To visualize minicell formation and validate that there are minicells present in the samples, we also performed various microscopy experiments.
1. Minicell size and formation
Firstly, we analyzed minicell size and formation using microscopy images and time-lapse videos of minicell cultures in a O.D600 = 0.05. By using 100x Oil Ph3 Objective Lens and TRANS wavelength in a Zeiss microscope with camera super focus, we could visualize minicells formation from the poles of parental cells (supplementary video 1). Microscope slides were 120x50mm, and coverslips were 25x25mm. 2 uL of TB43 bacterial culture at O.D.600 = 0.05 was used while incubating at 37°C in 80% agarose pads. Images such as these were used for our minicell counting software (see Dry-lab).
Figure 8) Microscopy image of the strain TB43. The formation of minicells is visible from the pole of the parental cell.
This image is a representative sample of 3 replicates that enables us to compare the size of minicells and their respective parental cells, as well as to observe their formation through the poles.
2. Membrane integrity of minicells
Secondly, we analysed the lifespan of minicells by observing their membrane integrity. According to Huang et al., 2020 these cells may have a longevity of 10 days on average.
Figure 9) Microscopy image through TRANS lens of TB43 after 6h. The cellular membrane of minicells is still intact, even though they are not metabolically active anymore. We can see the strain TB43 under a 100x Oil Ph3 Objective Lens and TRANS wavelength in a Zeiss microscope with camera focus.
The image above was taken as exploratory data, from a timelapse video of 12 hours (data not shown). Even after 6h, the minicell membrane is still intact. For more on the lifespan of minicells, see Side project.
3. Minicells fluorescence intensity
As minicells will be the host for our protein producing plasmids we sought to see the difference between protein expression between minicells and parental cells. In the following pictures, we compare population images of MG1655, TB43 and MinB, all of which have been transformed with a constitutively expressed plasmid containing GFP (see Design).
By looking under the GFP wavelength, we can see a difference between fluorescence strength visually between populations. Here we have data gathered from wt and min mutant strains under GFP and TRANS wavelengths. These images are representative of a sample of images taken in triplicate. They follow the same settings and slide sizes as the images above.
Figure 10A) Microscopy images of 3 initial populations of E. coli, expressing GFP. MG1655 is a wild-type strain, TB43 and MinB are minicell producing strains. Data shown of TRANS and fluorescence measurements.
The densities of populations have similar O.D. and were incubated at 37°C for 4 hours. As expected, there is a higher proportion of filamentous cells in TB43 and MinB samples. Minicells are hardly visible.
Figure 10B. Zoomed image with TRANS wavelength taken from TB43 strain. Black arrow indicates a minicell.
We hypothesized the small circular dots to be minicells in the TRANS images. However, they are not visible in the fluorescence pictures. This could be because the amount of GFP produced in minicells is below the detection limit or they do not express GFP at all.
4. Purification methods
Next, we purified the minicell containing samples by centrifugation and addition of antibiotics (see Protocol) and analysed them again by microscopy.
Figure 11) Microscopy images of TB43 and MinB supernatant, after the first step of purification by ultracentrifugation (15 min for .10.000 rpm).
The amount of rod-shaped cells has considerably reduced showing the efficiency of the purification step. However, some bacteria remain, even in their filamentous form.
As still, some parental cells remained by using only ultracentrifugation to separate both populations, we performed a second round of purification with Ceftriaxone - it acts inhibiting the synthesis of compounds from the cellular membrane of bacteria. It seems to have no effect on minicells due to differences in membrane composition (4).
Figure 12) Microscopy images of TB43 and MinB after addition of ceftriaxone antibiotic and 45 min incubation in 160 pm Shaker for 37°C.
After the second step of purification involving the Ceftriaxone antibiotic, there seemed to be no rod-shaped bacteria left. Interestingly, it was possible to visualize small dots in the GFP measurements. They could correspond to minicells expressing the GFP plasmid.
We hypothesize not seeing a large amount of minicells in our frame for 3 different reasons:
- The purification method removes a large quantity of minicells as well.
- Not all minicells have taken up the GFP producing plasmid;
- The yield of minicell production is low.
All 3 hypotheses could be true together.
5. Staining assays
a. Trypan blue (0.4%)
This stain is used for dyeing cells that have the membrane permeable. Microscopy images were taken with a Nikon Eclipse E200 microscope, using a 40x Ph2 objective lens. The image is representative of experiments conducted in triplicate.
In this experiment, it was possible to observe minicells viability, corroborating to findings from previous assays (see Timecourse).
Figure 13) Microscopy image of TB43 transformed with plasmid inducing FtsZ overexpression under IPTG (5mM) regulation. Magnification 40x (See Engineering success).
b . DAPI (4′,6-diamidino-2-phenylindole) staining
DAPI is a chromosomal DNA staining agent that allowed us to observe if our minicell sample was DNA-free. Due to not being purified, the sample presented cells with genomic DNA and without.
Figure 14) Microscopy image of unpurified TB43 lambda (see Engineering success). 100x Oil Ph3 Objective Lens in a Nikon Eclipse E200 microscope. Minicells are indicated by black arrow.