Composite

Part:BBa_K4316031

Designed by: Luca Donati   Group: iGEM22_Sorbonne_U_Paris   (2022-10-07)


UV-dependent killswitch

As a part of our partnership with UESTC_BioTech, we decided to help them create a killswitch for their strain of Chlamydomonas. When they evoked their safety concerns for their project, we first thought of our university’s 2020 project, where they had also developed a failsafe for the same reasons. Thus began our investigative journey.

This part is a MoClo level 2 multigenic construct, containing three transcriptional blocks: - The UVR8-TEV_Cter fusion protein - The COP1-TEV_Nter fusion protein - The cCA-TMD_HAP2-TEV_cleavage_site-NucA-NLS fusion protein

Our killswitch is an improved version of Sorbonne University (SU) 2020 iGEM team’s (BBa_K3373014), who were themselves inspired by Munich's 2013 iGEM team. They wanted to address Nuclease A to the plasma membrane by linking it to a transmembranary domain and ER translocation signal peptide. In between the nuclease and transmembranary domain would be a TEV-specific cleavage site. Therefore, under escape conditions, UV-sensitive dimerization could assemble both parts of the TEV protease, liberating the Nuclease A, which would then localize to the nucleus and provoke cell death. In practice, they ran into a few issues that will be discussed later. It can also be considered an improvement because it was entirely redesigned in order to make the basic parts compatible with the RFC10 standard and assemblable by specifying their part names and RFC10 scars (also working out a method of assembling with MoClo). We also coined the term « investigative design » when trying to find sources for SU2020’s parts. Using sequence alignment we were able to find equivalent, citable sources for SU2020s basic parts. Some discrepancies in reading frames were also fixed.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1658
    Illegal BamHI site found at 2327
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 883
    Illegal NgoMIV site found at 2152
    Illegal NgoMIV site found at 2212
    Illegal NgoMIV site found at 5220
    Illegal NgoMIV site found at 5926
    Illegal NgoMIV site found at 6040
    Illegal NgoMIV site found at 6100
    Illegal AgeI site found at 1480
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 2253
    Illegal SapI.rc site found at 2659
    Illegal SapI.rc site found at 6529


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Parameters
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