Difference between revisions of "Part:BBa K4642028"

Latest revision as of 14:56, 11 October 2023

Gen4 320b-320a-4640 Double AND-Gate Toehold Switch

Toehold switches are a class of de-novo-designed riboregulators that enable posttranscriptional activation of protein translation through mechanisms employed in artificial systems rather than natural ones. Unlike conventional riboregulators, the switches take advantage of toehold-mediated linear-linear interactions developed in vitro to initiate RNA-RNA strand displacement interactions. Furthermore, they rely on sequestration of the region around the start codon to repress protein translation, eschewing any base pairing to the RBS or start codon itself to regulate translation.

As a result, toehold switches can be designed to activate protein translation in response to a trigger RNA with an arbitrary sequence, enabling substantial improvements in component orthogonality. The absence of binding to the RBS and use of thermodynamically favourable linear-linear interactions also enables facile tuning of translational efficiency via RBS engineering. Consequently, these systems routinely enable modulation of protein expression over two orders of magnitude.

This toehold switch upregulates the translation of a reporter protein in the CDS downstream in the presence of micro-RNAs hsa-miR-320b, hsa-miR-320a and hsa-miR-4640 - miRNAs upregulated in ovarian cancer patients. When the miRNAs are present, they bind to parts BBa_K4642029 and BBa_K4642030, synthetic anti-miRNAs we designed, forming a complex which itself would act as a trigger.

Once the complex has formed, the 4640 section binds to the 4640 binding site, partially unfolding the switch, then the first anti-miRNA binds to the first anti-miRNA binding site, further unfolding the switch, then the hsa-miR-320a and the second anti-miRNA, before finally the 320b section unfolds the switch fully, exposing the RBS and start codon and allowing translation to occur at a ribosome. In a composite, a reporter gene should be placed downstream here which will report when in the 'on' state.

Sequence and Features