RiboFACSeq: A new method for investigating metabolic and transport pathways in bacterial cells by combining a riboswitch-based sensor, fluorescence-activated cell sorting and next-generation sequencing
Bibliographic record
Abstract
The elucidation of the cellular processes involved in vitamin and cofactor biosynthesis is a challenging task. The conventional approaches to these investigations rely on the discovery and purification of the products (i.e proteins and metabolites) of a particular transport or biosynthetic pathway, prior to their subsequent analysis. However, the purification of low-abundance proteins or metabolites is a formidable undertaking that presents considerable technical challenges. As a solution, we present an alternative approach to such studies that circumvents the purification step. The proposed approach takes advantage of: (1) the molecular detection capabilities of a riboswitch-based sensor to detect the cellular levels of its cognate molecule, as a means to probe the integrity of the transport and biosynthetic pathways of the target molecule in cells, (2) the high-throughput screening ability of fluorescence-activated cell sorters to isolate cells in which only these specific pathways are disrupted, and (3) the ability of next-generation sequencing to quickly identify the genes of the FACS-sorted populations. This approach was named "RiboFACSeq". Following their identification by RiboFACSeq, the role of these genes in the presumed pathway needs to be verified through appropriate functional assays. To demonstrate the utility of our approach, an adenosylcobalamin (AdoCbl)-responsive riboswitch-based sensor was used in this study to demonstrate that RiboFACSeq can be used to track and sort cells carrying genetic mutations in known AdoCbl transport and biosynthesis genes with desirable sensitivity and specificity. This method could potentially be used to elucidate any pathway of interest, as long as a suitable riboswitch-based sensor can be created. We believe that RiboFACSeq would be especially useful for the elucidation of biological pathways in which the proteins and/or their metabolites are present at very low physiological concentrations in cells, as is the case with vitamin and cofactor biosynthesis.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.000 |
| Meta-epidemiology (narrow) | 0.001 | 0.001 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.001 | 0.001 |
| Science and technology studies | 0.001 | 0.001 |
| Scholarly communication | 0.001 | 0.001 |
| Open science | 0.001 | 0.001 |
| Research integrity | 0.001 | 0.002 |
| Insufficient payload (model declined to judge) | 0.002 | 0.002 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".