Difference between revisions of "Part:BBa K2411004"
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The new classes of regulatory components offer wide dynamic range and low system crosstalk. SCUT_China 2019 selected four toehold switches, including Toehold Switch D, with widest dynamic range and highest orthogonality used for multiplexing systems and provided by the reference, which are switch A&B&C&D and trigger A&B&C&D, to regulate the expressions of four acid-resistance factors.<br> | The new classes of regulatory components offer wide dynamic range and low system crosstalk. SCUT_China 2019 selected four toehold switches, including Toehold Switch D, with widest dynamic range and highest orthogonality used for multiplexing systems and provided by the reference, which are switch A&B&C&D and trigger A&B&C&D, to regulate the expressions of four acid-resistance factors.<br> | ||
| − | We expressed this gene and tested the amount of leakage at E.coli MG1655-T7 RNAP (MGR).<br> | + | We expressed this gene and tested the amount of leakage at E.coli MG1655-T7 RNAP (MGR).(Fig. 1)<br> |
The functional gene Toehold Switch D was constructed on plasmid pACYC184-RFP and the functional gene Trigger DNA D was constructed on plasmid PUC19. Then the two plasmids were transformed into MGR. At the same time, MGR with plasmid pACYC184-RFP-Switch D was constructed for subsequent measurement.<br> | The functional gene Toehold Switch D was constructed on plasmid pACYC184-RFP and the functional gene Trigger DNA D was constructed on plasmid PUC19. Then the two plasmids were transformed into MGR. At the same time, MGR with plasmid pACYC184-RFP-Switch D was constructed for subsequent measurement.<br> | ||
LB medium containing antibiotics was inoculated with cells picked from individual colonies and incubated overnight with shaking at 37°C. Cells were then diluted 100-fold into fresh selective LB medium and returned to shaking at 37°C and 250 rpm in 96-well plates. For T7 RNA polymerase driven expression, cells were induced with 0.1 mM IPTG at 0.2-0.3 OD600 after 80 minutes of growth. measurements on cell cultures were taken 3 hours after addition of IPTG.<br> | LB medium containing antibiotics was inoculated with cells picked from individual colonies and incubated overnight with shaking at 37°C. Cells were then diluted 100-fold into fresh selective LB medium and returned to shaking at 37°C and 250 rpm in 96-well plates. For T7 RNA polymerase driven expression, cells were induced with 0.1 mM IPTG at 0.2-0.3 OD600 after 80 minutes of growth. measurements on cell cultures were taken 3 hours after addition of IPTG.<br> | ||
| − | <center>[[File:T--SCUT China--toe D zhengjiao.jpeg|500px]]</center><br> | + | <center>[[File:T--SCUT China--toe D zhengjiao.jpeg|500px]]<br> |
| + | Fig. 1 :test the amount of leakage at E.coli MG1655-T7 RNAP (MGR)</center><br> | ||
The results show that Trigger D and Switch D have certain orthogonality and that gene leakage is not too high for a single plasmid Switch D. This means that the experimental results shown by Switch D and Trigger D are more consistent with expectations. | The results show that Trigger D and Switch D have certain orthogonality and that gene leakage is not too high for a single plasmid Switch D. This means that the experimental results shown by Switch D and Trigger D are more consistent with expectations. | ||
Latest revision as of 03:34, 22 October 2019
Forward Engineered Toehold 1
This sequence is the forward engineered toehold from Green et. al. A toehold is an RNA based riboregulator that, in the presence of a trigger RNA, allows for the expression of a downstream gene. This part contains the hairpin loop sequence that with the RBS. There is no gene associated with this toehold.
Green, Alexander A., et al. "Toehold switches: de-novo-designed regulators of gene expression." Cell 159.4 (2014): 925-939.
Characterization from SCUT_China 2019:
In SCUT_China 2019's project, Toehold Switch 1 also named toehold switch D.
The new classes of regulatory components offer wide dynamic range and low system crosstalk. SCUT_China 2019 selected four toehold switches, including Toehold Switch D, with widest dynamic range and highest orthogonality used for multiplexing systems and provided by the reference, which are switch A&B&C&D and trigger A&B&C&D, to regulate the expressions of four acid-resistance factors.
We expressed this gene and tested the amount of leakage at E.coli MG1655-T7 RNAP (MGR).(Fig. 1)
The functional gene Toehold Switch D was constructed on plasmid pACYC184-RFP and the functional gene Trigger DNA D was constructed on plasmid PUC19. Then the two plasmids were transformed into MGR. At the same time, MGR with plasmid pACYC184-RFP-Switch D was constructed for subsequent measurement.
LB medium containing antibiotics was inoculated with cells picked from individual colonies and incubated overnight with shaking at 37°C. Cells were then diluted 100-fold into fresh selective LB medium and returned to shaking at 37°C and 250 rpm in 96-well plates. For T7 RNA polymerase driven expression, cells were induced with 0.1 mM IPTG at 0.2-0.3 OD600 after 80 minutes of growth. measurements on cell cultures were taken 3 hours after addition of IPTG.
Fig. 1 :test the amount of leakage at E.coli MG1655-T7 RNAP (MGR)
The results show that Trigger D and Switch D have certain orthogonality and that gene leakage is not too high for a single plasmid Switch D. This means that the experimental results shown by Switch D and Trigger D are more consistent with expectations.
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]