Rolling Circle Amplification (RCA)
SYBR™ Safe Experimentation
Our team ran the first round of experimentation to determine the success of our miRNA-1 biosensor in an in vitro reaction. Our goal was to determine the success of the ligation and rolling circle amplification. Instead of using a molecular beacon-based reporting mechanism, we utilized a SYBR™ Safe-based fluorescence readout to limit our reaction to the ligation and RCA steps.
Additionally, we ran three triplicates of controls: the first with the padlock probes, second with the miRNA, and the last with both the padlock and the miRNA. To analyze the results, we added SYBR™ Safe to all the samples and read the results in a plate reader.
We found one sample that showed some improvement over our controls. The plate reader’s readout from sample A6’s readout was 9899.64 which showed a significant increase from our third control which averaged 103.39. However, due to the lack of consistency, these results also conveyed the fact that either our padlock likely did not produce a successful RCP, or the SYBR safe detection mechanism did not succeed.
Gel Experimentation
After our first round of experimentation, we could not successfully detect a long strand of DNA. Therefore, we decided to run our RCP (rolling circle product) on a gel in order to determine if our output is a very long DNA strand.
By analyzing the results on the gel, our team concluded that a very long strand of DNA, likely the RCP, was produced (see Fig. 1). The gel 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 a result, we can infer that the RCA reaction allowed the creation of a really long DNA stand — our RCP.
Lettuce with Complement
From these results, we saw an increase in fluorescence with the presence of simulated RCP and lettuce in the reaction as compared to the controls (see Fig. 2). This shows that the lettuce aptamer is actually binding to the RCP and binding the dye causing fluorescence. A significant decrease in fluorescence is also observed in the lettuce reaction tube compared to the dye background. This decrease is most likely due to the opacity of the lettuce DNA sequences. However, overall, the lettuce with complement reaction greatly increased in fluorescence compared to the controls.
Lettuce with Rolling Circle Product (RCP)
The results of the split Lettuce reaction in conjunction with RCP suggested that this reporter mechanism can successfully correlate the presence of a specific miRNA to an increase in fluorescence through RCA and the Lettuce aptamer (see Fig. 3).
Linear Probes with Complement
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. 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.
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.
Linear DNA 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. We confirmed their efficiency through related experiments. As shown by Figure 7, 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 for our miRNA-1-3p and miRNA-133a-3p sensors and that this mechanism was an effective reporting method for our sensor.
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. 7. Moreover, the data shown above closely parallels the predictive ordinary differential equation (ODE) model (see Fig. 8) 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.
As shown by Figure 10, 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/BHQ-1 linear DNA probes is an effective sensing and reporting mechanism for miR-1-3p.
Rolling Circle Transcription (RCT)
Lambert iGEM attempted Rolling Circle Transcription but was unsuccessful. The products of RCT did not appear on a 1% agarose gel, shown in Figure 11, as it did with the RCA reaction gels, Figure 1, for example. To test if the secondary structure of the RCT padlock probe from the Broccoli aptamer complement was problematic to the transcription process by T7 RNA polymerase, we ran RCA with the RCT padlock probe. The results of a gel electrophoresis showed that RCA was successful with the RCT padlock probe, revealing that the folding of the RCT padlock probe is likely not the major problem (see Fig. 12). Considering the possibility that the RNA products may have degraded while loading and running RCT products on a gel, Lambert iGEM decided to continue testing RCT with the DFHBI-1T dye.
To test the efficacy of the miRNA-Broccoli-spacer design incorporated into the RCT padlock probe, the sequence (BBa_K4245210) was transformed, cloned, mini-prepped, and tested in a cell-free reaction. As seen in Figure 12, the results showed a significant increase in fluorescence in reactions containing the miRNA-Broccoli-spacer coding plasmid, revealing the potential of the use of fluorescent aptamers through RCT. However, when we added DFHBI-1T, the fluorophore that the Broccoli aptamer uses to induce fluorescence, to the RCT products, there was no significant increase in fluorescence observed as compared to the controls (see Fig. 13). Therefore, Lambert iGEM discontinued the experimentation with RCT and focused on improving and testing RCA.
Proof of Concept (POC)
As shown by Figure 15, 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/BHQ-1 linear DNA probes is an effective sensing and reporting mechanism for miR-1-3p.
Micro-Q
Micro-Q was tested using fluorescein from concentrations 0 - 500 µM. In order to determine its efficacy compared to a commercial fluorometer, we measured triplicates of several concentrations with sample sizes of 100µL in a plate reader and in Micro-Q. Since Relative Fluorescence Units (RFU) are relative, we scaled the output of Micro-Q to match the scale of the output from the Plate Reader so that they can be compared (see Fig. 16). In order to have an accurate comparison of the data from the Plate Reader and Micro-Q, we added points at the origin to our data set, and calculated a slope from a linear regression for each measurement device, now assuming a y-intercept of 0. The slopes of the data are ~0.9473 and ~0.9194 from Micro-Q, respectively, achieving a percent error of -0.02952%.
Micro-Q was also compared against a Plate Reader in the quantification of BBa_J428112 DH5-alpha and BL21 using the iGEM InterLab Experiment 1 Protocol. The InterLab study instructs to quantify the sample at a 0 hour and 6 hour time mark. To better align with members’ schedules, we only quantified fluorescence at a 0 hour time mark. As expected, BL21 exhibited statistically greater amounts of fluorescence than DH5-alpha (see Fig. 17). Moreover, to evaluate the accuracy of our hardware component, we quantified the same samples in both plate reader and Micro-Q. The values gathered from Micro-Q were scaled up by a factor of 104 to be comparable with those of the plate reader. The fluorescence/OD600 values are consistent between MicroQ and plate reader at 6766.657 and 7405.837 for DH5-alpha, and 16916.56 and 21328.17 for BL21. When comparing fluorescence in each cell strain individually, the error bars for MicroQ and plate reader overlap (see Fig. 17). Therefore, the outputs of MicroQ and plate reader are not significantly different, validating the fluorescence measurement of our hardware device.
