Home Team Team Attributions Collaborations Project Description Design Proof of concept Engineering Results Notebook Implementation Contribution Experiments Parts Safety Human Human Practices Communication Partnership Jamboree Organization Awards Education Model Sustainable Partnership Partnership ARIA-RECONBY: Our team has coworked with ARIA team from the Pompeu Fabra University (UPF) of Barcelona. The context of the forthcoming partnership has been due to the strong belief of both teams that the sum of the parts contributes much more than each of them separately. The two key alliance points are: technical expertise bidirectional support and collaborative design of combined proposed implementation. Hands on: Technical expertise bidirectional support: plasmid miniprep and nanopore sequencing PLASMID PURIFICATION Our 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 (for further information visit our project design). These newly formed nanobody gene fragments are cloned in plasmids and the nanobodies are expressed in E. coli membrane (1), allowing the final selection of those with higher binding affinity to the target antigen. For this, we are using low and medium copy plasmids from pSEVA plasmid repository that allow surface expression of the nanobodies. The plasmids are: pSEVA228-I, low copy plasmid with the intimin-based expression system. pSEVA238-I, medium copy plasmid with the intimin-based expression system. pSEVA228-AT, low copy plasmid with the autotransporter-based expression system. Figure 1. pSEVA228-I and pSEVA228-AT structure. Both of the systems allow displaying nanobodies on the surface of bacteria when cloned in the MCS (multiple cloning site). When trying to isolate the plasmid from the cells, we had some troubles. For this reason, ARIA team 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. 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 and 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 made. Figure 2. RECONBY's agarose electrophoresis gel. Molecular weigth marker (phage lambda genome digested with PstI restriction enzyme), pSEVA228-I, pSEVA238-I and pSEVA228-AT purification by Metabion protocol respectively, pSEVA228-I, pSEVA238-I and pSEVA228-AT purification by handmade protocol respectively (see protocols) Figure 3. ARIA's agarose electrophoresis gel. The gel is divided into two segments, the first 6 bands for the digested plasmids and the second 6 bands for the supercoiled ones. For each segment there are two biological replicas (colony 1 and 2) of the 3 plasmids: 228-AT, 228-I and 238-I. Results discussion: Table 1. Plasmid concentration results after purification. Recomby's results in pink and ARIA's in blue. By using NZYTech Kit, ARIA team obtained quite good DNA concentration values that allowed them to transform and plate culture the Miniprep resulting plasmids into NZY5α competent cells. To verify that the miniprep had been performed correctly, they digested the plasmids with the same restriction enzymes as us, HindIII and EcoRI, to obtain two fragments: one of 7600 bp and the other of 100 bp. Since they ran the samples on a 1% Agarose gel only the t 7600bp band could be observed. 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. This collaboration helped us determine that our purification method had some problems with this E. coli strain, so using a different approach is required. At first, we transformed E. coli DH5α with the little plasmid we were able to obtain and then purified it again from it, the results were higher. Finally, by applying Dr. Esteban Martínez method we obtained a great concentration value. NANOPORE SEQUENCING In addition, we needed nanopore DNA sequencing for verifying that all the possible combinations of the CDR fragments are obtained after in vitro recombination. For this, we could have contacted a company and sent their samples there, but we thought that we could also collaborate with ARIA in this sphere. In Universidad de Zaragoza’s lab, the equipment for doing such protocol is not available, but in Universitat Pompeu Fabra they have the proper tools for doing the nanopore DNA sequencing. 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 us the opportunity to bring this collaboration further in after iGEM project development steps. Protocol 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 antibiotics 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 RECONBY can act. We 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 cyclescould 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 (2). This system is similar to bacteriophages’ genetic material injection system. After conversations with Dr.Esteban Martínez from CNB who has already developed this system in a human microbiome Enterococus (4), we decided that this system is the best option for the ARIA-Reconby design. Figure 4. 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. (a) The system is activated by contact and the spike is launched towards the attacked cell trespassing it membranes, then the effectors (nucleases, proteases, toxins) are released in the cytoplasm and the system disassembled (3). Another strategy could be 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 and act in a wide spectrum of bacteria (5). With that, we could use ARIA’s kit diagnostic results to design and build nanobodies that 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 if is necessary. References: Veiga E, De Lorenzo V, Fernández LA. Autotransporters as scaffolds for novel bacterial adhesins: Surface properties of Escherichia coli cells displaying Jun/Fos dimerization domains. J Bacteriol. 2003;185(18):5585–90. Zoued A, Brunet YR, Durand E, Aschtgen MS, Logger L, Douzi B, et al. Architecture and assembly of the Type VI secretion system. Biochim Biophys Acta - Mol Cell Res. 2014;1843(8):1664–73. Hernandez RE, Gallegos-Monterrosa R, Coulthurst SJ. Type VI secretion system effector proteins: Effective weapons for bacterial competitiveness. Cell Microbiol. 2020;22(9). Ting SY, Martínez-García E, Huang S, Bertolli SK, Kelly KA, Cutler KJ, et al. Targeted Depletion of Bacteria from Mixed Populations by Programmable Adhesion with Antagonistic Competitor Cells. Cell Host Microbe. 2020;28(2):313-321.e6. Haney EF, Mansour SC, Hancock REW. Antimicrobial peptides: An introduction. Methods Mol Biol. 2017;1548:3–22.
