Coding

Part:BBa_K2022002

Designed by: Gavin Sutton   Group: iGEM16_UNSW_Australia   (2016-10-08)

TolR, Periplasmic Domain

Residues 44-117 of the tolR protein from E. coli, with added start and double stops. It is a modification of Paris 2009's BBa_K257005, which lacked these features, and in addition to these sequence improvements we also characterised the part's function. Overexpression of this part is known to stimulate formation of outer-membrane vesicles (OMVs) (Henry et al., 2004).

Usage and Biology

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!

Sequence and Feature Information

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


This part is simply an ORF encoding the E.coli tolR residues 44-117, i.e. not equivalent to the whole protein. Please note that this doesn't neatly correspond to any domains listed on Uniprot, instead falling in the middle of the periplasmic domain. We instead used the residues suggested and tested by Henry et al (2004).

Characterisation by UNSW Australia (2016)

On the NanoSight we ran two samples of OMVS harvested from ΔtolA E. coli, which is already known to hypervesiculate (Deatherage et al., 2009). This was done under two conditions: no plasmid, or expressing TolR (on the pRSF Duet plasmid under control of a T7 promoter, where T7 was induced by 50mM arabinose).

T--UNSW Australia--ParticleCount.png

The above figure essentially shows a normalised comparison of OMV production across a range of strains, in terms of the number of particles produced. Comparing ΔtolA to ΔtolA+tolR E. coli suggests that tolR expression roughly doubles increases the number of OMVs produced, which demonstrates that this part functions as expected in inducing hypervesiculation (Henry et al., 2004).

However, it should be noted that these data are in a specific KO strain of E. coli, and there may be an interaction between the two; tolA's efficacy should be tested in other strains or species. In addition, these data were generated by one run of the NanoSight, and thus should be replicated at a later point.

T--UNSW Australia--ParticleSizeTols.png

As seen above, expression of tolR shifts the peaks of particle size to 130nm, 170nm, and 310nm, which is higher than when tolR is not expressed where the peaks are at 17nm, 81nm, and 115nm. When we combine these data with the above, tolR increases both the number and size of particles, and thus the number and size of OMVs, hence it is an effective part for inducing hypervesiculation.

Characterization by TU Delft (2017)

This year, the TU Delft team also wanted to use hypervesiculation in their design. We discovered that UNSW Australia of 2016 already improved and characterized hyperversiculation by overexpressing the TolR gene. For this reason, we used this biobrick to let our bacteria hypervesiculate in our design. Furthermore, we utilized hypervesiculation optimally by designing and characterizing a fusion protein that translocates from the cytoplasm into the periplasm upon its folding.

Experiment Design

We performed a series of tests, to determine if we successfully induced hypervesiculation by deletion of TolA in the E.coli BW25133 strain from the Keio collection (KEIO) and the overexpression of TolR. As shown in Figure 1, the following combinations of plasmid and cells were used: pET-Duet with and without the insert of TolR both in the E. coli BW25133 strain with (KEIO) and without (WT) the deletion of TolA.


Figure 1 Overview of different combinations of strains and plasmids. Different plasmids were transformed into the parental strain of the Keio collection (WT) and the KEIO collection itself (KEIO). TolR-pET-Duet is the plasmid with the insert that overexpresses protein TolR which results in hypervesiculation of the bacteria. pET-Duet is the backbone in which TolR was placed into.

By measuring the size distribution of the vesicles with Dynamic Light Scattering (DLS), we wanted to confirm vesicle production and determine vesicle size. Further confirmation was obtained through negative stain Transmission Electron Microscopy (TEM). Lastly, the vesicles were quantified by staining them with the membrane dye FM4-64, which only shows fluorescence when bound to a membrane.

Confirmation of vesicle production and size

Dynamic Light Scatter (DLS) measurements were performed following the DLS Protocol. The vesicle size distribution in the KEIO strain and WT strain with either TolR or pET-Duet were compared at different time points after induction: three, four, five hours and overnight (approximately 20 hours).


Results

In Figure 2, the raw data of both TolR and pET-Duet in the KEIO strain clearly show a distribution of larger particles compared to the WT strain. Demonstrating that the WT strain produces a very low amount to no vesicles. Therefore, we decided to only focus on analyzing the DLS data of the KEIO strain.

Figure 2 Raw data of the size distributions of the pET-Duet and TolR in the KEIO (a and b) and WT (c and d) strain, measured with Dynamic Light Scatter (DLS). The samples are put against the size in nm and the color represents the size distribution in percentages.

