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.

Usage and Biology

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. The in vitro transcription/ translation reactions were done using the PURExpress® In Vitro Protein Synthesis kit. To enable translation of the protein from the toehold regulated construct, different amounts of trigger sequence were added in the reaction. Also, a reaction without a trigger sequence was included, as a negative control and a leakage measure. To reduce the cost of the reaction, we lowered the reaction volume from 25 to 7 μL.

After a 3-hour incubation in a PCR machine at 37℃, protein expression was measured using a plate-reader. To measure eGFP, an excitation step at 488nm was required before visualizing at 515nm, where the protein’s emission wavelength is. The results of the assay can be seen at the graph below.

As expected, the non-regulated eGFP emitted the highest fluorescence, providing a positive control and confirming the protocol we used was functional. The toehold-regulated construct produced robust signal in decreasing concentrations of trigger (75nM, 15nM, 7nM). It is interesting that the signal remained the same even with a 10-fold decrease in trigger concentration, while the no trigger control had an almost 2-fold lower fluorescence.

Figure 1: eGFP Fluorescence after in vitro protein expression.

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]