Part:BBa_K2973006

32B Toehold Switch_Enhanced Green Fluorescent Protein

This composite part consists of T7 Promoter (BBa_J64997) and T7 Terminator (BBa_K731721), the Ribosomal Binding Site (AGAGGAGA), the 32B Toehold Switch (Pardee et al., 2016) and the CDS of the Enhanced Green Fluorescent Protein(BBa_K1094400). Toehold switch systems are composed of two RNA strands referred to as the switch and trigger. In our system, the switch RNA contains the coding sequence of the gene being regulated and that is the Enhanced Green Fluorescent Protein . Upstream of this coding sequence is a hairpin-based processing module containing both a strong RBS and a start codon that is followed by a common 21 nt linker sequence coding for low-molecular-weight amino acids added to the N terminus of the gene of interest. A single-stranded toehold sequence at the 5’ end of the hairpin module provides the initial binding site for the trigger RNA strand. This trigger molecule contains an extended single stranded region that completes a branch migration process with the hairpin to expose the RBS and start codon, thereby initiating translation of the Enhanced Green Fluorescent Protein. This part is an improved version of the EPFL’s iGEM team 2017 part BBa_K2203005 because of the instant signal that is produced of the EGFP when the trigger is present. The part BBa_K2203005 contains the LacZa fragment which needs to unite with the ω-fragment in order to form a fully active LacZ enzyme that will afterwards hydrolyze its Substrate X-gal and give after some minutes optical signal. On the other hand, the EGFP protein produces fluorescent signal instantly and therefore saves a lot of time. EGFP (Enhanced Green Fluorescent Protein) is a version of GFP(green fluorescent protein) that produces enhanced bioluminescence signal in the green zone of the noticeable spectrum and is being used widely as a protein reporter.

During our experiments, one of the reporter genes that we tested was EGFP(Enhanced Green Fluorescent Protein). To test its functionality, we added its sequence downstream of Pardee's Toehold Switch 32B. We tested the efficiency of our regulatory system with 4 different concentrations of the Trigger 32B (BBa_K2973023): 70ng, 25ng, 7ng and 3ng. In addition, we tested both a positive control which corresponds to an unregulated T7 EGFP gene and a negative control which refers to the Toehold switch 32B with the gene of EGFP (BBa_K2973006), with no addition of the trigger construct. To do the experiments we followed the standard Protocol for a typical 7ul reaction with PURExpress® In Vitro Protein Synthesis Kit.

The incubation of the PURE reaction lasted 3 hours for each construct and we took fluorescence measurements immediately after that time.

The graph bellow shows that our toehold switch regulates the EGFP efficiently, even when low concentrations of trigger 32B are added.

Picture1: Fluorescence measurements of EGFP at excitation max = 488nm and emission max = 515nm

Usage and Biology

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]