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

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

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) Overview of GGA logistics.

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

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

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

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

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