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 Georgia Institue of Technology that was number 2 of the 144 generation orthogonal toehold switch collection from the Collins Paper titled, Toehold Switches: De-Novo-Designed Regulators of Gene Expression.

Discussion

Toehold Switches are biosensors that can activate gene expression in response to a chosen RNA sequence, comprised of a switch and a trigger. The switch includes a hairpin loop structure designed to be complementary to the trigger sequence along with a reporter protein downstream. The RBS and starting sequence are concealed in the toehold switch, preventing the reporter from being expressed. 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 X-gal (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

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Figure 1: (Left) The biobricked trigger transformation on chloramphenicol antibiotic resistance. (Right Top) The T7 Toehold LacZ switch transformed on chloramphenicol, carbenicillin, and Xgal; the construct is also not expressing blue color because the trigger is not present. (Right Bottom) The blue colonies demonstrated that the tigger induces LacZ expression by binding to the complementary toehold sequence.

As seen in the first image, 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 top image, the toehold can unbind 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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 104
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]