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 2. - Subcellular localization GroEL
Figure 3. - Subcellular localization MBP
Cas9 functionality assay
In order to determine the cytosolic functionality of our saCas9 constructs, and to examine if fusion with signal sequences would affect this functionality, we created a visual gRNA mediated Cas9 nuclease assay. This assay allowed for basic visualization of the Cas9’s functionality at both recognizing a cut site on a guide plasmid and actually cleaving the gene of interest on a cytosolic level. For this experiment we used BBa_K2019002 as the reporter plasmid to detect the functionality of Cas9.
Functionality was quantified by examining the fluorescence of samples containing a plasmid coding for mRFP before and after plasmids containing our Cas9 constructs were introduced. Since this examination relies on transforming a single sample of cells with both plasmids, and subsequently measuring whole cell florescence, this experiment is not designed to quantify periplasmic directing nature or periplasmic functionality.
As shown below, Figure 4 gives the results of a plate reader fluorescence analysis for the control samples: A JC8031 sample containing the gRNA-mRFP plasmid, a dually transformed sample with gRNA-mRFP and saCas9 without a signal sequence, and a sample containing only the saCas9 coding plasmid.
By examining Figures 5 through 9, one can observe how the functionality of the saCas9 is affected by the different signal peptides. This observation leads to the conclusion that despite the presence of signal peptides, Cas9 performed in a manner consistent with what was expected from analysis of the controls.
Quantitatively, in each plot there is a significant difference between the base-level gRNA-mRFP only transformed plasmid, and the co-transformed gRNA-mRFP and ss-Cas9 samples. Additionally, because the level of fluorescence in the co-transformed samples is also greater than the base-level fluorescence of only the Cas9, meaning that we were not measuring just the fluorescence of cells without mRFP, but with cells that had the mRFP gene cut by Cas9 and directed by the gRNA.
Although current results showing the functionality of our constructs are promising, there are many other factors of the experiment to consider. For example, because we carried out a dual transformation with the signaling sequence constructs, we should have also conducted a dual-transformation with the positive control, such as transforming the reporter plasmid with the backbone of the Cas9 construct, sans Cas9. Similarly, the negative controls should also have been transformed with a blank version of the reporter plasmid. Some additional considerations include testing the compatibility of the origin sequences between the two plasmids, as conflicting origins can affect the output of protein.
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):
Figure 9. - Nanosight results
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.
Figure 10. - TEM 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 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 incorporation in OMVs.
Figure 11. - OMV delivery experiment results - TEM visualization for different magnifications
Achievements
• Identified signal sequences that are good candidates for periplasmic targeting.
• Successfully cloned 5 new constructs for saCas9 periplasmic export.
• 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.
