Part:BBa_K2411007

Toehold 2

This sequence is the second best performing 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 with an 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 2 also named toehold switch B.
The new classes of regulatory components offer wide dynamic range and low system crosstalk. SCUT_China 2019 selected four toehold switches, including Toehold Switch B, 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).

The functional gene Toehold Switch B was constructed on plasmid pACYC184-RFP and the functional gene Trigger DNA B was constructed on plasmid PUC19. Then the two plasmids were transformed into MGR. At the same time, MGR with plasmid pACYC184-RFP-Switch B 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.

The results show that Trigger B and Switch B do not have good orthogonality and that gene leakage is high for a single plasmid Switch B. That means that Switch B may not be working as well as it should.

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]