Team:UNIZAR/Design







Design

Project design

This section describes the design of the components that build up the project Reconby, all together lead us to our results. The strength of the project lies in the ability of the genes to reassembly in vitro forming new nanobodies that are capable of recognizing the target more efficiently.

Our goal

Our goal is to change the way nanobodies are produced aiming to reduce or even avoid the use of animals, the current unique manufacturing process available.
  • We want to develop a high-quality library of nanobodies sequences obtained by recombination of nanobodies from different origins and even species.
  • We want to achieve a more efficient, faster, and cheaper production system.
  • We want to generate new nanobodies with enhanced charactersitics such as higher affinity to their targets.
But how can be this possible? Below are the five main phases in which the project is divided; so… let´s start the journey:

  1. SELECTION AND ADAPTATION OF THE SEQUENCES

    2. Selecting the different sequences of the nanobodies we were going to use and synthesizing them chemically (IDT synthesis) with the accurate variations was done before we started working in the laboratory. This was a fundamental part of the whole project since the sequences needed to be assembled together and further inserted in the expression vector. Any mistake in the overlapping parts would have implied an unreached recombination process. The carried out design is explained in the engineering cycle.
    An important fact in the adaptation of the sequences is the codon usage that refers to differences in the frequency of occurrence of synonymous codons in coding DNA. Codon optimization (1) was implemented taking into account that the expression of the Nbs was going to be in bacteria and the sequence of the different genes came from mammals (alpaca, camel, llama) or were synthetic. Mammal codons were replaced with synonymous ones in order to increase protein expression in E. coli.
    The different parts used in the project are described in the part collection.
    We had three different parts that will build up the nanobody sequence called: CDR1, CDR2, and CDR3.
    Figure 1
    Figure 1. Representation of the three different sequences that when combining will build up the Nb primary sequence.

  2. RECOMBINATION

    3. Gibson Assembly (2,3) was the assembly method used to create our nanobody sequences for the following reasons:
    • It is an elegant and robust seamless technology that allowed us to assemble in the desired order different parts, so the directionality is maintained.
    • This method does not rely on the presence of restriction sites, which simplifies the construct design as it does not need compatible restriction enzyme recognition sites.
    • Robust reaction: even if the conditions of the reaction are modified the different parts are assembled.
    • Everything can be done in a simple tube which makes things much easier, using a single polymerase, exonuclease activity, and a ligase under isothermal conditions.
    • It is faster and more efficient than traditional cloning. It is seamless and avoids the need for multiple rounds of cloning and screening. It can build constructs with nanogram quantities of DNA. And thanks to the proof-reading polymerase, the method minimizes the incorporation of errors at cloning junctions.
    First, the pool of the different parts (CDR1, CDR2 y CDR3) was randomly combined in order to create different sequences of Nbs.
    Figure 2 Figure 3
    Figure 2. Sequences of the Nbs after the recombination process using Gibson Assembly Figure 3. Gibson assembly process
    After the recombination, there is a needed step before inserting the sequence into the plasmid. In order to do another cycle of Gibson assembly (where the whole Nb sequence is inserted into the plasmid) it is necessary to insert two tails at the ends of the fragments (in the 5' and 3' ends), so there are overlapping ends that could recombine with the linearized plasmid.
    To add these tails we carried out PCRs in which the primers used are annealed with part of the CDR1 and CDR3 sequences, and the sequence of the primer that does not hybridize is a part of the plasmid.
    Figure 4
    Figure 4. In order to insert the nanobody sequence into the plasmid, a PCR is needed. The primers used are shown on the image; the grey part corresponds to the sequence of the primer that hybridizes to CDR1 and CDR3 sequences and the green and orange parts correspond to the overlapping sequence for the Gibson assembly reaction, as the sequence is equal to a part of the plasmid.

    After the PCR in which the tails were inserted, we were ready to proceed and carry out the Gibson Assembly method and, thus, introduce the sequences into the linearized plasmid (to do so we digested the plasmid with EcoRI and HindIII)
    Figure 5 Figure 5.1 Figure 5.2 Figure 5.3
    Figure 5. Representation of the Gibson Assembly method inside the lined-squares there are the overlapping sequences that the method requires.
    Figure 6 Figure 6.0
    Figure 6. Plasmid used in the whole project; in them we can see the different components that will alllow us express the Nb, and select them.

