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Biosensor Parts & Microbial Database Integration Platform. Explore, select, and compare genetically encoded biosensors.
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Sensor Selection Guide
Answer a few questions to find the best sensor for your experiment
Find Your Sensor
Our guided quiz helps you navigate 87 sensors across 17 analytes and 8 sensor types.
Sensor Database
Fluorescent proteins, biosensors, and biological parts
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How to Use Biosensors
A practical guide for getting started with genetically encoded biosensors
A genetically encoded biosensor is a molecular tool designed to detect and measure biological molecules (e.g., ions, metabolites) or environmental conditions within living cells. These biosensors are encoded by synthetic genes that can be introduced into cells through genetic engineering.
Key Features
- Specificity: Designed to bind or interact with a particular target molecule (Ca2+, glucose, neurotransmitters) or respond to conditions (pH, oxidative stress)
- Reporting Mechanism: Generate detectable signals (fluorescence/luminescence) upon binding to their target
- Non-invasive: Continuous, real-time monitoring in natural cellular context without external probes
Applications
- Cell signaling pathway studies
- Metabolic activity monitoring
- Drug discovery and screening
- Neuroscience research
- Environmental biosensing
Fluorescence-Based Biosensor Components
Fluorescence-based genetically encoded biosensors convert the detection of specific targets (ions, metabolites, or protein interactions) into measurable fluorescence changes.
Fluorescent Proteins
Core component that emits light when excited. Common variants include:
- GFP variants: EGFP, cpGFP, LSSmGFP
- Blue/Cyan: CFP, cpCerulean3, cpT-Sapphire
- Yellow/Orange: YFP, LSSmApple, LSSmOrange
- Red: mCherry, LSSmKate2
Sensing Domain (SBP)
Specifically binds the target molecule, undergoing conformational change. Examples:
- Calcium: Calmodulin (used in GCaMP sensors)
- Sucrose: ThuE receptor
- Iron: DtxR binding protein
- NADH/NAD+: Rex domain
Fluorescence Modulation Mechanisms
- FRET: Donor and acceptor FPs - energy transfer efficiency changes with distance/orientation
- cpFP: Circularly permuted FP - fluorescence intensity changes due to structural alterations
- Single FP: Direct intensity, lifetime, or spectral changes upon target binding
Linker Regions
Flexible peptide sequences connecting FPs and sensing domains. Critical for:
- Proper conformational changes
- Effective fluorescence modulation
- Sensor functionality and dynamic range
Design Considerations
| Property | Importance |
|---|---|
| Brightness | Bright FPs improve signal-to-noise ratio |
| Photostability | Resists photobleaching during time-lapse imaging |
| Dynamic Range | Significant change upon target detection |
| Response Time | Must capture biological process dynamics |
| Biocompatibility | Should not interfere with cellular functions |
Guidelines for Biosensor Application in Your System
Follow these steps to successfully apply biosensors to your research system. Remember: Know your system! This is the first key to success.
Register & Use MibiSense
Explore the MibiSense database to find information about available biosensors. The database provides plasmids, strains, KD values, and literature references but NOT about your specific system - you need to adapt the sensor to your organism.
Define Your Target & System
Identify what you want to measure (sucrose, ATP, Fe2+, calcium, etc.) and clarify:
- What is your target system/organism?
- Is there an expression system available?
- What is known about protein production in your host (rare codons, etc.)?
Check the Literature
Research what is known about your target metabolite and relate it to your system:
- KD values, concentration levels, stability
- What is known about your biological system?
- What do you want to know and why is it important?
- How can biosensors help answer your question?
Check for Substrate Binding Proteins
Identify potential sensing domains for your target:
- Molecular Recognition Element: Should ideally be monomeric, up to 100 kDa
- Structurally characterized (X-ray, cryo-EM, AlphaFold)
- Biochemically analyzed
- Are there pre-existing biosensors or detection methods?
