Team:Freiburg/Production Nanocillus
Nanocillus Production
To combine our targeting and drug delivery system, we used the spores of Bacillus subtilis as a carrier. The developed product is called Nanocillus.But before any experiments with the spores can be performed, they need to be produced!
The following video shows the production of Nanocillus, beginning from the routine culture, transformation of competent B. subtilis, the sporulation process and finally the preparation of capsules containing our Nanocillus.
During the process of production, western blots and flow cytometric analyses were performed to verify the generated spores. In doing so, it is confirmed that the Nanocillus is expressing our constructs and can be used for the spore production and in further applications.
To start with the production of the Nanocillus, competent cells of B. subtilis are needed.
The Bacillus subtilis WT and the different protein surface knock outs: dCotZ, dCotB, dCotG and dCgeA were inoculated in 4 ml of LB medium to grow overnight at 37°C at 200 rpm . The next day, the samples were diluted in 10 ml of minimal medium (MNGE) to an optical density (OD600) of around 0.1/ml and the incubation was continued. The OD was measured every 20 minutes until the samples reached an OD600 of around 1.0-1.3/ml, which takes around 4 hours depending on the different strains and the status of the original culture.
The Bacillus subtilis WT and the different protein surface knock outs: dCotZ, dCotB, dCotG and dCgeA were inoculated in 4 ml of LB medium to grow overnight at 37°C at 200 rpm . The next day, the samples were diluted in 10 ml of minimal medium (MNGE) to an optical density (OD600) of around 0.1/ml and the incubation was continued. The OD was measured every 20 minutes until the samples reached an OD600 of around 1.0-1.3/ml, which takes around 4 hours depending on the different strains and the status of the original culture.
Figure 1: Growths curve if the WT strain.
Figure 2: Competent Bacillus subtilis cells.
The competence was checked under the microscope (the cells should be very motile) and 400 µl were filled in different tubes. Each tube can be used for one transformation.
If not used directly, glycerol was added to each tube to a final concentration of 10% and the samples were frozen at -80°C for optimal storage.
For the transformation of the constructs into the host B. subtilis, 600 ng of the construct DNA, is added to each tube. The negative controls were left untreated. All the samples were incubated for 1 hour at 37°C at 200 rpm and then 100 µl of the expression mix (see protocol) was added to each sample, followed by another incubation for one more hour.
To check if the transformation was successful, 400 µl of the samples were plated on selective chloramphenicol plates.
After 14 hours of growth, single colonies could be identified and be tested for the insertion into the correct locus. Another test was performed to make sure that the DNA was inserted in the right locus. For the amyE- locus, the picked colonies were put on starch plates to grow. The next day lugol’s iodide was dropped on each colony. The WT and wrong inserted DNA colonies were still able to express the a-amylase and consequently can reduce the starch and decolourize the surrounding area. Only colonies with positively inserted DNA remain coloured from the lugol’s iodide and therefore are used for further experiments
For another approach, DNA was inserted in the thrC- locus. The positive colonies no longer own the threonine synthase and are not able to grow on minimal medium without threonine. Picked colonies were then plated on two different minimal mediums. One with threonine and one without the threonine to determine which colonies have the correct integrated DNA. Additionally, for the sacA- and lacA- locus a colony PCR was performed to confirm the right integration.
Positive colonies were picked and incubated in 4 ml LB to grow overnight at 37°C at 200 rpm. To induce the sporulation of vegetative cells, the samples were once again diluted in 10 ml LB to an OD600 of 0.1 - 0.2/ml and let grown untill an OD600 of 0.8-1.0/ml. At that point the samples reached their exponential growth phase. They were washed once with 1x PBS (Phosphate-buffered saline) and resuspended in 5 ml of DSM (Difco sporulation medium) to incubate for 24 hours at 37°C and 200 rpm.
If not used directly, glycerol was added to each tube to a final concentration of 10% and the samples were frozen at -80°C for optimal storage.
