APPLICATION OF FLUORESCENT

PROTEINS IN iGEM

Fluorescent Proteins (FPs) are a unique class of proteins capable of emitting bright fluorescence upon excitation by light of specific wavelengths. Hailed as "beacons" in the life sciences, they have revolutionized how we observe, understand, and demonstrate biological processes. The initial discovery of fluorescent proteins dates back to 1962 [1], when Japanese scientist Osamu Shimomura first isolated Green Fluorescent Protein (GFP) from Aequorea victoria. He discovered that this protein could emit green fluorescence under blue light excitation solely relying on its self-formed chromophore, without the need for substrates or coenzymes. This property laid the foundation for subsequent biomarking technologies. For this pioneering work, Shimomura was awarded the 2008 Nobel Prize in Chemistry.

Subsequently, in the 1990s, Chalfie and others further elucidated the crystal structure and luminescence mechanism of GFP. By altering amino acid sequences to adjust GFP's fluorescent properties, they also created fluorescent proteins emitting different colors, such as Blue Fluorescent Protein (BFP),Cyan Fluorescent Protein (CFP) and Yellow Fluorescent Protein (YFP) [2].

With the continuous enrichment of fluorescent protein resources and ongoing innovation in protein engineering technologies, their application scope in synthetic biology research has increasingly expanded. In the International Genetically Engineered Machine (iGEM) competition, fluorescent proteins serve as crucial reporter tools and visualization elements, and their varieties and functional designs show a highly diverse trend. This article focuses on three classic fluorescent protein tags used in our team's project—Yellow Fluorescent Protein (YFP), Green Fluorescent Protein (GFP), and monomeric Red Fluorescent Protein (mCherry)—systematically elaborating on their optical properties, functional advantages, and representative applications in past iGEM projects, aiming to provide references for the design and optimization of related genetic circuits.

Green Fluorescent Protein (GFP) was initially isolated from the jellyfish Aequorea victoria. It is a fluorescent protein with an endogenous chromophore (formed by the spontaneous cyclization and oxidation of the Ser65–Tyr66–Gly67 tripeptide). Its maximum excitation wavelengths are 395 nm/475 nm, and its emission wavelength is 509 nm, emitting green fluorescence upon blue light excitation without requiring substrates or cofactors [3]. Chromophore formation is an autocatalytic cyclization process: through the folding of the protein sequence, the core three amino acid residues undergo an autocatalytic reaction, form covalent bonds with surrounding amino acids, cyclize to form an imidazolin-5-one intermediate structure, and subsequently, through oxidative dehydrogenation, form a stable cyclic conjugated structure of 4-(p-hydroxybenzylidene)-imidazolin-5-one [4]. GFP possesses good genetic encodability, low cytotoxicity, and high spatiotemporal resolution. Since its first successful heterologous expression in 1994 [5], it has become one of the most fundamental and widely used reporter proteins in synthetic biology and molecular biology. A schematic diagram of GFP's three-dimensional structure and core chromophore is shown in Fig. 1 [6]. and core chromophore is shown in Fig. 1 [6].

Fig.1 Schematic diagram of the three-dimensional structure and core chromophore of GFP

In iGEM projects, due to its maturity, stability, and high degree of standardization, GFP is widely used in the following areas:

1.1 As a Universal Reporter Gene

Green Fluorescent Protein (GFP), as the most classic and widely used reporter gene in synthetic biology, finds its core application in serving as a real-time, non-destructive reporter for gene expression levels. This function provides iGEM teams with an intuitive, quantitative, and efficient experimental tool, greatly accelerating the characterization of genetic parts and system optimization.

In specific applications, iGEM teams use molecular cloning techniques to precisely fuse the GFP coding sequence with the genetic element under study:

• Placing the GFP gene downstream of different candidate promoters allows for real-time, dynamic comparison of the transcriptional activity of these promoters under specific conditions.
• Under the control of the same promoter, linking different RBS sequences or strengths to the GFP gene means GFP expression levels directly reflect differences in translation initiation efficiency, enabling rational design and high-throughput screening of RBS parts.

