Part:BBa_K3328000:Design

OFF-switch1;switch of NOT gate

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

We hope that the logic gates and some of the basics we designed will be widely used. Therefore, we add these structures after the promoter to assemble them into the entire circuit.

We added 3WJ to the T7 promoter (BBa_K2150031) committed by the 2016 UCAS team. Our new part (BBa_K3328000) is an OFF-switch to regulate the expression of downstream gene. This design changes the original functionality of the T7 promoter and gives it new features. In addition, it is an integral part of NOT and IMPLY boolean calculation.

Our part can be turned off by the trigger. In addition, it is an integral part of NOT and IMPLY boolean calculation.

The OFF-switch

We chose three-way Junction (3WJ) repressor as the OFF-switch. Three-way junction (3WJ) repressors switch RNA employs an unstable hairpin secondary structure that contains an RBS in the loop region and a start codon in the stem region.

Despite its high secondary structure, this unstable hairpin was previously demonstrated to be translationally active in toehold switch mRNA sensors. However, When the trigger RNA is expressed, the trigger will bind to the switch RNA. The resulting trigger–switch complex has a stable 3WJ structure that effectively sequesters the RBS and start codon within the loop and stem of the switch RNA, respectively, and strongly represses translation.

NOT gate

We used 3WJ repressor to build NOT gate. Its unstable hairpin was previously demonstrated to be translationally active. When a complementary trigger RNA is expressed, the trigger will bind to the switch RNA, making the originally unstable 3WJ structure stable, and represses translation.

IMPLY gate

We combined the 3WJ switch and toehold switch to realize the IMPLY Boolean calculation.

When no trigger expressed, this logic gate just likes a 3WJ switch. When trigger A expressed, the trigger will bind to the switch RNA. The binding allows for a branch migration process, exposing AUG and RBS for translation initiation. When trigger B expressed, the trigger will bind to 3WJ switch RNA. The resulting trigger–switch complex has a stable 3WJ structure that effectively sequesters the RBS and start codon within the loop and stem of the switch RNA, respectively, and strongly represses translation. When trigger A and B both expressed, the binding of trigger RNA to the toehold sequence allow the RNA polymerase binds to the former RBS and break open 3WJ stable hairpin.

Source

synthesize from company

References

Kim, J., Zhou, Y., Carlson, P. D., Teichmann, M., Chaudhary, S., Simmel, F. C., … Green, A. A. (2019). De novo-designed translation-repressing riboregulators for multi-input cellular logic. Nature Chemical Biology. doi:10.1038/s41589-019-0388-1