Proof of Concept
During our brief time in the biology lab, we were able to show proof of concept that: RPA amplified target region, RPA is specific to target region, and CRISPR is successful in the detection of Bd. Below are more in-depth results:
RPA amplified and is specific to target region
We confirmed our RPA reaction was able to amplify both the Bd and Bsal target regions. In addition, our RPA primers are specific towards its corresponding target gene: our Bd RPA primers are specific for only the Bd target gene and our Bsal RPA primers are specific for only the Bsal target gene.
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 Bd DNA that matched the Bd primers. No amplification detected when Bsal primers were used against Bd DNA
CRISPR successful detection of Bd
CRISPR shows successful detection of Bd. The overall specificity of our detection system is significantly increased through the coupling of our CRISPR/Cas system with our RPA reaction, both of which exhibit specificity.
Triplicate run of CRISPR DETECTR on Bd. Fluorescence in the Bd CRISPR of the reaction works as intended.
Fluorescence detection calibration
We built the final setup and used the TCS RGB34735 sensor along with a Arduino micro to perform tests to see whether the sensor will be able to differentiate between Blue and Green lights. Experiment 1 (refer Results) clearly showed that the sensor can differentiate between the two different wavelengths and display the results in a tabular format. We will now use this similar setup to perform Experiment 2 and 3 where we will calibrate it with SYBR green first and then with CRISPR. Due to these encouraging results, we are certain that we would be able to calibrate the sensor to our fluorescence reading with both SYBR Green (Experiment 2) and CRISPR (Experiment 3).