In 2011, the Freiburg iGEM team constructed GFP expression units regulated by promoters and RBSs of different strengths, using GFP as the reporter protein to quantitatively evaluate the activity of these genetic parts. To precisely quantify promoter and RBS strength, researchers used a microplate reader for fluorescence analysis of this reporter system. GFP, when excited by light at 509 nm, emits fluorescence around 520 nm. In the experiment, the microplate reader's high-energy xenon flash lamp provided the excitation light source, and specific wavelengths were precisely selected via filters or monochromators to ensure excitation and detection specificity. The fluorescence signal intensity is proportional to the expression level of GFP in the sample, thus allowing the assessment of promoter and RBS transcriptional and translational activity by measuring fluorescence values.

1.2 Genetic Logic Gate Circuit Functional Validation:

Genetic logic gate circuits in synthetic biology are gene regulation systems inspired by electronic engineering, enabling cells to perform basic logic operations (like AND, OR, NOT) on internal or external signals, much like miniature computers. These circuits achieve precise spatiotemporal control of specific gene expression by integrating biological processes such as transcription, translation, and protein interactions, thereby endowing cells with decision-making capabilities to respond to complex environmental signals and execute predefined functions.

GFP serves as the output terminal of the logic circuit. By applying different input signals (e.g., chemical molecules, light signals) and observing the "on/off" or "high/low" state of GFP expression, the functional correctness, signal-to-noise ratio, and response sensitivity of logic gates (e.g., AND, OR, NOT) can be quantitatively evaluated. In logic gate circuits, the application of GFP as a core reporter output module mainly includes the following aspects:

1.2.1 Functional Validation and Visual Output:

The GFP coding sequence is placed at the final output of the logic circuit as the reporter gene. GFP is expressed only when the circuit receives the correct combination of inputs and completes the intended logical operation.

The 2013 ETH_Zurich team designed a logic gate system based on the LuxI-LuxR quorum sensing mechanism. It utilized the logic of "sender cells (miner cells) expressing LuxI to synthesize AHL signal molecules, which diffuse and bind to LuxR in receiver cells (non-miner cells) to form a complex, subsequently activating the PLux promoter." This achieved precise "signal input-recognition-output" transmission, and the AHL response threshold could be adjusted by mutating the PLux promoter, providing a molecular basis for game grid design. GFP served as the core reporter molecule: its fluorescence intensity changes helped characterize AHL diffusion properties to determine game parameters; it monitored PLux promoter leakage for system optimization; and it was a key component in building the game prototype with wild-type PLux+GFP and GFP-RFP dual fluorescence, validating the feasibility of the "miner-non-miner cell" interaction logic.

1.2.2 Dynamic Quantification and Performance Characterization:

Utilizing the quantifiable nature of GFP fluorescence, parameters such as signal-to-noise ratio (SNR), response kinetics, leakage expression levels, and transfer characteristics can be precisely assessed by measuring fluorescence intensity using flow cytometry or a microplate reader. This quantitative output provides crucial data for model calibration and circuit iterative optimization.

• Signal-to-Noise Ratio (SNR): The ratio of fluorescence intensity under high input conditions to that under no input conditions, reflecting the gate's ability to distinguish between "on" and "off" states.
• Response Kinetics: Includes response time, rise curve, and half-life, characterizing the dynamic process from input reception to output signal generation.
• Leakage Expression: The basal fluorescence level when no expression should occur (without input or with partial input), an important metric for assessing circuit stringency.
• Transfer Characteristic: The quantitative relationship between input signal strength and output fluorescence intensity, often represented as a dose-response curve, used to calculate the linear range, dynamic range, and threshold of the logic gate.

Such quantitative data not only provides key calibration parameters for mathematical models, supporting the prediction and simulation of genetic circuit behavior, but also guides teams in rational design and iterative optimization, thereby enhancing the reliability, robustness, and predictability of logic gates, truly realizing engineered synthetic biology design.

