Difference between revisions of "Part:BBa K2974400"

Revision as of 05:42, 21 October 2019

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.

Description

Toehold switches are riboregulators that activate translation in response to a distinct RNA sequence. It is comprised of a switch and a trigger. The switch is composed of a hairpin loop structure that represses translation through its complementary bases in between the ribosome binding site and start codon, which is followed by a 21 nucleotide linker sequence. These sequences ensure that the toehold switch structure will be maintained while coding for low-molecular weight amino acids that would not interfere with the switch’s function. The toehold domains at the beginning of the hairpin are 12 to 18 nucleotides long and are designed to be complementary to the trigger in order to initiate linear RNA binding. The trigger contains complementary sequences to the toehold domain that once it is in the presence of the switch, it will bind to the hairpin stem and unbind the loop. This exposes the ribosome binding site and start codon, allowing translation of the reporter protein to occur.

Results

Figure 1:

Sequence and Features