Team:Lambert GA/Basic Part

BBa_K2974410

C. elegans Trigger

The C. elegans trigger is an RNA sequence used in conjunction with the T7 promoter to induce GFP expression for Lambert iGEM’s C. elegans Toehold switch Biosensor. When the trigger RNA sequence is present, it binds to the complementary sequence on the toehold switch, expressing the GFP reporter gene and producing a green fluorescence. The trigger RNA sequence was designed using NUPACK (Nucleic Acid Package) software. Our trigger (36 base pairs) assembled with the T7 promoter was then inserted into pSB1A3, which is a high copy plasmid obtained from the parts registry.

BBa_K2974310

C. elegans Toehold Switch

This C. elegans toehold was built using BBa_K2550202 as the stem-loop domain for the toehold, and the target sequence domain is derived from the C. elegans lin-4 gene pre-mRNA primary transcript F59G1.6 (non-toxic). This part encodes Switch Number 1 of the C. elegans Toehold Series developed by Lambert iGEM. The described structure was optimized in design using the parameters described by Pardee et al. and validation using the toehold designer software developed by To et al. The design of this switch involved consideration of numerous components including free energy of RBS-Linker, MFE of various structures, illegal stop codons, etc. in order to optimize the secondary structure sequence. The corresponding sequence was then processed using the NUPACK (Nucleic Acid Package) to determine whether the appropriate secondary structure formed and whether the RBS was sufficiently enclosed in the upper loop. Following analysis of the sequence and structure, this part was experimentally tested.