In figure 3, the analyzed data of the KEIO strain is shown. In this graph, the mean and width of the size distribution are plotted per time for TolR and pET-Duet. Overall, neither time nor the presence of TolR shifted size distribution of the vesicles. The only exception is the 4h time point, which might possibly be a statistical outlier (maybe to a 2-tailed t-test or ANOVA test on the results to establish whether these are significantly different). Therefore, the KEIO strain is mostly responsible for vesicle production. Pertaining to size, the average we found was around 18 nm in diameter (d.nm), which differs from the 80-100 d.nm sized vesicles found by the 2016 iGEM team from University of New South Wales (UNSW) Australia. Furthermore, they showed that the size distribution shifts to larger vesicles when TolR is overexpressed in the KEIO strain, which was not evident in our data. A possible reason could be the low amount of IPTG we added. However, due to time constraints, we were not able to test a range of different IPTG concentrations.

Figure 3 Vesicles size after induction of TolR (orange) and pET-Duet (blue) in the KEIO strain obtained by DLS measurements. The mean for each measurement was represented with dots and the width of the distribution with bars. The time points in hours are set against the size in nm.

Visualisation of vesicles

As shown in previous experiments, both pET-Duet and TolR in KEIO seem to produce vesicles. To confirm that the objects measured by DLS are vesicles and not, for example, parts of the cell, we made negative stain Transmission Electron Microscopy (TEM) images following the TEM protocol. We expected that vesicles have a different shape than the cell debris, namely spherical. Furthermore, when vesicles are big enough you should be able the see the lipid bilayer of the membrane.


Results

As shown in the raw DLS data in Figure 2, the range of vesicles goes up to approximately 70 d.nm. Due to the resolution limitations of the TEM, we could only visualize vesicles above the average size of 18 d.nm.


Figure 4 Transmission Electron Microscop (TEM) images of TolR (left) and pET-Duet (right) in the KEIO strain. Figure 4 shows PET-Duet in KEIO and TolR in KEIO. Vesicles were identified in the images, indicating that the measured objects were simply not cell debris.

Quantification of vesicles

From the previous DLS experiment we concluded that vesicle size distribution does not change over time or upon introduction of the TolR plasmid. However, the experiment does not provide any information about the concentration of produced vesicles. Therefore, vesicle concentration was determined by staining the DLS samples with the membrane dye FM4-64 and subsequent fluorescence measurements in a plate reader. The experiment followed the membrane staining protocol.


Results

In order to calculate the concentration of vesicles from the measured intensity, a calibration curve of synthetic liposomes was made. Figure 5 shows the following linear function which was fitted with the measured data.

I = 4.7865 * C + 950.0159

In which I is the intensity of the fluorescence in arbitrary units and C, the concentration of the liposomes in µg/µL.


Figure 5 Calibration curve of liposomes. The intensity (a.u.) is put against the concentration of liposomes. The liposomes were stained with the membrane dye FM4-64, which only fluoresce when it is bound to the membrane. A linear fit is made, with the formula I = 4.7865 * C + 950.0159.

As can be seen in Figure 6, little to no vesicles are produced in the wild-type strain compared to the KEIO strain. Furthermore, the concentration of vesicles after growing overnight is much higher than 3 hours after induction, thus showing that more vesicles are produced over time. Also, the concentration of vesicles with and without induction differ only with around 2 mg/uL which is not that significant considering the error bars. The reason for this could be the use of a high copy plasmid in combination with a leaky promoter. Besides this, it is confirmed that the presence of the TolR plasmid does not result in large differences in vesicle production.

Figure 6 The concentration of vesicles present in the purified samples of TolR and pET-Duet in the KEIO strain and pET-Duet in the WT strain. The concentration, in mg/uL, plotted for the different samples at 3h and 20h after induction.

Conclusions

We put the pET-Duet plasmid with and without TolR into the KEIO and WT strains, with the goal of inducing hypervesiculation.

TEM images show that the objects present in the DLS samples are vesicles and not other parts of the cell. In the membrane staining experiment, the concentration of vesicles was quantified confirming the DLS results. The experiment also showed that more vesicles are produced over time. The DLS experiments show that the KEIO strain produces vesicles and that the size distribution does not change with either TolR overexpression or over time. Produced vesicles have a size of around 18 d.nm. These results contradict the data UNSW Australia 2016 obtained during their project.

References

Deatherage, B.L., Lara, J.C., Bergsbaken, T., Barrett, S.L.R., Lara, S. and Cookson, B.T., 2009. Biogenesis of bacterial membrane vesicles. Molecular Microbiology, 72(6), pp.1395-1407.

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.

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