Plasmid

Part:BBa_K5196001:Design

Designed by: Sriram Garapati, Joel Groves, Avi Patel   Group: iGEM24_Michigan   (2024-09-30)


THFMO Expression Plasmid

Design Notes

We considered maintaining the integrity of the araBAD promoter site and gentamicin resistance sequence during our construct formation. We decided to use the EcoRI-HF and XbaI restriction sites to insert the THFMO sequence. However, we realized that by using the EcoRI-HF enzyme, we would be excising the consensus RBS (ribosome-binding site) AGGAGG, meaning the Gibson assembly must regenerate the Shine-Dalgarno sequence via the first THFMO fragment and its forward primer [2,3].

Source

The source of the THFMO gene is Pseudonocardia dioxanivorans strain CB1190 [1]. The pJN105-NicA2 plasmid was derived from Bordetella bronchiseptica; specifically, it is a derivative of the pBBR1MCS-5 plasmid originating from this organism.

References

[1] Sales, C. M., Grostern, A., Parales, J. V., Parales, R. E., & Alvarez-Cohen, L. (2013). Oxidation of the Cyclic Ethers 1,4-Dioxane and Tetrahydrofuran by a Monooxygenase in Two Pseudonocardia Species. Applied and Environmental Microbiology, 79(24), 7702-7708. https://doi.org/10.1128/AEM.02418-13

[2] Biolabs, N. E. (n.d.). Nebuilder. NEBuilder. https://nebuilder.neb.com/#!/add/

[3] Integrated DNA Technologies. IDT. (2024, September 11). https://www.idtdna.com/page

Sequence and Features:


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 6405
    Illegal PstI site found at 6777
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 6015
    Illegal PstI site found at 6405
    Illegal PstI site found at 6777
    Illegal NotI site found at 2
    Illegal NotI site found at 2915
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 3715
    Illegal BglII site found at 8905
    Illegal BamHI site found at 5954
    Illegal BamHI site found at 6182
    Illegal BamHI site found at 8233
    Illegal BamHI site found at 9500
    Illegal XhoI site found at 4701
    Illegal XhoI site found at 7107
    Illegal XhoI site found at 7236
    Illegal XhoI site found at 8097
    Illegal XhoI site found at 8955
    Illegal XhoI site found at 10274
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 6405
    Illegal PstI site found at 6777
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 6405
    Illegal PstI site found at 6777
    Illegal NgoMIV site found at 643
    Illegal NgoMIV site found at 7245
    Illegal NgoMIV site found at 9642
    Illegal NgoMIV site found at 9976
    Illegal AgeI site found at 483
    Illegal AgeI site found at 5789
    Illegal AgeI site found at 6585
    Illegal AgeI site found at 6928
    Illegal AgeI site found at 7629
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 3237
    Illegal BsaI.rc site found at 6630
    Illegal BsaI.rc site found at 7857
    Illegal BsaI.rc site found at 9564
    Illegal BsaI.rc site found at 9876
    Illegal SapI site found at 5771
    Illegal SapI site found at 7658