Download S6. pClone Genetics-Overview of Golden Gate

Using Synthetic Biology and pClone Red for
Authentic Research on Promoter Function:
Genetics (analyzing mutant promoters)
Todd T. Eckdahl and A. Malcolm Campbell
Synthetic Biology
Promoter Discovery
with pClone Red
developed by Todd Eckdahl and A. Malcolm Campbell
Goal: Clone a mutant promoter
and test its effectiveness.
Golden Gate Assembly (GGA) Method
• Mix promoter + receiving plasmid
• GGA Ligation Protocol
– Bsa I + ligase in tube with DNA
– 37° C for 1 minute (optimal for Bsa I)
– 16° C for 1 minute (optimal for ligase)
– Total of 20 - 30 cycles (time dependent)
– 37° C for 15 minutes (optimal for Bsa I)
pClone Red = J119137
receiving
plasmid
Promoter Made of Self-Assembled Oligos
top strand
3’
5’
5’
bottom strand 3’
boil & cool
5’
3’
3’
5’
add to GGA mixture
left
sticky
end
right
sticky
end
Golden Gate Assembly
GGA = Bsa I and ligase
left
sticky
end
new promoter
annealed oligos
right
sticky
end
Promoter Ready for Transformation
Red Colonies Contain Functional
Experimental Promoter