Vector Maps and Primers Flashcards
What is the recombinant protein production approach?
What are the 2 ways the DNA can be produced?
Use of samples of DNA encoding the target genes and vectors/plasmids for expressing those genes
- Direct synthesis of the gene (formerly very complex and expensive but now more routine for smaller proteins)
- PCR amplification of the gene sequences using primers
What features can vectors provide?
- Give examples if you can
Overexpression - Enable production of the protein in large amounts by a host cell through elevated levels of mRNA transcripts arising from the genes inserted into the vectors
Tags that enable downstream processing, localisation or purification
- Can add another protein that helps product fold; Can add a cleavage site to remove this protein after folding
- Can add sequences so it forms disulphide bonds in certain locations
What do we use vector ‘maps’ for?
Check that the correct features are going to be present in the final product
Insertion of the DNA gene into the vector is carried out properly using the right restriction enzymes
Where in the vector is the gene/DNA inserted?
Multiple cloning region
What is the first codon/amino acid of most multiple coding regions?
ATG - Methionine
Look at first multiple cloning region in (7); How can we get a protein starting with that methionine and ending with His tag with protein of interest in between?
Chop for example from Nde I site (just after methionine) to Xho I site and replace it with our gene
Look at first multiple cloning region in (7); pelB leader allows protein to be translocated and point into periplasmic space, so what restriction enzymes would we not use if we wanted the protein in periplasmic space and why?
What restriction enzymes could we use?
bspM I as its cleavage site would truncate this region, or Nde I as it would remove it completely
Use EcoR V and for example Xho I to replace this region with our gene
What is rbs?
Why is it necessary?
Ribosome binding site
Needed for ribosome to bind so protein is translated
What is meant by restriction sites not neatly corresponding with amino acids/codons?
Why is this important for final protein product?
They span multiple codons for amino acids/parts of amino acids
Need to keep amino acid sequence in frame so we don’t get a different protein sequence
What are thrombin and kinase sites in vectors used for? (What is thrombin?
Allows us to cleave at this thrombin site with thrombin (protease)
Allows us to phosphorylate at the kinase site
How can stop codons be utilised in inserted gene sequence?
Can put a stop codon in gene of interest if there’s something after it we don’t want
How big are restriction enzyme sites?
What are the 2 ways it can cut a gene sequence?
What is typical restriction enzyme structure?
Why do we need to use 2 different restriction enzymes when inserting a gene?
Typically between 6 and 8 base pairs
Can cut the DNA site at the centre or towards one end to leave a symmetric nucleotide overhang
Nucleases often dimeric to cut each strand
So that the genome is cut in 2 places; Otherwise genome closes up on its own
What can we do with our gene so that it easily anneals to restriction enzyme cut strand?
How?
We can put a sticky overhanging end on our gene that corresponds so that it will easily anneal to the cut strand
Do this by putting restriction enzyme sites on DNA insert so that it will be cut by same restriction enzymes we are using to cut vector
Why may we need to add extra nucleotides to a sequence that will be cut by restriction enzymes?
So it is still in frame, and you get the correct protein sequence; Could get start or stop codon in different places, making truncated protein
How is a vector selected for?
Using antibiotic that it has resistance gene for
