Team:Northwestern/Results
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).
Figure 1. - Subcellular localization of saCas9 when fused to YcbK ss (Tat-specific)
Figure 1. - Subcellular localization MBP
Figure 1. - Subcellular localization GroEL
Cas9 functionality assay
OMV visualization
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 conclusive evidence of the purified vesicles fusing with the target cells, instances of budding/fusion were observed (Figure 4). However, the directionality of the event remains under 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 fluorescent proteins (such as GFP) after its incorporation in OMVs.
Figure 4. - OMV delivery experiment results - TEM visualization for different magnifications
Achievements
