Biology Experimental Results
Stage 1 - gBlock cloning, Transformation and Quality Control
Goal:At the inception of our experimental period, our first goal is to generate stock solutions of plasmids containing either our Bd gBlock or Bsal gBlock to be used for all our downstream experiments for amplification and CRISPR detection. To give an experimental overview, this was achieved by:
- Amplifying our gBlock by PCR
- Ligating the amplified PCR product into a pJET vector
- Transforming the ligated vector into competent E. coli cells
- Inoculating the successfully transformed bacteria into Lb Borth in order to purify the large quantities of the plasmid using the miniprep protocol
- PCR was performed on the purified plasmids to ensure our product was successfully ligated
Note: When our PCR primers were designed, two sets for each Bd gBlock and Bsal gBlock were created. Considering we only had 3 weeks to experiment, it is crucial that this stage is completed as soon as possible to ensure that we can start testing the the main aspects of our project: amplification and CRISPR detection. To ensure that this stage happens successfully, we used two sets of primers for each gBlock in the case that one set of primers did not work.
Quality Control Check
Quality control check on the production of purified Bd plasmid. Bd1 refers to the Bd gBlock amplified by our first Bd PCR primer set, and Bd2 refers to the Bd gBlock amplified by our second Bd PCR primer set. Bd1 Primers amplicon length: 364 bp. Bd2 Primers amplicon length: 373 bp.
Quality control check on the production of purified Bsal plasmid. Bsal3 refers to the Bsal gBlock amplified by our third Bsal PCR primer set. Bsal4 refers to the Bsal gBlock amplified by our fourth Bsal PCR primer set. Bsal3 Primers amplicon length: 476 bp. Bsal4 Primers amplicon length: 486 bp.
The first set of PCR reactions were successful. The bands observed were in agreement with the amplicon length for each corresponding primer set. pJET was used as a reference for the bands of the miniprep. For each miniprep, the bands were indicative of successful ligation, transformation, inoculation, and miniprep. This is true because for each miniprep, a heavily concentrated band that travelled faster than the pJET band is observed. This is indicative of our circular plasmid containing the corresponding gBlocks. In addition, a fainter band that travelled slower than the pJET band is observed. This is indicative of our linear plasmid containing the corresponding gBlocks. To ensure that our plasmid clones contained the correct gBlocks, we ran another PCR test on the minipreps. For the Bsal plasmids, we were able to get successful amplification. Bands that were observed matched that of the initial bands from the first PCR reaction. There were also another set of bands that matched that of our plasmid. Since a band was observed in the negative control of primer set 3, which was most probably a primer dimer, we decided that all our downstream reactions will strictly work with Bsal plasmid amplified by primer set 4. While on the other hand, for the Bd plasmids, we were not able to get conclusive results and required us to troubleshoot our PCR reaction for Bd.
PCR Reaction of minipreps after making a new working stock of Bd primers. Bd1 refers to the Bd gBlock amplified by our first Bd PCR primer set, and Bd2 refers to the Bd gBlock amplified by our second Bd PCR primer set. Bd1 Primers amplicon length: 364 bp. Bd2 Primers amplicon length: 373 bp.
Suspected to be due to primer contamination, the Bd PCR reaction after the miniprep was repeated with a new set of diluted primers. Contamination in Bd1 was observed again, but successful results were observed for Bd 2. Two bands were observed: a 370 bp band, which is the same length as the amplicon length of Bd Primer set 2, and a band at the top, which is the plasmid that was used to amplify the DNA. That said, all downstream reactions worked with minipreps that were produced from Bd Primer set 2.
Stage 2 - Amplification with RPA
Now that we had confirmed successfully inserting our gblock into our plasmid, transforming it into E. coli, and miniprepped our Bsal and Bd plasmids, it was time to move on to amplifying our product.
Goal: Amplify the Bd or Bsal product to then be used for CRISPR detection.
Due to limited time in the lab, we decided to test amplification using RPA and later test and compare amplification techniques with LAMP. We began with RPA specificity tests against Bd and Bsal primers that simultaneously confirmed that our RPA functioned as intended to amplify exclusively our region of interest. We initially ran into problems with one Bd RPA primer set and so switched to another set. We then confirmed RPA specificity. Going forward, we plan on testing for RPA sensitivity and testing LAMP as an amplification technique. See results below:
Protocol needs adjustment: there should be no band seen when Bd primers are used with Bsal DNA. As it is a similar length to the Bd primer length, we decided to use our alternate Bd RPA primer set. The right-most Bsal primer with Bsal DNA served as an initial proof of concept that RPA worked.
