Part:BBa_K3100011
toehold switch A
Usage and Biology:
The toehold switches are classes of riboregulators that are de-novo-designed based on influence of secondary structure on mRNA translation and knowledge of RNA thermodynamics and kinetics. (Fig. 1)Toehold switch systems are composed of two RNA strands referred to as the switch and trigger. The switch RNA contains the coding sequence of the gene being regulated. Upstream of this coding sequence is a hairpin-based processing module containing both a strong RBS and a start codon. The trigger RNA can specifically bind to a single-stranded toehold sequence at the 5’ end of the hairpin module, and expose the RBS and start codon by a branch migration process that depends on the complement of an extended single-stranded region and the hairpin, thereby initiating translation.
The new classes of regulatory components offer wide dynamic range and low system crosstalk. We selected four toehold switches 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.
Characterization:
We expressed this gene and tested the amount of leakage at E.coli MG1655-T7 RNAP (MGR).(Fig. 2)
The functional gene Toehold Switch A was constructed on plasmid pACYC184-RFP and the functional gene Trigger DNA A was constructed on plasmid PUC19. Then the two plasmids were transformed into MGR. At the same time, MGR with plasmid pACYC184-RFP-Switch A 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. 2 :test the amount of leakage at E.coli MG1655-T7 RNAP (MGR)
The results show that Trigger A and Switch A do not have good orthogonality and that gene leakage is high for a single plasmid Switch A. That means that Switch A may not be working as well as it should 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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