Team:Lambert GA/Measurement

BACKGROUND & OVERVIEW

Background

In order to diagnose C. elegans presence in fecal samples, Lambert iGEM constructed a toehold switch biosensor to produce a reporter protein when a C. elegans RNA trigger was detected. The switch expresses the reporter protein green fluorescent protein (GFP). GFP displays a bright, green fluorescence when excited by UV light, and therefore provides visual identification for C. elegans presence. GFP does not require substrates or cofactors to express, nor is it inhibited by environmental factors such as temperature, pH, or denaturant concentration. We utilized the specific chromophore arrangement of the GFP known as enhanced GFP.

A low amount of C. elegans DNA in the sample would yield a low level of GFP expression.

A high amount of C. elegans DNA in the sample would yield a high level of GFP expression.

Overview

As our toehold switch only unbinds in the presence of a target RNA strand from C. elegans, the level of fluorescence in a liquid culture is correlated to the amount of C. elegans genetic material. While visual detection of fluorescence may alert health care workers to the possibility of helminthiasis diagnosis, we intended to create a more practical manner to quantify fluorescence. Keeping in mind the standards of frugality, accessibility, and portability, we introduce FluoroCents: a portable, ultra-low cost fluorometer that can be used in the field by healthcare workers.

FluoroCents is the most portable fluorometer in the world - at just 7 grams, Fluorocents will provide a rapid and accessible platform for in-field detection. The physical portion of FluoroCents is a 3-D printed light excitation and filtration mechanism.

The carousel depicts multiple views of the FluoroCents system.


A cuvette containing the tested liquid culture will be placed in the main insert. A light source is attached to the square opening on the device. We required light filters to select for the narrow bands of wavelengths that would excite GFP, and the wavelengths of light that GFP would fluoresce to, respectively. To filter for the excitation and emission filters, we tested a variety of different plastic, affordable filters based on their transmission rate and the wavelengths they filtered for. The device is paired with our FluoroCents android application in order to quantify the fluorescence. Most modern smartphones contain built-in ambient light sensors. The ambient light sensor provides a method of detecting the fluorescence with no previous technical knowledge, and no additional attachment. The ambient light sensor thus becomes the tool for fluorescence data collection from liquid cultures in terms of lux. Lux is the SI unit for illuminance, which measures luminous flux: a measure of brightness based on the energy emitted.

DESIGN

Product Construction

Our FluoroCents device consists of two parts: a tangible sample holder and an android application. The tangible sample holder was constructed on the bases of frugality, accessibility, and portability. For this reason, we printed the FluoroCents using a 3D Printer, to make the device accessible to mass production, and created the device to weigh less than 7 grams and cost less than $1 to create. Furthermore, the device is printed using black PLA, with the black coloration used to absorb external disruptive light sources. Due to the prevalence of phones across the globe, we have coded an application accessible on the Google Play Store.

Light Source

When considering the UV light source for FluoroCents, a battery powered, small light was needed to make the device mobile, and at an effective price. We chose a miniature LED keychain flashlight from LUMAND, found here, which we felt was suitable in terms of price and size for building our hardware device.

This is the LED light source used by Fluorocents.

Ambient Light Sensor

The ambient light sensor is a photodetector found at the top of most smartphones. The sensor is intended to sense ambient light near the surface of the phone and dim the brightness of the phone accordingly. The ambient light sensor picks up electromagnetic energy in a limited range of wavelengths of visible light, but includes the wavelengths emitted by GFP. We will manipulate the sensor and phone device to collect data about luminescence emitted by fluorescent samples, giving us tangible information about the total fluorescence found in a sample. Furthermore, the prevalence of sensors within smartphones allow for an accessible mechanism for this data collection.

The ambient light sensor location on most smartphones is located on the top.


Angle

The final version of the FluoroCents device was designed to operate at a 90 degree angle, so the exciting light, which comes from our UV light source, will not interfere with the emitted light. We found that when the light source was positioned directly in the path of the light emissions, the incident light polluted the emitted light and affected our results. Thus, we opted to have the exciting and emitted light at a 90 degree angle to each other, the same way that most industrial fluorometers operate.

