Part:BBa_K2550000

T7 promoter Toehold Ribosome Switch with LacZ expression

T7 Toehold LacZ is a construct that was developed to be applied as a biosensor. The part BBa_I732005 was submitted by 2007 UTSC iGEM that singularly included the LacZ gene encoding the Beta-galactosidase protein. In effort to build on this biobrick and implement LacZ blue color expression as a biosensor, Lambert iGEM obtained a LacZ toehold switch construct assembled with a T7 promoter from the Styczynski Lab at Georgia Institute of Technology. When assembled with a distinct RNA sequence complementary to the trigger sequence, the produced blue pigment expression can be characterized based on a Red, Green, Blue (RGB) scale.

Description

Toehold switches are biosensors that can activate gene expression in response to a chosen RNA sequence that is 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 from being expressed. When the trigger RNA sequence is present, it will bind to the complementary sequence in the toehold switch and unravel the hairpin loop allowing the reporter protein to be expressed. The toehold switch applied in the construct conceals the RBS and starting sequence that codes for the LacZ gene. When the trigger sequence is present, the toehold unbinds and the LacZ gene is expressed, subsequently producing blue color.

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 switch construct to identify the presence of specific substances.

Results

pSB3C5 containing the T7 Toehold LacZ sequence produced blue colonies when plated without the trigger sequence onto chloramphenicol, carbenicillin, and Xgal antibiotic resistance. Since the hairpin loop in the toehold switch conceals the RBS and starting sequence, the LacZ gene should be repressed; therefore, no blue color should be observed. However, with further research and collaboration with Yan Zhang, a researcher at the Georgia Institute of Technology, the unexpected color can be explained by the toehold leakiness. This occurs when a reporter gene is transcribed despite the bound toehold due to a strong promoter (T7). When the same construct was transformed in a dual plasmid transformation with the trigger sequence (obtained from the Styczynski Lab at Georgia Institute of Technology), a darker blue color was produced.

Update A new part has been designed by Lambert 2019 BBa_K2974500 to improve the function of the toehold switch by eliminating over expression when the trigger sequence is not present. It is hoped that this will lead to less false positives.