  3. TRANSFORMATION

    4. Once the nanobody sequence is inserted into the plasmid, it is time for bacteria transformation. The strain chosen to carry out the expression of the nanobody is E. coli UT5600 (this was a cession of the CNB (National Biotechnology Centre))., but first to obtain a Nb library we used E. coli DH5α.
    E. coli UT5600 strain is deficient in the OmpT protease, a periplasmic protease. This protease can degrade cytoplasmically expressed proteins when the cells are lysed to make a crude extract, and it may degrade proteins expressed in the periplasm as well. UT5600 is EcoK r+ m+, so plasmids have to be modified in order to get transformants (4).
    UT5600 genotype (F−ara-14 leuB6 azi-6 lacY1 proC14 tsx67 Δ(ompT-fepC266) entA403 trpE38 rfbD1 rpsL109 xyl-5 mtl-1 thi-1)
    To introduce the plasmid into the bacteria it is necessary to follow an appropriate transformation protocol, for example electocompetent E. coli UT5600 cells; and then follow the electroporation protocol.
    Figure 7
    Figure 7. Diagram showing how plasmids are introduced in the bacteria

  4. EXPRESSION

    5. Once the nanobody sequences are inserted in the plasmid and the bacteria are transformed, how can we obtain them?
    The plasmids that we are using have the Pm promoter so to induce the expression of the nanobody it is required the transcription regulator XylS, that stimulates the expression of the Pm promoter in the presence of 3-metilbenzoate.
    Once the protein is expressed, due to the signal peptide pelB, it goes to the periplasm and then there are two ways to express the protein on the cell surface. Although their mechanism of secretion remains uncertain, AT (autotrasporter) and Intimin proteins are translocated into the periplasm (thanks to the pelB peptide) and then use the β-barrel assembly machine (BAM) complex for insertion into the external membrane and translocation of the passenger region to the cell surface:
    • Intimin pathway
    • Autotransporter pathway:
      They possess common features necessary for transport to the cell surface: an N-terminal signal peptide that enables the transport across the inner membrane, a passenger domain that is either surface displayed or secreted to the extracellular medium, a linker for a complete surface exposure, and a β-barrel domain that is inserted into the outer membrane(5,6).
    Figure 8 Figure 8
    Figure 8. Expression of the Nbs using the intimin and autotransporter methodology

  5. NANOBODY TESTING

    6. After expressing the nanobody, testing their ability to join their target is a great importance. To test this we used a whole ELISA assay. We chose this method because it is sensitive and selective. It is also relatively simple to perform and it is quantitative. This allows us to compare the affinity between the new Nbs produced with the recombination method and the Nbs produced by the traditional way using animals. This assay also allows us to select Nbs with higher affinity.
    • First, it is important to attach the cells to the surface of the plate wells, as the Nbs are expressed on the cell surface they are already viable and capable of recognizing their target without performing additional steps.
    • Afterwards they are incubated with their target, GFP. GFP is a fluorescent protein, that when exposed to light in the blue to ultraviolet range, radiates green fluorescence.
    Assuming that the fluorescence is proportional to the amount of protein-bound, the higher the affinity, the greater the amount of protein-bound. That is why by using this test and measuring the obtained fluorescence we can know which nanobody has a better affinity for protein.
    Figure 9 Figure 9
    Figure 9. Whole-cell ELISA assay

    Although we tested this ability with a purified GFP antigen, other techiniques will be implemented to prove and verify that the nanobodies are able to recognize their target in real samples.

References:

  1. Athey J, Alexaki A, Osipova E, Rostovtsev A, Santana-Quintero L V., Katneni U, et al. A new and updated resource for codon usage tables. BMC Bioinformatics. 2017 Sep 2;18(391):391.
  2. Gibson Assembly. CLONING GUIDE 2 ND EDITION RESTRICTION DIGESTTFREE, SEAMLESS CLONING.
  3. GeneArt Gibson Assembly Cloning | Thermo Fisher Scientific - ES [Internet]. [cited 2021 Oct 16]. Available from: https://www.thermofisher.com/es/es/home/life-science/cloning/seamless-cloning-and-genetic-assembly/gibson-assembly.html
  4. E. coli K12 UT5600 | NEB [Internet]. [cited 2021 Oct 17]. Available from: https://international.neb.com/products/e4129-ecoli-k12-ut5600#Product Information
  5. Salema V, Marín E, Martínez-Arteaga R, Ruano-Gallego D, Fraile S, Margolles Y, et al. Selection of Single Domain Antibodies from Immune Libraries Displayed on the Surface of E. coli Cells with Two β-Domains of Opposite Topologies. PLoS One [Internet]. 2013 Sep 23 [cited 2021 Oct 18];8(9):e75126. Available from: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0075126
  6. Sichwart S, Tozakidis IEP, Teese M, Jose J. Maximized Autotransporter-Mediated Expression (MATE) for Surface Display and Secretion of Recombinant Proteins in Escherichia coli. Food Technol Biotechnol [Internet]. 2015 [cited 2021 Oct 18];53(3):251. Available from: /pmc/articles/PMC5068381/