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SynPhage Specifications: Engineer a phage therapy scaffold that can easily be programmed to deliver CRISPR-based genetic circuits targeting bacterial antibiotic resistance and virulence factor genes.
M13 has previously been shown to deliver CRISPR-based genetic circuits that kill bacteria based on the presence of antibiotic resistance genes. However, M13 has limited host range, meaning the number of bacterial strains M13 can infect. However, T7 and T3 bacteriophages can infect strains of E. coli, including lipopolysaccharide (LPS) and other mutant strains, or related smooth strains, and other enteric microbes. T7 and T3 phages are strictly lytic phages, meaning infects and lyses bacteria and does not enter the lysogenic life-cycle [2]. Thus, these phages have attracted strong interest as phage therapies. Moreover, T7 and T3 can readily be programmable to expand host range to target a wide array of bacterial pathogens, including E. coli, Yersinia, Klebsiella, Salmonella, and Shigella. Therefore, T7 and T3 will be used as therapeutic phage scaffolds for delivering CRISPR-based genetic circuits.
We are keen on using Escherichia coli as the bacterial host for quantitative and phenotype tests. E. coli provides the benefits of versatility, ease of handling, a collection of environmental phenotypes and applicability within human hosts. Researchers have built a vast number of E.coli genomes varying among strains and among techniques, reinforcing the model of E.coli in the field of genetic engineering or various academic settings [1].
As a result of its well established precedence throughout most scientific disciplines, we would like to use the E.coli as the cells to exercise the desired gene transformation from the phage. From there we will monitor the effects of phage infection through growth tests, using optical density (OD) as a proxy, and plaquing assays.
Previously, T7 and T3 host range can be altered by swapping out the gene encoding the tail fiber complex, gp17, with tail fiber genes from other phages. However, this strategy is not straightforward, and requires many iterations to obtain a successful phage with altered host range. This is because phage tail fibers are very diverse and have many different sizes and structures. Therefore, we propose engineering T3 and T7 to express a nanobody at the terminus of the tail fiber complex to alter phage host range.
The nanobody is small in size and possesses the same general
structure. Therefore, engineering phage host range simply requires exchanging out a nanobody that
binds to the target bacterium. Our initial experiments aimed to synthesize a direct fusion of the
nanobody and gp17 tail fiber protein. However, Alphafold2 modeling predicted complex misfolding of
such peptides, regardless of various linker insertions. Therefore, we propose functionalizing the
tail fiber complex post assembly, thus preserving the functionality of both components by letting
them fold prior to functionalization.
To view the models, visit our model page.
Sortase A (SrtA) is a transpeptidase, recognizing the sorting signal LPXTG-motif at the C-terminal domain (CTD) and cleaving at the peptide bond between the Glycine and Threonine residue. The resulting amino-acyl intermediate is then resolved via nucleophilic attack by a second peptide containing a hydrophobic poly-glycine fragment at the N-terminal domain (NTD). Sortase catalyzed reactions are utilized in natural gram-positive bacterial systems to ligate peptides, mostly to modify bacterial surface proteins to aid in pathogenic attacks against its host (Figure 1) [3]. The simple addition of short polypeptide tags makes Sortase A useful for modifying target proteins with a variety of functional and chemical groups (e.g. biotin, fluorophore, etc.) [4]. We utilized the Sortase A pentamutant, an optimized mutant for experimental and ex vivo reactions, to catalyze the ligation reaction of target protein to phage tail fiber and ultimately produce an optimal phage vector for delivery of CRISPR-Cas 13 components into a specific bacterial species. Through analysis of enzyme kinetics, this mutant was found to significantly increase efficiency of ligation compared to wild type Sortase found in Staphylococcus aureus as shown by higher kcat/Km value. As a result of these findings, sortase mutant can be found listed as eSrtA in pET29 [5].
Figure 1. The mechanism of the Sortase A nicking two ends of peptides based on the sensing of LPXTG and Glycine-rich region.
