1. Todd T. Eckdahl and A. Malcolm Campbell
Using Synthetic Biology and pClone Red for Authentic Research on Promoter Function: Genetics (analyzing mutant promoters) 
Todd T. Eckdahl and A. Malcolm Campbell

2. Synthetic Biology Promoter Discovery with pClone Red
This is a simple overview of using GGA to replace one known promoter with a mutated version of the target promoter.
developed by Todd Eckdahl and A. Malcolm Campbell

3. Goal: Clone a mutant promoter and test its effectiveness.

4. 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 cycles (time dependent)
37° C for 15 minutes (optimal for Bsa I)
Overview of GGA logistics.

5. pClone Red = J119137 receiving plasmid ampicillin resistance gene
Starting plasmid with a strong backwards promoter (PlacIQ1) driving the transcription of GFP. This promoter will be cut out during GGA.
ampicillin resistance gene
origin of
replication

6. Promoter Made of Self-Assembled Oligos
top strand 5’ 3’
bottom strand 3’ 5’
boil & cool 5’ 3’ 3’ 5’
add to GGA mixture
Overview of how students will assemble their dsDNA promoters from two ssDNA oligonucleotides synthesized by a company. Students will choose the sequences to be synthesized.
left sticky end
right
sticky end

7. Golden Gate Assembly GGA = Bsa I and ligase right left sticky end
Shows the two DNA components: pClone Red on top and the students’ experimental promoter below.
right
sticky end
left sticky end
new promoter annealed oligos

8. Promoter Ready for Transformation
ampicillin resistance gene
After GGA, the desired outcome of students’ promoter potentially driving transcription of RFP.
origin of
replication

9. Red Colonies Contain Functional Experimental Promoter
Typical student results after GGA and transformation if the promoter is a strong one (see red colonies).