Domain: Natural Science — Microbiology
Target concept: E. coli was transformed with a GFP plasmid and exhibited green fluorescence under blue light.

Video generation prompt: Two E. coli agar plates placed side by side: the plate on the left transformed with a GFP plasmid by heat shock, and the plate on the right untransformed; both were cultured and placed under blue light in a dark laboratory.

Evaluation guide:
Target concept: Plasmid-mediated heterologous gene expression in prokaryotes, visualized via GFP fluorescence as a binary, spatially resolved phenotypic readout. Minimum mechanistic assumptions: (1) GFP is non-functional unless encoded, transcribed, and translated; (2) heat shock enables plasmid entry into E. coli but does not confer expression in untransformed cells; (3) GFP fluorescence requires blue-light excitation and appears green; no green signal occurs without both the plasmid and the correct light. Phased storyline: [Phase 1: Setup] Two identical agar plates (GFP+ on the left, GFP− on the right) placed side by side in total darkness. [Phase 2: Activation] Blue light switches on, uniformly illuminating both plates. [Phase 3: Verification] Rapid emergence of bright green fluorescence only from discrete colonies on the left (GFP+) plate; the right (GFP−) plate stays completely non-fluorescent, showing similarly-shaped off-white colonies. Visibility constraints: Must be filmed from directly above with colonies large and clear enough to tell apart by eye; the green fluorescence on the transformed plate must be strongly and unmistakably visible against the dark background, while the untransformed plate's colonies stay visibly dark with no green tint.

Manual:
I. Basic Demonstration Information
Demonstration Title: GFP Transformation in E. coli (Transformed vs. Untransformed Agar Plates Under Blue Light) Demonstration
Subject: Natural Science - Microbiology - Genetic Engineering
Demonstration Purpose: To visually demonstrate plasmid-mediated gene expression via green fluorescent protein (GFP) by comparing side-by-side E. coli cultures — one transformed with pGFP plasmid via heat shock, the other untransformed — under blue light in darkness; enables recognition of functional gene expression through phenotype (green fluorescence) and reinforces core concepts of molecular cloning, selectable markers, and reporter genes.

II. Demonstration Carrier/Equipment
Core Carrier: Two sterile 90-mm LB-agar plates: (a) plated with E. coli DH5α transformed with pGFP (ampicillin-resistant, GFP-expressing), (b) plated with untransformed E. coli DH5α (ampicillin-sensitive control); both incubated 16–18 h at 37°C to yield discrete, isolated colonies (30–100 CFU/plate).
Auxiliary Equipment: Blue LED transilluminator (470 nm, 10–20 mW/cm²), fully darkened laboratory chamber (light-tight enclosure or blackout curtains), matte black bench surface, waterproof labeling tape (‘GFP+’, ‘GFP−’), standard lab gloves (non-reflective).

III. Demonstration Content Description
Layout: Place both unlidded plates side-by-side (≤1 cm gap), parallel and centered on matte black surface inside darkened lab. Orient so lids are off and agar surfaces face upward. Label left plate ‘GFP+’, right plate ‘GFP−’ on outer dish rim (not lid).
State Control: Ensure plates are dry (no condensation), at room temperature, and colonies are well-isolated (no merging). Avoid touching agar surface. Confirm blue light is the sole illumination source — no ambient light leakage.
Comparison Baseline: Colonies must be morphologically identical (circular, convex, smooth, off-white/cream) in size (0.8–1.5 mm), density, and distribution across both plates — fluorescence is the only distinguishing feature.

IV. Observation of Core Phenomena
GFP+ Plate Fluorescence: Discrete, bright green punctate fluorescence localized strictly to individual E. coli colonies; emission is vivid, saturated green (not yellowish or bluish-green), with sharp boundaries matching colony edges; no background or medium fluorescence.
GFP− Plate Appearance: Identical colony morphology and distribution, but absolutely zero green fluorescence — colonies appear off-white/cream under blue light, with no emission; background remains black.
Characteristic Stability: Fluorescence appears instantly upon blue light onset and vanishes immediately upon shutoff; persists unchanged over 5 s observation; no fading, bleaching, or spread.
Auxiliary Comparison: Side-by-side placement ensures direct chromatic and spatial comparison; identical colony traits confirm that fluorescence difference arises solely from plasmid presence — validating genetic causality.

V. Summary of Key Knowledge Points and Principles
Core Concept Definition: GFP is a genetically encoded reporter protein that fluoresces green when excited by blue light; its expression in E. coli requires successful plasmid transformation, transcription, and translation — serving as a visible, non-destructive phenotypic marker of recombinant DNA function.
Scientific Significance:
• Transformation Validation: Green fluorescence provides rapid, visual confirmation of successful plasmid uptake and functional gene expression — bypassing lengthy antibiotic selection or PCR validation.
• Gene Expression Principle: Demonstrates central dogma in action — DNA (plasmid) → RNA → functional protein (GFP) → observable phenotype (fluorescence).
• Experimental Control Logic: The untransformed plate serves as a critical negative control, confirming that fluorescence is plasmid-dependent and not due to auto-fluorescence, contamination, or lighting artifact.

