Our understanding of translation underpins our capacity to engineer living systems. The canonical start codon (AUG) and a few near-cognates (GUG, UUG) are considered as the ‘start codons’ for translation initiation in Escherichia coli. Translation is typically not thought to initiate from the 61 remaining codons. Here, we quantified translation initiation of green fluorescent protein and nanoluciferase in E. coli from all 64 triplet codons and across a range of DNA copy number. We detected initiation of protein synthesis above measurement background for 47 codons. Translation from non-canonical start codons ranged from 0.007 to 3% relative to translation from AUG. Translation from 17 non-AUG codons exceeded the highest reported rates of non-cognate codon recognition. Translation initiation from non-canonical start codons may contribute to the synthesis of peptides in both natural and synthetic biological systems.
Synthetic biology needs to adopt sound scientific and industry‐like standards in order to achieve its ambitious goals of efficient and accurate engineering of biological systems.
The abundance of bacteria in liquid culture is commonly inferred by measuring optical density at 600 nm. Red fluorescent proteins (RFPs) can strongly absorb light at 600 nm. Increasing RFP expression can falsely inflate apparent cell density and lead to underestimations of mean per-cell fluorescence by up to 10%. Measuring optical density at 700 nm would allow estimation of cell abundance unaffected by the presence of nearly all fluorescent proteins.
Nucleic acid based affinity reagents (e.g., aptamers) offer several possible advantages over antibodies as specific recognition elements in biochemical assays. Besides offering improved cost and stability, aptamers are ideal for rapid electrophoretic analysis due to their low molecular weight and high negative charge. While aptamers have proven well-suited for affinity-shift electrophoretic analysis, demonstrating a fully integrated aptamer-based assay platform represents an important achievement toward low-cost point-of-care analysis, particularly for remote or resource poor settings where cost and ambient stability of reagents is a key consideration. Here we perform and evaluate the suitability of aptamer-based affinity assays for two clinically relevant target analytes (IgE using a known aptamer and NF-κB using a thio-modified aptamer) in an integrated electrophoretic gel-shift platform. Key steps of (i) mixing sample with aptamer, (ii) buffer exchange, and (iii) preconcentration of sample were successfully integrated on-chip upstream of a fluorescence-based gel-shift analysis step. This approach, utilizing a size-exclusion membrane optimized here for aptamer retention and preconcentration with sample, enables automated sample-to-answer for trace analytes in 10 min or less. We addressed notable nonspecific interference from serum proteins by adding similar nucleic acid competitors to suppress such interactions with the aptamer. Nanomolar sensitivities were demonstrated and integrated preconcentration of sample provides an important means of further improving detection sensitivities. Aptamers proved superior in many respects to antibody reagents, particularly with regard to speed and resolution of gel-shifts associated with specific binding to target.
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