Microfluidic cell sorter-aided directed evolution of a protein-based calcium ion indicator with an inverted fluorescent response
Bibliographic record
Abstract
We demonstrate a simple, low cost and disposable microfluidic fluorescence activated cell sorting system (μFACS) for directed evolution of fluorescent proteins (FP) and FP-based calcium ion (Ca(2+)) indicators. The system was employed to pre-screen libraries of up to 10(6) variants of a yellow FP-based Ca(2+) indicator (Y-GECO) with throughput up to 300 cells per s. Compared to traditional manual screening of FP libraries, this system accelerated the discovery of improved variants and saved considerable time and effort during the directed evolution of Y-GECO. Y-GECO1, the final product of the μFACS-aided directed evolution, has a unique fluorescence hue that places it in the middle of the spectral gap that separates the currently available green and orange FP-based Ca(2+) indicators, exhibits bright fluorescence in the resting (Ca(2+) free) state, and gives a large response to intracellular Ca(2+) fluctuations in live cells.
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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 | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.001 | 0.000 |
| Open science | 0.001 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
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".