths, each ending in a ddNTP marked with a color.
- Run the fragments through capillary gel electrophoresis, and illuminate each fragment with a laser from small to big. The marked base at each fragment length will allow for detection and that way you can base call from each detected signal to get the sequence.
Explain the term “heterologous expression”, why is it useful?
Heterologous expression is when you use a non-natural cell for protein expression. It is useful as it has allowed us to express things that would otherwise be very unethical, like overexpressing a protein causing disease in humans.
If you would want to express a eukaryotic protein in E. Coli, what would you need?
- First you need cDNA from the eukaryotic host, then you sequence it to confirm that it is your target DNA.
- Then you need to ligate a ribosomal binding site (RBS) seq and terminator seq to the cDNA so that it can be translated in the host cell)
- Then you insert the donor cDNA into a vector (often plasmid) containing a marker (antibiotic resistance), an ori, and a promoter that functions in the host cell.
- The promoter is often inducible, so that you can control when expression occurs, as you might want the cells to grow and proliferate first (and not putting energy toward expression) and then induce expression. Eg the lac operon and IPTG.
Now you can express the protein in the non-natural host!
How do you generate cDNA from a eukaryotic cell?
First, you would need to isolate the mRNA from eukaryotic cells by using an oligo dT primer that hybridize to the polyA tail (selection for only mRNA), then you use reverse transcriptase to yield a complimentary DNA strand, then you degrade the mRNA with alkali solution and ligate a sequence of known seq to the 3’end of the DNA. Then use DNA polymerase to extend the primer and synthesize a complimentary DNA strand and boom you have converted mRNA to cDNA.
Explain in short how you would perform directed mutagenesis with PCR.
To make a base substitution mutation via PCR, we can construct primers that include the mutation. When we run the PCR, we get many copies of the plasmid strands containing the mutation. Then, we digest the parental strands by using a restriction endonuclease that only cleaves methylated strands. Then we can anneal the daughter strands and transform them into the host, now containing the mutation.
You can also add thing you might need to the primers, like his tag or restriction site, super useful!
Explain how PCR works in short.
- Denaturation: from dsDNA to 2 ssDNA (first cycle longer time in this step)
- Annealing: Primers binding to either ssDNA
- Extension: DNA polymerase taking ssDNA to dsDNA –> double the amount of DNA (final extension in the end that everyone do but not sure if necessary)
Then cycle starts over and going for about 30 cycles, resulting in an exponential increase in DNA!
What is protein engineering and what is it used for?
Protein engineering is the process of changing protein structure through substitution, insertion, or deletion of nucleotides in the encoding gene, with a specific purpose/to achieve a certain goal.
Purpose:
– Structure/function studies of existing proteins
– Re-design of protein function
Give three examples of goals you could have with protein engineering.
The goal varies, but could be that you want to study:
- molecular interactions
- changing active site residues to elucidate the reaction mechanism of an enzyme
- changing the binding interface to see if it affects something in the cell or
- Facilitate the expression of a protein, for example changing a codon in a eukaryotic gene to a codon more used in E. Coli.
- Facilitate purification of a protein by adding stuff (adding things to C/N terminal rarely affects protein function)
When performing mutagenesis, you can utilize either directed or random mutagenesis. When do you choose what?
When you know a lot about a protein, you can perform directed mutagenesis, which generally give you specific information
When you know very little about a protein, you can use random mutagenesis. This generally provides less specific information, but is a good starting point.
The task/goal of protein engineering determines how many and how big changes you make. Give two examples of what change you want to make and what corresponding methodology you would use.
Level - Methodology
Insertions/deletions – PCR
Point mutations, like changing single (or a few) residue(s) – Oligo-directed, PCR
Multiple mutations, like replacing a whole double helix – PCR, DNA shuffling (oligo-directed)
Chimeric proteins – PCR, DNA shuffling
Explain in detail how you would make point mutations with oligo-directed PCR.
To create point mutations with oligo-directed PCR, you would need four primers, 1. one that starts where the gene starts upstream of your wanted mutation, 2. one oligo that contains the mutated codon. 3. one that overlaps with the mutated primer on the other strand and 4. one that starts where the gene ends.
During the first round of PCR, you add all four primers, which will lead to the sequence upstream of the mutation being single stranded and an overlap containing the mutation (double stranded (primer 1+2) and single stranded after the mutation (primer 3+4).
In the second round, you take the resulting DNA from round one and add primer 1 and 4, resulting in many copies of double stranded DNA containing the mutation! For more mutations you construct more primers and run more rounds of PCR.
