Part:BBa_K2550001:Experience

Applications of BBa_K2550001

Results

In the 2018 Lambert iGEM project, CAPTIVATE, the results for the trigger were confirmed through a dual plasmid transformation in BL21 E. coli. The dual plasmid transformation presented darker blue colonies in comparison to the single switch transformation confirming the function of the trigger as it induces the toehold switch to produce blue pigment in response to the presence of Xgal in conjunction with the LacZ reporter gene. In addition, sequencing analysis results shown below also confirms the presence of the trigger in comparison to the trigger sequences in the reference paper (Green et al. 2014). The biobricked trigger's (BBa_K2550001) function has been validated through experimentation and sequencing.

Figure 1: Image showing the sequencing results of the T7 Toehold LacZ RNA Trigger Sequence. Annotated in a white arrow is the Biobricked T7 promoter (BBa_I719005); this is followed by the Biobricked trigger sequence, which is 182 base pairs long, starting at 50 bp and ending at 231 bp.

The illustration justifies that the trigger sequence is present along with the T7 promoter.

Updates

In the 2019 Lambert iGEM project, LABYRINTH, the team utilized the toehold switch and trigger mechanism for proof of concept for an RNA regulatory toehold biosensor specific to C. elegans. The 2019 project proof of concept utilized promoter J23106, reporter eGFP, and the toehold and trigger sequences outlined in the reference paper (Green et al. 2014). The toehold was also built using promoter J23106, the strong promoter used with Lambert’s 2018 toehold, to characterize and improve upon leaky overexpression observed in the CAPTIVATE project. The trigger sequence was obtained through a 2018 glycerol stock that contained the previously sequenced and confirmed part, and it was transformed both individually in Dh5a cells and in a dual plasmid transformation with the toehold sequence in BL21 cells. Fluorometer readings of light intensity (lux) values prove eGFP expression in the dual plasmid and confirm the function of the trigger. When used in conjunction with a strong promoter toehold, there was greater fluorescence measured than in a dual plasmid containing a medium-strength promoter toehold.

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