The 2022 iGEM project vilnius-lithuania successfully developed and validated an innovative nanoplastics detection system based on synthetic biology principles. The system aimed to address the core challenges of high technical barriers and difficulty in popularization in current nanoplastic pollution detection. Its design cleverly integrated protein engineering and materials science: by designing two functionalized fusion peptides—CBD-PBD (for immobilization and capture) and GFP-PBD (for reporting signals)—and relying on a cellulose membrane platform, a four-step detection process (immobilize-capture-report-detect) was constructed. Proof-of-principle results showed that the system could not only achieve quantitative detection of commercial nanoplastics (fluorescence signal positively correlated with concentration) but also significantly reduce background noise through a key "saturation optimization" step. It successfully detected mechanically prepared nanoplastics closer to real environments, demonstrating its reliability, high signal-to-noise ratio, and great application potential in practical scenarios.

1.2.3 Multi-level Circuit Integration and Debugging:

In complex circuits containing multiple logic gates, GFP can serve as a reporter for intermediate or final nodes, helping researchers debug subsystem functions step by step. For example, by connecting GFP after different modules, the functionality of each unit can be independently verified before integration into a complete system, thereby reducing design complexity. The 2017 Tsinghua University iGEM team successfully constructed an aflatoxin (AFT) biosensor based on a yeast two-hybrid system. By fusing specific single-chain antibodies (ScFv) with transcription activation domains (AD) and DNA binding domains (BD), specific activation of the GAL promoter was achieved in the presence of AFT. In this system, Green Fluorescent Protein (GFP), as a key multifunctional reporter gene, was used in the sensor module validation phase to monitor the activation efficiency and kinetics of the pGALS promoter in real-time, quantitatively evaluating the correspondence between AFT concentration and gene expression levels through fluorescence intensity changes. In the system integration and debugging phase, GFP served as a visual indicator to optimize compatibility and signal transmission efficiency between modules, providing crucial intermediate validation data for the final use of hexose transporter (HXT) and glucose concentration changes as output signals, significantly accelerating biosensor development and improving system reliability.

1.2.4 Enabling Biological Computation and Programming:

GFP serves as the optical representation of binary output ("1" for fluorescence on, "0" for off), which is fundamental for constructing advanced applications like cellular calculators, state machines, and biosensors. By combining with fluorescent proteins of different spectral characteristics (e.g., RFP, YFP), multi-channel output can be achieved, expanding the parallel processing capability of logic circuits. The iGEM 2014 Heidelberg team developed a standardized protein engineering toolbox based on split inteins. Its core contribution was achieving modular operation of protein cyclization and protein ligation by establishing the RFC[i] new assembly standard and novel plasmid backbones (like the pSB1X30 series). The project's innovation was prominently demonstrated by using superfolder GFP (sfGFP) as a key validation tool: by co-expressing split sfGFP fused with inteins, the invisible process of protein trans-splicing was successfully converted into an intuitive fluorescence signal, not only proving system reliability but also establishing a platform for quantitatively evaluating splicing efficiency. This design of using fluorescent proteins as "molecular probes" provided a visual verification method for intein function (e.g., proving correct protein splicing through fluorescence recovery) and simplified the clone screening process via mRFP marking, fully embodying the "design-build-test-learn" cyclic engineering mindset in synthetic biology, providing a standardized, scalable protein modification platform for subsequent research.

Therefore, GFP, as an "optical ruler" for genetic part functional activity, runs through the entire "Design-Build-Test-Learn" cycle of iGEM projects and is an indispensable core tool in standardized and engineered synthetic biology research.

Yellow Fluorescent Protein (YFP) is a genetically encoded fluorescent reporter protein that has become a core tool in synthetic biology and iGEM projects. It was developed through directed mutagenesis (e.g., S65G/V68L/Q69M/S72A/T203Y) of the Aequorea victoria Green Fluorescent Protein (GFP). Its excitation and emission spectra are red-shifted compared to GFP, with main excitation and emission peaks at 515 nm and 525 nm, respectively [7]. YFP features fast maturation, high quantum yield, and good stability, making it particularly suitable for live-cell quantitative studies.

