Protein Expression

Question 1

What are then benefits associated with ligation independent cloning?

Answer 1

Engineer in exactly what you want. 
Commercial or homemade (cheap for homemade) 
Suitable for large scale 
PIPE has no kits 
Inexpensive to scale up

Question 2

What are image clones and their benefits?

Answer 2

chunks of DNA in vectors, search sequence database to see if protein you’re interested in is within.
~£100 per image clone.
May still need to modify protein to add tags / restriction sites.
Good for LARGE proteins.

Question 3

What is TOPO cloning?

Answer 3

DNA TOPOi = restriction enzyme and ligase - cleaves and rejoins supercoiled DNA ends.
Sent a plasmid with topoisomerase enzyme coupled to one end.
Do PCR –> add extra CACC sequence on forward primer –> overhang compatible with vector’s overhang.
If no tag –> CACC not expressed –> no extra residues.

Question 4

Why is TOPO cloning advantageous?

Answer 4

Quick and RELIABLE

Can only clone into certain ready prepped vectors - expensive.

Question 5

qWhat must you consider when adding a signal sequence ?

Answer 5

If also adding an N-terminal tag, don’t leave signal peptide in sequence as will not get cleaved, or if it does - tag lost!
Signal sequence can also affect bacterial expression levels!