Part:BBa_K5403017
Fusion protein for truncated porinMspA-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 porinMspA (PDB ‘1UUN’, 2004), truncated by 13 amino acids at the C-terminus, a flexible linker (BBa_K5403002) and GFP (BBa_K5403997). The mycobacterial porin MspA 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). PorinMspA is an assembly of several identical domains, see figure 1. To construct this fusion protein, one domain was incorporated in the fusion protein. The aim being to have the GFP localized on the membrane, the fusion protein should assemble with other porin-MspA domains and reside in the membrane.
The reason for the 13 amino acid truncation at the C terminus, is that these amino acids fold inwards (towards the membrane). Therefore, immediate elongation of a domain of porinMspA, as it is done in part BBa_K5403018, might be prone to misfolding or being unable to assemble with other domains of porin MspA, because of steric hindrance. The removal of the 13 C-terminal amino acids is thus hypothesized to have a higher chance of success, since it leaves the new C-terminus pointing away from the membrane.
Two other porinMspA-based fusion proteins with alterations in the sequence were designed (BBa_K5403016& BBa_K5403018).
Figure 1: Structure of PorinMspA (PDB ‘1UUN’, 2004)
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
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
RCSB (2004) PDB - 1UUN: Main porin from Mycobacterium smegmatis (MspA). https://www.rcsb.org/structure/1UUN
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 958
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 457
Illegal AgeI site found at 511 - 1000COMPATIBLE WITH RFC[1000]
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