Team:UPF Barcelona/Partnership
Human practices
RECONBY-ARIA Partnership
The context of the forthcoming partnership has been due to the strong belief of both teams that the sum of the parts contribute much more than each of them separately. The two key alliance points are: technical expertise bidirectional support and collaborative design of combined proposed implementation.
1. Technical expertise bidirectional support
1.1 Plasmids purification
RECONBY team’s goal is the creation of a library of newly formed nanobodies that bind to a specific antigen (GFP as 'proof-of-concept'). This is achieved by random in vitro recombination of the different CDRs genetic sequences of already characterized nanobodies that bind to that same antigen. These newly formed nanobody gene fragments are cloned and nanobodies are expressed in E. coli membrane, allowing the final selection of those with higher binding affinity to the target antigen.
For this, they are using a plasmid from pSEVA plasmid repository which expresses the nanobody in the cell membrane. When trying to isolate the plasmid from the cells, the RECONBY team is having some troubles. For this reason, we tried to replicate their miniprep experiments using a kit from a different company in order to compare our results with theirs. In that way, RECONBY obtained the miniprep results comparison to check if their technique was being properly applied, and ARIA got the chance to contemplate a new approach to our experiments using RECONBY’s plasmid.
i) Results comparison
- RECONBY preliminary results: when using a commercial kit such as Metabion Kit (as well as Roche kit, Biotool kit) the concentration of plasmid obtained was low and further molecular biology protocols (digestion, clonation) couldn’t be performed due to this lack of high plasmid concentration. With the handmade protocol the concentration obtained was higher, however RNA contamination was high as it is shown in the agarose gel in Figure 1. The quantitative results are shown in table 1.
- Results discussion: using NZYTech Kit, we replicated RECOBY’s plasmids purification experiments. The values obtained when comparing our results with their Metabion kits results are much more positive (see table 1), since the DNA concentration is higher and allowed us to transform and plate culture the Miniprep resulting plasmids into NZY5α competent cells (Figure 2). To verify that the miniprep had been performed correctly, we digested the plasmids with HindIII and EcoRI restriction enzymes to obtain two fragments: one of 7600 bp and the other of 100 bp. Since we ran the samples on a 1% Agarose gel only one of the two bands (the 7600bp band) could be observed. However, it was possible to distinguish between plasmids that were digested and those that were not. As shown in Figure 3, the digested plasmids ran less, while the undigested plasmids, being able to adopt a supercoiled conformation, were able to advance more. So, we can conclude that it is possible to achieve acceptable DNA concentration results using a commercially available kit, even though it is not always easy.
| Sample | Plasmid concentration (ng/μL) | |
|---|---|---|
| Metabion Kit - RECONBY | 228-AT | 12.1 |
| 228-I | 25.6 | |
| 238-I | 12.5 | |
| Handmade - RECONBY | 228-AT | 260.5 |
| 228-I | 266.6 | |
| 238-I | 310.4 | |
| NZYTech - ARIA | 228-AT | 45.0 |
| 228-I | 44.8 | |
| 238-I | 45.0 |
ii) ARIA’s new approach
In our original approach, the biosensors built for AR sample detection are grown inside living E. coli and then a lysis is performed in order to extract the sensors from the cell so that they are in contact with the patient’s sample.
Using RECONBY’s plasmids, we could try to express the Cas12 protein in the cell membrane so that the lysis step is avoided. This would solve two imperfections of our approach. Firstly, the lysis protocol slows down the biosensor production and even though we are contemplating trying to program cell autolysis we have not shown that it works and Cas12 keeps functional. Secondly, the fact of using the cell lysate directly provokes the presence of lots of DNases from the cell in the medium, and they react with our reporter producing a high background noise which complicates our results visualization.
The main basic design of the new approach would consist of cloning our LbCas12 protein and a protease cutting point inside RECONBY’s plasmid. In this way, one Cas12 is expressed in the cell membrane, it could be cut by a protease and once liberated be functional. The specific gRNAs could be synthetised in vitro or we could use our cell lysate.
1.2 Nanopore DNA sequencing
In addition, RECONBY team needs nanopore DNA sequencing for verifying that all the possible combinations of the CDR fragments are obtained after in vitro recombination. For this, they could have contacted a company and sent their samples there, but we thought that we could also collaborate in this sphere. In Universidad de Zaragoza’s lab, the equipment for doing such protocol is not available, but in Universitat Pompeu Fabra we have the proper tools for doing the nanopore DNA sequencing. For this reason, they sent us the samples to sequence and we performed the wet lab experiments. In this way, a win-win collaboration strategy was achieved, where RECONBY obtained the sequencing results and ARIA wet lab team learned and practiced the nanopore DNA sequencing protocol.