Length

To decide the most optimal specific length for channels of light around sample, we tested on multiple varying 3-D printed versions of the device. The channel connected to the ambient light sensor was kept constant and with a minimum distance, with the incident light channel being varied. Results from our testing are below:

Length 0.5cm, Fluoroscein 1.0cm, Fluoroscein 1.5cm, Fluoroscein 2.0cm, Fluoroscein 2.5cm, Fluoroscein
Average Lux 107 48.5 41.4 25 20.4
Variance 41.8 2.94 7.7 1.2 1.8
n 27 10 19 6 8

From this data, we can see that the highest lux values are created with a 0.5 cm chamber. However, the 1.0 cm chamber still created a high average lux value, and at a much lower variance and error than the 0.5 cm chamber. Thus, we opted to use the 1.0 cm as the standard chamber for FluoroCents.

Filters

The FluoroCents device uses both an emission and excitation filter. In commercial filters, the filtration system is meant to narrow down the bandpass lengths that excite the sample and are then emitted by the sample. The emission filter is meant to narrow down the light wavelength to the band that excites fluorophores within a sample, and this excitation leads to fluorescence. This fluorescence occurs at a specific wavelength band, the wavelength that we hope to measure fluorescence at.

Filtration Purpose Transmission Rate eGFP Peak (nm) Actual Peak (nm) Filter Name
Excitation 39% 488 480 Calcolor 90 Cyan
Emission 40% 509 502 Chroma Green

All filters are courtesy of Rosco Laboratories.

Phone Case

During the development of FluoroCents, we had difficulty with aligning the location of the ambient light sensor on the phone with the FluoroCents hardware device. Thus, we developed a phone case/frame with an opening for the device to fit into in order to facilitate the use of FluoroCents. This will allow us to remove another variable that could affect our data.

The FluoroCents Phone Case holds Android Phones to facilitate the data collection of the FluoroCents App.

Modularity

In addition, the FluoroCents has built-in modularity with filters that can be swapped out according to excitation and emission wavelength of different fluorescent reporters. In this way, the applications of FluoroCents can be expanded outside of the eGFP specifications Lambert iGEM used in this project.

Assembly

Our product was designed with ease of use and build in mind. A smartphone fits easily into our Phone Case but the screen remains case-free, allowing users to access their phones while attached to the FluoroCents Device. The Light Channel can then be affixed onto the Phone Case. An opening in the top of the channel allows for both the insertion of cuvettes for testing samples and both an excitation and emission filter. The Light Case houses our light source, which can be turned on and connected to the light filter to excite the sample. When testing, place the printed cuvette cap on top of the cuvette and hit record on the FluoroCents application.

This diagram shows the different components of the FluoroCents hardware.

Click here to access the STL files.

FluoroCents App

At its base, FluoroCents provides a simple method for detecting the presence of fluorescence in a liquid culture. However, with the revolutionary FluoroCents application, our fluorometer can go beyond this simple test and provide analytics on the level of fluorescence in a sample and use this data to extrapolate information about the relative level of helminth DNA in a sample.

The FluoroCents app makes collecting fluorescence data easy.

The FluoroCents Android app takes repeated measurements of the sample's lux value by capturing the reading from the ambient light sensor as changes in the lux value occur. FluoroCents was developed in Java using the Android Studio IDE. The app outputs the mean lux value and variance of the lux onto the user interface over a 30-second interval. The number of lux measurements taken during the interval is also displayed according to user preference to run trials in a scientific setting. To ensure in-field functionality, the mobile app comes with a saving mechanism that stores the results of fluorescence tests in the cloud. By utilizing Amazon Web Service's DynamoDB NoSQL database infrastructure, many data points can be recorded in one test including the following:


  • Latitude
  • Longitude
  • Timestamp
  • Disease Status
  • Name of User
  • Nearest Body of Water
  • Source of Sample
  • Additional Notes


The latitude and longitude coordinates for each test can then be retrieved from the DynamoDB database and displayed within the FluoroCents Android app using Google Maps API. Each location marker for its respective test is labelled with characteristics as inputted by the user. These features make up the helminth diagnosis mapping tool (HDMT). This tool is built into the testing app and accessible on mobile phones across the globe. Healthcare workers can easily access data where helminth infections are prevalent and which areas are in immediate need of aid. On a broader scale, this enables healthcare workers to allocate resources and plan distribution pipelines in a way that is efficient and resourceful. Housing all parts of the diagnosis workflow, including the measurement of fluorescence, storage of data, and map access all in one app serves as a highly useful capability in the field.

