Hammerhead Ribozyme Insulator

CchJ

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Description

Part of a collection of Hammerhead ribozyme-based insulators, which are used for combinatorially insulating NOT gates in the Cello CAD, developend by the Voigt lab at MIT. RiboJ, a hammerhead ribozyme derived from the satellite RNA of tobacco ringspot virus, was initially validated in another study by showing its ability to insulate gene expression from promoter 5'UTR interference. Variants were then found by part mining or created through mutation of the RiboJ scaffold.

Characterization

Parts were characterized on E. coli NEB10-beta in M9 glucose media at 37°C in 96-well flow citometer assays.The parts were characterized similarly to a previous work, by comparing gene expressions with different promoter-CDS combinations. Specifically, the authors tested the ribozymes by expressing GFP and cI-GFP with either PLacO1 or PTac promoters. All insulators were sufficiently effective, recovering the gene expression from the uninsulated state and making expression predictable.

Sequences

Insulator

AGTTCCAGTCGAGACCTGAAGTGGGTTTCCTGATGAGGCTGTGGAGAGAGCGAAAGCTTTACTCCCGCACAAGCCGAAACTGGAACCTCTACAAATAATTTTGTTTAA

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Reference

Nielsen, A. A. K. et al. Genetic circuit design automation. Science 352, aac7341 (2016).