Genetically Modified M13 Bacteriophage Nanonets for Enzyme Catalysis and Recovery

Kadiri, Vincent Mauricio; Alarcón‐Correa, Mariana; Ruppert, Jacqueline; Günther, Jan-Philipp; Rothenstein, Dirk; Fischer, Peer

doi:10.3390/catal9090723

Cited by 5 publications

(3 citation statements)

References 40 publications

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“…For instance, Kadiri et al . demonstrated the formation of nanonets using genetically modified M13 bacteriophages, highlighting the potential of genetic engineering to create novel phage-based structures with unique properties ( 16 ). Additionally, the study by Gosh et al .…”

Section: Discussionmentioning

confidence: 99%

The enhancement of M13 phage titration by optimizing the origin of replication

Darvishali,

Fadaie,

Khanahmad

2024

Research in Pharmaceutical Sciences

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Background and purpose: M13KO7, a modified M13 phage variant, carries the p15A replication origin and Tn903 kanamycin resistance gene. This study aimed to optimize M13KO7's replication by substituting the p15A origin with the higher-copy pMB1 origin (500-700 copy numbers). Experimental approach: A 6431-nucleotide fragment from the M13KO7 plasmid lacking the p15A replication origin and kanamycin resistance gene was amplified using a long polymerase chain reaction (PCR). The modified M13AMB1 plasmid was created by adding adenine to the 3’ ends of this fragment and ligating it to the pMB1-containing fragment using T/A cloning. Afterward, to prepare the phage, pM13AMB1 was transformed into E. coli TG1 bacteria, and then, using the PEG-NaCl precipitation, the modified phage was propagated. The modified phage titer was determined utilizing the serial dilution and the qPCR methods, compared with the M13KO7 phage. Findings/Results: The results showed that in the serial dilution method, the titers of modified phage and M13KO7 phage were 4.8 × 1014 and 7 × 1012 pfu/mL, respectively. Besides, the phage titer calculated by the qPCR method for the modified phage was equal to 1.3 × 109 pfu/mL, whereas it was 4.08 × 108 pfu/mL for the M13KO7 phage. Conclusion and implications: This study provides evidence that replication origin replacement led to a significant increase in phage titers. It highlights the importance of replication optimization for molecular biology applications.

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Section: Discussionmentioning

confidence: 99%

The enhancement of M13 phage titration by optimizing the origin of replication

Darvishali,

Fadaie,

Khanahmad

2024

Research in Pharmaceutical Sciences

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“…More site-specific chemical conjugation methods relying on an initial genetic unnatural amino acid incorporation have been used to modify phages with biotin 71 – 74 but suffer from the complexity and inefficiency of the unnatural amino acid incorporation process 75 , 76 . Phages have been genetically modified with biotin carboxyl carrier proteins, but this requires an extra step for the proteins to be biotinylated by a biotin ligase enzyme within the system for phage production 36 , 77 – 80 . Additionally, this would require streptavidin modification of the immobilization surface which reduces commercial viability due to the cost.…”

Section: Conculsionsmentioning

confidence: 99%

Monomeric streptavidin phage display allows efficient immobilization of bacteriophages on magnetic particles for the capture, separation, and detection of bacteria

Carmody,

Nugen

2023

Sci Rep

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Immobilization of bacteriophages onto solid supports such as magnetic particles has demonstrated ultralow detection limits as biosensors for the separation and detection of their host bacteria. While the potential impact of magnetized phages is high, the current methods of immobilization are either weak, costly, inefficient, or laborious making them less viable for commercialization. In order to bridge this gap, we have developed a highly efficient, site-specific, and low-cost method to immobilize bacteriophages onto solid supports. While streptavidin–biotin represents an ideal conjugation method, the functionalization of magnetic particles with streptavidin requires square meters of coverage and therefore is not amenable to a low-cost assay. Here, we genetically engineered bacteriophages to allow synthesis of a monomeric streptavidin during infection of the bacterial host. The monomeric streptavidin was fused to a capsid protein (Hoc) to allow site-specific self-assembly of up to 155 fusion proteins per capsid. Biotin coated magnetic nanoparticles were functionalized with mSA-Hoc T4 phage demonstrated in an E. coli detection assay with a limit of detection of < 10 CFU in 100 mLs of water. This work highlights the creation of genetically modified bacteriophages with a novel capsid modification, expanding the potential for bacteriophage functionalized biotechnologies.

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“…Nanonets are systematically aggregated viruses, which serve as a more tractable material for large-scale applications of virus-based platforms, as they are larger and more easily isolated. [90,91] Using bacteriophage M13, Fischer and co-workers fused AviTags to the termini of the coat protein to allow for biotinylation on the phage surface. Avidin and streptavidin were introduced to the system to create colloidal M13 nanonets.…”

Section: Nanonets and Bacteriophage Materialsmentioning

confidence: 99%

Protein Mediated Enzyme Immobilization

Caparco

Dautel

Champion

2022

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Enzyme immobilization is an essential technology for commercializing biocatalysis. It imparts stability, recoverability, and other valuable features that improve the effectiveness of biocatalysts. While many avenues to join an enzyme to solid phases exist, protein‐mediated immobilization is rapidly developing and has many advantages. Protein‐mediated immobilization allows for the binding interaction to be genetically coded, can be used to create artificial multienzyme cascades, and enables modular designs that expand the variety of enzymes immobilized. By designing around binding interactions between protein domains, they can be integrated into functional materials for protein immobilization. These materials are framed within the context of biocatalytic performance, immobilization efficiency, and stability of the materials. In this review, supports composed entirely of protein are discussed first, with systems such as cellulosomes and protein cages being discussed alongside newer technologies like spore‐based biocatalysts and forizymes. Protein‐composite materials such as polymersomes and protein–inorganic supraparticles are then discussed to demonstrate how protein‐mediated strategies are applied to many classes of solid materials. Critical analysis and future directions of protein‐based immobilization are then discussed, with a particular focus on both computational and design strategies to advance this area of research and make it more broadly applicable to many classes of enzymes.

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Genetically Modified M13 Bacteriophage Nanonets for Enzyme Catalysis and Recovery

Cited by 5 publications

References 40 publications

The enhancement of M13 phage titration by optimizing the origin of replication

The enhancement of M13 phage titration by optimizing the origin of replication

Monomeric streptavidin phage display allows efficient immobilization of bacteriophages on magnetic particles for the capture, separation, and detection of bacteria

Protein Mediated Enzyme Immobilization

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