Composite

Part:BBa_K5403015

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


Fusion protein for membrane anchoring of GFP based on OmpAtb and OmpA

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 signal sequence of outer membrane protein A of M. tuberculosis (OmpAtb) (BBa_K5403006), outer membrane protein A of E. coli (OmpA) (BBa_K5403004), a flexible linker (BBa_K5403002) and GFP (BBa_K5403997). OmpAtb 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, Rv_0899). Because of it virulent functions, the full proteins is not approved for use in BSL-I laboratories, but the signal sequence (consisting of the 72 N-terminal amino acids) is safe to use. Therefore, the fusion protein was constructed with the non-virulent E. coli analogous outer membrane protein instead of the main body of OmpAtb. This fusion protein was designed for expression in M. smegmatis, to test the hypothesis that the signal sequence will cause localization of the GFP to the membrane. Another fusion protein was designed that only makes use of the OmpAtb signal sequence without the E. coli analogous protein OmpA, see part (BBa_K5403014).

For cloning and expression, the plasmid pCHERRY3 can be used. This shuttle plasmid is designed such that it can be multiplied in standard laboratory competent E. coli cells such as TOP10 or dH5α strains, 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/Rv0899

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 1696
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 30
    Illegal AgeI site found at 169
    Illegal AgeI site found at 628
    Illegal AgeI site found at 782
    Illegal AgeI site found at 807
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 16
    Illegal BsaI.rc site found at 744
    Illegal SapI.rc site found at 1043


[edit]
Categories
Parameters
None