Team:Lambert GA/Composite Part
BBa_K2974316
BBa_J23106 C. elegans Toehold GFP
BBa_J23106 C. elegans Toehold GFP is a construct that was developed by the Lambert iGEM 2019 team that can be applied as a proof of concept for our biosensor specific to nematodes, such as parasitic helminths. A combination of BBa_J23106, a moderate strength promoter, and BBa_B0032, a moderate Ribosomal Binding Site (RBS), is used to ensure the expression of the downstream reporter. Used as a quantitative reporter, the green fluorescent protein can be expressed using Fluorocents, a low-cost fluorometer developed by Lambert iGEM.
The construct above displays the proof of concept T7 GFP switch and trigger. The switch consists of medium constitutive promoter BBa_J23106, along with GFP as the reporter gene. The trigger was cloned into a high copy plasmid while the switch was cloned into a low copy plasmid to ensure the replication of two different plasmids for the E. coli cell to produce proportional amounts.
BBa_K2974400
T7 C. elegans Trigger
The T7 Toehold Trigger is an RNA sequence to be used in conjunction with part BBa_K2974316 to induce expression of GFP within a biosensor system. When this trigger RNA sequence is present, it binds to the complementary sequence in the toehold switch and unravels the hairpin loop allowing the reporter protein (GFP) to be expressed; As a result, this mechanism produces fluorescence. The complementary sequence was calculated using NUPACK (Nucleic Acid Package) software, and the trigger was experimentally tested with part BBa_K2974316 to determine whether the toehold’s secondary structure formed correctly and whether the RBS was sufficiently enclosed in the upper loop.
BBa_K2974700
BBa_J23100 Toehold GFP
BBa_J23100 Toehold GFP is a construct designed to be utilized as a color biosensor and to be used in conjunction with part BBa_K2550001. In the 2018 project, Lambert iGEM developed part BBa_K2550000 which was comprised of promoter J23100, a standard Toehold Switch, and a LacZ reporter. However, this part experienced overexpression of the LacZ due to the strong promoter, J23100, of the Anderson series. In order to avoid this problem in Lambert iGEM’s 2019 project, the team looked to replace the J23100 with the medium-strong promoter of the same series, J23106. The team assembled this promoter with a toehold switch and the Green Fluorescent Protein (eGFP) and has been used to compare promoter strength with part BBa_K2974500. When transformed this construct with the complementary trigger sequence, part BBa_K2550000, the colonies produced green fluorescence which was then characterized based on light intensity (lux) values.
BBa_K2974101
BBa_J23106 Toehold GFP
BBa_J23106 Toehold GFP is a construct that was developed by the Lambert iGEM 2019 team to be applied as a proof of concept for our biosensor. A combination of BBa_J23106, a moderate strength promoter, and BBa_B0032, a moderate Ribosomal Binding Site (RBS), is used to ensure the expression of the downstream reporter. Used as a quantitative reporter, the green fluorescent protein can be expressed using Fluorocents, a low-cost fluorometer developed by Lambert iGEM.
BBa_K2974500
BBa_J23106 Toehold LacZ
Building upon the 2018 Lambert iGEM part BBa_K2550000, the team looked to improve upon the overexpression of LacZ when not induced by the corresponding trigger sequence. The team began by modeling and testing the effect of changing Promoter sequences and Ribosomal Binding Sites on a similar construct using Biobuilder’s BBa_J10050- BBa_J10058 Promoter and RBS combinations. Those results led us to change the promoter in BBa_K2550000 from BBa_J23100 to BBa_J23106. The improved part is BBa_K2974500 and is currently, as of wiki freeze, in the cloning stage, but the team expects to update the registry with the final results after the 2019 Giant Jamboree.