Part:BBa_K2411008:Design
Forward Engineered Toehold 2 with deGFP gene
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
The gene cassette in this device uses the promoter, deGFP coding sequence, and terminator that Shin et. al. used in their cell free system. In place of the RBS we inserted the second best performing forward engineered toehold sequence designed by Green et. al.
It was important to ensure that including the toehold did not introduce a frame shift between the RBS contained within the toehold sequence and the start of the GFP gene. If improperly design or inserted, the GFP will not be produced properly and the fluorescent response will not be detectable.
Source
The toehold in this part was synthetically designed by Green et al* and the sequence was retrieved from the supplemental information.
The remaining parts were found in the pBEST-OR2-OR1-Pr-UTR1-deGFP-T500 plasmid. This plasmid was used for expression in E. coli and in an E. coli derived TX-TL system.
References
Shin, Jonghyeon, and Vincent Noireaux. "Efficient cell-free expression with the endogenous E. Coli RNA polymerase and sigma factor 70." Journal of biological engineering 4.1 (2010): 8.
Green, Alexander A., et al. "Toehold switches: de-novo-designed regulators of gene expression." Cell 159.4 (2014): 925-939.