Part:BBa_K2411008
Forward Engineered Toehold 2 with deGFP gene
This device encodes for a toehold that when activated expresses deGFP. It has an OR2-OR1 promoter, followed by the second best forward engineered toehold sequence designed by Green et al. The deGFP follows the toehold, and the device ends with a T7 terminator. The part has been designed for and tested in an E. coli derived transcriptional translation cell free system. In order for the part to function, it needs to be used in conjunction with a trigger RNA.
Usage and Biology
Expression was achieved when activating the toehold switch with RNA trigger at 10,000X concentrations relative to the toehold. Activation is achieved when trigger RNA binds with the homologous region of the toehold. This causes a hairpin loop to unbind, revealing a previously sequestered start codon, and allowing transcription of a downstream deGFP gene.
In this experiment, fluorescence from reactions with plasmid toeholds and RNA trigger was compared to fluorescence from reactions containing plasmid toehold switches alone, no DNA at all, and a plasmid containing a constitutively active deGFP. Measurable fluorescence is achieved from two different toehold switch/trigger pairs, but the expression is lower than that seen from the constitutively active deGFP. Larger amounts of RNA may allow for better fluorescence, but the methods we employed for transcribing the trigger DNA to RNA was not able to produce higher concentrations.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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