In iGEM projects, due to its compatibility with other fluorescent proteins like CFP and mCherry in multiplexed detection, YFP is widely used for characterizing genetic parts (promoters, RBS), building biosensors, and implementing logic gate circuits [8]. The development of the optimized variant Venus (F46L/F64L/M153T/V163A/S175G) further improved its performance in both prokaryotic and eukaryotic systems [9].

In iGEM projects, YFP is far more than a simple color alternative; it is a powerful, versatile reporter and key tool whose applications permeate the entire project cycle of design, construction, testing, and demonstration. The main application scenarios of YFP in iGEM are as follows:

2.1 As a Reporter Gene: Visualizing Gene Expression

Like GFP, YFP is a core tool for gene expression detection, enabling precise quantification of key parameters like promoter activity through its fluorescence intensity. Simultaneously, leveraging its unique spectral properties, YFP can be used in conjunction with GFP, RFP, and other fluorescent proteins to achieve in situ synchronous monitoring and analysis of multiple genetic signals.

In the part BBa_K2991016 designed by the Nantes 2019 iGEM team, the core role of YFP was to serve as a high-intensity, quantitative reporter gene for precisely characterizing and validating the activity of their synthetic promoter pRbsD under oxidative stress conditions. This promoter is inducible by sublethal concentrations of antibiotics. By driving YFP expression, its fluorescence intensity directly reflected the activation level of the promoter, allowing the team to quantify the induction efficiency of different antibiotics and providing crucial debugging and optimization basis for subsequent construction of more complex antibiotic-induced apoptosis circuits.

2.2 Differentiating Signals in Multi-Output Systems

In multi-output systems, YFP, with its unique spectral characteristics, becomes an ideal marker for distinguishing complex genetic signals. For example, in iGEM projects, teams often combine YFP with GFP, RFP, and other fluorescent proteins. Multi-color fluorescence output visually represents different logical states or pathway activities, enabling advanced functions like full-adder operations or multi-factor environmental detection.

The 2017 Bielefeld-CeBiTec team used a CFP-YFP fusion protein containing an amber codon within the linker to convert the performance of synthetases into precisely measurable fluorescence signal changes. When the synthetase effectively and specifically incorporated the ncAA into the amber codon site, translation proceeded to completion, forming the full CFP-YFP fusion protein, resulting in FRET (primarily emitting YFP fluorescence at 525 nm). Conversely, if translation terminated prematurely, only the CFP unit was produced (emitting at 475 nm). By calculating the ratio of the two fluorescence signals (negative readout vs. positive readout), the specificity (CFP/YFP ratio without ncAA) and efficiency (YFP/CFP ratio with ncAA) of the synthetase could be efficiently and reliably quantified, providing a powerful tool for optimizing artificial translation systems.

2.3 As an Output Signal in Biosensors

In biosensors, the core function of Yellow Fluorescent Protein (YFP) as an output signal is to convert invisible molecular recognition events into visible, quantifiable optical signals, thereby enabling highly sensitive detection of target analytes. Its typical mode of operation is as follows: when the sensor recognizes a specific target molecule (such as a pathogen, heavy metal ion, metabolite, or signaling molecule), it triggers a pre-set intracellular genetic circuit that ultimately activates a promoter driving YFP expression. The expression level of the YFP protein is directly related to the concentration of the target molecule; thus, measuring the fluorescence intensity allows for precise quantitative analysis of the target.

The 2020 RDFZ-China team designed a biosensor to detect the presence of the activator PcaUAM. In this biosensor system, YFP served as the core output signal, successfully converting the invisible molecular recognition event (specific binding of the PcaUAM protein to the p3B5B promoter) into a quantifiable optical signal. By precisely measuring changes in YFP fluorescence intensity, the team not only quantified the sensitivity and background leakage level of the mutant promoter p3B5B but also, through performance comparison with other variants (like p3B5c), verified its optimized characteristics of "moderate sensitivity and low noise."

