Difference between revisions of "Part:BBa K4586007"

Latest revision as of 21:01, 11 October 2023

Sensor of the DART V ADAR switch

Part Description

This part is our novel sensor, which is a single-stranded RNA that recognizes the specific complementary part and initiates the downstream transcription process to activate the CRISPR-Cas system.

Usage

In our project, this part is implemented in the DART V ADAR switch to be complementary to ACPA variable region mRNA with mismatched adenosine (A) group within the stop codon (UAG) that hinder the translation of the CRISPR-Cas system as shown in figure 1.

Figure 1: This figure illustrate the activity of our DART V ADAR tissue specific switch that is designed to be in the on state after recognition of the autoreactive B-cells,this recognition based on mismatched base editing in the level of transcribed RNA that is mediated through ADAR enzyme activity.

Literature Characterization

The study investigated whether a DART VADAR sensor directed towards a point mutation of interest could specifically activate translation in cells expressing a disease biomarker. To this end, the study focused on the single-base mutation (c.658 T > C) in the human p53 tumor suppressor gene, which results in a Y220H substitution and is known to destabilize p53's DNA binding domain, making it a key factor in the development of breast cancer. The study co-transfected plasmids expressing either the wild-type or the Y220H mutant p53 gene with a DART VADAR sensor that is specifically made to detect the p53 mutant in HEK293FT cells.

The sensor was engineered to be entirely complementary to the Y220 codon and the surrounding sequence, preventing the target adenosine from being edited by ADAR. In cells expressing p53-Y220H, we noticed a fivefold activation of the reporter gene downstream of the sensor, demonstrating the selectivity of DART VADAR sensors.

Experimental Characterization

In order to amplify this DNA part, we used PCR amplification to reach the desired concentration to complete our experiments using specific forward and reverse primers, running the parts on gel electrophoresis as this part presents lane (p9) including second part of MS2, the sensor and cas12k , and then we measured the specific concentration of the running part using Real-Time PCR as shown in the following figure.

We performed the double digestion method for this part in the prefix and suffix with its specific restriction enzyme and applied this part to gel electrophoresis as shown in the following figure lane(P9).

References

Gayet, R. V., Ilia, K., Razavi, S., Tippens, N. D., Lalwani, M. A., Zhang, K., ... & Collins, J. J. (2023). Autocatalytic base editing for RNA-responsive translational control. Nature Communications, 14(1), 1339. Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 31: / Line 31: @@
 							padding-bottom:25px;
 							padding-top:25px;
-							"src="hhttps://static.igem.wiki/teams/4586/wiki/literature-characterisation-parts/sensor-of-the-switch.png">
+							"src="https://static.igem.wiki/teams/4586/wiki/literature-characterisation-parts/sensor-of-the-switch.png">
 <p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span
 lang=EN style='font-size:11.0pt;line-height:115%'>The sensor was engineered to be entirely complementary to the Y220 codon and the surrounding sequence, preventing the target adenosine from being edited by ADAR. In cells expressing p53-Y220H, we noticed a fivefold activation of the reporter gene downstream of the sensor, demonstrating the selectivity of DART VADAR sensors.
   </span></p></div></html>
+==Experimental Characterization==
+In order to amplify this DNA part, we used PCR amplification to reach the desired concentration to complete our experiments using specific forward and reverse primers, running the parts on gel electrophoresis as this part presents lane (p9) including second part of MS2, the sensor and cas12k , and then we measured the specific concentration of the running part using Real-Time PCR as shown in the following figure.
+<html><div align="center"style="border:solid #17252A; width:80%;float:center;"><img style="                              	max-width:850px;
+							width:100%;
+							height:auto;
+								position: relative;
+							top: 50%;
+							left: 50%;
+							transform: translate( -50%);
+							padding-bottom:25px;
+							padding-top:25px;
+							"src="https://static.igem.wiki/teams/4586/wiki/parts-experiments/pcr-ampli.png">
+<p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span
+lang=EN style='font-size:11.0pt;line-height:115%'>
+ </span></p></div></html>
+<br><br><br><br>
+We performed the double digestion method for this part in the prefix and suffix with its specific restriction enzyme and applied this part to gel electrophoresis as shown in the following figure lane(P9).
+<html><div align="center"style="border:solid #17252A; width:80%;float:center;"><img style="                              	max-width:850px;
+							width:100%;
+							height:auto;
+								position: relative;
+							top: 50%;
+							left: 50%;
+							transform: translate( -50%);
+							padding-bottom:25px;
+							padding-top:25px;
+							"src="https://static.igem.wiki/teams/4586/wiki/parts-experiments/digestion-2.png">
+<p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span
+lang=EN style='font-size:11.0pt;line-height:115%'>
+ </span></p></div></html>
 ==References==
 Gayet, R. V., Ilia, K., Razavi, S., Tippens, N. D., Lalwani, M. A., Zhang, K., ... & Collins, J. J. (2023). Autocatalytic base editing for RNA-responsive translational control. Nature Communications, 14(1), 1339.