Part:BBa_K4245131:Experience

Applications of BBa_K4245131

Lambert_GA 2022
The middle sequence was incorporated into the padlock probe designs for RCA and then RCA protocols were run. The RCP was then tested with both the linear probes mechanism as well as the split Lettuce mechanism. Both mechanisms were also tested with the complement of the middle sequence. Both mechanisms were successful in binding the middle sequence and producing the desired output.

Experimental data with the use of hsa-miR-1-3p RCA Padlock Probe (BBa_K4245200), hsa-mir-133a-3p RCA Padlock Probe (BBa_K4245204), and hsa-mir-451a RCA Padlock Probe (BBa_K4245209) containing the middle sequence:

Rolling Circle Amplification (RCA) was successful with the RCA Padlock Probes mentioned above, which all contained the middle sequence. The products of RCA are long DNA strands composed of repeating complementary sequences of the used padlock probe. Therefore, one way in which the success of RCA can be determined is by running the rolling circle products (RCP) on an agarose gel, as a fluorescent band very close to the wells would indicate the presence of an extremely long DNA strand. The result was a really long DNA strand close to the well (see Fig. 1, 2, 3).

Figure 1. Image of gel ran with miRNA-1 padlock probe RCP product.

Figure 2. Image of gel ran with miRNA-133a padlock probe RCP product. RCP is visible in wells 7 and 8.

Figure 3. Image of gel ran with miRNA-451a padlock probe RCP product.

By analyzing the results on the gel, our team concluded that a very long strand of DNA, likely the RCP, was produced for all of the padlock probes used. The gels exhibited a fluorescent band of DNA very close to the well, which indicates that a long strand of DNA, greater than 1 kB was produced due to our reaction, as seen in Figure 1, Figure 2, and Figure 3. As a result, we can infer that the RCA reaction was successful with the padlock probes containing the middle sequence.

Experimental data with the use of the linear probes 6-carboxyl-fluorescein (FAM) Labeled DNA Probe (BBa_K4245130) and Black Hole Quencher 1 (BHQ1) DNA Probe (BBa_K4245132): Lambert iGEM collected data for basic parts BBa_K4245130 and BBa_K4245132 using the characterization protocol linked here.

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 4. Linear Probe Complement Fluorescent Readout

Figure 4 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.

Figure 5. Characterization curve for parts BBa_K4245130 and BBa_K4245132 showing a negative logarithmic relationship between RFU and complement concentrations ranging from 0.1-100 μM. Note: 0-0.1 μM shows a positive relationship, but large error bars at 0 μM suggest this was due to faulty pipetting.

Figure 6. Deterministic ODE Model Simulation of RFU output dependent on the concentration of linear DNA probe complement concentration.

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. 5). 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. 6) correlating complement concentration to RFU (see Model). Therefore, the overall data collected depicts an accurate relationship between the complement concentration and RFU.
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)

Figure 7. Fluorescent Read of Rolling Circle Product for miRNA-133-3p and miRNA-1-3p

As shown by Figure 7, 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 8. Fluorescent reading of RCP produced by miR-451a padlock probe (generated via Probebuilder). BHQ-1 and FAM probes compared to solely FAM probes.

Figure 8 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.

Figure 9. Characterization curve for showing a negative logarithmic relationship between RFU from linear DNA probes and miRNA concentrations

Figure 10. Deterministic ODE Model Simulation of RFU output dependent on concentration of miRNA concentration.

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. 9). Moreover, the data shown above closely parallels the predictive ordinary differential equation (ODE) model (see Fig. 10) 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.

Figure 11. Linear DNA Probe Fluorescence from Serum Extracted miRNA-1-3p Rolling Circle Amplification. Results show significant decrease in fluorescence, indicating a successful Proof of Concept.

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 11, 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.

Experimental data with the use of the split Lettuce DNA fluorescent aptamer Lettuce left (BBa_K4245134) and Lettuce right (BBa_K4245135):

The Lettuce split aptamer has been tested with RCP produced through Rolling Circle Amplification (RCA), as well as a sequence complementary to the middle sequence.

We attempted to simulate the binding of the split Lettuce to the RCP by testing the split Lettuce with a sequence complementary to the middle sequence ordered from Integrated DNA Technologies. The complement, split Lettuce, and DFHBI-1T were mixed and heated at 70°C for 5 minutes, then cooled and held at 41°C for 1 hour. The fluorescence before and after were measured on the plate reader. From the results, we saw an increase in fluorescence in the presence of the complementary middle sequence and lettuce in the reaction as compared to the controls (see Fig. 12). This suggests that the RCP produced through this middle sequence would allow for successful hybridization between the split Lettuce and RCP.

Figure 12. Graph of split Lettuce reaction with middle sequence complement.

RCA was run with hsa-miR-1-3p RCA Padlock Probe BBa_K4245200. The products, split Lettuce, and DFHBI-1T were mixed and heated at 70°C for 5 minutes, then cooled and held at 41°C for 1 hour. The fluorescence before and after were measured on the plate reader. (see Fig. 13)

Figure 13. Graph of split Lettuce reaction with RCP. The values represent the change in fluorescence before and after the reaction with DFHBI-1T took place.

The increase in fluorescence of the RCP + Lettuce + dye was significantly greater than the controls, which suggests that the split Lettuce was successfully bound to the RCP and induced fluorescence in DFHBI-1T.

Lambert_GA 2023
Lettuce
Lettuce was tested as a reporter 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. Utilizing an on-state reporter such as Lettuce would result in a direct relationship, in which a higher concentration of fluorescence will correspond to a higher concentration of miRNA.

We characterized and quantified RCP through the Lettuce reporting mechanism (see Experiments: Lettuce with RCP). There is a positive logarithmic correlation between the miRNA concentrations and the relative fluorescence units (RFU) (see Fig. 14).

Figure 14. Characterization curve showing a positive logarithmic relationship between miR-1 concentrations and RFU from split Lettuce aptamer.

Linear DNA Probes

Additionally, 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. 15).

Figure 15. Characterization curve showing a negative logarithmic relationship between miR-1 concentrations and RFU from linear DNA probes

Comparison Both reporter mechanisms resulted in significant SEM overlap between the lower miRNA concentrations, making accurate differentiation of miRNAs difficult. However, there was no indication that Lettuce outperformed linear DNA probes. Therefore, we continued to conduct further experiments with linear probes. In the future, we hope to find another on-state reporter that would make reading RCA results more comprehensible and accurate. Such reporters include molecular beacons and other DNA aptamers.

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