Week 5 Wednesday

Question 1

How does Golden-Gate assembly work?

Answer 1

• Uses special restriction enzymes (type IIS) that cut DNA outside of the recognition motif
• Complementary sticky ends (NNNN in example) are designed around cut sites
• Backbone & inserts are digested & ligated in same reaction

Question 2

What is the efficiency of Golden-Gate assembly and why?

Answer 2

Very high efficiency due to the inability of correctly ligated DNA to be further digested

Question 3

How is GFP cut out during Golden-Gate?

Answer 3

On either side of the GFP gene, it has a recognition sequence for the enzyme Bsa1. The Bsa1 is what is in the master mix. It also has DNA ligase. At 37 degrees, the enzyme finds the sites on the plasmid and cuts out the GFP. The DNA ligase will then stitch the ends back together or insert the piece.

Question 4

What stops ligase from adding GFP back in and being stitched back together?

Answer 4

The Bsa1 recognition is on the part of the plasmid that gets cut out, so it no longer has that site. It will cut it out every time until ligase puts the piece in, then it can’t be cut out because there is no recognition site. The more cycles you run, the more efficient it will be. Generally, 100 percent efficient.

Question 5

What are the 4 steps of DNA assembly in a plasmid?

Answer 5

DNA assembly, Propagation in E. coli, plasmid extraction, and delivery to host (S. cerevisiae)

Question 6

How do we know if GFP is still in the ecoli?

Answer 6

If it glows green

Question 7

Why do we use pJV4 in Golden Gate Assembly?

Answer 7

pJV4 is a GFP dropout, so it is easy to know when it works

Question 8

What would happen if we plated on unselective media?

Answer 8

it could kick the plasmid out and the cells would go from orange to white