Part:BBa_K4245130:Experience
Linear Probes with Complement
Lambert_GA 2022
We initially tested linear probes with the complement of the middle sequence to ensure that linear probes were an effective and characterizable means of quantifying miRNA (see RCA Protocols)
Figure 1 displays a significant decrease in the fluorescence intensity of a triplicate with FAM Probe, BHQ Probe, and Linear Probe Complement as compared to a triplicate of just FAM tagged Probes. Therefore, we concluded that linear probes were an efficient means of reporting the output of our biosensor.
In order to quantify the relationship between linear probe complement concentration and fluorescence, we further characterized these parts with varying linear probe complement concentrations. There is a negative logarithmic correlation between the complement concentrations ranging from 0.1-100 mM and the relative fluorescence units (RFU) (see Fig. 2). The 0 mM complement concentration outputs less RFU than 0.1 mM, which does not align with the model. However, the large error bars at 0 mM suggests that there was some degree of significant error. Thus, this data point is insignificant and further trials should be performed to achieve more accurate results. Moreover, the data from 0.1-100 mM closely parallels the predictive ordinary differential equation (ODE) model (see Fig. 3) correlating complement concentration to RFU (see Model). Therefore, the overall data collected depicts an accurate relationship between the complement concentration and RFU.
Linear Probes with RCP
We use linear probes as a means to quantify and report the miRNAs that we sensed through rolling circle amplification (RCA) reactions. To go beyond verifying that linear probes are efficient means to do the aforementioned tasks through testing with the complement of the linear probes, we wanted to confirm that they are able to quantify the miRNAs experimentally (see RCA Protocols)
As shown by Figure 4, there is a statistically significant decrease in the fluorescent output of a triplicate with FAM Probe, BHQ Probe, and RCP as compared to a triplicate of just FAM tagged Probes. This confirms that we did produce our desired RCP in the RCA reaction for our miRNA-1-3p and miRNA-133a-3p sensors and that this mechanism was an effective reporting method for our sensor.
Figure 5 displays a significant decrease in the fluorescence intensity of a triplicate with FAM Probe, BHQ Probe, and the RCP produced as compared to a triplicate of just FAM tagged Probes. This finding experimentally validates the use of ProbeBuilder as a means of producing effective padlock probes.
In order to quantify the relationship between miRNA concentration and fluorescence, we further characterized these parts with varying linear probe complement concentrations. There is a negative logarithmic correlation between the complement concentrations and the relative fluorescence units (RFU) (see Fig. 6). Moreover, the data shown above closely parallels the predictive ordinary differential equation (ODE) model (see Fig. 7) correlating complement concentration to RFU (see Model). Therefore, the overall data collected depicts an accurate relationship between the miRNA concentration and RFU, further validating that RCA coupled with linear probes are an effective and efficient means of quantifying miRNA concentrations.
In addition, Lambert iGEM also tested linear probes in spiked human serum (where we added miRNA-1 to serum) in order simulate human serum. As shown by Figure 8, there is statistically significant decrease in the fluorescent output of a triplicate with FAM Probe, BHQ Probe, and RCP as compared to a triplicate of just FAM tagged Probes. This confirms that we did produce our desired RCP in the RCA reaction performed on our miRNA-1-3p spiked serum. This further validates that biosensors utilizing RCA coupled with FAM and BHQ-1 linear DNA probes is an effective sensing and reporting mechanism for miR-1-3p.
Lambert_GA 2023
Linear DNA Probes
Linear DNA probes were tested as reporters for rolling circle amplification (RCA) and was used for the successful characterization of hsa-miR-1-3p in the 2022 season (see Lambert iGEM Wiki RCA, 2022). Linear DNA probes are an off-state reporter, therefore they produce an indirect relationship between microRNA (miRNA) concentration and fluorescence output.
Similar to last year, we characterized and quantified RCP through the linear probes reporting mechanism (see Experiments: Linear DNA Probes with RCP). There is a negative logarithmic correlation between the miRNA concentrations and the relative fluorescence units (RFU) (see Fig. 1).
Figure 9. Characterization curve showing a negative logarithmic relationship between miR-1 concentrations and RFU from linear DNA probes
Comparison
Linear DNA Probes resulted in significant SEM overlap between the lower miRNA concentrations, making accurate differentiation of miRNAs difficult. Therefore, we continued to conduct further experiments with linear probes.
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