After choosing the Sortase variant for the mechanical ligation-based modification of the phage tail fiber, we needed to design the phage tail and nanobody. Two options, shown below, were the hypothesized ways to assemble the phage tail and nanobody fusion based on placement of Sortase recognition sequences.
The first was to encode poly-glycine in the NTD of one peptide and the Sortase tag (LPXTG motif) on the CTD of the other. When we mix the two types of self-folded peptides together with the purified Sortase protein followed by our tailless T7 phage (IYPH60), we expect the assembly of a functional T7 phage with our target protein, properly folded and functional, attached to its tail fiber.
Figure 2 and Figure 3 depict the various possibilities for arrangement of nanobody, tail fiber, and linker sequences. Between these options, we concluded that the first would likely be more successful as compared to the second which would result in the N-terminal domain being oriented towards the phage head, making it mechanically inaccessible. Therefore, we chose to design the truncated phage tail fragments to contain the motif LPETGG on the CTD.
Figure 2. First scheme of the sortase coupling with the motif attached on the CTD of the phage. This is our chosen methodology.
Figure 3. Second scheme of the soratase coupling with the motif attached on the CTD of the nanobody fragment.
Compare the infectivity of both truncated and non-truncated versions of T7 and T3
Truncated T7, non-truncated T7, truncated T3 and non-truncated T3 plasmids were constructed by Gibson assembly using appropriate primers. (See parts registry)
Plasmids were then transformed into DH5-𝞪 competent cells through heat shock and transformants were plated on respective antibiotic selection plates. Engineered phages were then made by using plasmid containing bacterial strains and infecting with IYP60 (Δgp11, 12, 17) for the assembly of the different tail fiber constructs.
Plaquing assays were then used as a means of screening and quantifying the engineered phage species by using respective plasmid containing bacterial strains. The formation of plaques would suggest the successful transformation, expression of desired plasmid, and the formation of a number of engineered phages that can be quantified through counting plaques at the highest serial dilution.
Plaquing was observed with all phages but truncated T3. Conducted additional plaquing assays using the remaining three.
Design and build:
Phage lysates were acquired from liquid culture based infection of the 3 plasmid containing bacteria and characterized via plaquing assays. In the plaquing assays, serial dilutions of all three phage lysates were aliquoted on the following bacterial lawns:
Of the three remaining phages, Truncated T7 phage was chosen as the ideal phage for future experimentation since it had the most robust plaquing results.
The ligation of the nanobody and tail fiber required the preservation of functionality of both components.The goal was to achieve a customizable template phage which is capable of having its infectivity and specificity adapted, according to the target of interest, via sortase A tail fiber alterations. The proof-of-concept system utilized for this was the nanobody-antigen system expressed in strains DSG289 (nanobody) and DSG419(antigen).
Our initial experiments aimed to synthesize a direct fusion of the nanobody and gp17 tail fiber protein by cloning respective genes into a plasmid, using Gibson assembly (see plasmid design below). This fusion plasmid would lack the Sortase A component. Once constructed, the plasmid would be transformed into DH5-𝞪 competent cells in order to test for potential functionality or use as a control in reference to Sortase A mediated reaction.
However, Alphafold2 modeling predicted complex misfolding of such peptides, regardless of various linker insertions. We concluded, therefore, that Sortase A coupling would likely be necessary to encourage proper folding and formation of stable tertiary structures.
Plaquing assay using a serial dilution of T7 Δgp11, 12, and 17 phage lysate, previously collected from plaques on T7 transformed production strain, plated on bacterial lawn of T7 tail fiber with nanobody fusion bacteria.
Phage lysate was made through IYP60 amplification in fusion plasmid transformants would then be used to test infectivity against DSG419 transformants expressing EPEA tag nanobody protein as compared to production strain of T7 tail fiber as well as BL21 and BW25113. Similar infectivity in all strains would suggest unsuccessful attachment and functionality of fusion protein, indicating the need for the Sortase enzyme catalyzed reaction.
