Part:BBa_K2550010

T7 Toehold LacZ RNA Trigger Sequence

Ribonucleic Acid sequence designed to be used in conjunction with the T7 Toehold LacZ switch. 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 Institute 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.

Description

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 Ribosomal Binding Site (RBS) and starting sequence are concealed in the toehold switch, preventing the reporter, which is the LacZ gene, 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 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: (Left) The biobricked trigger transformation on chloramphenicol antibiotic resistance. (Right) 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. When the trigger is present, as seen in the third 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. The blue colonies demonstrated that the trigger induces LacZ expression by binding to the complementary teohold sequence. This proves that the trigger part successfully

As seen in the image, no color is being produced because toehold is not present.

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]