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 Results NANOBODIES (NBs) SEQUENCE ANALYSIS Analysis of NBs sequences revealed the existence of two constant regions between CDRs of different NBs. These regions are responsible for nanobodies' 3D structure and are perfect to be used for assembly. Thanks to these preserved regions, we were able to design independent nanobody fragments composed of CDR and overhanging regions, which are capable of overlapping and combining with any other CDR with the same overhanging region. These fragments were chemically synthesized and from now on we will refer to them as CDR1, CDR2 and CDR3, respectively, although they do not correspond exactly with the actual CDR (see part collection). Figure 1. Sequence allignment of different anti-GFP nanobodies from different species. Mismatches to the reference sequence are shown in red. Two regions with common sequences can be observed in the middle of every nanobody sequence. CDR ASSEMBLY A12 nanobody We demonstrated that we were able to recombine a CDR1 with a CDR2 and a CDR3 to create an in-vitro-generated nanobody by overlapping their constant regions. This implies that our original design was successful and that the overlapping sequences we designed did work. A12 nanobody's three fragments: CDR1, CDR2 and CDR3 were combined through CloneEZ PCR cloning kit. 1 µL of each CDR at 10 ng/µL were mixed with 1 µL of buffer, 1 µL of enzyme and 5 µL of nuclease free water. The solution was then kept 30 minutes at 22 ºC. A sample was taken in order to check through PCR amplification that the nanobody had been correctly assembled, using the complete NB A12 as positive control (fig. 2). Figure 2. Agarose gel electrophoresis showed that nanobody A12 was obtained from its CDRs through Clone EZ PCR cloning. Original NB A12 and CloneEZ product show a band for the same molecular weight. CDR pools We demonstrated that we were able to generate nanobodies through recombination of three different pools of CDR. We combined all CDR1, CDR2 and CDR3 from different nanobodies, generating new ones. In this case, CloneEZ PCR cloning DNA samples consisted on three mixtures of all CDR1, all CDR2 and all CDR3, respectively. Each pool at a final concentration of 10 DNA ng/µL. 1 µL of each mix was added to the assembling reaction with the CloneEZ PCR cloning kit. Assembly was checked through PCR amplification of newly formed complete NBs as explained above. It was afterwards loaded in a agarose gel electrophoresis together with a secondary control of amplification: a NBs pool composed of NBs mixed in equal proportions (Figure 3). Figure 3. 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. EcoRI + HindIII PLASMID DOUBLE DIGESTION The next step consisted in cloning nanobodies' sequences into plasmids. In order to achieve this, we first needed to digest our purified plasmids with EcoRI and Hind>III restriction enzymes. We intented to find the most appropiate digestion conditions testing two different buffers: Red and TANGO buffers from ThermoFisher and three digesting times: 1 h, 4 h and overnight. In addition, we checked the individual activity of both enzymes in the different tested conditions to ensure none of them could degrade the plasmid. Figure 4. Visualization on agarose gel electrophoresis of the digested plasmids on different conditions. There is no sign of degradation in any of them although the band is barely visible in some single digestion samples. As overnight digested plasmids were well digested and not degraded we decided to use overnight digestions in our future experiments to reduce as much as possible the amount of undigested plasmid.(Figure 4) PLASMID CLONING After following the whole process of plasmid purification, digestion, insert cloning and transformation, we found out that most of our competent cells had no insert at all. Seeing the transformation results, we could concluded that many of the bacteria had been transformed with undigested plasmid with no insert (Figure 5). This hypothesis was also confirmed by PCR amplification of the plasmid MCS. As it can be seen in Figure 6, two bands appear in the transformed cells samples. A lower one (lighter) corresponding to the undigested MCS (200 bp) and a series of higher ones (heavier) that could correspond to inserted nanobodies (≈500 bp). These ones are absent in the original plasmid control (228 AT). Figure 5. Negative control of transformation with numerous grown colonies. This negative control was obtained by transforming digested, theoretically linear, plasmid directly into competent cells, with no insert. The rest of the transformation plates with insert had a non-significantly greater amount of colonies. Figure 6. PCR amplification of liquid cultures of transformed competent cells. The amplified fragments correspond to the MCS, with or without insert, of the transformed plasmids. GeneRuler 100 bp DNA ladder was used. TROUBLESHOOTING With the aim of dealing with the undigested plasmids, we thought that an effective solution could be purifying digested plasmid directly from its electrophoresis agarose gel band. Nevertheless, we were not able to follow this strategy since our original plasmid quantity was too low (Figure 7) to recover a significant amount of it from the gel. Consequently, we tried to increase plasmid purification efficiency. PLASMID PURIFICATION Plasmids pSEVA228-I, pSEVA238-I and pSEVA228-AT were originally purified from liquid culture of E. coli XL1-Blue. As the plasmid concentration obtained was unsatisfactory (Figure 7), different miniprep kits were used. Similar results were obtained with all of them, indicating they were probably not the cause of the low plasmid concentration. We decided to keep using the same miniprep kit and to change the plasmid expression strain to another well-known laboratory strain, DH5α, which is commonly used for transformation and plasmid expression. Even though there was a mild increase, the plasmid concentration results remained below our expectations. To improve our results we asked for advice to other iGEM teams. We sent samples of our plasmids to UPF Barcelona iGEM with the aim of knowing if they could obtain higher concentrations (see Partnership). Moreover, we asked Dr. Esteban Martinez for guidance and he recommended as to elute different columns with the same buffer in order to get higher concentrations of plasmid. Figure 7. Comparative results of plasmid concentration nanodrop measurements obtained from XL1 strain liquid culture, DH5α strain liquid culture and XL1 strain using Dr. Esteban Martinez's strategy. This strategy allowed us to work with a greater volume of liquid culture and so to obtain more plasmid. Using homemade maxiprep for plasmid purification, we obtained DNA concentrations over 200 ng/µL or even 300 ng/µL. Unfortunately, when observed at agarose gel electrophoresis , these samples turned out to be degraded or contaminated with RNA (Figure 8). Figure 8. Agarose gel after electrophoresis of plasmid samples contaminated with RNA. Samples showed concentration values of 260.5 ng/µL, 266.6 ng/µL and 310.4 ng/µL, respectively. Over the RNA, the band of the plasmid could be seen but its quantity was too low for band purification. PLASMID PCR Another strategy to deal with undigested plasmids was amplifying by PCR our purified plasmids except the MCS. In this way we would ensure to obtain linear plasmid, ready to be used for cloning with no need to improve plasmid purification results. Furthermore, PCR amplified plasmids would never close without the insert, securing no more bacterial growth in our transformation negative controls. We wanted to find the most appropiate PCR conditions, so we assayed three different DNA polymerases: NEB Hifi, PrimeStar and MyTaq Bioline at three different annealing temperatures: 56 ºC, 58 ºC and 60 ºC and with our three plasmids: 228I, 238I and 228AT. Only one of the conditions tested allowed linear plasmid amplification. However, such amplification was not reproducible on further attempts. Figure 9. Agarose gel electrophoresis showing the PCR results, with no positive results except one, that was proved to be not reproducible.
