Results

Fractionation controls and protein expression

Figures 1-3 depict our fractionation controls and blot for the identification of saCas9 protein (expected size ~130 kB). The rightmost column of the blot, labelled "control" is a purified His-tagged protein and was used to ensure that the anti-His antibody could properly bind the protein in question. The fractionation results indicated that although Maltose-binding periplasmic protein (MBP) was exclusively present in the periplasmic fraction, showing strong bands of the expected size (42.5 kDa), GroEl was visible in both the cell's periplasm and cytoplasm (60 kDa) suggesting leakage from the cytoplasm to the periplasm. Although the positive control was successful, there was no evidence of saCas9 in the anti-His6 blot.

To prevent leakage between the two compartments, the current protocol (cold osmotic shock) could be optimized (e.g by minimizing incubation time of the cells on ice and proceeding to the centrifugation step earlier) or alternative protocols could be attempted (e.g. protocols utilizing lysozyme).

Cas9 functionality assay

OMV characterization

This summer, we received an OMV isolation kit from System Biosciences which helped us in the isolation and purification of OMVs in our own lab space. The kit makes use of the charged surface of the vesicles, as well as precipitation, to concentrate OMVs. The size distribution and vesicle concentration were determined using Nanoparticle Tracking Analysis (NTA). NTA utilizes the Stokes-Einstein equation to track the motion of particles.

The mean diameter of the vesicles was found to be 99.7nm with a standard deviation of 50.6nm. Since the vesicles were isolated by both charge and precipitation, the concentration of the samples was high. Following a 1:50 dilution, the concentration was 1.83e+8 +/- 2.35e+7 particles/mL. The vesicle distribution is portrayed graphically below (Figure 9):

Additionally, we made an attempt to visualize our OMV samples by carrying out TEM with undiluted vesicles. Samples were stained with uranyl acetate and air dried. The sample was very dense and the vesicles aggregated. Although vesicles were recognizable, the quality of the images was not ideal. To obtain better pictures plunge freezing and cryo-TEM were suggested as alternatives; we would also need to optimize sample preparations by identifying the correct dilution required.

Vesicle delivery to Top10 cells - TEM visualization

Top10 cells grown overnight were incubated with purified outer membrane vesicles for a duration of 30 minutes. Following washing, the samples were prepared for TEM imaging. Postfixation was performed with OsO4 and sections were stained with uranyl acetate and lead citrate. Although there was no convincing evidence of the purified vesicles fusing with the target cells, instances of budding/fusion were observed (Figure 11). However, the directionality of the event remains in question. Circular discolorations of the expected size were also frequent on the images. Alternative ways to investigate this phenomenon, with more promising outcomes, include the tagging of the vesicles with synthetic gold nanoparticles or the use of fluorescence microscopy with proteins such as GFP after incorporating it in OMVs.

Achievements

‌• Used computational techniques to identify signal sequences that are good candidates for periplasmic targeting.

• Successfully cloned 5 new constructs for periplasmic targeting.

‌• Verified Cas9 functionality by performing a Cas9 nuclease assay for all submitted constructs

• Built a mathematical model to investigate the quantity of Cas9 that can be packaged in OMVs by utilizing Tat export and identified parameters to tune this export pathway.

• Purified and characterized nano-scaled vesicles released by a hypervesiculating E.coli strain using Nanoparticle Tracking Analysis (NTA).

• Confirmed the presence of OMVs in our samples by TEM visualization.

Additional work