VI. Demonstration Precautions
• Darkness Integrity: Seal all light leaks; verify darkness with smartphone camera (no visible pixels on screen) before illumination.
• Colony Quality: Use freshly streaked, well-isolated transformants — overgrown or confluent lawns prevent discrete fluorescence resolution.
• Light Source: Use narrow-band 470 nm LED only; avoid UV or broad-spectrum blue-white lights that induce nonspecific autofluorescence.
• Timing: Observe within first 5 seconds of illumination to prevent photobleaching; do not exceed 10 s per trial.
• Labeling: Labels must be on dish base (not lid) and legible without obstructing colonies.

Prompt Grounding rubric:
level_1: The two agar plates are missing, or shown one at a time rather than together; no blue light is shown; the room isn't dark; no E. coli colonies are visible on either plate; or there's nothing in the shot distinguishing a transformed plate from an untransformed one; the scene doesn't resemble this demonstration.
level_2: Two petri dishes appear, but they're stacked, overlapping, or misaligned instead of placed together; the light shown looks white or generally ambient rather than blue; the background is bright or looks like a normal room, not dark; colony shapes may be barely visible, and there's no fluorescence to be seen.
level_3: Two agar plates sit together on a bench and the room appears dim, though not fully dark; a blue light source is present but weak or diffuse, giving only a faint, hard-to-read glow; colonies are visible, but it isn't clear which plate is glowing green or the glow isn't clearly confined to one side.
level_4: Two similarly-sized agar plates sit side by side on a dark surface in a visibly darkened room; a blue light shines directly on both; the left plate's colonies show a visible green glow while the right plate's colonies show none; labels resembling '+pGFP' and '−pGFP' are present and legible, though maybe slightly off-center or blurred.
level_5: Two identical agar plates, lids off, sit close together side by side on a dark surface in a fully darkened room; a blue light illuminates both evenly from above; the left plate's colonies show a clear, vivid green glow while colonies on the right plate look completely unlit; colonies on both plates otherwise match in size and spacing; labels reading 'GFP+' (left) and 'GFP−' (right) are clearly visible and don't cover any colonies.

Scientific and Causal Correctness rubric:
level_1: The fluorescence color is wrong (e.g., red, yellow, or white instead of green); both plates glow the same way; colonies glow without the blue light being on; or a broad glow covers the whole plate or agar rather than being confined to individual colonies; any of these contradicts how GFP actually behaves.
level_2: A green glow appears but is spread diffusely across the plate rather than confined to distinct colonies; the glow appears under ordinary room light rather than requiring the blue light; the untransformed plate also shows a faint green tint; there's no clear link between which plate was transformed and which one glows.
level_3: Green fluorescence appears only on the left (transformed) plate under blue light, matching the correct plate; however, the glowing colonies look blurred, merged together, or overgrown into a lawn; or the green glow keeps showing for several seconds after the blue light is switched off; or a stray greenish glow appears somewhere that isn't a colony (e.g., a smear on the dish rim).
level_4: Green fluorescence is crisp and confined to distinct, round, pale colonies on the left plate only, with no fluorescence anywhere on the right plate; the glow turns off essentially as soon as the blue light is removed; colony spacing and shape look like a normal streaked culture; only small imperfections remain, such as a faint halo around a colony or a slightly uneven label.
level_5: Fluorescence is unambiguously green and confined to separate, well-defined round colonies on the left plate; the right plate shows no green signal anywhere; the glow appears essentially as soon as the blue light turns on and disappears essentially as soon as it turns off, with no lingering afterglow; aside from the fluorescence, colony size, shape, and spacing look identical between the two plates, with no stray glowing spots or light bleeding from one plate onto the other.

Spatiotemporal Consistency rubric:
level_1: Plates jump in and out of frame, or the lighting flickers wildly or switches color mid-clip; the plates rotate or slide out of place; a hand appears and disappears with no visible action; or events happen out of order (e.g., the blue light appears to already be on before the plates are placed); the whole sequence feels physically broken.
level_2: Plates stay in frame but the lighting pulses or its angle shifts noticeably; there's visible jitter in the camera or plate position; a hand reaches in as if to switch on the lamp but nothing visibly changes afterward; the transition from dark room to blue light is missing or abrupt rather than a clear on/off event.
level_3: Plates stay fixed and aligned, and the blue light turns on smoothly with the dark room established beforehand; however, there's noticeable blur on the colonies during the shot, or focus differs between the two plates; a hand may enter to switch on the light, but its timing feels disconnected from when the fluorescence actually appears.
level_4: The overhead shot stays steady with both plates rigidly fixed on the dark surface; the blue light brightens smoothly to a steady level and stays there for the rest of the clip; the dark background is maintained throughout; no hand or stray object crosses the frame; colony positions and relative brightness look essentially unchanged from frame to frame, with at most a barely perceptible flicker or lens flare.
level_5: Everything in the 4-point description holds, and in addition, once the blue light reaches its steady level there's no visible jitter in the plates, background, or lighting; colony positions and the green glow stay in the same spots frame to frame; the blue illumination looks steady and consistent in tone for the remainder of the clip, and the green glow's color and brightness stay visually constant with no flickering or color drift; the contrast between the brightly glowing colonies and the dark background stays sharp and consistent for the whole clip.