Difference between revisions of "Part:BBa K3328000"
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an OFF-switch to regulate the expression of downstream gene;an integral part of NOT and IMPLY boolean calculation | an OFF-switch to regulate the expression of downstream gene;an integral part of NOT and IMPLY boolean calculation | ||
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| − | + | 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. | |
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| − | + | ===Lower Leakage and Higher ON/OFF Ratio=== | |
| + | Our experiments were done with both the improved part (BBa_K3328000) and the original part (BBa_K2150031) as a control. Our part has lower leakage and higher ON/OFF ratio than the T7 promoter. | ||
| − | + | https://2020.igem.org/wiki/images/thumb/6/60/T--OUC-China--part_im_1.jpg/652px-T--OUC-China--part_im_1.jpg | |
| − | = | + | In the blank control (IPTG=0 M), the fluorescence of T7 promoter (BBa_K2150031) is higher than 3WJ Switch1 (BBa_K3328000). This indicates that our part (BBa_K3328000) has the advantage of low leakage. When the inducer is added (IPTG=0.1 M), 3WJ Switch1 showed a high ON/OFF ratio of up to 15-fold, and the T7 promoter has the ON/OFF ratio of only 3-fold. |
| − | + | ||
| − | + | ===New functions=== | |
| + | 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. | ||
| − | + | https://2020.igem.org/wiki/images/a/ad/T--OUC-China--part_fig2.png | |
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| + | 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. | ||
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| + | https://2020.igem.org/wiki/images/thumb/a/af/T--OUC-China--design_fig4.png/800px-T--OUC-China--design_fig4.png.jpeg | ||
https://2020.igem.org/wiki/images/thumb/c/c6/T--OUC-China--result_fig3.jpg/653px-T--OUC-China--result_fig3.jpg | https://2020.igem.org/wiki/images/thumb/c/c6/T--OUC-China--result_fig3.jpg/653px-T--OUC-China--result_fig3.jpg | ||
| − | In the validation of 3WJ, as with toehold, we also set blank control (IPTG=0 M, aTc=0 mg/ | + | 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). |
https://2020.igem.org/wiki/images/thumb/3/31/T--OUC-China--result_fig4.jpg/768px-T--OUC-China--result_fig4.jpg | https://2020.igem.org/wiki/images/thumb/3/31/T--OUC-China--result_fig4.jpg/768px-T--OUC-China--result_fig4.jpg | ||
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. | 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. | ||
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==== NOT gate==== | ==== 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. | ||
| + | |||
| + | https://2020.igem.org/wiki/images/thumb/1/1f/T--OUC-China--design_lunbo_not.jpg/799px-T--OUC-China--design_lunbo_not.jpg | ||
https://2020.igem.org/wiki/images/thumb/4/4b/T--OUC-China--result_fig7.jpg/800px-T--OUC-China--result_fig7.jpg | https://2020.igem.org/wiki/images/thumb/4/4b/T--OUC-China--result_fig7.jpg/800px-T--OUC-China--result_fig7.jpg | ||
| − | 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/ | + | 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==== | ==== IMPLY gate==== | ||
| + | We combined the 3WJ switch and toehold switch to realize the IMPLY Boolean calculation. | ||
| + | |||
| + | https://2020.igem.org/wiki/images/3/3b/T--OUC-China--part_fig5.png | ||
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| + | 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. | ||
| + | |||
| + | https://2020.igem.org/wiki/images/thumb/8/88/T--OUC-China--design_fig6.jpg/799px-T--OUC-China--design_fig6.jpg | ||
| − | https://2020.igem.org/wiki/images/thumb/ | + | https://2020.igem.org/wiki/images/thumb/8/85/T--OUC-China--result_fig9.jpg/800px-T--OUC-China--result_fig9.jpg |
| − | + | 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). | |
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<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here | ||
Latest revision as of 07:55, 27 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
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.
Lower Leakage and Higher ON/OFF Ratio
Our experiments were done with both the improved part (BBa_K3328000) and the original part (BBa_K2150031) as a control. Our part has lower leakage and higher ON/OFF ratio than the T7 promoter.
In the blank control (IPTG=0 M), the fluorescence of T7 promoter (BBa_K2150031) is higher than 3WJ Switch1 (BBa_K3328000). This indicates that our part (BBa_K3328000) has the advantage of low leakage. When the inducer is added (IPTG=0.1 M), 3WJ Switch1 showed a high ON/OFF ratio of up to 15-fold, and the T7 promoter has the ON/OFF ratio of only 3-fold.
New functions
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.
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]