Reporter

Part:BBa_K4244024:Design

Designed by: Arthur Vancolen   Group: iGEM22_Wageningen_UR   (2022-10-08)


Three-step inducible system with three fluorescent genes


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 151
    Illegal XbaI site found at 157
    Illegal XbaI site found at 2147
    Illegal SpeI site found at 30
    Illegal PstI site found at 169
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 151
    Illegal NheI site found at 7
    Illegal NheI site found at 307
    Illegal SpeI site found at 30
    Illegal PstI site found at 169
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 151
    Illegal BglII site found at 181
    Illegal BamHI site found at 2690
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 151
    Illegal XbaI site found at 157
    Illegal XbaI site found at 2147
    Illegal SpeI site found at 30
    Illegal PstI site found at 169
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 151
    Illegal XbaI site found at 157
    Illegal XbaI site found at 2147
    Illegal SpeI site found at 30
    Illegal PstI site found at 169
    Illegal AgeI site found at 914
    Illegal AgeI site found at 1026
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

These three fluorescent genes are constitutively expressed, only when they are repressed by dSpyCas9 they are not. This is why during spacer introduction and during cloning when the double repression plasmid is not present, they are not repressed, the burden is very high on the cells. Due to this, the growth is very slow and they are prone to get mutated. A solution for this was introducing it on a low copy number plasmid, like a artificial chromosome or introducing it on the chromosome. This plasmid is a composite of BBa_K4244024, BBa_K4244025, BBa_K4244026, Here BBa_K4244025 is placed on the lagging strand.


Source

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References