Part:BBa_K5403016

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 mycobacterial protein porinMspA (PDB ‘1UUN’, 2004), 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. Each domain has a signal sequence at its N-terminus, that is responsible for its localization. Therefore, the 50 N-terminal amino acids are used to create this fusion protein, with the hypothesis that the signal sequence will still cause localization to the membrane, despite most probably not assembling with any other domains of porinMspA. Two other porinMspA-based fusion proteins with alterations in the sequence were designed (BBa_K5403017 & 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