Protocol needs adjustment: Bands were faint. We optimized by using higher concentration miniprep plasmids.
Initial protocol needed adjustment. The primary issue seemed to be the Bd RPA primer set, so we substituted it for an alternate Bd RPA primer set
Positive result: RPA showed specificity by amplifying only the Bsal DNA that matched the Bsal primers. No amplification detected when Bd primers were used against Bsal DNA.
Positive result: RPA showed specificity by amplifying only the Bsal DNA that matched the Bsal primers. No amplification detected when Bd primers were used against Bsal DNA.
Adjusted protocol showed optimal results.
SYBR green (5X) detection of RPA reactions
As another method to detect RPA amplification, SYBR green was used. We found an optimal concentration of SYBR green to be 5X. At this level, there was minimal noise from the negative control and sufficient fluorescence from the experimental.
Stage 3 - CRISPR Detection
After evidence of successful RPA amplification of our genes, including our specificity experiments, we moved on to CRISPR to add a second layer of specificity and in order to observe the fluorescence using our reporter. The optimization and use of CRISPR and RPA compared to the traditional PCR and gel is that in our method, we have reduced the time from 2 hours (PCR) plus 30 minutes (loading gel + running gel) to 20 min (RPA) and 20 min (CRISPR).
Goal: Confirming proof of concept for CRISPR Cas12a.
To give an experimental overview, this was achieved by:
- Performing RPA to amplify our gene of interest
- Performing DETECTR RPA CRISPR Cas12a protocol on the RPA product
- Repeating and testing to troubleshoot and optimize protocol
The first CRISPR experiment did not produce florescence for either Bd or Bsal samples, even when concentrations of reagents were tripled and left to incubate overnight. However, we saw detection using SYBR green on the RPA, indicating it was the CRISPR protocol that needed optimizing.
We then ran CRISPR using the optimized protocol for our last year’s experiments that had slightly varied the reagents’ concentrations. Results were successful for our Bd target gene; however, Bsal did not fluoresce. Future experiments will investigate the issue further. Both RPA samples were detected using an optimized 5X concentration of SYBR green. See results below:
CRISPR optimization required: Fluorescence of CRISPR products of Bd. CRISPR had slight fluorescence indicating the method works but there may be an error in the CRISPR protocol section
CRISPR optimization required: No fluorescence was detected.
The first initial set of CRISPR experiments did not provide successful results; thereby, our team decided to re-asses the protocol and test out the optimized version our 2019 NYUAD iGEM team performed. When ran again, Bsal again did not fluoresce and will require further analysis and troubleshooting. However, our Bd target gene provided successful results displaying successful amplification and detection. Further troubleshooting and protocol optimization is our next step for our Bsal target gene.
Triplicate run of CRISPR DETECTR on Bd. Fluorescence in the Bd CRISPR of the reaction works as intended.
RPA amplification of Bd target gene in addition to CRISPR DETECTR protocol displayed successful detection under UV light. This suggests that using this method of detection can be applied to our point-of-care device to be used in the field. Future work will be done to test sensitivity, optimize protocols, and compare the best methods, such as LAMP versus RPA, that will integrate well into our final product.
Future Biology Work
Our future work will work in the following general path: testing and optimizing amplification and detection techniques, testing sensitivity and specificity, comparing different techniques, comparing results to the golden standards, and working throughout the process to integrate our chosen biological techniques into the engineering design. The work we have done up until this point is a humble beginning in showing our project's promise and potential. The work we have ahead of us is to verify, optimize, and integrate.
Refer to the design page for the overview of our project's timeline and how far we have come!
Engineering Research Progress
Engineering product development timeline
Sample collection is most commonly performed in the field on adult amphibians via the swabbing method. Swabbing the infection sites (hands, feet, thighs, and venter) minimizes the occurrence of false-negatives. The swabbing method is thus far the golden standard in non-lethal sample collection of Chytrid fungi from infected amphibians. Several other methods have been developed in recent years, providing alternative routes to obtaining the fungi samples. Mouthparts swabbing of tadpoles is less sensitive than lethal sampling (extraction of mouthparts) but does not cause mortality, unlike ordinary excision methods done on tadpoles. Non-lethal sampling is also performed via biopsy punches from toe webbing from live amphibians. This method does not decrease survival after one year and has been validated via microscopy. Due to the difficult circumstances, field samples could not be obtained for this project, but if they were, they would be obtained via the swab method.