2.4 Serving as a Visible Signal for Intercellular Communication

In iGEM projects, Yellow Fluorescent Protein (YFP) acts as a carrier of visible signals for intercellular communication. The mechanism involves visualizing and quantifying quorum sensing systems: first, "sender cells" express quorum sensing signal molecules; subsequently, "receiver cells" detect these signal molecules through built-in sensing circuits and activate downstream YFP expression; finally, the fluorescence intensity of YFP directly reflects the concentration of the signal molecules, enabling spatial mapping and quantitative analysis of the range and intensity of intercellular communication.

The 2023 BostonU-HW team designed a part, which was an ingeniously designed composite promoter capable of simultaneously responding to dual regulation by Quorum Sensing and LacI repression. Its core function was to act as the "command center" of a genetic circuit within an intercellular communication system, synchronously driving the expression of both the signal molecule synthase (RhlI) and a reporter gene (CFP), endowing engineered bacteria with dual capabilities of "sending" and "receiving" signals. In this system design, although YFP was not directly driven by this part, it was the ultimate validation of its function. Its core role was to serve as the specific response signal in receiver cells, converting the biological effects produced by the part (i.e., the synthesis and release of the C4HSL signal molecule) into a quantifiable, observable optical output. When sender cells containing this part successfully produced and released C4HSL, they activated surrounding receiver cells, triggering their YFP expression. Therefore, the YFP fluorescence intensity directly quantified the efficiency and reliability of the intercellular communication link mediated by this part.

2.5 As a Lineage Tracing or Labeling Tool

In iGEM projects, the working principle of YFP as a lineage tracing and labeling tool is based on its stable and heritable fluorescence expression characteristics: by coupling the YFP coding sequence with a specific promoter (such as constitutive or cell-type-specific) and integrating it into the cell genome, the target cell population and all its progeny will continuously express the yellow fluorescent protein, forming a unique "optical tag." Researchers can then observe the spatial distribution and migration trajectories of labeled cells in real-time using fluorescence microscopy, or precisely sort the target population using Fluorescence-Activated Cell Sorting (FACS), achieving long-term quantitative tracking of cell lineage dynamics, population interactions, and spatial localization.

The 2023 Tsinghua-TFL team designed a biological system using Chlamydomonas reinhardtii sgt1 mutant (motility-deficient) as the chassis. They constructed a fusion expression vector containing a strong promoter (pSAD), the SGT1 gene, a self-cleaving F2A peptide, and the target foreign gene (YFP/GCaMP in this model) to achieve functionality. In this system, YFP (in the form of GCaMP) served as a key lineage tracing and expression marker tool: its fluorescence signal directly indicated successfully transformed and expressing cell clones, allowing researchers to quickly identify, screen, and track positive cell lines via microscopy; simultaneously, the fluorescence intensity semi-quantitatively reflected protein expression levels, providing intuitive optical evidence for verifying the reliability of the SGT1 phenotype screening (motility restoration) results. Together, these built an efficient detection system ranging from initial screening (motility) to verification (fluorescence).

mCherry is an important variant in the Red Fluorescent Protein (RFP) family obtained through directed evolution. Its development reflects the sophisticated design of protein engineering. Initially developed by the Tsien laboratory through a series of modifications to DsRed protein, leading to variants like mRFP1 and mStrawberry, mCherry was finally obtained with faster maturation and higher photostability [10]. Its maximum excitation/emission wavelengths are 587/610 nm, with a large Stokes shift beneficial for reducing autofluorescence interference. As a strict monomer, mCherry does not cause erroneous aggregation of target proteins when fused [11]. Particularly noteworthy is its pKa of approximately 4.5, allowing it to maintain fluorescence stability within the pH range of 4.5-10. This characteristic gives it a unique advantage in labeling acidic intracellular environments, such as lysosomes [12].

mCherry, as an outstanding representative of red fluorescent proteins (RFP), has become an indispensable core tool in the iGEM competition due to its bright red fluorescence, excellent photostability, low cytotoxicity, and mature monomeric properties. Its applications extend far beyond simple labeling, deeply penetrating various stages of project design, validation, and optimization.