Collect & Sort All Information
Gather data on:
- Targeted metabolite (KD, levels, stability, physiological role)
- Substrate binding protein (structure & properties)
- Background (pre-existing sensors or detection methods)
- Expression requirements
- Host properties (autofluorescence, growth conditions, etc.)
Approach MibiNet Z01 for Biosensor Creation
If no suitable sensor exists, collaborate with Project Z01:
- Initial screens for expression of sensory cassettes in YOUR system
- Biosensor creation pipeline and characterization (in vitro) in parallel
- Distribution of the finalized biosensor
Apply the Biosensor
When you receive your biosensor:
- Check fluorescence properties (FRET or Matryoshka) → Spectra
- Determine what you want to observe
- Test systemic properties (autofluorescence of media)
- Perform in vitro titration and in vivo imaging of targeted metabolite
- Receive assistance with data analysis and experiment refinement
| Design | Principle | Readout | Advantages | Count in DB |
|---|---|---|---|---|
| Matryoshka | cpFP reporter + large Stokes-shift reference FP nested inside | Ratiometric (reporter/reference) | Built-in normalization, robust quantification | - |
| FLIP (FRET) | Donor and acceptor FPs flanking a binding domain | FRET ratio change | Well-established, good dynamic range | - |
| Single cpFP | Circularly permuted FP with inserted sensing domain | Intensity change | Simple design, bright signal | - |
| FRET | Direct FRET between two FPs | FRET ratio | Ratiometric, distance-sensitive | - |
| Semi-synthetic FRET | Protein-based FRET with synthetic fluorophore component | FRET ratio | Extended spectral range | - |
Host Organism Considerations
Before choosing a biosensor, thoroughly evaluate your target system:
Bacterial Systems
- E. coli BL21(DE3): Most common for protein expression
- E. coli K-12: General laboratory strain
- Corynebacterium: Industrial production host
- Check for autofluorescence in growth media
Genetic Compatibility
- Codon usage: Verify rare codons match host
- Promoter availability: T7, pBAD, native promoters
- Plasmid origin: pUC, pBR322 compatibility
- Selection markers: Antibiotic resistance
Expression Optimization
- Temperature: 18-37°C for folding
- Induction timing: OD600 ~0.6-0.8
- Inducer concentration: IPTG (0.1-1 mM)
- Expression time: 2-24 hours
Expression Requirements Checklist
- Vector compatibility: Is the plasmid compatible with your host strain?
- Promoter strength: Match promoter to desired expression level
- Codon optimization: Rare codons may require special strains (e.g., Rosetta)
- Protein folding: Lower temperatures often improve folding for complex sensors
- Maturation time: Allow sufficient time for chromophore maturation (typically 1-4 hours)
Imaging Techniques for Biosensors
Widefield Fluorescence
Fast, simple imaging suitable for routine monitoring. Best for detecting bulk fluorescence changes in cell populations.
- Speed: High frame rates possible
- Resolution: ~250 nm lateral
- Best for: Population studies, plate assays
Confocal Microscopy
Provides optical sectioning to eliminate out-of-focus light. Essential for detailed cellular and subcellular imaging.
- Speed: Moderate
- Resolution: ~150 nm lateral
- Best for: Subcellular localization, 3D imaging
FLIM (Fluorescence Lifetime)
Measures fluorescence decay time rather than intensity. Enables multiplexing with sensors having similar spectra but different lifetimes.
- Speed: Slower, but improving
- Advantage: Intensity-independent, environment-insensitive
- Best for: Multiplexing, quantitative measurements
Plate Reader Assays
High-throughput measurement of fluorescence in multi-well plates. Ideal for screening and dose-response curves.