Sample collection via swabbing.
Sample collection via biopsy punch
Another recently emerging sample collection that the team looked at is eDNA, a brief description of which is given below:
Currently, the team is focusing on making the device compatible with the standard swabbing method, however, we plan to work more on eDNA in the future.
Sample preparation, which is nucleic acid extraction in our device, is one of the most crucial stages in the diagnostic pathway for nucleic acid detection based point of care devices (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4943866/). It is critical that we get high quality DNA to be used for detection. As our device deals with fungal cells which are generally harder to lyse, this stage is even more important and challenging. We started out with compiling research about all the available nucleic acid extraction options , and then upon researching the feasibility of these options of POC Fungal Cell lysis, we mapped the filtered options as below:
Mapping of sample preparation/nucleic acid extraction options (note: the options we are trialing are marked green, the ones we need to research more marked yellow, and the ones we will not be proceeding with red)jpg
A brief overview of these filtered options is given below:
Using silica matrices is a well known method for purifying DNA by exploiting its negatively charged backbone. Positively-charged silica acts as a selective filter to DNA by tightly binding to it. Sodium cations in the solution can break the hydrogen bonds between the hydrogen in water and the negatively charged oxygen ions in silica under high salt conditions (pH ≤ 7). The DNA is tightly bound, and extensive washing removes all contaminations. The purified DNA molecules can be eluted under low ionic strength (pH ≥ 7) later by using TE buffer or distilled water. Tan. 2009
Whatman FTA cards
Whatman FTA cards are fibrous cards impregnated with chelators and denaturants that would lyse and inactivate most microorganism. It is used for sample collection, long-term storage, and purification of the nucleic acid. Once the eukaryotic cells are in contact with the card, it lyses the cell and the DNA is bound to the matrix of the card and protected from the environmental and enzymatic damages. Trinh et al., 2019 uses Whatman FTA card accompanied with LAMP in POC device. The research team applied the FTA card in the bottom layer of the microdevice and use FTA paper for DNA extraction and purification. It took 30 minutes for the extraction.
Bead beating is a mechanical method for the lysis of thick-walled organisms. As opposed to chemical methods such as enzymatic treatment or detergents, bead beating uses high-energy cell disruption in order to release nucleic acids.
The most common method of implementing bead beating is through high-power, benchtop devices such as the gold standard Biospec Mini-BeadBeater. However, for the purposes of our application, we intended to incorporate bead beating in a more portable, low-energy device that was also inexpensive and required little expertise to operate. As such, our research focused on the options mapped below:
Bead Beating options mapping
OmniLyse and AudioLyse, are low-power, low-cost alternatives to benchtop bead beating devices. They devices are also disposable, more suited for commercial production and significantly less expensive. OmniLyse is a battery-powered bead blender while AudioLyse is an audio-powered mechanical lysis device. In terms of other options, we also considered fabricating something on-Chip according to the papers.
As per the color coding in the map above, however, we have decided to follow OmniLyse and AudioLyse instead of fabricating a lab-on-a-chip bead beating device. The factors we kept in mind while making a decision between these beat beating options included viability of the method, time taken and lysis efficiency.
We have also looked at Magnetic bead extraction as an option for nucleic acid extraction and narrowed down our research to the methods in the map below. The factors we kept in mind while deciding include input volume, time taken and lysis efficiency.
Magnetic extraction options mapping
Each of these methods is listed in Nucleic Acid Extraction.
As can be seen from the mapping, we are in the process of making decisions on the options we will be prototyping and testing in tandem with the reaction medium options to develop our final concepts. On the options we have marked green, i.e. the ones where we have made the decision to prototype, we expect to start the process very soon.
We define reaction medium as the platform where the processes in the diagnostic device such as sample preparation and sensing happen, such as microfluidic chips, paper-based bio sensors etc. The constraint of the device being usable in the field plays a great role in reaction medium. By researching numerous case studies of literature about POC diagnostic devices compiled in Case Studies, we were able to map out the possible options for the reaction medium of our device:
Mapping of reaction medium options
PMP Droplet Cartridge
LabOnAChip Integrated Method
For reaction mediums, currently, we are in the process of ideation and concept development where we will be researching more options for the reaction medium type based on the compatibility with sample preparation options and will be prototyping and testing the already formed options.
