Composite

Part:BBa_K5276029

Designed by: Yuming Luo   Group: iGEM24_UCAS-China   (2024-10-01)


Patch

cymR_Promoter-Cre-J23111-loxP-LuxI-PobR_promoter-fimE-J23111-LuxR

img
Figure. Patch system using Cre/loxP system and fimE inversion system

Description

Cre/loxP: The Cre/loxP system originally comes from the P1 bacteriophage in bacteria and consists of two key components: the Cre recombinase and the loxP sequence.
  • Cre recombinase: Cre (Cyclization Recombination) is an enzyme that can recognize specific DNA sequences (loxP sites) and carry out site-specific recombination. It can excise, invert, or insert the DNA fragment located between two loxP sites, depending on the relative position and orientation of the loxP sequences.
  • loxP sequence: loxP (locus of X-over P1) is a specific DNA sequence recognized by Cre recombinase. It is 34 base pairs long, consisting of inverted repeats on either side and an asymmetric sequence in the middle. The orientation of the loxP site is determined by this asymmetric central region. If two loxP sites are on the same DNA strand but in opposite orientations, Cre recombinase will invert the DNA fragment between them.
FimE System: The FimE system is another site-specific recombination system.
  • FimE recombinase: FimE is a site-specific recombinase that recognizes a specific DNA sequence within the fim gene cluster and carries out the inversion of the DNA fragment.
  • fimS site: fimS is the DNA sequence recognized by FimE recombinase. FimE recombinase binds to the fimS site and performs recombination at this location.
We introduced a cumate-inducible cymR promoter to express the Cre recombinase in the pathway; upon adding cumate, the expressed Cre recombinase recognizes the loxP sequence, allowing the LuxI gene sequence to flip and be expressed by the constitutive promoter. This constitutes the Patch portion of Register A. Similarly, in the Register B pathway, we incorporated the PobR promoter used by the 2023 UCAS-China team and the fimE inversion system from Escherichia coli (Ham et al., 2006) to complement the expression of the LuxR gene.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 3690
    Illegal SpeI site found at 5067
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1135
    Illegal NheI site found at 1158
    Illegal NheI site found at 3003
    Illegal NheI site found at 3718
    Illegal NheI site found at 3741
    Illegal NheI site found at 5217
    Illegal SpeI site found at 5067
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 4684
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 3690
    Illegal SpeI site found at 5067
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 3690
    Illegal SpeI site found at 5067
    Illegal NgoMIV site found at 2405
    Illegal NgoMIV site found at 5343
    Illegal NgoMIV site found at 5376
    Illegal AgeI site found at 640
    Illegal AgeI site found at 2097
    Illegal AgeI site found at 2618
    Illegal AgeI site found at 4998
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2538
    Illegal BsaI.rc site found at 5228


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