Difference between revisions of "Part:BBa K3328000"

Revision as of 07:00, 25 October 2020

OFF-switch1;switch of NOT gate

an OFF-switch to regulate the expression of downstream gene;an integral part of NOT and IMPLY boolean calculation

Design

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.

3WJ repressor

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.

3WJ repressor switch RNA employs an unstable hairpin secondary structure. This 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.

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.

Result

3WJ repressor

In the validation of 3WJ, as with toehold, we also set blank control (IPTG=0 M, aTc=0 mg/ml). Compared with the group without trigger expression (IPTG=0.1 M, aTc=0 mg/ml), the group with trigger expression (IPTG=0.1 M, aTc=0.25 mg/ml) showed inhibitory effect.

Crosstalk was determined by dividing the arithmetic mean of the GFP fluorescence from a given trigger switch pair by the arithmetic mean of the GFP fluorescence for the cognate trigger switch interaction. GFP fluorescence was measured from n=9 biologically independent samples.

3WJ repressor showed remarkable ON/OFF ratio and had good orthogonality, we choose it as OFF switch. And we used it in the construction of logic gates and the design of some more complex circuits.

NOT gate

The left figure shows that when INPUT=0, the fluorescence of GFP is high. And the fluorescence intensity of GFP was low when INPUT=1. This corresponds to the situation described in the truth table on the right. INPUT=1 means that aTc (0.25 mg/ml) is added.

IMPLY gate

The left figure shows that when INPUT A=0, INPUT B=1, the fluorescence of GFP is low. And the fluorescence intensity of GFP was high in the other three groups. This corresponds to the situation described in the truth table on the right. INPUT A=1 means that aTc (0.25 mg/ml) is added, INPUT B=1 means that HSL (0.1 mg/ml) is added.

Sequence and Features