Part:BBa_K2022000
Attachment Protein g3p, N-terminal Domain
Expression of this part is suggested to induce hypervesiculation in E. coli (Henry et al., 2004), i.e. direct the bacterium to overproduce outer membrane vesicles (OMVs).
Contents
Usage and Biology
Attachment Protein g3p has been shown to interact with TolA, a highly-abundant protein in the bacterial outer-membrane that confers membrane stability. In doing so, it destabilises the membrane, helping promote OMV production. Interaction is mediated by both g3p's N1 and N2 domains (Lubkowski et al., 1999), but, to date, only the former has been assessed for its OMV-inducing capacity; we thus made N1 an ORF, submitting it as this part
The intended use of this part is achieved via simple overexpression, and doing so stimulates OMV formation. Thus far, this has only been demonstrated in E. coli, so please try it out in other organisms! Remember that it may require an interaction with TolA to exert its effects; ensure, then, that there is a homologue of TolA in your chassis
Sequence and Feature Information
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
In short, this part is just an ORF encoding the N1 domain of g3p (residues 1-71). Please note that we didn't use the definition of N1 provided by Uniprot (residues 19-85), but instead that suggested by Henry et al (2004) as that is what they tested for OMV-inducing efficacy; one reason why may be that there's a putative signalling peptide in residues 1-18. Please also see the design page for all considerations involved in designing the sequence, both the good and the regrettable
Characterisation by UNSW Australia (2016)
The NanoSight is a machine that can track the number and size of particles in a sample, and past research has demonstrated its utility in doing so for OMVs.
We thus took an uninduced and induced (by 1mM IPTG for 12h, 15m in the pRSF Duet Vector) sample of BL21 E. Coli bearing g3p, and harvested the OMV fraction from these. This harvested fraction was then read by the NanoSight.
The above graphs show the distribution of particle sizes, where the above-left shows the uninduced sample, while the induced sample produced the above-right. First and foremost, both samples produce some kind of particle, likely OMVs; this is not controversial, and is expected. However, expression of g3p increases the mean size of these OMVs, to a peak at 102nm diameter, compared to several smaller peaks at 23, 47, and 77nm diameter. It's worth noting here that the NanoSight overestimates the size of all particles. Further, due to the expense and lateness that characterised this experiment, only one read was performed. All the same, we believe these data suggest that bigger OMVs are produced by g3p expression.
To extend upon this, it seems that a greater quantity of OMVs are produced when g3p is expressed (below):
This graph shows the corrected particle count between a range of conditions, produced from the same run of the NanoSight. Inducing g3p expression appears to more than double the number of OMVs produced per cell, which, in combination with the above data that show an increase in mean particle size, suggests that the mass of OMVs produced is greatly improved by g3p's expression. This is in line with predictions from Henry et al (2004), and suggests that the BioBrick works as intended. Again, we note that this was shown by a single run of the machine, and should be subject to further testing to show reliability and to perhaps gauge the extent of a dosage-bleb relationship.
References
Henry, T., Pommier, S., Journet, L., Bernadac, A., Gorvel, J.P. and Lloubès, R., 2004. Improved methods for producing outer membrane vesicles in Gram-negative bacteria. Research in Microbiology, 155, pp.437-446.
Lubkowski, J., Hennecke, F., Plückthun, A. and Wlodawer, A., 1999. Filamentous phage infection: crystal structure of g3p in complex with its coreceptor, the C-terminal domain of TolA. Structure, 7, pp.711-722.
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