Difference between revisions of "Part:BBa K2411004"

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

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]

@@ Line 11: / Line 11: @@
 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).(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>
+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]]<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.