Exploring the structural engineering of phages


In a recent study published in Proceedings of the National Academy of Sciencesthe researchers showed the containment of biological bacteria and synthetic engineering.

Study: Synthetic engineering and biological containment of phages.  Image credit: MattLphotography/Shutterstock
Stady: Synthetic engineering and biological containment of phages. Image credit: MattLphotography/Shutterstock

The serious dangers posed by drug-resistant bacterial diseases and recent advances in synthetic biology have led to great interest in transgenic phages with therapeutic potential. To date, many studies of modified phages have been limited to proof-of-concept (POC) or foundational research using phages with relatively short genomes or ‘phage display populations’. In addition, no precautions were implemented to ensure an effective translation of the practical application.

about studying

The current study created a platform for cell-free and recombinant phage engineering.

First, the team sought to reboot several wild-type (WT) phages that infected Gram-negative bacteria and acid-fast mycobacteria using the collected genomes. in the laboratory. Polymerase chain reaction (PCR) was used to amplify the entire assembled phage genome obtained from either deoxyribonucleic acid (DNA) or template genome. Primers were developed to generate 28 bp to 65 bp homologous regions in each flanking PCR fragment. These pieces were aggregated and electrified into bacterial hosts, reactivating the phages.

The team developed fragments that enabled the end-joining of reactivated phages derived from the circular genomes. Coliphage lambda was effectively reactivated from five fragments in Salmonella phage P22 and E. coli, along with four fragments present in S. Typhimurium LT2. The mycophage model D29, which infects acid-fast Mycobacterium spp and has a high guanine and cytosine genome, was also restarted. Five segments of DNA were produced to create a circular genome.

consequences

The T7 coliphage model, which contains a linear genome, showed linear sequences at the time of replication and was used to evaluate the study strategy. The linearized genome was constructed from four fragments by polymerase chain reaction (PCR) and then electrophoretically stacked in E. coli to reactivate the T7 phage. Coliphage T3, Pseudomonas phage gh-1 and Salmonella phage SP6 were predicted to have similar genetic structures and life cycles to those of T7. The team also succeeded in regenerating T3 from E. coli DNA fragments, SP6 from Salmonella Typhimurium LT2 DNA fragments, and gh-1 from Pseudomonas putida DNA fragments. These results showed that the study design guidelines and methodology were effective for rebooting phages from the assembled linear genomes.

Functional mycophage D29, non-tagged mycophage B1, and GS4E were rebooted from M. smegmatis mc2 155. Mycophage TM4 was constructed from six chemically produced DNA segments based on publicly available sequencing data. DNA fragments underwent amplification to demonstrate 28-bp to 38-bp overlap with adjacent fragments, purified, and then spliced in the laboratory. The genome was inserted into M. smegmatis mc2 155. The active TM4 synthetic complex, whose genome was biochemically produced, was rebooted. The entire synthetic TM4 genome was sequenced by MiSeq and shown to be 100% identical to the indicated TM4, indicating efficient phage replay from the sequencing data.

The premium Phage T7 is well selected as the model for POC realization. The T7 machine selected a packaging signal (Pac) sequence to ‘encapsulate’ or ‘store’ its genome in the T7 head autosome. Pac consists of PacB and PacC. The small gp18 subunit binds with the gp19-prohead complex to translocate DNA to the head of the phage. After head packaging, the team observed that gp19 cleaves into PacC in the genome of the sequenced T7 phage, leading to T7 head maturation.

Next, a plasmid containing Pac and your ‘favorite gene’ (yfg) was generated and injected into Escherichia coli. Electroporation of the genome into plasmid-containing Escherichia coli cells resulted in the production of T7-dependent synthetic transforming molecules (T7Pac). In the cell, the Pac-null genome generated progeny virions before packaging of the Pac-containing plasmid into head particles, yielding T7Pac-yfg. Using this method, the team synthesized T7Pac-lacZ, a plasmid harboring lacZ. The lysate was combined with lacZ-deficient E.coli, followed by plating with LB X-gal plates. Thus, 1.6 x 104 CFUs/ml of T7Pac-lacZ were generated, and all colonies formed were blue. On the bacterial lawn, T7Pac-lacZ did not develop plaques, indicating biological containment.

The team constructed the genome of phage SP6 minus the gene encoding the capsid protein 31. The genome was inserted into the LT2 strain of S. Typhimurium, which expresses gene 31. Using PCR, gene 31 was deleted from the synthetic SP6.Δ head confirmed. A high caliber SP6 was obtainedΔ head Lysate developed plaques on the LT2 grass that express gene 31, which was not present in the LT2 parents.

Overall, the results of the study showed the generation of natural and engineered phages that infect Gram-negative bacteria and acid-fast mycobacteria by in the laboratory Genome assembly.



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