Part:BBa_K5242023
sensorRNA_Pre
1.Introduction
This part can express the sensor RNA used in the pre-experiment, and the overall design is relatively simple and does not have any practical application significance, serving only as a proof of concept.
2.Design
The structure of the part is illustrated in FIG. 1, where the ogRNA contains a GSG linker and an E2A peptide with a termination codon at the end of the eGFP. the GSG linker is used to connect the front and back parts, whereas the E2A peptide separates the front and back fluorescent proteins and avoids interference.
In addition, we designed the gRNA for the assay based on the sequence of the part, and the gRNA binds to the part to generate an editable A-C mismatch site. Activation of downstream genes after ADAR editing (expression of blue fluorescent protein here)
3. Experimental Characterization
We sets 4 groups for the pre-experiment totally. To make sure our sensor RNA expresses with gRNA at the same time, we loaded them into the same plasmid, which is called pSensor1. Also, we set another 2 groups as negative control and 1 group as positive control. In pSensor2, gRNA is mutated; it can bind with sensor RNA perfectly, without a A-C mismatch. We set up this control group to verify the effect of A-C mismatch on ADAR editing. In sensor 3, gRNA is replaced by another gRNA, which will not form dsRNA with our sensor RNA. We set up this control group to verify the effect of dsRNA on ADAR editing. In pSensor4, sensor RNA is mutated; the terminal codon UAG was already edited to UGG. We set this control group to check if the 2A peptide and downstream eBFP could work normally.
According to our experimental design, the predicted results are as follows: The pSensor1 group, which serves as the experimental group, is expected to exhibit blue fluorescence; the pSensor2 group will not display blue fluorescence because there are no A-C mismatch sites available for editing; the pSensor3 group will not show blue fluorescence due to the lack of double-stranded RNA formation; and the pSensor4 group, because the UAG codon has been edited to UGG, will display blue fluorescence with the highest intensity.
3.1 Plasmid Construction and Plasmid Transformation
We used Gibson Assembly to build our plasmid, and the sequencing results are shown below.
Figure 2. The Sequencing Results
After that, successful yeast transformation were done.
3.2 The Results of Observation
3.2.1 LSCM (laser scanning confocal microscope) Analysis
After going through the procedures of transformation, incubation and sample preparation, we put the four groups of samples under the microscope and observed the results as shown in Figure 3 and Figure 4.
We observed blue fluorescence in both pSensor1 and pSensor4. The result indicated that both our ADAR1_p150 and ADAR2_MCP could edit the sensor in yeast cells. However, the blue fluorescent in pSensor1 is much weaker than pSensor4. Therefore, we need to improve our sensing system to enhance its editing efficiency.
Figure 3: The LSCM rusult of ADAR1_p150.
Figure 4: The LSCM rusult of ADAR2_MCP.
3.2.2 Flow cytometry analysis
The Figure.5 has shownd how we processed our raw data, and Figure.6 has shown the result. Unfortunately, for both ADAR1_p150 and ADAR2_MCP, between pSensor1, psensor2 and pSensor3, we did not observe significant differences. While the eBFP/eGFP value of pSensor4 is close to 100%. The result showed that we need to improve the editing efficiency.
Figure 5: The process how we processed our raw data.
Figure 6: The result of Flow cytometry analysis
3.3 Conculsions
In summary, the results basically proved that our RNAssay is feasible. ADARs is correctly expressed in yeast and play certain functions, but the editing efficiency is relatively low. In subsequent experiments, the part was not able to meet our requirements due to its rudimentary design, and we then designed a new sensor RNA, which was then discarded.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1163
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 175
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