3.1 As a Universal Reporter Gene and Selection Marker

This is the most basic and widespread application of mCherry.

3.1.1 Promoter and Circuit Output Characterization:

By placing mCherry downstream of the promoter or logic gate under test, the performance of genetic parts, such as strength, leakage expression level, and dynamic range, can be quantitatively characterized by measuring fluorescence intensity.

The 2016 USP_UNIFESP-Brazil team used mCherry as the core reporter protein, primarily for visualizing and quantitatively evaluating the performance of a transgenic expression and secretion system in Chlamydomonas reinhardtii. Its red fluorescence directly confirmed successful expression of the foreign gene and correct protein folding; by measuring fluorescence intensity of colonies and supernatants, high-yield clones were screened, and expression levels were quantitatively assessed; simultaneously, it served as a natural tracer, guiding protein secretion verification and purification process development, ultimately proving the system could efficiently achieve functional protein expression and secretion.

3.1.2 Positive/Negative Clone Selection Marker:

mCherry is often used as a visual selection marker in vectors. In Golden Gate or Gibson assembly, successful insertion of the target gene replaces the mRFP expression cassette, causing colonies to change from red to white, thus easily screening for correct recombinant clones.

In the 2019 TU_Darmstadt team's project, mCherry was used as a reporter protein to validate Sortase enzyme activity: by constructing GGGG-mCherry and mCherry-LPETGG fusion proteins, catalyzed connection by Sortase formed an mCherry dimer (~57 kDa). Fluorescence imaging of SDS-PAGE gels directly showed the connected band, visually confirming Sortase's catalytic function.

3.2 Core Role in Multi-Color Systems

The emission spectrum of mCherry (~610 nm) is well separated from those of GFP (~510 nm), CFP (~480 nm), and YFP (~530 nm), making it an ideal choice for multi-color reporter systems.

3.2.1 Multi-Parameter Reporting and Logical Operations:

In complex biological computing circuits, different output states can be represented by different colored fluorescent proteins. For example, the four output states (00, 01, 10, 11) of a two-input AND gate can be visually displayed by no fluorescence, green, red, and yellow (GFP + mCherry) fluorescence.

The 2022 YiYe-China team designed a system where the fluorescent proteins (GFP and mCherry) together constituted a precise genetic mutually exclusive switch, used to directly report the status of intracellular RNA splicing. mCherry served as the indicator signal for "normal splicing," its red fluorescence indicating the successful excision of the key exon. GFP served as the alarm signal for "aberrant splicing," its green fluorescence activating only when splicing was blocked and the exon was retained. These two fluorescent signals appeared mutually exclusively. The switch between red/green fluorescence allowed for intuitive, quantitative assessment of splicing functionality integrity, providing a powerful visualization tool for disease mechanism research and drug screening.

3.2.2 Ratiometric Sensing and Internal Standard:

To eliminate errors caused by cell-to-cell variation and experimental operations, teams use dual-reporter systems. For example, using mCherry driven by a constitutive promoter as an internal reference, and GFP driven by a conditional promoter as the signal reporter. By calculating the GFP/mCherry fluorescence ratio, the signal can be normalized, obtaining more accurate and reliable data.

The system designed by the 2023 SYSU-SLS-CHINA team utilized both GFP and mCherry fluorescent proteins. In this system, Green Fluorescent Protein (GFP) served as the functional reporter gene, its expression strictly controlled by SNIPR receptor activation, directly indicating successful target recognition and initiation of downstream signaling pathways. Red Fluorescent Protein (mCherry), driven by a constitutive promoter, served as an internal reference marker, used to monitor transfection efficiency, locate engineered cells, and provide a standardization baseline for quantitative analysis. Together, they enabled visual interpretation and functional validation of this synthetic biological system.

3.3 As a Protein Tag and Localization Tool

The monomeric nature of mCherry means it does not cause aggregation when fused to other proteins, faithfully reflecting the localization and dynamics of the target protein.

3.3.1 Subcellular Localization:

By constructing fusion proteins of the target protein with mCherry, teams can visually observe the distribution of that protein within the cell (e.g., localization to the cell membrane, nucleus, or organelles) under a microscope.

