Demonstration of Hypervesiculation
Our results showed that the knock-in mutation of g3p successfully induced hypervesiculation of E. coli with an OMV count of 2.8x108 per mL per OD600, significantly higher than the count obtained from the negative control sample (0.29x108 per mL per OD600) (Figure 1). Knock-out of TolA also showed an impressive increase in OMV production, over 36 times the amount in wildtype E. coli (Figure 1). This affect was all the more pronounced when TolR overexpression was added to this same knockout strain (Figure 1). Knockout of DegP also resulted in increased OMV production compared to the wildtype, nevertheless production fell very short of the production rates seen in the other mutants. Most importantly, however, knockout of DegP was associated with a dramatic increase in vesicle size, if not vesicle number (Figure 2).
Figure 1 OMV count x 10^8, per mL, per OD600, as measured by NanoSight LM20. Samples are: Control = NEB T7 E. coli; g3p (non-induced) = NEB T7 + g3p; g3p (induced) = NEB T7 E. coli + g3p + 1mM IPTG; ΔdegP = ΔdegP E. coli; ΔtolA = ΔtolA E. coli; ΔtolA + tolR (induced) = ΔtolA E. coli + TolR + 50mM arabinose
Figure 2 Demonstrate a wide distribution of vesicle sizes produced
Through all of our experiments and results collated, we were able to create novel strains of E. coli as per our aim. We were also able to characterise and compare the way in which these strains created vesicles; with regards to both the number of vesicles produced and the distributions of sizes (of the vesicles). The use of the nanosight was able to show that different strains varied in their capacity to produce quantities of OMVs, with tolA+tolR being the best at this. To combine with this, though, we were able to compare the size of OMVs produced, showing again that tolA+tolR produced the biggest OMVs. We thus conclude that tolA+tolR is the ideal strain to use as a platform for OMV production, at least amongst those we tested. In the future we anticipate testing many more, including all the possible permutations