dynamic mutually inhibitory switch

Stages

1. 1.
   Core isolation from native type I TA pairs(library_design)

   This stage extracts the reusable core architecture from native systems before artificial pair reconstruction.

   Selection: Isolation of the core of type I TA pairs for reuse as synthetic regulatory modules.

2. 2.
   Artificial pair reconstruction(library_build)

   This stage converts isolated native cores into engineered RNA device pairs.

   Selection: Reconstruction of artificial TA-derived RNA regulator pairs.

3. 3.
   Constraint-based generation of orthogonal portable pairs(library_design)

   The stage exists to produce orthogonal and portable regulator pairs rather than only reconstructed native-like pairs.

   Selection: Introduce structure and energy constraints to generate orthogonal SRTS-OPRTS pairs with cross-species application.

4. 4.
   Functional characterization in gene regulation and circuit applications(functional_characterization)

   This stage demonstrates that the designed RNA devices work as practical regulatory tools and support downstream circuit behaviors.

   Selection: Test quantitative regulation, mutually inhibitory switch construction, and selective enrichment applications.

Steps

1. 1.
   Isolate the core of type I toxin-antitoxin pairs

   Extract the minimal reusable regulatory core from native type I TA systems.

   The abstract presents core isolation as the first step before artificial pair reconstruction.

2. 2.
   Demonstrate independence between structure and repression function

   Establish that the reusable design can separate structural features from repression function for engineering.

   The abstract places this demonstration immediately after core isolation and before reconstruction of artificial pairs, implying it justifies the engineering step.

3. 3.
   Reconstruct artificial TA-derived RNA pairs as SRTS-OPRTSengineered RNA regulator pair

   Create synthetic post-transcriptional regulatory devices from the validated TA core logic.

   Artificial reconstruction follows core isolation and structure-function analysis in the abstract's reported order.

4. 4.
   Introduce structure and energy constraints to generate orthogonal cross-species pairsdesigned RNA regulator pair set

   Generate orthogonal SRTS-OPRTS pairs that remain portable across multiple bacterial species.

   Constraint-based design is applied after artificial pair reconstruction to improve orthogonality and portability.

5. 5.
   Test quantitative gene regulation using SRTS with cognate 3' UTR OPRTSengineered regulatory RNA elements

   Validate that the designed RNA elements can quantitatively regulate target genes.

   Functional regulation testing follows design and pair generation to confirm practical activity.

6. 6.
   Construct dynamic mutually inhibitory switches from tagged genesRNA-enabled circuit construct

   Use portability of the RNA devices to build reciprocal regulatory circuits.

   The abstract states that portability enabled this downstream circuit construction after regulatory function was established.

7. 7.
   Construct a selective lethal system to enrich high-fluorescent mutantsselection-linked application construct

   Apply the RNA-device framework to phenotype enrichment.

   The abstract describes this as a further application leveraging the established approach after regulation and portability were demonstrated.