| MiamiU| MiamiU
The goal of the Miami U iGEM 2022 project was to design and characterize a programmable biological system for combating antibiotic resistant infections. Currently progress includes completion of the initial cloning and transformations for our desired T7 phage tail fiber constructs. We eventually hope to incorporate CRISPR-Cas13a for specific targeting of antibiotic resistance genes [1].
An additional component of our design involved the incorporation of a nanobody to the tail fiber of the T7 bacteriophage. There are two methods we designed and prepared for implementation in order to compare transcriptional versus post translational engineering of the gp17 phage tail fiber protein. The first method we pursued was direct fusion of a nanobody to a truncated version of tail fiber protein gp17. The second method was adding the nanobody and T7 through an enzymatic reaction with Sortase pentamutant, eStrA. We hypothesized that engineering a fusion protein expressed from combined genes that encode two separate proteins would pose many challenges when it comes to proper folding and maintenance of function. Therefore, we expected that fusion plasmid would result in non-functional phages whereas, catalytic ligation following translation and proper folding of each protein would allow proper phage assembly with functionalized tail fiber.
Phage tail fiber engineering was conducted hand-in-hand with modeling. AlphaFold 2 was used to model seven different sequences. These include a wild type T7 tail fiber, wild type T7 tail fiber with nanobody attached, wild type T7 tail fiber with Sortase linker, truncated T7 tail fiber with no linker, truncated T7 tail fiber with Sortase linker, truncated T7 fiber control, and a nanobody control.
Figure 1: This shows the three control models of wild type T7, truncated T7, and the nanobody control vs the fusion of the truncated T7 and nanobody with the sortase motif linker.
For all the primers, engineered plasmids, and their functions please refer to the parts registry. Parts Registry
Figure 2. Gel electrophoresis for the amplicons of the fusion plasmid (left) and T7/T3 with truncated or non-truncated tail fiber (right).
The gels in figure 2 are examples of the many gel electrophoresis that we ran. Both of them have examples of each amplicon that was made for their method. Primers BBa_K4514002, BBa_K4514019, BBa_K4514020, BBa_K4514021, BBa_K4514022 were used on the left for the fusion plasmid. While primers BBa_K4514002, BBa_K4514004, BBa_K4514000, BBa_K4514001, BBa_K4514057, BBa_K4514058, and BBa_K4514005 were used for the sortase-mediated method.
PCR and gel electrophoresis took most of our group's time. Figure 2 shows a successful gel for the Truncated T7 fused to the nanobody with the sortase linker, or fusion plasmid. There were two iterations of this fusion plasmid: 1) A three-piece gibson combining the 390bp nanobody, 3711bp of 3RCF main genes, and 4212bp of the backbone. 2) A two-piece gibson with a 390bp nanobody being added to the 7550bp truncated T7 backbone. Figure 2 shows an example of a gel of amplicons for this fusion plasmid. It has two 1kb ladders in well 1 and well 5. One 50bp ladder in well 8. Well 2 is for the 3B plus 27 primers(3711bp), well 3 is for primers 29 plus 4(4212bp). Well 4 was the 7550bp of primers 27 plus 29 which did not work. Well 7 was the nanobody amplicon.
The results (figure 2) showed that PCR for the three-piece Gibson assembly of fusion plasmid was successful. Additionally, integrity of plasmids synthesized via Gibson Assembly can be further tested by using an antibiotic marker which allows for selective survival of bacteria with said antibiotic following transformation. This same procedure would be done for designing and synthesizing the Sortase plasmid.
Figure 3: Assembled plasmids using Gibson assembly
Figure 4: Transformation of the fusion plasmid (left) and the truncated T7 (right).
After the gel electrophoresis, gel extraction, and DNA concentration measurements we moved on to Gibson Assembly.We used Gibson Assembly to make 5 different plasmids. These include T7 with sortase motif, T3 with sortase motif, truncated T7 with Sortase motif, fusion plasmid, and the nanobody inserted into the eSrtA in pET29 plasmid. Figure 3 shows two example plasmids that were created. Figure 4 shows the confirmation of these through transformation. These plasmids were the fusion plasmid and the truncated T7. For more examples, please refer to the lab notebook. Virtual lab notebook
Figure 5 (left to right):
A) Plaque assay performed with T3 Reg Bacterial strain with the phage
lysates: T3 reg, T7 reg, and T7 truncated
B) Plaquing assay using our truncated T7 + Nanobody Fusion phage
lysate on previously transformed truncated T7 + Nanobody Fusion bacterial strain
Formula for the PFU (plaque-forming unit):
Table 1. The plaque-forming unit/mL
| Phage lysate/ strain | # plaques | Lysate Volume (mL) | Dilution | PFU per mL |
|---|---|---|---|---|
| Phage 60/ truncated T7 | 15 | 0.002 | 10-6 | 5*109 |
| T7/ fused protein | 12 | 0.002 | 10-5 | 4*108 |
| Truncated T7/ fused protein | 21 | 0.002 | 10-4 | 7*107 |
Truncated T7 was designed to expose the C-terminus of gp17, T7 tail fiber. The regular C-terminus folds towards the phage capsid which could cause problems. During the design phase of iGEM the hypothesis was that truncated T7 would fail to infect; however, it was still given a chance. The modeling showed results which confirmed this hypothesis, showing the tail fiber completely unfolded. Normally this would result in a loss of function. The experiment was still carried out and the plaquing assay had unexpected results. The truncated T7 had the highest plaquing (see plaque assays). T7 having the best plaque assays means that although potential unfolding occurred, the phage could still infect. This breakthrough prompted the new models for the truncated T7 fused with the nanobody. This new model is depicted in figure 1 and shows promising result. However, no clear conclusion about nanobody integration and function can be drawn solely from plaquing assays conducted at this time.
Fast Protein Liquid Chromatography (FPLC) was our method of choice for purifying Sortase, however, the product has been unobtainable due to a manufacturing shortage. Future plans include purification of the Sortase enzyme which, once achieved, would be used to carry out the ligation reaction of the T7 tail fiber to the nanobody ex vivo. Hypothetically, the product of this reaction would be introduced to a tailless phage, allowing it to assemble with our engineered components. Various plaquing assays on different bacterial strains, such as the DSG419 strain expressing the nanobody binding antigen, would allow us to determine viability of this engineered phage as a transducing particle. Then, we would experiment with different Cas RNAs to ultimately determine optimal gene targeting to maximize on the antimicrobial abilities of our proposed phage therapy.