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 Proof of Concept Project objetive: Concept Figure 1. Representation of the anti-GFP nanobodies from the four different origins. The overall goal of the project is to create a library in E. coli of artificially developed nanobodies by random in vitro recombination of the different CDR genetic sequences, mimicking what happens in vivo. Nanobodies have a wide range of applications in diagnosis, therapeutics and pollutant biosensing, however, their production is difficult, expensive and requires animals. Hence, our system improves the traditional method for nanobody production, being faster and cheaper. Also, this approach allows recombining CDR from different species, which provides greater variability than just randomizing CDRs as it also changes the nanobody structure, thus creating new nanobodies with different targets and affinities that would be impossible to form naturally. In order to demonstrate the viability of this project we have designed a proof-of-concept using anti-GFP nanobodies. We have selected these specific nanobodies against GFP due to fluorescence, since it facilitates the detection and allows to detect affinity differences. These anti-GFP nanobodies were chosen from 4 different origins: arabian camel (Camelus dromedarius), llama (Llama glama), alpaca (Vicugna pacos) and synthetic. Once the nanobodies sequences to be used were selected, their codon sequence was modified so as to optimize their expression in E. coli. An aligment all of the sequences was done in Benchling (1) in order to find the FR conserved regions. These regions are located right between the CDR regions, which allows the division of each nanobody sequence into three fragments, each one containing one CDR, and with the FR regions at the ends as overhangs for recombination. Also, EcoRI and HindIII restriction sites were introduced in each CDR sequence by chemical synthesis, in 3´and 5´respectively. This is needed to clone the sequences in the MCS of the plasmids for future nanobody display in E. coli. Moreover, in order to manatian the ORF in frame with the signal peptide, which guides the system to the bacterial periplasm, some nucleotides were added. Figure 2. Representation of recombination step. FR regions used for recombination in blue and pink. Each green fragments represents a nanobody fragment, with this technology fragments from different nanobodies recombine creating a whole new nanobdy with its owns characteristics. The proof-of-concept can be divided into four sections: in vitro recombination viability, variability generation, nanobody expression in E. coli and nanobody improvement. In vitro recombination viability Our team has the entire sequence of the nanobody anti-GFP A12 as well as its separate CDR fragments. When recombining A12 CDR fragments in vitro with the CloneEZ PCR cloning kit and amplifying the result by PCR, it is observed the same electrophoresis band than the entire sequence of nanobody A12. This shows that recombination of CDR fragments can occur in vitro. Figure 3. Electrophoresis of nanobodies PCR-amplified nanobodies (NBs). Negative controls are labelled as c-. A12 NB was obtained from Clone EZ assembly of three fragments CDR1, CDR2 and CDR3. Cloning was successful as its band after PCR amplification Cl-Ez corresponds to the A12 nanobody sequence, generated in vitro through the combination of its CDRs. The rest of bands correspond to complete chemically synthesized nanobodies, which act as positive controls for the PCR. Variability generation Knowing that the in vitro CDR recombination is viable we also would like to show that variability is generated. For this, we carry out the recombination of CDR fragments of different anti-GFP nanobodies. The result it is amplified by PCR and observed in a electrophoresis gel. The presence of different bands could be an indicator of variability, in order to confirm this it would be necessary to sequence the PCR products obtained after recombination. This sequencing was going to be performed by Aria iGEM Team from Barcelona (link partnership), however, this step couldn't be done. Figure 4. Agarose gel electrophoresis of PCR amplification of NB A12, the NBs pool and the assembly of the CDRs pool. PCR amplifications were developed at 5 different temperatures, from 58ºC to 62ºC. Nanobody expression in E. coli The ultimate goal is to be able to express these newly formed antibodies in bacteria in an easy way, using intimins and autotransporters, proteins used to display small proteins in the surface of bacteria, allowing the nanobody screening. Figure 5. Simplified representation of transformation and expression step. Three plasmids are used, the differences between them are the copy number and the nanobody expression strategy, two of them contain the gene for intimin and the other one the gene for autotransporter. The nanobodies sequences are clone into the MCS of these plasmids using EcoRI and HindIII restriction sites. Finally, E. coli is transformed with the construct and its growth in plaque is evaluated in presence of kanamycin. Figure 6. Representation of MCS of plasmids (blue). The restriction sites used are highlighted in yellow. Figure made in SnapGene. Figure 7. Representation of E. coli chemical transformation protocol and selection in plaque in presence of kanamicine. Nanobody improvement Also, this strategy could be useful for improving the affinity and kinetics properties of existing nanobodies. Starting from the anti-GFP nanobodies generated in the previous section, affinity essays could be carried out in order to determine if any of the new CDR combinations has better characteristics binding to GFP than the already existing anti-GFP nanobodies. To do so, a whole-cell ELISA could be considered, the bacteria would be fixed to the well, and the antigen, GFP, would be added afterward. If GFP binds to the nanobody displayed in the bacterial membrane, we could detect the fluorescence signal, which is proportional to the binding affinity. The results would be compared with the affinity of the already existing anti-GFP nanobodies. Figure 8. Whole-cell ELISA for nanobody affinity assays. Apart from all of these experiments, recently a paper has been published with a similar approach (2). In search of potent neutralizing nanobodies against SARS-CoV-2, by accident, they discover that CDR swapping by overlap PCR can be used to rapidly increase both the affinity and the neutralization activity of isolated nanobodies (2). Actually, they expect that "this approach of combining multiple low-affinity clones via CDR swapping holds great potential for rapidly generating nanobodies and, more generally, antibodies with high affinity with much less effort than is typically require". This discovery shows, once more, the viability, as well as the utility of our project. References: Editor · Benchling [Internet]. [cited 2021 Oct 10]. Available from: https://benchling.com/editor Zupancic JM, Desai AA, Schardt JS, Pornnoppadol G, Makowski EK, Smith MD, et al. Directed evolution of potent neutralizing nanobodies against SARS-CoV-2 using CDR-swapping mutagenesis. Cell Chemical Biology. 2021;28(9):1379-88.e7.
