triple brake design

Stages

1. 1.
   Design of light-responsive trans-acting factors and promoter architectures(library_design)

   This stage creates the candidate design space for transcriptional control by specifying the trans-acting factor and cis-element combinations to be tested.

   Selection: Generate candidate light-inducible or light-repressive transcription systems by combining WC-1-based trans-acting factors with LRE/endogenous promoter chimeras.

2. 2.
   Configuration testing for promoter performance(broad_screen)

   This stage narrows the design space to promoter configurations with better performance characteristics.

   Selection: Test various trans-acting factor/LRE pairs and different LRE positions and copy numbers for optimal promoter performance.

3. 3.
   Reporter-based characterization of selected transcription systems(functional_characterization)

   This stage provides functional evidence for the best-performing transcriptional designs.

   Selection: Characterize selected promoter systems using GFP expression and compare against PGAP.

4. 4.
   Construction and evaluation of light-repressive translation control(functional_characterization)

   This stage extends control beyond transcription to translation and tests whether light can repress protein synthesis without altering mRNA expression.

   Selection: Build a rare-codon brake translation system controlled by light-regulated pLRE-tRNA expression and assess leakage, protein synthesis repression, and mRNA impact.

Steps

1. 1.
   Link WC-1 to activation domains of endogenous transcription factors

   Create light-responsive trans-acting factors for transcriptional control.

   The trans-acting factor is needed before promoter pairings can be explored because it provides the light-sensing regulatory input.

2. 2.
   Construct chimeric LRE-containing endogenous promotersengineered promoter candidates

   Generate light-inducible and light-repressive promoter architectures.

   Promoter candidates are designed after defining the light-responsive trans-acting factors so the cis and trans components can be paired.

3. 3.
   Test trans-acting factor/LRE pairings and vary LRE position and copy number

   Identify promoter configurations with optimal performance.

   Configuration testing follows design because multiple candidate architectures must be compared to find the best-performing systems.

4. 4.
   Evaluate selected promoter systems with GFP and benchmark against PGAPpromoter systems under characterization

   Quantify expression strength, light/dark response, and repression behavior of selected transcription tools.

   Reporter characterization is performed after configuration screening to quantify the behavior of the best candidate promoter systems.

5. 5.
   Construct a rare-codon brake translation system controlled by light-regulated pLRE-tRNA expressiontranslation-control construct

   Create a light-repressive protein synthesis system operating at the translation level.

   This step extends the campaign from transcriptional control to translation control after establishing light-regulated transcription design logic.

6. 6.
   Assess leakage, protein synthesis repression, and mRNA impact of the translation systemtranslation-control system under characterization

   Determine whether the translation module represses protein synthesis by light while avoiding transcriptional effects.

   Functional assessment follows construction to verify that the translation-control design achieves repression without altering mRNA expression.