Part:BBa_K2550001
T7 promoter with a trigger sequence to match the toehold sequence of part BBa_K2550000
T7 Toehold LacZ Trigger is an RNA sequence to be used in conjunction with part BBa_K22550000 to induce LacZ expression for 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 (LacZ gene) to be expressed; this mechanism produces a blue pigment when inoculated in Xgal. The RNA sequence was obtained from the second entry in the 144 first generation orthogonal toehold switches collection from the 2017 Green etal paper. .
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.
The lac operon is induced by lactose and IPTG (isopropyl β-D-1-thiogalactopyranoside); this region of the genome is responsible for transporting and metabolizing lactose. Within the lac operon, the gene, LacZ, codes for the B-galactosidase protein. When this protein is expressed, it breaks down Xgal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) into galactose and an insoluble blue pigment. Therefore, when the gene is synthesized in the chassis, the colonies will appear blue. This mechanism was implemented in the toehold construct to identify the presence of specific substances.
Results
Figure 1: (Right) Dual Plasmid Transformation of pSB6A1 T7 Trigger Sequence and pSB6A1 T7 Toehold LacZ on chloramphenicol, carbenicillin, and Xgal antibiotic resistance. (Left) The pSB6A1 T7 Toehold LacZ switch transformed on chloramphenicol, carbenicillin, and Xgal; the construct is not expressing blue color because the trigger is not present.
As seen in the image on the right, no color is being produced because this transformation singularly included the trigger sequence and the toehold is not present. When the trigger is present, as seen in the left image, the toehold unbound and the reporter protein is expressed. The trigger sequence was used in a dual plasmid transformation with BBa_K2550000, and as a result, the colonies produced a blue pigment as expected. This proves that the trigger part induces the toehold switch successfully. Sequence and Features
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