The first result that was useful for us was linked with our model. Indeed, our model permitted us to have a first apprehension of how our tool works. You can find all the creation fo it in the Model page and the python code used is available on our gitlab.
We have these followed parameters:
\( R(t) \), population of resistant bacteria at time \(t\),
\(S(t)\), population of sensitive bacteria at time \(t\),
\(Rc(t)\), population of resistant bacteria containing the CRISPR tool plasmid at time \(t\),
\(Sc(t)\), population of sensitive bacteria that are recipients containing the CRISPR tool plasmid at time \(t\),
\(C(t)\), population of donor bacteria containing the CRISPR tool plasmid at time \(t\).
We ended up with the next evolution of our parameters, with at t=2 there is induction by anhydrotetracycline:
The results are conclusive regarding the effectiveness of our tool. Indeed, the population of sensitive bacteria then becomes significantly dominant, leading to the extinction of resistant bacteria. Thus, we observe a pronounced growth curve for the S population. The model that permits us to have an in silico study of our CRISPR dCas9 plasmid tool concludes that it works ! Thanks to it, by modifying the parameters, we are able to predict the different behavior possible of our tool, which is of great importance for the wet lab part and also for the safety aspect. This is therefore a major support for predicting the behaviour of bacterial populations in the laboratory testing phase!
Our software also created some important results for our SuperBugBuster creation ! To get an idea of what is the principle of it, you can look at the Software page and at the python code available on our gitlab.
Indeed, thanks to it and to the run of our program, we had as a result the motifs of our RNAg. We ended up with those next motifs:
These results were really important for our plasmids creation and construction. Indeed, it is thanks to that, that we target the antibiotic resistance gene, to induce its modification and inactivate it.
For our BacProtac part, our results lie on the finding of 3 potential nanobodies that will permit protein deterioration. These Nanobodies will permit us to create our BacPROTAC system specific for our interest protein. Thanks to docking experiments, we ended up with the 10 most compatible Nanobodies, but decided to perform our tests only on the top 3. The next list presents sequences of these ones, that seem to be good candidate for our tool:
Next step is to test them, to see if they are actually working on our protein. To do so, we thought of 3 different tests such as: Fluorescent, NMR and ELISA (you can find more information on that on our Experiments page).
During this project we achieved the build of much than 14 different plasmid, here are our success results : To check our result, for each plasmid we realized the check of PCR on gel and sequencing. These two steps permitted us to see if our plasmids created were the good ones and the ones expected.
Gel on PCR :
The first step of the construction of pEDIT1 was the PCR product, which was checked in this gel. (0,7% agarose, loading dye (6X), 1kb+ ladder)
Here is our gel result :
The lengths obtained from the PCR amplification products were as expected.
Then we check out the length of the linearised plasmid from the isolated clone, and it was the good one:
Sequencing :
After sequencing, we can say that we obtained the good plasmid. That could have been pretty cool, but we realized that we needed other verification tools (RifR resistance, ccdB cassette) in order to really select our final plasmid at the end of the final transformation.
Gel on PCR :
We are expecting these fragments after enzyme digestion of pEDIT1 version 2 (with ccdB).
Here is our gel result:
We have obtained that good length for the PCR, and as we ordered this plasmid, we can say that what we have in our colonies is the plasmid we want. So we have obtained the host plasmid of our construction, with the origins of replication and transcription and the CmR resistance. The double resistance (CmR, RifR) and the ccdB gene will allow us to eliminate cells that kept this plasmid during the final assembly. Indeed, the RifR and ccdB cassette should be lost during the process, so cells having a resistance to RifR won’t be the good ones so we will eliminate them, and on top of that, the ccdb cassette should kill them. This double verification is necessary since we’re at the final stage of our construction.
Gel on PCR :
Sequencing :
We obtained the right length for the pEDIT2-Cm plasmid without digestion (ND) for each of the 4 clones. After digestion by EcoRI, we had two fragments as expected. So we can conclude that we obtained the right pEDIT2-Cm plasmid.
Gel on PCR :
Sequencing :
We obtained the right length for the pEDIT2 plasmid without digestion (ND). After digestion by KnpI and SpeI, we had two fragments as expected corresponding to the correct lengths. So we can conclude that we obtained the right pEDIT2-Cm plasmid.
Gel on PCR :
Sequencing :
We obtained the correct length for the PCR pT1 and PCR INS-rif that allow us to build the pHost-spacer1. So we can conclude that we obtained the right pHost-spacer1 plasmid after ligation. So it’s ready to host the backbone of the 1st gRNA that will target the oxa gene.
Gel on PCR :
Sequencing :
We obtained the correct length for the PCR pT1 and PCR INS-rif that allow us to build the pHost-spacer1. So we can conclude that we obtained the right pHost-spacer2 plasmid after ligation. So it’s ready to host the backbone of the 2nd gRNA that will target the oxa gene.
Gel on PCR :
Sequencing :
We obtained the correct length for the PCR pT1 and PCR INS-rif that allow us to build the pHost-spacer1. So we can conclude that we obtained the right pHost-spacer3 plasmid after ligation. So it’s ready to host the backbone of the 3rd gRNA, that will target our plasmid so it will be inactivated.
Gel on PCR :
Sequencing :
We obtained the correct length for the PCR pT1 and PCR INS-rif that allow us to build the pHost-spacer1. So we can conclude that we obtained the right pHost-spacer4 plasmid after ligation. So it’s ready to host the backbone of the 4th gRNA, that will target our plasmid so it will be inactivated.
Gel on PCR :
Sequencing :
pEX-gRNA1-oxa48
pEX-gRNA2-oxa48
We obtained the right length for the pEx-gRNA1-oxa48 and pEx-gRNA2-oxa48 plasmids without digestion (ND). After digestion by ZraI, we had one fragment meaning that the plasmid has been linearized as expected and the fragment obtained is corresponding to the correct lengths. So we can conclude that we obtained the right pEx-gRNA1-oxa48 and pEx-gRNA2-oxa48 plasmids, the plasmids with the gRNA that will guide our cas9 to the oxa gene.
Gel on PCR :
Sequencing :
We obtained the right length for the pENTR4-Delta-BbsI plasmid without digestion (ND). After digestion by BbsI and ApaI, we had two fragments as expected and not one like in the original pENTR4 and they are corresponding to the correct lengths. So we can conclude that we obtained the pENTR4-Delta-BbsI plasmid wanted.
Gel on PCR :
Sequencing :
We chose 2 different regions because we cannot sequence only one (length regulation) and we see that we have the expected plasmid since mismatches and gaps / insertions located at the ends are consistent with the sequence. This shows that errors are often made at the beginning and the end during a sequencing but with two different regions, we checked that these ends were correct. We obtained the right length for the pV2-mScarlet plasmid without digestion (ND). After digestion by BbsI, we had two fragments as expected and they are corresponding to the correct lengths. So we can conclude that we obtained the right pV2-mScarlet plasmid.
Gel on PCR :
We obtained the right length for the pet28-oxa48 plasmid without digestion (ND). After digestion by BbsI, we had two fragments as expected and they are corresponding to the correct lengths. So we can conclude that we obtained the right pet28-oxa48 plasmid.
Sequencing :
We obtained the right length for the pet28-oxa48 plasmid without digestion (ND). After digestion, we had two fragments as expected and they are corresponding to the correct lengths. So we can conclude that we obtained the right pet28-oxa48 plasmid.
Our results ended up positive, all our plasmids were successfully built.