For future Sortase screening, Sortase and histidine tag components would be cloned into a
plasmid
by a similar procedure. Extraction and purification of the enzyme would follow an affinity
chromatography column protocol. The G-block peptide, which expresses the nanobody with the LPETG
motif, will be connected to a fluorophore which will be used to test activity and efficiency of
the Sortase reaction. Purified Sortase will be used to functionalize the tail fiber with the
nanobody, ex vivo, and the infectivity would then be tested through plaquing assay on DSG419
antigen strain.
To view the models, visit our model page.
We designed primers to produce the PCR amplicons encoding the full tail fiber protein, and a truncated tail fiber protein (the 252 base pairs on the 3' end encoding the 84 C-terminal amino acids were deleted) for both T7 and T3 bacteriophages. The amplicons were cloned from the plasmids GEM3RCF(T7) and MGP4185 (T3), respectively. Amplicons were assembled into plasmids by Gibson Assembly with certain antibiotic resistance genes: Kanamycin for pMGP4185 and Carbapenem for pGEM3RCF. A more detailed description of our primers and plasmids provided in our parts registry.
We followed the protocol provided by BioLabs inc. in the NEBuilder HiFi DNA Assembly Cloning Kit Manual [6].
The continued development of CRISPR-Cas systems have provided us with numerous ways to tackle problematic aspects of microbes, including antibiotic resistance. CRISPR-Cas systems offer the ability to specifically target genes of interest for degradation. Well-characterized systems to achieve this include those involving Cas9 or Cas3.[7]
CRISPR-Cas components can be delivered through a phage vector and target antibiotic resistance and virulence factor genes, which can either be located on the bacterial chromosomal or plasmid. We think Cas13a, a type VI class II CRISPR can be quite promising. Cas13 exhibits promiscuous RNA cleavage activity which is highly detrimental to a microbial system when sequence complementarity is accomplished.
[8]This allows for post-transcriptional interference with the gene of interest (antibiotic resistance genes, pathogenicity genes, virulence factor genes etc.) and the other non-specific bacterial RNA as well [9]. Additionally, this allows for a level of selection towards bacteria expressing pathogenicity or AMR genes (disease/problem-causing genes) while essentially harmless bacteria are able to escape RNA damage due to lack of expression of pathogenic genes regardless of their presence in their genome, making it an eduactive component to be packages into our engineered phages. Similar to Cas9, the Cas13a system utilizes a guide RNA; after the gRNA binds to the target RNA, the Cas13a would cleave both the target and other non-specific ssRNA as well as the region is in close distance to the ribonucleoprotein complex (figure 4) [10].
Figure 4. Schematic representation of Cas13a broad cleavage activity on RNA sequences.
After determination of target genes, plasmids and respective primers are designed through Benchling software. Amplicon size and primer melting temperature noted for proper adjustment of procedures.
The modeling of the nanobody and tail fiber fusion revealed that there will not be proper folding of the nanobody. As a result, this will be used as a control when constructed. The improper folding suggests improper or malfunction of the nanobody leaving the phage fusion as unable to infect the DSG419 (strain expressing nanobody antigen).
Negligible detection of the transformed colonies on selective antibiotic agar plates can potentially indicate toxicity of engineered plasmids or the exhaustion of cellular machinery. Other obstacles we faced included difficulty with PCR amplification of large amplicons as well as the low efficiency of 3-piece Gibson assembly.
Because we set out to make a therapeutic for human systems, we were faced with inherently complicated design considerations. These included promoting robust silencing of antimicrobial resistance genes while avoiding disruption of the commensal gut microbiome. In addition, we sought to incorporate emerging synthetic biology techniques such as sortase mediated protein engineering and nanobody recognition. Extensive trial and error is still required, therefore, before all aspects of our desired system are fully optimized.