- Throughput: 96, 384, or 1536 wells
- Best for: KD determination, screening
Common Filter Sets for Biosensors
| Sensor Type | Fluorophore | Excitation (nm) | Emission (nm) | Filter Set |
|---|---|---|---|---|
| Reporter (Matryoshka) | cpGFP | 488 | 505-520 | Standard GFP |
| Reference (Matryoshka) | LSSmOrange/LSSmApple | 560-590 | 610-630 | Texas Red |
| FRET Donor | CFP | 430-450 | 470-490 | CFP/YFP |
| FRET Acceptor | YFP | 514 | 525-550 | YFP |
Quantitative Analysis Methods
Ratiometric Analysis
For Matryoshka and FRET sensors, calculate the ratio between two emission channels:
Ratio = Reporter / Reference
- Advantage: Normalizes for expression level and optical variations
- Required for: Matryoshka sensors, FRET sensors
Dose-Response Curves
Fit sensor response to determine KD and dynamic range:
Response = Rmin + (Rmax - Rmin) × [L] / (KD + [L])
- KD: Concentration at half-maximal response
- Dynamic range: (Rmax - Rmin) / Rmin
Data Processing Steps
- Background subtraction: Subtract autofluorescence from media and cells
- Flat-field correction: Correct for illumination unevenness (if needed)
- ROI selection: Define regions of interest for analysis
- Ratio calculation: Compute reporter/reference ratios
- Normalization: Normalize to baseline or control conditions
- Statistical analysis: Apply appropriate statistical tests
Common Software Tools
- ImageJ/Fiji: Free, widely used for image analysis
- CellProfiler: Automated image analysis pipelines
- Python: scikit-image, NumPy, pandas
- GraphPad Prism: Curve fitting and statistics
- Origin: Advanced data analysis
- FLIMfit: Lifetime analysis software
- OMERO: Image data management and sharing
Common Problems and Solutions
Possible Causes:
- Low expression levels
- Incorrect induction conditions
- Poor chromophore maturation
- Wrong filter sets
- Protein not folding properly / inclusion body formation
Solutions:
- Try lower temperature (18-25°C) for better folding
- Increase induction time (up to 24 hours)
- Verify plasmid sequence and transformation
- Check microscope settings and filter sets
- Use a positive control (e.g., standalone GFP)
- Check for inclusion bodies - solubility issues indicate folding problems; consider fusion tags or chaperone co-expression
Possible Causes:
- Media autofluorescence
- Cellular autofluorescence
- Improper background subtraction
- Crosstalk between channels
Solutions:
- Use minimal media when possible
- Always include negative control (cells without sensor)
- Properly subtract background from each image
- Check for spectral bleed-through in FRET experiments
- Consider using FLIM to avoid intensity-based issues
Possible Causes:
- Sensor not functional in host organism
- Target analyte not present in accessible form
- pH or redox conditions affecting sensor
- KD outside physiological range
Solutions:
- Test sensor in original host organism as control
- Verify analyte is reaching the cells
- Check intracellular pH conditions
- Perform in vitro titration to confirm functionality
- Contact MibiNet Z01 for sensor optimization assistance
Solutions:
- Reduce expression temperature (18-25°C)
- Lower inducer concentration
- Use chaperone co-expression strains
- Test different promoters with weaker expression
- Consider solubility tags (MBP, SUMO)
Solutions:
- Reduce laser/illumination intensity
- Use anti-fade mounting media (for fixed samples)
- Limit exposure time
- Use more photostable fluorescent proteins
- Consider acquiring time-lapse images at lower frequency
External Databases & Resources
FPbase
Comprehensive database of fluorescent proteins with spectral data, references, and sequence information.
BiosensorDB (UCSD)
Database of genetically encoded biosensors with characterization data and references.
RCSB PDB
Protein Data Bank - archive of 3D structural data for proteins and nucleic acids.
Imaging & Analysis Software
- Fiji/ImageJ - Free image processing software
- CellProfiler - Automated image analysis
- MolStar - 3D structure visualization
- GraphPad Prism - Statistical analysis and curve fitting
- OMERO - Image data management and sharing platform
Community Support
- Submit feedback - Ask questions or report issues
- Request new sensors - Contact MibiNet Project Z01
Microscopy & Results
Fluorescence microscopy images and sensor characterization results
Fluorescent Protein Collection
Overview of fluorescent proteins expressed in E. coli BL21(DE3), including EBioFP2, LSCerulean, cpCerulean3, cpT-Sapphire, eGFP, LSSmGFP, NowGFP, NovaCFP, cpCherry, and more.