The engineering-based research regarding sensing is compiled in the Measurement section of our Notion space. However, a brief overview is given here.
Upon deciding the objective of creating a point of care diagnostic device, the engineering team started the research with a broad overview of all the reporting and sensing techniques that have been successfully implemented in point of care devices by mapping them as below:
Sensing Methods Mapping Board
As the mechanism of the device was narrowed to a nucleic acid detection based diagnostic device based on the CRISPR-Cas systems, we focused our research on sensing methods compatible with CRISPR-Cas which are mapped with detailed options as below:
CRISPR-Cas compatible Sensing Methods (note that the options we are trialing are marked as green)
We decided not to follow E-CRISPR after comparing it with the other two methods and discussing with faculty experts in POC devices who pointed out the demerits of the techniques such as fragile and expensive electrodes.
More information about our further research regarding the other two methods is given as below:
Lateral Flow Assays
LFA is the use of immunoassay technology using nitrocellulose membrane, coloured nanoparticles (or labels), and typically antibodies to detect the presence of a target substance in a liquid sample without the need for specialized and costly equipment. For our device, we are testing CRISPR-Cas based LFA detection where the technique is used to detect a cleaved single stranded DNA (ssDNA) reporter.
How does it work?
The expected results for LFA is the successful detection and analysis (quantification) of the CRISPR-Cas reaction product through the reporter. This is indicated by the control and test line of the LFA strip showing coloration, and this is illustrated by the mechanism below.
LFA Mechanism - Due to the recognition and presence of The target analyte, the sgRNA/Cas- complex unleashes collateral activity. This activates the Cas complex therefore, the Cas protein cleaves the dual labelled FAM or FITC/Biotin reporter. The complexed DNA analyte, labeled with FITC/FAM and biotin, binds first to the gold-labeled FITC/FAM-specific antibodies in the sample application area of the dipstick. The gold complexes travel through the membrane, driven by capillary forces. Only the analyte captured gold particles will bind when they pass the line with the immobilized biotin-ligand molecules and generate a red-blue band over the time. Unbound gold particles migrate over the control band and will be captured by species-specific antibodies. With prolonged incubation time, the formation of an intensely colored control band appears. Taken from: https://www.milenia-biotec.com/en/tips-lateral-flow-readouts-crispr-cas-strategies/
What have we worked on?
Having performed extensive research, we decided to quantify the results as well as perform qualitative analysis (which is what is primarily used for LFA). The following description is an extensive assessment on the experimental design which was formulated for both quantitative and qualitative assessment.
For qualitative results, the following methodology will be conducted:
Methodology Qualitative results
- After completion of the pre-amplification step, there will be a 20 μL of reaction mix which is a result of the CRISPR reaction. This also included the FAM/Biotin reporter. This will be added to a 2-mL Eppendorf tube after which 80 μL of Hybridetect 1 Assay Buffer should be added into it. A nuclease-free 2-mL 96- well block can be used instead of Eppendorf tubes.
- A lateral flow strip (Milenia HybriDetect 1, TwistDx) should then be added to the reaction tube and a result should be visualized after approximately 2 min. The lateral flow strip must be placed in an upright position and must be incubated at room temperature for at least 5 minutes. (unto 15 minutes). Results must then be analysed.
- single band, close to the sample application pad (the control band) will indicate a negative result, whereas a two bands close to the top of the strip (test band) and the control band should indicate a positive result. The bands are clearly formed red bands. In case of very high concentrations of hybridisation product, control band’s intensity may be affected (during a positive test) Nevertheless, the control band should be still visible clearly.
- After completion, remove the lateral flow strip and place it on a white background for visual inspection. View Figure 1 and 2 for qualitative assessment.
For quantitative results, we created a software that will be able to analyse images of the control test band. The analyte quantity in the sample is expected to be proportional to the intensity of the red colored test line region. Hence, a weighted summation of red color in the test line region can be considered as the parameter for the detection of analyte quantity, where the weight is considered to be the color intensity of each pixel. The intensity of the red could then be quantified by image acquisition followed by an image processing software to measure the band intensity and to determine a positive detection against a positive-control dilution series. Image-processing software was then created and designed using Python.
