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.
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.
Summary During heavy exercise in chronic obstructive pulmonary disease (COPD), dynamic airways compression leads to a progressive fall in intrabreath flow. This is manifested by concavity in the ...spontaneous expiratory flow–volume (SEFV) curve. We developed a method to quantify the SEFV curve configuration breath-by-breath during incremental exercise utilizing a computerized analysis. The flow signal was digitized at 100 Hz. For each breath's SEFV curve, points of highest flow ( V ˙ max ) and end-expiration ( V ˙ EE ) were identified to define a rectangle's diagonal. Fractional area within the rectangle below the SEFV curve was defined as the “rectangular area ratio” (RAR); RAR <0.5 signifies concavity of the SEFV. To illustrate the utility of this method, time courses of RAR during incremental exercise in 12 healthy and 17 COPD individuals (FEV1 %Pred. = 39 ± 12) were compared. SEFV in healthy individuals manifested progressively more convex SEFV curves throughout exercise (RAR = 0.56 ± 0.08 at rest and 0.61 ± 0.05 at peak exercise), but became progressively more concave in COPD patients (RAR = 0.52 ± 0.08 at rest and 0.46 ± 0.06 at peak exercise). In conclusion, breath-by-breath quantification of SEFV curve concavity describes progressive shape changes denoting expiratory flow limitation during incremental exercise in COPD patients. Further studies are warranted to establish whether this novel method can be a reliable indicator of expiratory flow limitation during exercise and to examine the relationship of RAR time course to the development of dynamic hyperinflation.
Highlights • The spontaneous flow-volume curves become concave in COPD patients during exercise. • Some show marked, others moderate concavity depending on their resting FEV1. • The degree of ...concavity of the spontaneous flow-volume curves during exercise correlates with the developing dynamic hyperinflation in patients with COPD.
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.
This paper demonstrates a proof-of-principle for a new signal transduction method for protein detection called Bead Assembly Magnetorotation (BAM). BAM is based on using the target protein to mediate ...the formation of aptamer-coated 1μm magnetic beads into a bead assembly, formed at the bottom of a 1μL hanging droplet. The size, shape and fractal dimension of this bead assembly all depend on the protein concentration. The protein concentration can be measured in two ways: by magnetorotation, in which the rotational period of the assembly correlates with the protein concentration, or by fractal analysis. Additionally, a microscope-free magnetorotation detection method is introduced, based on a simple laser apparatus built from standard laboratory components. In this paper, we chose to focus on the protein thrombin, a popular choice for proof-of-principle work in this field.
•Bead Assembly Magnetorotation is demonstrated as a viable signal transduction method.•Limit of detection for thrombin in buffer is 80fM.•Portable laser-and-photodiode system demonstrated for microscope-free detection.•Fractal analysis demonstrated as a secondary signal transduction method.
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.
We use fractal analysis to calculate the protein concentration in a rotating magnetic assembly of microbeads of size 1 μm, which has optimized parameters of sedimentation, binding sites and magnetic ...volume. We utilize the original Forrest-Witten method, but due to the relatively small number of bead particles, which is of the order of 500, we use a large number of origins and also a large number of algorithm iterations. We find a value of the fractal dimension in the range 1.70-1.90, as a function of the thrombin concentration, which plays the role of binding the microbeads together. This is in good agreement with previous results from magnetorotation studies. The calculation of the fractal dimension using multiple points of reference can be used for any assembly with a relatively small number of particles.
This paper presents a new signal transduction method, called label-acquired magnetorotation (LAM), for the measurement of the concentration of proteins in solution. We demonstrate the use of LAM to ...detect the protein thrombin using aptamers, with a limit of detection of 300 pM. LAM is modeled after a sandwich assay, with a 10 μm nonmagnetic “mother” sphere as the capture component and with 1 μm magnetic “daughter” beads as the labels. The protein-mediated attachment of daughter beads to the mother sphere forms a rotating sandwich complex. In a rotating magnetic field, the rotational frequency of a sandwich complex scales with the number of attached magnetic beads, which scales with the concentration of the protein present in solution. This paper represents the first instance of the detection of a protein using LAM.
This paper presents a novel application of magnetic particles for biosensing, called label-acquired magnetorotation (LAM). This method is based on a combination of the traditional sandwich assay ...format with the asynchronous magnetic bead rotation (AMBR) method. In label-acquired magnetorotation, an analyte facilitates the binding of a magnetic label bead to a nonmagnetic solid phase sphere, forming a sandwich complex. The sandwich complex is then placed in a rotating magnetic field, where the rotational frequency of the sandwich complex is a function of the amount of analyte attached to the surface of the sphere. Here, we use streptavidin-coated beads and biotin-coated particles as analyte mimics, to be replaced by proteins and other biological targets in future work. We show this sensing method to have a dynamic range of two orders of magnitude.
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