Difference between revisions of "Part:BBa K2550000:Design"

Revision as of 15:43, 15 October 2018

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 lactose operon encoding the Beta-galactosidase protein. In effort to build off of this biobrick and implement LacZ blue color expression as a biosensor mechanism, Lambert iGEM obtained a LacZ toehold 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 HSV (hue saturation value) RGB (red green blue) scale. 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 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 sequence used in the LacZ construct was obtained from the 144 first generation orthogonal toehold switches collection from the 2017 Collins paper titled Toehold Switches: De-Novo-Designed Regulators of Gene Expression. Following this unique toehold sequence is the LacZ operon. We introduced a base pair wobble in the LacZ gene that substituted an Adenine for a Guanine. The wobble mutation sequence was obtained from the Styczynski Lab at the Georgia Institute of Technology and was used to eliminate the illegal EcoRI site in the LacZ operon. 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.

Design Notes

This part can be draining on the metabolism of E coli cells and toxic in high copy number plasmids. The actual toehold sequence in BBa_k2550100 comes from the 2 of 144 Orthogonal switches designed by Alexander A. Green, Pamela A. Silver, James J. Collins, Peng Yin, Toehold Switches: De-Novo-Designed Regulators of Gene Expression in their work on Ribo regulators.

Source

References

Alexander A. Green, Pamela A. Silver, James J. Collins, Peng Yin, Toehold Switches: De-Novo-Designed Regulators of Gene Expression, Cell, Volume 159, Issue 4, 2014, Pages 925-939, ISSN 0092-8674, https://doi.org/10.1016/j.cell.2014.10.002