Difference between revisions of "Part:BBa K4245200:Experience"

Revision as of 12:58, 12 October 2023

Applications of BBa_K4245200 with RCA

Lambert_GA 2022
Rolling Circle Transcription (RCA) was successful with this part. 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. Since a fluorescent band very close to the wells would indicate the presence of an extremely long DNA strand, our RCP was run on a gel. The result was a really long DNA strand close to the well.

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

By analyzing the results on the gel, our team concluded that a very long strand of DNA, likely the RCP, was produced. 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 (see Fig. 1). As a result, we can infer that the RCA reaction allowed the creation of a really long DNA stand -- our RCP.

BBa_K4245200 Produced RCP with FAM/BHQ1 Tagged Linear DNA Probes

RCA and RCT was tested with the FAM and BHQ1 labeled linear probes.

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

As shown by Figure 3, 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 and that this mechanism was an effective reporting method for our sensor.

Figure 4. Characterization curve for showing a negative logarithmic relationship between RFU from linear DNA probes and miRNA concentrations

Figure 5. Characterization curve for showing a negative logarithmic relationship between RFU from linear DNA probes and miRNA concentrations

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. 4). Moreover, the data shown above closely parallels the predictive ordinary differential equation (ODE) model (see Fig. 5) correlating complement concentration to RFU. 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 6. 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 to simulate human blood. As shown by Figure 6, 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.

BBa_K4245200 Produced RCP with Lettuce Aptamer

The RCP was also tested with the split lettuce aptamer. DFHBI-1T and the lettuce right and the modified lettuce left was added to the RCP, and the fluorescence was read on the plate reader.

Figure 2. Graph of split Lettuce reaction with RCP. The values represent the change in fluorescence before and after the reaction with DFHBI-1T took place.

As seen in Figure 2, 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. In addition, the DFHBI-1T was also successfully bound within the split lettuce secondary folding. According to these results, RCA was successful, and the reaction between the split lettuce and RCP was successful as well.

Therefore, RCA created RCP that can be quantified by our chosen reporting mechanisms.

Lambert_GA 2023
Exponential Rolling Circle Amplification (eRCA) was successful with this part. The products of eRCA are short 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 exponential rolling circle products (eRCP) on an agarose gel. Since a fluorescent band very close to the wells would indicate the presence of an extremely long DNA strand, no/dim bands near the top of the well indicate that short DNA was produced(see fig. 1).

Figure 1. Image of gel ran with miRNA-1 RCP product; A: eRCA with 40.8 pM miR-1; B: negative control (no enzymes)

By analyzing the results on the gel, our team concluded that short strands of DNA were produced, likely the eRCP.
The eRCP was also tested with DFHBI-1T dye as the RCP would consist of Lettuce Aptamer sequences. The fluorescence was read on the plate reader (see fig. 2).

Figure 2. Graph of fluorescence after DFHBI-1T was added

As seen in Figure 2, the increase in fluorescence of the eRCP+dye was significantly greater than the controls, which suggests that Lettuce aptamers were produced. According to these results, eRCA was successful.

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