Composite

Part:BBa_K5403020

Designed by: Lizette van der Ziel   Group: iGEM24_TU-Eindhoven   (2024-10-02)


Fusion protein for FtsQ-based membrane anchoring of GFP

Context

iGEM Eindhoven 2024 created a library of parts (BBa_ K5403012 - BBa_K5403020) that encode fusion proteins that are designed to functionalize the membrane of the bacterial membrane vesicles (BMVs) of M. smegmatis and E. coli with a protein. These fusion proteins all consist of an N-terminal domain that is known to exist in the membrane of the BMVs, a flexible linker (BBa_K5403002) and a C-terminal GFP (BBa_K5403997). The team’s aim is to express these proteins in M. smegmatis, analyze the expression by FACS, isolate vesicles from the bacteria and determine the presence of the fusion protein in the vesicles by western blotting. GFP was chosen as the C-terminal protein for initial characterization, but the flexible linker contains an NheI-restriction site that allows you to replace the GFP with a gene for any protein of your interest.


Part description

This part is a fusion protein that consists of the mycobacterial protein FtsQ, a flexible linker (BBa_K5403002) and GFP (BBa_K5403997). The mycobacterial protein FtsQ is thought to be a cell division protein and was shown to be part of the vesicles of M. smegmatis (Prados-Rosales et al., 2011). The protein is documented in the mycobacterial database MyCoBrowser (MyCoBrowser, MSMEG_4225). For cloning and expression, the plasmid pCHERRY3 can be used. This shuttle plasmid is designed such that it can be multiplied in standard laboratory E coli competent cells such as TOP10 or dH5α, and subsequently used for expression in M. smegmatis. pCHERRY3 was a gift from Tanya Parish (Addgene plasmid # 24659 ; http://n2t.net/addgene:24659 ; RRID:Addgene_24659).

References

Addgene plasmid # 24659. Sensitive detection of gene expression in mycobacteria under replicating and non-replicating conditions using optimized far-red reporters. Carroll P, Schreuder LJ, Muwanguzi-Karugaba J, Wiles S, Robertson BD, Ripoll J, Ward TH, Bancroft GJ, Schaible UE, Parish T. PLoS One. 2010 . 5(3):e9823. 10.1371/journal.pone.0009823 PubMed 20352111

MyCoBrowser. (n.d.). https://mycobrowser.epfl.ch/genes/MSMEG_4225

Prados-Rosales, R., Baena, A., Martinez, L. R., Luque-Garcia, J., Kalscheuer, R., Veeraraghavan, U., Camara, C., Nosanchuk, J. D., Besra, G. S., Chen, B., Jimenez, J., Glatman-Freedman, A., Jacobs, W. R., Porcelli, S. A., & Casadevall, A. (2011). Mycobacteria release active membrane vesicles that modulate immune responses in a TLR2-dependent manner in mice. Journal Of Clinical Investigation, 121(4), 1471–1483. https://doi.org/10.1172/jci44261

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 41
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 41
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 614
    Illegal XhoI site found at 1441
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 41
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 41
    Illegal NgoMIV site found at 708
    Illegal AgeI site found at 151
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 375
    Illegal BsaI.rc site found at 136
    Illegal SapI.rc site found at 203
    Illegal SapI.rc site found at 287


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