The nanopore sequencing protocol is available in the following button. Finally, the results are not available due to issues with RECONBY's experiments. However, this gives ur the opportunity to bring this collaboration further in after igem project development steps.
2. Combined proposed implementation
ARIA is a project which can be categorized inside clinic stages as diagnostic, since the final aim of it is to be used in clinical scenarios in a simple way: taking a liquid biopsy from a patient, putting it into our paper-based array so that it shows a fluorescence pattern, and finally taking a picture of it in order to give a final assessment to the clinician based on the found resistances by our second AI layer.
But, what comes next? What may happen in case there was no antibiotic to treat the patient’s bacterial infection? Or, even if there is an antibiotic, could we think of a better therapeutic approach to handle this problem? Here is where our partnership comes up and RECONBY can act.
They propose that once the resistance is identified, the sample is cultivated in presence of the antibiotic so as to isolate the resistant bacteria. The bacteria are incubated with the library in order to find a nanobody that specifically recognizes this bacterium. Multiple improvement cycles could be done by recombining the CDRs of these nanobodies to obtain the best one.
After the nanobody selection, the next step is to specifically attack the resistant bacteria. Several approaches are proposed:
- The plasmid with the selected nanobody would be transferred to an E. coli strain with the type IV protein secretion system. The bacteria are administered to the patient and when the nanobody recognizes the resistant bacteria, the system would be activated and it would kill the resistant bacteria. This type IV secretory system consists of a machinery similar to a crossbow that is expressed constitutively and once our bacteria contacts another bacteria (thanks to the nanobody) the system is activated and the “arrow” is launched towards the resistant bacteria [1]. This system is similar to bacteriophages’ genetic material injection system.
The type VI protein secretory system is a protein translocation nanomachine widespread among Gram-negative bacteria and used as a means to deliver effectors directly into target bacterial or eukaryotic cells. When the machinery is expressed and the effectors are attached to the tip of the spike, the system is activated by contact. Then, a contraction in the proteins forming the tube is produced and the spike is launched towards the attacked cell trespassing its membrane. Finally, the effectors (nucleases, proteases, toxins) are released in the cytoplasm and the system disassembled [2].
Figure 3: Type VI protein secretory system.
After conversations with Esteban Martínez from CNB who has already developed this system in a human microbiome Enterococus [3], we decided that this system is the best option for the ARIA-Reconby design. - Another available strategy is based in phage therapy. A lithic phage is modified by replacing the spicules sequences by the selected nanobody sequence, the phage would be specifically directed towards the resistant bacteria.
- The last strategy consists of introducing the nanobody linked to an antimicrobial peptide. These molecules act altering the membrane permeability [4].
With that, RECONBY could use ARIA’s kit diagnostic results to design and build nanobodies which are able to attack the bacterial pathogens of a specific patient in order to save its life.
To show the awareness of the described joint-efforts approach, we have explained above the design pipeline of the first small proof of concept based on the inicial technologies that both teams are working on. The next upcoming steps of our collaboration will be continuing with the engineering cycle by building and testing our designed implementation in order to find out possible weak points and redesign what is necessary.
References
[1] Zoued A, Brunet YR, Durand E, Aschtgen MS, Logger L, Douzi B, et al. (2014). Architecture and assembly of the Type VI secretion system. Biochim Biophys Acta - Mol Cell Res. 1843(8):1664–73. doi:10.1016/j.bbamcr.2014.03.018
[2] Hernandez RE, Gallegos-Monterrosa R, Coulthurst SJ. (2020). Type VI secretion system effector proteins: Effective weapons for bacterial competitiveness.Cell Microbiol. 22(9). doi:10.1111/cmi.13241
[3] Ting SY, Martínez-García E, Huang S, Bertolli SK, Kelly KA, Cutler KJ, et al. (2020). Targeted Depletion of Bacteria from Mixed Populations by Programmable Adhesion with Antagonistic Competitor Cells. Cell Host Microbe. 28(2):313-321.e6. doi:10.1016/j.chom.2020.05.006
[4] Haney EF, Mansour SC, Hancock REW. (2017) Antimicrobial peptides: An introduction. Methods Mol Biol. 1548:3–22. doi:10.1007/978-1-4939-6737-7_1
