Composite

Part:BBa_K5403019

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


Fusion protein for RbsC-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 RbsC, a flexible linker (BBa_K5403002) and GFP (BBa_K5403997). The mycobacterial protein RbsC is a ribose transport system permease 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_1710).

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_1710

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 146
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 146
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 1339
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 146
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 146
    Illegal AgeI site found at 712
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
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