The FluoroCents app helps map locations of helminthiasis incidences.

By measuring the lux value using a phone’s ambient light sensor and our FluoroCents app, we can generate more complex information regarding the detection of a helminth organism.

PROTOCOLS

Materials
  • LB
  • Plain Cells (if possible)
  • Potentially eGFP fluorescent cells
  • Pipette capable of pipetting 1 ml
  • Standard 3.5 ml cuvettes
  • 1x iGEM Standard Fluorescein
  • Android phone with FluoroCents installed
  • FluoroCents Device
Preparation
  1. Obtain potentially fluorescent cells and grow in a 5ml LB culture for 24-48 hours, and if possible, grow a 5 ml LB culture of non-fluorescent plain cells.
  2. Lightly vortex cells for 1-2 seconds or simple invert/shake culture to ensure cells are evenly distributed
Data Collection
  1. Reduce as much external light in the testing environment
  2. Set up Android phone with FluoroCents device and phone case
  3. Take 1 ml of LB, and pipette into a sterile cuvette
  4. Place the sterile cuvette with the sample and place into the FluoroCents hardware device opening
  5. Place the necessary excitation and emission filters for GFP detection into the FluoroCents device on its proper channels
  6. Align emission light channel with the placement of the ambient light sensor and run the FluoroCents app
  7. Record average lux value, variance, and number of measurements for the trials
  8. Repeat steps 3-7 for the plain cell culture if available, GFP samples in question, and iGEM Standard for Fluorescein
  9. Perform further analysis with data as needed to determine presence of fluorescence/relative level of fluorescence

MATHEMATICAL RELATIONSHIPS

In order to increase the accuracy of FluoroCents and standardize its values for comparison across different conditions and labs, the peripheral calculations include the lux value for LB or a non-fluorescent culture and the iGEM Fluorescein standard.

Taking the lux value for our GFP sample, we subtract the lux value for either a plain culture of non-fluorescent cells or LB, preferably the plain cells if available. Thus, the final output lux value will be the lux contributed by the fluorescence rather than LB or cell debris. We can call this value modified Lux, or mLux. The variance for this new mLux value is created by adding the variances together.


In order to standardize the lux values across different conditions, we used the iGEM standard fluorescein and its lux value (fLux for fluorescein lux) to create a ratio of a sample’s lux to the fluorescein’s lux to compare two different samples. We can call this value standardized Lux, or sLux. The new variance is computed by multiplying by the square of the fluorescein Lux by itself. The final 2SEM is calculated from this variance.

RESULTS & MODELING

Preliminary Results

In our primary testing of our FluoroCents device, we took basic lux values without a filter using an arbitrary culture with GFP-expressing cells. For our negative control, we used LB, and for our positive control, we used the iGEM standard for Fluorescein that we received. We recorded these values with our app and received the results shown below.

(Note: these values were recorded during an early version of the app, one without variance, so error bars are unavailable)

This graph was made using GraphPad Prism. The graph shows the lux values as measured by an earlier FluoroCents model. As seen, more fluorescent samples yielded greater lux.



From these basic preliminary tests, we discerned that there was a range of possible values for a fluorescent culture of GFP that lies between the minimum and maximum ranges of LB and fluorescein. This information suggested that if a culture did fluoresce, the lux values would be between these values. Thus, we decided to do further testing with different levels of GFP cultures to see the variation in lux as we vary the density of our culture.

GFP Testing & Model for sLux to Relative Fluorescence to Fluorescein

As part of our testing, we wanted to find the relationship between lux values and the relative level of fluorescence in our sample in relation to the fluorescein standard.

Using the protocol established above, measurements were taken for a plain cell culture (puc19) and several two fold dilutions of a GFP culture, along with 1x Fluorescein. The values for GFP mean lux were than standardized using the further calculations mentioned to get sLux values and SEM to create a potential model for our data.

