Part:BBa_K3328004
trigger of OFF-switch1 and NOT gate;triggerB of IMPLY gate
trigger of 3WJ repressor binding to 3WJ switch (BBa_K3328000) to inhibit the expression of downstream gene;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.
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. Error bar: SD (n=9).
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.
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.
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. Error bar: SD (n=9).
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.
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. Error bar: SD (n=9).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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