Matryoshka Sensors Overview
Reporter FP vs Reference FP channels and merged views for ATPlyze_7uGA, ATPlyze_2mGA, ReNatoR, and MSucMatec_5 sensors.
IronSenseR WT
Merge, cpsfGFP (G), LSSmApple (A), ratio G/A, and zoom views at PBS, 20 uM BPD, and 250 uM BPD concentrations.
Fe2+ Matryoshka
IronSenseR H79A (Binding Deficient)
Control mutant showing minimal ratio change across Fe2+ concentrations.
Fe2+ Matryoshka
Co-Culture: eGFP & cpmVenus
Fast FLIM imaging of fixed E. coli BL21(DE3) co-cultures expressing eGFP (green) or cpmVenus (red). Scale bar: 2 um.
Fluorescent Bacteria
Wide-field fluorescence microscopy of E. coli expressing fluorescent proteins. Excitation 488 nm. Scale bar: 10 um.
Co-Culture: Blue & Green Channels
Fluorescence microscopy of E. coli co-cultures showing blue and green channels. Scale bar: 10 um.
FLIM Phasor Analysis: eGFP & cpmVenus
Phasor plot from fast FLIM measurements of E. coli co-cultures expressing eGFP and cpmVenus.
FLIM Phasor Analysis: cpmVenus
Phasor plot analysis of cpmVenus fluorescence lifetime. Used for lifetime-based multiplexing in biosensor experiments.
IronSenseR BPD Dose Response
Normalized cpsfGFP/LSSmApple ratio for MDtxR-G149 variants (WT, H79A, H98A, C102A) at PBS, 20 uM, and 250 uM BPD.
Fe2+
FLIM Iron Sensor Measurement
Fluorescence lifetime imaging microscopy (FLIM) of iron sensor before analyte addition.
Fe2+
MATP_2mGA Sensor Response
cpsfGFP/LSSmApple ratio under various starvation and metabolic inhibitor conditions (glucose, KCN, CCCP, oligomycin).
ATP Matryoshka
MSucGA Sucrose Sensor Response
pBAD-MSucGA-D192N-W283C cpsfGFP/LSSmApple ratio in M9 medium with and without sucrose at 37 C.
Sucrose Matryoshka
AlphaFold: eGFP Biosensor (Front)
AlphaFold2-predicted structure of a FRET biosensor showing the sensing domain (gray), cpGFP reporter (green), and red fluorescent protein reference (red).
AlphaFold
AlphaFold: eGFP Biosensor (Side)
Side view of the same biosensor structure. The beta-barrel folds of the fluorescent proteins are clearly visible alongside the alpha-helical sensing domain.
AlphaFold3D Protein Structures
Interactive 3D visualization of fluorescent protein structures from PDB.
Fluorophore Testbase
20 fluorescent proteins available for biosensor construction and testing.
About Fluorescent Proteins
The fluorophore testbase contains a collection of fluorescent proteins (FPs) used in biosensor construction. These FPs serve as reporters, references, or FRET partners in genetically encoded biosensors.
Available Fluorophores
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Statistics & Analytics
Database analytics, visualization, and community dynamics data.
Biosensor Analytics
Statistical analysis and trends from 453 fluorescent biosensors
Fluorescent Protein Analysis
Spectral properties and database statistics from 1,040 proteins
Sample Species Interactions
Sample microbial community interaction data from in silico models.
| Species A | Species B | Interaction Type | Mechanism | Strength |
|---|
Unified Database Search
Search across all open-access biosensor and synthetic biology databases.
Cross-Database Search
Search Results
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About External Databases
Individual Database Browsers
Browse fluorescent proteins from FPbase. Click any entry to view on FPbase.
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Browse biosensors from UCSD. Click any entry to view full details.
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