Difference between revisions of "Part:BBa K4586008"

 
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<partinfo>BBa_K4586008 short</partinfo>
 
<partinfo>BBa_K4586008 short</partinfo>
  
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It is single stranded RNA forming hairpin structure have high affinity to MCP.
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==Part Description==
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This part is a highly specific RNA hairpin loop structure that has a high affinity for MCP to navigate the MCP-ADAR2 to the sequence of interest by flanking the sequence with two MS2 hairpin structures.
  
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==Usage==
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This part is used in our design to direct the activity of MCP-ADAR2  to the sensor sequence that is flanked by two MS2 hairpin structures by the RNA binding domain MS2 coat protein (MCP).  Therefore, the sensitivity of this system is markedly enhanced as shown in figure 1.
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<html><div align="center"style="border:solid #17252A; width:100%;float:center;"><img style="                              max-width:850px;
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width:100%;
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height:auto;
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position: relative;
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top: 50%;
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left: 45%;
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transform: translate( -50%);
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padding-bottom:25px;
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padding-top:25px;
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"src="https://static.igem.wiki/teams/4586/wiki/parts/ms2-sensor-mcp-adar-7.png
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">
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<p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span
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lang=EN style='font-size:11.0pt;line-height:115%'>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.  </span></p></div></html>
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==Literature Characterization==
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The study tested if MCP-ADAR activated the translation of cargo, specifically if the sensor contained MS2 RNA hairpins that encoded this cargo.
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<html><div align="center"style="border:solid #17252A; width:50%;float:center;"><img style="                              max-width:850px;
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width:55%;
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height:auto;
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position: relative;
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top: 50%;
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left: 25%;
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transform: translate( -50%);
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padding-bottom:25px;
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padding-top:25px;
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"src="https://static.igem.wiki/teams/4586/wiki/literature-characterisation-parts/ms2.png">
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<p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span
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lang=EN style='font-size:11.0pt;line-height:115%'>Off-state refers to mNeonGreen expression in the absence of iRFP720 trigger mRNA, while on-state refers to mNeonGreen expression in the presence of iRFP720 trigger mRNA. Orange points refer to the sensor with MS2, and blue points refer to the sensor without MS2. They found that MS2 increased the specificity of the switch.
  
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</span></p></div></html>
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==Experimental Characterization==
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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 in 2 lanes as its length is than 3000 bp which is the maximum bp that coul be ordered from IDT. it present in (P8) including first part of MS2 and in (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;
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width:100%;
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height:auto;
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position: relative;
 +
top: 50%;
 +
left: 50%;
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transform: translate( -50%);
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padding-bottom:25px;
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padding-top:25px;
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"src="https://static.igem.wiki/teams/4586/wiki/parts-experiments/pcr-ampli.png">
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<p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span
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lang=EN style='font-size:11.0pt;line-height:115%'>
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</span></p></div></html>
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<br><br><br><br>
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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 (P8) and (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%);
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padding-bottom:25px;
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padding-top:25px;
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"src="https://static.igem.wiki/teams/4586/wiki/parts-experiments/digestion-2.png">
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<p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span
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lang=EN style='font-size:11.0pt;line-height:115%'>
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</span></p></div></html>
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<br><br><br><br>
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After the ligation step, we cultured the ligated product to specifically select the optimum colonies to screen it using Colony PCR to make sure that our parts were correctly ligated in the pCDNA3(-) plasmid vector containing insert parts.
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<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/results/3.png">
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<p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span
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lang=EN style='font-size:11.0pt;line-height:115%'>
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</span></p></div></html>
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<!-- Add more about the biology of this part here
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===Usage and Biology===
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<!-- Uncomment this to enable Functional Parameter display
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===Functional Parameters===
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<partinfo>BBa_K4586006 parameters</partinfo>
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<!-- -->
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==References==
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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.
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Latest revision as of 13:42, 12 October 2023


MS2

Part Description

This part is a highly specific RNA hairpin loop structure that has a high affinity for MCP to navigate the MCP-ADAR2 to the sequence of interest by flanking the sequence with two MS2 hairpin structures.

Usage

This part is used in our design to direct the activity of MCP-ADAR2 to the sensor sequence that is flanked by two MS2 hairpin structures by the RNA binding domain MS2 coat protein (MCP). Therefore, the sensitivity of this system is markedly enhanced 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 tested if MCP-ADAR activated the translation of cargo, specifically if the sensor contained MS2 RNA hairpins that encoded this cargo.

Off-state refers to mNeonGreen expression in the absence of iRFP720 trigger mRNA, while on-state refers to mNeonGreen expression in the presence of iRFP720 trigger mRNA. Orange points refer to the sensor with MS2, and blue points refer to the sensor without MS2. They found that MS2 increased the specificity of the switch.

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 in 2 lanes as its length is than 3000 bp which is the maximum bp that coul be ordered from IDT. it present in (P8) including first part of MS2 and in (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 (P8) and (P9).





After the ligation step, we cultured the ligated product to specifically select the optimum colonies to screen it using Colony PCR to make sure that our parts were correctly ligated in the pCDNA3(-) plasmid vector containing insert parts.



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]