Introducing a class of standardized and interchangeable parts utilizing programmed ribosomal frameshifts for synthetic biology applications

Abstract

Synthetic biology and the rational design of biological devices depend on the availability of standardized and interchangeable biological parts with diverse range of functions. Reliable access to different reading frames during translation has largely been overlooked as functionality for bioengineering applications. Here we report the construction and initial characterization of the first member of such a class of biological parts that conforms to the BioBrick Standard (RFC25), allowing its interchangeable use in biological devices. Using our standardized frameshifting signal consisting of a UUUAAAG slippery sequence, a 6 nt spacer and an engineered pseudoknot based on the infectious bronchitis virus pseudoknot PK401 embedded in a dual reporter construct, we confirm that the frameshifting activity is comparable to the previously published frequency despite the introduced sequence changes. The frameshifting activity is demonstrated using SDS-PAGE and fluorescence spectroscopy. Standardized programmable ribosomal frameshift parts with specific frameshifting frequencies will be of utility for applications such as double coding DNA sequences by expanding the codable space into the -1 frame. Programmed shifting into the -1 frame to bypass a stop codon allows labeling of a protein pool with a fixed stoichiometry of fusion protein, as well as the construction of multi-enzyme expression constructs with specific expression ratios. A detailed understanding of the structural basis of programmed frameshifting will provide the opportunities to rationally design frameshifting elements with a wide range of applications in synthetic biology, including signals that are regulated by small ligands.

Genetic selection for mistranslation rescues a defective co-chaperone in yeast

“…The tRNAs we have identified are relatively efficient at missense suppression and these tRNAs will continue to be useful tools for genetic analyses and synthetic biology applications (e.g. ( 75 )). The spontaneous nature of the selection process and the limited consequences of the genetic code change on growth suggest that tRNA alleles resulting in mis-aminoacylation may become fixed in a population where they confer advantage and may be more common than previously assumed.…”

Ribosomal frameshifting and transcriptional slippage: From genetic steganography and cryptography to adventitious use

“…There have been two recent studies pertinent to synthetic biology ( 699 , 700 ). One generated synthetic ligand-responsive stimulators for eukaryotic −1 frameshifting at U_UUA_AAC.…”

<p>INPP4B promotes colorectal cancer cell proliferation by activating mTORC1 signaling and cap-dependent translation</p>

“…Interestingly, we found that INPP4B significantly promoted the expression of cyclinD1 protein, but had little effect on the RNA expression levels of CCND1. These results may be attributed to the fact that CCND1 contains relatively long and structured 5′-untranslated regions, which makes it more dependent on the unwinding activity of eIF4A within the eIF4F complex, and thus highly sensitive to the levels of eIF4E in the cell 22. Since both INPP4B and phosphorylated 4E-BP1 have been reported to be overexpressed in CRC tissues, and it was found that INPP4B enhanced 4E-BP1 phosphorylation in CRC cells, it is interesting to explore the correlation between INPP4B and phosphorylated 4E-BP1 expression in clinical tissues.…”