RESULTS

Cas9 Translocation to Periplasm

Bradford Assay:

Before we ran a Western blot to test all of our constructs, we performed a Bradford assay to normalize the total protein loaded into each well of the protein gel, as the whole cell lysis, periplasm fractionation, and OMV preparation were all performed very differently. Protein concentrations in ug/uL were calculated using a bovine serum albumin standard curve. These results were initially promising.

The total protein concentration present in the OMV fractions of our hypervesiculating cells were overall less than that of the periplasm fraction, indicating that the OMV filtration procedure we obtained from UNSW-iGEM was more selective than the periplasm procedure alone (Figure 1).

Additionally, the total protein present in both un-tagged Cas9 periplasm fractions was considerably less than in tagged Cas9 fractions, indicating that naked Cas9 was (predictably) likely not in the periplasm. The total protein in the INP and ClyA-GFP periplasm fractions was very high, which could have been explained by the large size of both of these protein fusions (Table 1).

Figure 1: Average total protein concentration of Tat/Sec fusions in periplasm and OMV fractions in hypervesiculating strain JC8031.

 

 

Table 1: Total protein concentrations in ug/uL.

Western Blots:

Our first Western blot of whole-cells with cytosolic Cas9 indicated that Cas9 was being successfully expressed in our cells, though many smaller products with our N-terminus His6 tag were also present. These may have been synthesis truncation products; however, we did have an expected band for whole-Cas9 being expressed.

Figure 2: Our first Western blot confirmed Cas9 expression in the cytosol.

Our second Western blot was run on periplasm-directed Cas9. We ran samples from whole cells, collected OMV lysates, and periplasm fractions. This indicated that our Cas9 part was not successfully translocated into the periplasm or incorporated into OMVs by any of the signal sequences tried, nor by the outer-membrane protein fusions that were successful with our controls.

Figure 3: Our second Western blot indicated that while smaller proteins (GFP) targeted to OMVs in previous work ¹ are present in both periplasm fractions and in OMV lysates, our Cas9 was not successfully translocated by any of the signal sequences or protein-fusions constructed.

Cas9 Functionality

Nuclease assay:

This assay shows that both our Cas9 and gRNA devices are functional. To reiterate, in this experiment, we co-transformed saCas9 and a guide RNA targeting 20 base pairs of mRFP in the same plasmid into TOP10 E. coli cells. We also co-transformed saCas9 and a guide RNA with a template guide that did not target any sequence on either plasmid. In the first experiment, if Cas9 and the guide RNA are active, Cas9 will make a double-stranded break in the mRFP gene and knockout its expression (Figure 4). We would expect to see no red colonies and no fluorescence in the red light spectrum. In the control construct, Cas9 and guide RNA are not active and should not cut mRFP, and we would expect to see normal mRFP expression. We transformed all cells with minipreps that were verified by Sanger sequencing.

Figure 4: Cas9 and guide RNA devices.

Figure 5 shows that the cells with the template guide transformed normally with reasonable efficiency and expressed RFP. In Figure 6, the transformation was much less efficient—Cas9 activity could possibly be stressful to the cell. It produced mostly white colonies and seven very large pink colonies. This mix of phenotypes is likely a result of differences in DNA repair among individual cells--some cells may have repaired the double-stranded break with a variety of indels, or not at all.

Figure 5: Cotransformation of BBa_K2019000 (saCas9) in SB1C3 and BBa_K2019001 (guide RNA template) in SB1T3 in TOP10 E. coli strain

Figure 6: Cotransformation of BBa_K2019000 (saCas9) in SB1C3 and BBa_K2019002 (guide RNA mRFP targeting sequence) in SB1T3 in TOP10 E. coli strain

To verify further that Cas9 knocked out mRFP expression, we grew 15 mL cultures of each experimental condition for fluorescence measurement. We also included cultures of TOP10 with only the Cas9 device as a non-fluorescing positive control. We previously observed severe inhibition of culture growth with the co-transformed cells, probably from the stress of two plasmids. Therefore, we grew up cultures for this test without antibiotic using careful sterile technique to a standard optical density of .5 absorbance units. We included six biological replicates for each guide RNA condition, three biological replicates of Cas9-only, and three technical replicates for all of the above. Figure 7 shows that gRNA with mRFP-targeting guide did not fluoresce, as we expected. We can conclude that our saCas9 device is present and active.

Figure 7: Cotransformation of BBa_K2019000 (saCas9) in SB1C3 and BBa_K2019002 (guide RNA mRFP targeting sequence) in SB1T3 in TOP10 E. coli strain

Going forward for this experiment, we would need to sequence the mRFP insert from the cells affected by Cas9 to see what specific edits were made. This can be done by sequencing PCR fragments from colony PCR or by deep sequencing.