This graph was made using GraphPad Prism. The graph shows our FluoroCents Device's measured values for GFP dilutions, now standardized into sLux based on the Fluoroscein constant.



As seen above, the sLux values seem to increase significantly in the first slight increases in concentration, tailing off slightly after that. For this data, a natural logarithmic model was the best fit for relative GFP concentration to sLux, at a R^2 value of 0.971. The equation for the model is:


In order to modify this model so a user can input an sLux value, we have to divide the relative GFP Concentration variable by its specific value for fluorescein, which will yield a relative ratio of fluorescence to fluorescein and receive a relative value of fluorescence in relation to fluorescein, we first rearrange the equation to isolate Relative GFP Concentration:


This new equation model reveals an exponential relationship between sLux and relative concentration, and any taken sLux value can be inputted to find a relative concentration of GFP to the culture we used, but this equation needs to be modified so that the output value is the fluorescence in relation to fluorescein. So solving for the Relative GFP Concentration value for Fluorescein, which is obtained by plugging in an sLux value of mLux/fLux = 14/18 = 0.78, we get a concentration of e^(.78-0.325)/0.0912) = 146.79. At first glance, this value seems very high, but Fluorescein is near the highest range of fluorescence at GFP excitation and emission wavelengths. We hope to do further testing in the future with many more data values to refine our model.

Now modifying the equation with our found concentration value, we get:


With this final exponential model equation we can plug in any sLux value and get a fluorescence value in a ratio of fluorescence to fluorescein.

Verification of FluoroCents with a Plate Reader

As part of our testing, we wanted to verify that FluoroCents was a legitimate device to measure fluorescence by comparing it with an industrial plate reader. We wanted to graphically compare the lux measurements taken with FluoroCents to the arbitrary values from a plate reader for several two fold dilutions of the iGEM fluorescein standard.

This graph was made using GraphPad Prism. The graph shows the measurement of Fluoroscein dilutions by both a commercial plate reader and our very own FluoroCents Device. The graph has a log2 x-axis.



With the graph of the data we collected, it was clear that there was a real correlation between lux from FluoroCents, seen on the right y-axis, and the raw fluorescence data from a plate reader, seen on the left column. This correlation to an industrial-grade instrument means that FluoroCents does have a valid claim as a fluorescence tool.

FUNCTIONS OF FLUOROCENTS

FluoroCents as a Detection Tool

When using FluoroCents for detection, the sLux values are NOT used. Only the primary lux values are used.

To confidently say that there is a presence of fluorescence in the sample, the lux value of a fluorescent sample minus 2*SEM needs to be above the lux value for LB/plain cells plus 2*SEM. If this statement holds true, the user can verify that there is indeed fluorescence present in the sample.

Throughout our testing, the SEM values received were often less than 0.1, meaning that simply seeing if the lux value of your sample is at least 0.4 lux greater will usually be a good indicator for the presence of fluorescence. This can always be verified by manually calculating the SEM value from the variance and n value outputted from the FluoroCents application.

For an example of this function, see the main section directly below, “FluoroCents’ Use in Lambert iGEM’s LABYRINTH”

FluoroCents as a Tool for Measuring Fluorescence (Relative to Fluorescein)

To use FluoroCents to get a concrete fluorescence value (in relation to fluorescein), sLux values are necessary to make sure the value received is similar across many different conditions.

With the model developed above relating sLux values to fluorescence, as long as the proper protocol for using FluoroCents is followed, an accurate sLux value can be calculated and plugged into our developed equation (which needs refinement and more testing) to get a value for fluorescence in an amount of fluorescein.

FluoroCents’ Use in Lambert iGEM’s LABYRINTH

C. elegans Toehold Switch and Trigger Verification

FluoroCents was used in this year’s iGEM project as part of the Wet Lab process, specifically to verify that our C. elegans toehold was functional. If the cells were transformed with either the trigger sequence or the toehold switch plasmid separately, they should not fluoresce. However, in a dual plasmid transformation, the cells should fluoresce, as the trigger is present and would activate our toehold switch.

