Dual stop codon suppression in mammalian cells with genomically integrated genetic code expansion machinery.

Stop codon suppression using dedicated tRNA/aminoacyl-tRNA synthetase (aaRS) pairs allows for genetically encoded, site-specific incorporation of non-canonical amino acids (ncAAs) as chemical handles for protein labeling and modification. Here, we demonstrate that piggyBac-mediated genomic integration of archaeal pyrrolysine tRNA (tRNAPyl)/pyrrolysyl-tRNA synthetase (PylRS) or bacterial tRNA/aaRS pairs, using a modular plasmid design with multi-copy tRNA arrays, allows for homogeneous and efficient genetically encoded ncAA incorporation in diverse mammalian cell lines. We assess opportunities and limitations of using ncAAs for fluorescent labeling applications in stable cell lines. We explore suppression of ochre and opal stop codons and finally incorporate two distinct ncAAs with mutually orthogonal click chemistries for site-specific, dual-fluorophore labeling of a cell surface receptor on live mammalian cells.

Protein allocation and utilization in the versatile chemolithoautotroph Cupriavidus necator.

Bacteria must balance the different needs for substrate assimilation, growth functions, and resilience in order to thrive in their environment. Of all cellular macromolecules, the bacterial proteome is by far the most important resource and its size is limited. Here, we investigated how the highly versatile 'knallgas' bacterium Cupriavidus necator reallocates protein resources when grown on different limiting substrates and with different growth rates. We determined protein quantity by mass spectrometry and estimated enzyme utilization by resource balance analysis modeling. We found that C. necator invests a large fraction of its proteome in functions that are hardly utilized. Of the enzymes that are utilized, many are present in excess abundance. One prominent example is the strong expression of CBB cycle genes such as Rubisco during growth on fructose. Modeling and mutant competition experiments suggest that CO2-reassimilation through Rubisco does not provide a fitness benefit for heterotrophic growth, but is rather an investment in readiness for autotrophy.

Universal Single-Residue Terminal Labels for Fluorescent Live Cell Imaging of Microproteins.

Genetically encoded fluorescent tags for visualization of proteins in living cells add six to several hundred amino acids to the protein of interest. While suitable for most proteins, common tags easily match and exceed the size of microproteins of 60 amino acids or less. The added molecular weight and structure of such fluorescent tag may thus significantly affect in vivo biophysical and biochemical properties of microproteins. Here, we develop single-residue terminal labeling (STELLA) tags that introduce a single noncanonical amino acid either at the N- or C-terminus of a protein or microprotein of interest for subsequent specific fluorescent labeling. Efficient terminal noncanonical amino acid mutagenesis is achieved using a precursor tag that is tracelessly cleaved. Subsequent selective bioorthogonal reaction with a cell-permeable organic dye enables live cell imaging of microproteins with minimal perturbation of their native sequence. The use of terminal residues for labeling provides a universally applicable and easily scalable strategy, which avoids alteration of the core sequence of the microprotein.

Expression and light-triggered movement of rhodopsins in the larval visual system of mosquitoes.

During the larval stages, the visual system of the mosquito Aedes aegypti contains five stemmata, often referred to as larval ocelli, positioned laterally on each side of the larval head. Here we show that stemmata contain two photoreceptor types, distinguished by the expression of different rhodopsins. The rhodopsin Aaop3 (GPROP3) is expressed in the majority of the larval photoreceptors. There are two small clusters of photoreceptors located within the satellite and central stemmata that express the rhodopsin Aaop7 (GPROP7) instead of Aaop3. Electroretinogram analysis of transgenic Aaop7 Drosophila indicates that Aaop3 and Aaop7, both classified as long-wavelength rhodopsins, possess similar but not identical spectral properties. Light triggers an extensive translocation of Aaop3 from the photosensitive rhabdoms to the cytoplasmic compartment, whereas light-driven translocation of Aaop7 is limited. The results suggest that these photoreceptor cell types play distinct roles in larval vision. An additional component of the larval visual system is the adult compound eye, which starts to develop at the anterior face of the larval stemmata during the 1st instar stage. The photoreceptors of the developing compound eye show rhodopsin expression during the 4th larval instar stage, consistent with indications from previous reports that the adult compound eye contributes to larval and pupal visual capabilities.