Measuring differential fitness costs and interactions between genetic cassettes using fluorescent spectrophotometry
Bibliographic record
Abstract
ABSTRACT In this article, we present a method for designing, executing, and analyzing data from a microbial competition experiment. We use fluorescent reporters to label different competing strains and resolve individual growth curves using a fluorescent spectrophotometer. Our comprehensive data analysis pipeline integrates multiple experiments to simultaneously infer sources of variation, extract selection coefficients, and estimate the genetic contributions to fitness for various synthetic genetic cassettes (SGCs). To demonstrate the method, we employ a synthetic biological system based on Escherichia coli . Strains carry 1 of 10 different plasmids and one of three genomically integrated fluorescent markers. All strains are co-cultured to obtain real-time measurements of optical density (total population density) and fluorescence (sub-population densities). We identify challenges in calibrating between fluorescence and density and of fluorescent proteins maturing at different rates. To resolve these issues, we compare two methods of fluorescence calibration and correct for maturation by measuring in vivo maturation times. We provide evidence of genetic interactions occurring between our SGCs and further show how to use our statistical model to test some hypotheses about microbial growth and the costs of protein expression. IMPORTANCE Fluorescently labeled co-cultures are becoming increasingly popular. The approach proposed here offers a high standard for experimental design and data analysis to measure selection coefficients and growth rates in competition. Measuring competitive differences is useful in many laboratory studies, allowing for fitness cost-correction of growth rates and ecological interactions and testing hypotheses in synthetic biology. Using time-resolved growth curves, rather than endpoint measurements, for competition assays allows us to construct a detailed scientific model that can be used to ask questions about fine-grained phenomena, such as bacterial growth dynamics, as well as higher-level phenomena, such as the interactions between synthetic cassette expression.
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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 | 0.003 | 0.005 |
| 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 | 0.000 |
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".