Thus, FluoroCents was used in order to detect any presence of fluorescence. The results obtained from our testing are below:

This graph compares the mean lux values of the C. elegans toehold switch, trigger, and the dual plasmid. The toehold switch and trigger have very similar lux values to LB and plain cells, whereas the dual plasmid has a higher lux value, indicating fluorescence.



From the relative comparisons of the cell types shown, the culture of cells with our C. elegans toehold and trigger separately did not appear to fluoresce because their lux values fell in the range of the values of LB/Plain cell mean lux. However, the dual plasmid transformation had a mean lux value with an error range above all the other cell types, including the plain cells and each of the individual trigger and toehold cell cultures, signifying that fluorescence was present and that the C. elegans toehold switch and trigger together functioned properly.

Leaky Toehold Improvement Verification

As an improvement from last year’s project, Lambert iGEM used a different promoter this year (BBa_J23106) from last year (BBa_J23100) in their toehold constructs in order to prevent the leakiness that existed in last year’s project. If the new promoter reduced the leakiness, the cells with just the toehold switch and old promoter will fluoresce and have a mean lux above the plain cells and cells with the trigger due to leakiness, and the cells with the toehold switch and new promoter will not fluoresce and will have similar mean lux to the plain cells and cells with the trigger. Both dual plasmid transformations with both promoters should simply have mean lux values above their respective promoter cell types as a positive control to show the switch/trigger is functional. The data collected is here:

This graph shows the comparison of lux values between promoters BBa_J23100 and BBa_J23106 in strains of plain LB, plain E.coli cells, trigger-pSB6A1, toehold-pSB3C5, and dual plasmid transformation.



From this data, we can see the trigger-transformed cells for both promoters have similar lux values and overlapping error ranges with the plain cells, as expected The dual plasmid cells for both promoters having mean lux and error ranges outside of both their respective toehold only and trigger only cells, signifying that the system is functional. The toehold only cell with the old promoter did have a higher mean lux value than the trigger and plain cells, signifying fluorescence and leakiness, while the new toehold cells matched the mean lux values and error ranges of the trigger and plain cells, signifying that the new promoter construct did not fluoresce and was not leaky; however, the error bars for the old promoter toehold cells do overlap with the toehold, so one cannot say with certainty that there was leakiness in the old promoter and that the new promoter removed this leakiness without performing more trials.

LIMITATIONS & FUTURE WORK

From our development and use of FluoroCents, it appears to be a device that has a lot of potential in the field of fluorescence testing and quantification. However, there are a lot of limitations regarding the device, lots of room for improvement, and more tests to run. While FluoroCents has been tested with our toehold switch and trigger constructs, it has not been tested with actual C. elegans trigger RNA, so there is no data regarding the correlation between C. elegans and fluorescence. We hope to set up FluoroCents along with a full test of our LABYRINTH project workflow to collect this data in the future. Another aspect that we would like to improve is creating a better characterization of the device with more extensive testing with a plate reader in order to refine our model. Pertaining to the actual device, we want to modify the design to make inserting/removing the filters easier, as currently it is placed in with the cuvette and makes the process tedious. We also want to collect data with FluoroCents in different lighting conditions to see how the calculations, standardization, and model we developed hold up. Another point of concern is the light source used in FluoroCents, as the light we chose would often lose its intensity quickly; we want to see if a normal LED light outside of UV in combination with the filters could give us even more accurate results. If possible in the future, testing with other fluorescent proteins to see how the device operates outside of GFP/fluorescein would help define the role of FluoroCents in fluorescent testing.

REFERENCES

[1] Robić, G., Lacorte, C., & Miranda, E. A. (2009). Fluorometric quantification of green fluorescent protein in tobacco leaf extracts. Analytical Biochemistry, 392(1), 8–11. doi: 10.1016/j.ab.2009.05.016

[2] Pereira, V. R., & Hosker, B. S. (2019). Low-cost (<€5), open-source, potential alternative to commercial spectrophotometers. PLOS Biology, 17(6). doi: 10.1371/journal.pbio.3000321

[3] Excitation and Emission of Green Fluorescent Proteins: February 20, 2001. (0AD). Retrieved from https://www.biotek.com/resources/technical-notes/excitation-and-emission-of-green-fluorescent-proteins/