Methodology Quantitative results
The article "Analyte Quantity Detection from Lateral Flow Assay Using a Smartphone," by the authors Foysal, Kamrul H et al. was carefully studied and a similar procedure was followed in the design of the image processing software. The software was altered to suit the aims of this study accordingly.
In order to feed calibration data into the software, a series of experimental results will have to be calculated. This would allow accurate quantification of the data during the test, with our method for experimentation. The following method was used for the data acquisition required to build the calibration curve which is to be implemented in the software.
Feature extraction- The number of red pixels accumulated on the test line is considered the feature. Assuming that the cumulative sum of red pixels’ intensities in the test line varied proportionally with analyte quantity under a specific lighting environment, we carried out the test. In order to do this:
The testing procedure is as follows:
- - A test strip is cut according to consistent measurements with only the test line and control line regions visible. It is placed under the smartphone on a white background. Using the grid view of the camera, the ROI was positioned inside the center box.
- - The image was captured. The application then created a mask using the preprocessing technique mentioned in the proposed algorithm (Otsu's method).
- - In the masked region, the weighted sums of red pixels’ intensities of the test and the control line regions were calculated.
- - T/C ratio values that corresponded to the quantity of the analyte were calculated.
- - A regression curve is plotted which will be used as the calibration curve.
Currently, the future plans for the project include successfully detecting the CRISPR-Cas reactions, using LFA and ssDNA reporter. We hope to conduct the above qualitative and quantitative assessments. For quantitative assessment, we hope to develop the quantitative detection method by creating the software using the above proposed method. We then hope to use this software to conduct quantitative analysis in the future, with high accuracy.
Further plans for the project include developing the quantitative detection method even further. For example, instead of having to take an accurately fixed image of both regions A and B that have been identified, we could simply take a photo of the whole LFA strip. The software could then go on to crop the photo itself, to define regions A and B independently. The current method involves us feeding the images of the software independently.
Furthermore, currently, we try to maintain a specific lighting and background of the LFA strip. However, when it comes to point-of-care testing, it may be difficult to ensure this sense of consistency in the lighting and background atmosphere. Therefore, a plan for this project could involve training the software to ignore background noise thereby training it to overlook changes in lightning and adapt to the changes.
Citation: Foysal, Kamrul H et al. “Analyte Quantity Detection from Lateral Flow Assay Using a Smartphone.” Sensors (Basel, Switzerland) vol. 19,21 4812. 5 Nov. 2019, doi:10.3390/s19214812
A basic fluorescence detection platform has the following components :
- An excitation source
- A sensor
- Microcontroller to relay output
- Occasionally has filters to increase effectiveness
Fluorescence detection schematic
Over the course of the few months with limited lab access we researched different components that could be used for each of the above units. Talking to experts in the field and research of our own, we made a comprehensive list taking into account multiple factors like sejpnsitivity, cost effectiveness and the time taken for the entire process to occur. This full list can be found on the Fluorescence with CRISPR page along with the advantages and disadvantages of each method.
What did we decide
After looking at all our available options, we decided that we will be trying 2 possible methods:
- No filters
- - Uses a Blue LED or Laser for exciting the fluorophore
- - Photodiode for detection
- - We use statistical models to calculate the change in the fluorescence detected by the photodiode
- Dichroic mirror
- - Uses a Blue LED or laser for exciting the fluorophore
- - Photodiode for detection
- - Uses a dichroic filter to filter out all wavelengths except that emitted by the excitation of the fluorophore
These are the methods we decided to test because the No filters method is extremely cost effective while the Dichroic mirror method is expensive but effective. We finalized on testing both these methods to check if the results are comparable.
Where are we now?
After getting access to our lab spaces, we developed a rough prototype to test our No Filters method. Please refer to Hardware for more details about our current setup. We ran some initial tests to check if the sensor can give us different readings for Blue and Green light successfully and are now preparing to test our samples.
The code we used to test the sensor can be found here
Now that we have a working prototype, we plan on using SYBR green as the fluorophore and qualitatively test our samples. For this, we must first calibrate our device using statistical models to sense the difference between a positive and negative sample. We will then use the samples with CRISPR and calibrate it to give a quantitative result.
Once the steps mentioned above are completed and we have a fully functioning model for the No Filters method we will create a similar prototype to check the effectiveness of the dichroic mirror method. Finally, we intend to compare the results and close in on the method that we will be using in our final model.