For the transformation of the constructs into the host B. subtilis, 600 ng of the construct DNA, is added to each tube. The negative controls were left untreated. All the samples were incubated for 1 hour at 37°C at 200 rpm and then 100 µl of the expression mix (see protocol) was added to each sample, followed by another incubation for one more hour.
To check if the transformation was successful, 400 µl of the samples were plated on selective chloramphenicol plates.
After 14 hours of growth, single colonies could be identified and be tested for the insertion into the correct locus. Another test was performed to make sure that the DNA was inserted in the right locus. For the amyE- locus, the picked colonies were put on starch plates to grow. The next day lugol’s iodide was dropped on each colony. The WT and wrong inserted DNA colonies were still able to express the a-amylase and consequently can reduce the starch and decolourize the surrounding area. Only colonies with positively inserted DNA remain coloured from the lugol’s iodide and therefore are used for further experiments
For another approach, DNA was inserted in the thrC- locus. The positive colonies no longer own the threonine synthase and are not able to grow on minimal medium without threonine. Picked colonies were then plated on two different minimal mediums. One with threonine and one without the threonine to determine which colonies have the correct integrated DNA. Additionally, for the sacA- and lacA- locus a colony PCR was performed to confirm the right integration.
Positive colonies were picked and incubated in 4 ml LB to grow overnight at 37°C at 200 rpm. To induce the sporulation of vegetative cells, the samples were once again diluted in 10 ml LB to an OD600 of 0.1 - 0.2/ml and let grown untill an OD600 of 0.8-1.0/ml. At that point the samples reached their exponential growth phase. They were washed once with 1x PBS (Phosphate-buffered saline) and resuspended in 5 ml of DSM (Difco sporulation medium) to incubate for 24 hours at 37°C and 200 rpm.
Figure 3: Bacillus subtilis spores
To purify the cultures of spores from remaining vegetative cells, they were treated with the enzyme lysozyme. The lysozyme affects the freely accessible peptidoglycan of the gram-positive vegetative B. subtilis cells , while In contrast, the stable spores aren't affected or harmed. The samples were centrifuged, resuspended in 1x PBS and the lysozyme (15 mg/ml) was added at a dilution of 1:6. After the spores were inoculated at room temperature (RT) with vigorous shaking, all the vegetative cells should be lysed. Repeated washing and centrifugation of the samples separated the spores from the remaining cell fragments.
To set a norm for the following experiments, the remaining spores were counted in the Neubauer improved counting chamber and aliquots were made for an easier storage at -80°C.
To set a norm for the following experiments, the remaining spores were counted in the Neubauer improved counting chamber and aliquots were made for an easier storage at -80°C.
The wild type spores of Bacillus subtilis were analyzed using flow cytometry. The set gate for spores includes 75.5% of the sample. The spores were treated with lysozyme for 1 h. The flow cytometry analysis shows that after purification the amount of spores is considerably higher with 91.9%.
By including a hemagglutinin epitope tag to the introduced protein the detection can be verified via western blot or flow cytometry. Spores successfully displaying either anti-GFP nanobodies or GST can be visualized by surface immunostaining with anti-HA-Alexa647 conjugated antibodies (Figure 2).
Transformed B. subtilis spores displayed GST or nanobodies and a HA epitope tag on their surface, which could be verified by staining with anti-HA antibodies conjugated to Alexa Fluor 647. The stained spores exhibited a higher fluorescence intensity.
Chemical decoating of endospores enabled the analysis of extracted surface proteins.
Lysates of spore coats were analyzed by western blots (figure 4). For spores containing the construct BBa_K2114001 (anti-GFP nanobody fused to the coat protein CotZ and a HA tag), the HA tag could be detected in the spore coat lysate.
The spores of B. subtilis containing the construct BBa_K2114001 (anti-GFP nanobody fused to the coat protein CotZ and a HA tag) were chemically decoated. The coat lysates were analysed using western blots by detecting the HA tag.
After these verifications, the spores were lyophilized. This meant that the majority of medium from the samples removed and the capsules were filled with our final product: the Nanocillus.
Figure 7: The Nanocillus capsule
