Reporter

Part:BBa_K4244024

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


Three-step inducible system with three fluorescent genes

This plasmid contains the three entry places to introduce a genetic circuit and induce it at a different concentrations of rhamnose. Currently these three genetic circuits are substituted by fluorescent proteins to test the behaviour of the system. These are mRFP, uvGFP and BFP, with a target site for dSpyCas9 from the double repression plasmid in between the promoters and coding sequences of these proteins. Additionally, an introduction template for sgRNA spacers was introduced, this template contains two type IIS restriction sides, this way spacers can easily be introduced and tested.


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Figure 1: Three separate Escherichia coli Nissle 1917 KO strains were created, each containing the double repression plasmid and a plasmid containing mRFP and a different length of spacer P1. The 7nt version did not show any repression by dCas9. The 9 and 20 nucleotide versions both show repression till 250 µM and are expressed after this. Furthermore, a positive contro containing both plasmids but with a non-targeting spacer was added. This measurement was obtained in the same plate reader and experiment but on a different day.


Sequence and Features


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]


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