Translational_Unit

Part:BBa_K4586025

Designed by: Mohamed Mohamed Saad Aboelghare   Group: iGEM23_AFCM-Egypt   (2023-09-15)
Revision as of 16:33, 11 October 2023 by Ahmed A Rezk (Talk | contribs) (→‎literature charactrization of MCP/ADAR)


Cargo (guide RNA - switch- Cas12k- MCP/ADAR)

Description and Usage

The first component of Cargo is the CRISPR system, where we uses guide RNA to direct our Cas12k toward the BAFF-R gene in auto-reactive B-cells, and the second component is the DART V ADAR switch, where we uses ADAR enzyme to convert Adenosine into Inosine, causing a change in the switch condition from off to on status, but ADAR enzyme works only in the presence of complementary mRNA in the target cell to the sensor within the switch that contains the stop codon (UAG) of auto-reactive B-cells. This stop codon contains a mismatched Adenosine (A)group with the complementary mRNA; this group is hybridized by ADAR enzyme activity and converts to an Inosine group (I); thus, the stop codon sequence is disturbed in this condition and our cargo will be translated as shown in figure 1.

Figure 1: This figure illustrates the design of our biological circuit expressing our therapeutic agent under the control of the VP64 transcription module that regulates the activity of the ZF21.16minCMV promoter.

literature charactrization of MCP/ADAR

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.



The study tested the action of sensors containing MS2 hairpins without ADAR, with ADAR p150, or with MCP-ADAR2dd.

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. They found that constitutive expression of MCP-ADAR causes an increase in sensor activation in the absence of the trigger.


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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 5988
    Illegal AgeI site found at 6100
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1456


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Parameters
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