The 2017 Manchester team constructed a fusion protein system where mCherry, as the core reporter module, performed multiple key functions: It first served as an intuitive fluorescence indicator, using its red fluorescence to report the protein's subcellular localization in real-time, verifying the targeting efficiency of the PduD(1-20) domain to bacterial microcompartments. Simultaneously, during expression optimization, its fluorescence intensity was used as a rapid proxy measure for soluble protein yield, greatly improving screening efficiency. During the purification stage, its visible red color allowed researchers to visually track the protein's behavior in chromatography columns. Finally, in thermal stability analysis, the mCherry domain showed an independent and sharp denaturation peak at 95.2°C, not only confirming its own correct folding but also providing a crucial internal reference benchmark for characterizing the thermal stability of the entire fusion protein.

3.3.2 Cell Lineage Tracing:

In microbial communities or synthetic co-culture systems, marking specific cell types with mCherry allows long-term tracking of their proportion within the population, spatial distribution, and dynamic changes.

The 2022 UESTC-BioTech team, in cell imaging and localization research, used mCherry as a high-performance red fluorescent protein reporter gene. Its stable and bright fluorescence signal enabled real-time dynamic tracing and precise localization of target proteins within living cells. By fusing it with β-2-transferrin (bTF) and expressing the fusion protein, the distribution of the bTF-mCherry fusion protein within cells, such as enrichment on membrane structures or specific organelles, could be intuitively observed under a fluorescence microscope. This fusion strategy not only allowed preliminary judgment of successful protein expression by visually observing the solution color but also provided high-contrast fluorescence signals at the cellular level. Through co-localization analysis with chlorophyll autofluorescence or other cellular structure markers, it precisely revealed the subcellular localization behavior of the target protein, providing a powerful visualization tool for protein function research and cellular process analysis.

3.4 Application in Biosensors

The output of mCherry can directly serve as the carrier for the sensor's "readout."

The core principle of the biosensor designed by the 2017 Evry_Paris-Saclay team was based on the regulatory mechanism of transcription factors (like PsiR or MprA): in the absence of the target molecule (e.g., allulose or salicylic acid), the transcription factor represses the expression of the downstream reporter gene; once the target molecule is present, it binds to the transcription factor and inactivates it, thereby lifting the repression and initiating gene expression. The function of mCherry was precisely to be the product of this regulated reporter gene. Its emitted red fluorescence intensity is proportional to the concentration of the target molecule, thus converting the invisible chemical signal into a quantifiable optical signal, enabling sensitive, efficient detection and high-throughput screening of the target molecule.

In summary, Green Fluorescent Protein (GFP), Yellow Fluorescent Protein (YFP), and monomeric Red Fluorescent Protein (mCherry), as the most classic and widely used fluorescent reporter tools in synthetic biology, play irreplaceable roles in iGEM projects. They not only provide intuitive and reliable optical output methods for functional validation, dynamic quantification, and system optimization of genetic parts but also greatly promote exploration in cutting-edge directions such as biosensor design, parallel detection of multiple signals, intercellular communication, and even biological computation. With their well-defined photophysical properties, good genetic encodability, and high engineering compatibility, these fluorescent proteins have become indispensable core tools for iGEM teams in the "Design-Build-Test-Learn" cycle.

It is worth noting that the innovative application of fluorescent proteins in iGEM extends far beyond this. Over the years, numerous teams have continuously expanded their functional boundaries—for example, developing new fluorescent protein variants to expand spectral coverage, constructing Fluorescence Resonance Energy Transfer (FRET) systems for biomolecular interaction analysis, or combining fluorescent output with other output modalities (like sound, light, electricity) to enhance system readability and controllability. These collective contributions from global iGEM teams have collectively enriched the application ecosystem of fluorescent proteins and provided valuable resources and inspiration for our current and future projects. It is within this open, shared, and iterative community effort that fluorescent proteins continue to illuminate the innovative path of engineered research and application in synthetic biology.