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Ff-nano, short functionalized nanorods derived from Ff (f1, fd, or M13) filamentous bacteriophage.

Sattar S, Bennett NJ, Wen WX, Guthrie JM, Blackwell LF, Conway JF, Rakonjac J - Front Microbiol (2015)

Bottom Line: In contrast to standard Ff-derived vectors that replicate in E. coli and contain antibiotic-resistance genes, Ff-nano are protein-DNA complexes that cannot replicate on their own and do not contain any coding sequences.These nanorods show an increased resistance to heating at 70(∘)C in 1% SDS in comparison to the full-length Ff phage of the same coat composition.We demonstrate that functionalized Ff-nano particles are suitable for application as detection particles in sensitive and quantitative "dipstick" lateral flow diagnostic assay for human plasma fibronectin.

View Article: PubMed Central - PubMed

Affiliation: Institute of Fundamental Sciences, Massey University Palmerston North, New Zealand.

ABSTRACT
F-specific filamentous phage of Escherichia coli (Ff: f1, M13, or fd) are long thin filaments (860 nm × 6 nm). They have been a major workhorse in display technologies and bionanotechnology; however, some applications are limited by the high length-to-diameter ratio of Ff. Furthermore, use of functionalized Ff outside of laboratory containment is in part hampered by the fact that they are genetically modified viruses. We have now developed a system for production and purification of very short functionalized Ff-phage-derived nanorods, named Ff-nano, that are only 50 nm in length. In contrast to standard Ff-derived vectors that replicate in E. coli and contain antibiotic-resistance genes, Ff-nano are protein-DNA complexes that cannot replicate on their own and do not contain any coding sequences. These nanorods show an increased resistance to heating at 70(∘)C in 1% SDS in comparison to the full-length Ff phage of the same coat composition. We demonstrate that functionalized Ff-nano particles are suitable for application as detection particles in sensitive and quantitative "dipstick" lateral flow diagnostic assay for human plasma fibronectin.

No MeSH data available.


Related in: MedlinePlus

The system for production of functionalized Ff-nano.Escherichia coli cells containing the Ff-nano production plasmid pNJB7 were infected with the helper phage Rnano3FnB containing the coding sequence of a “probe” or “detector” protein fused to pIII. Upon infection, pII from the helper phage induces positive strand replication from the pNJB7 Ff-nano origin of replication and also provides all other phage proteins and assembly machinery for production of the Ff-nano particles. All five copies of pIII are fusions to the probe (only three copies of pIII fusions are shown).
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Figure 1: The system for production of functionalized Ff-nano.Escherichia coli cells containing the Ff-nano production plasmid pNJB7 were infected with the helper phage Rnano3FnB containing the coding sequence of a “probe” or “detector” protein fused to pIII. Upon infection, pII from the helper phage induces positive strand replication from the pNJB7 Ff-nano origin of replication and also provides all other phage proteins and assembly machinery for production of the Ff-nano particles. All five copies of pIII are fusions to the probe (only three copies of pIII fusions are shown).

Mentions: In this work we have constructed an Ff-derived phage display system for production of functionalized nanorods (50 nm × 6 nm) we named Ff-nano (Figure 1). In the first stage, we modified a setup for production of short phage originally published by Specthrie et al. (1992) to increase the amount (per cell) of the 221-nt circular ssDNA available as a template for assembly of the short particles. This was achieved by inserting the 303-nt short-phage (microphage or Ff-nano) origin of replication (Supplementary Figure S1) into the high-copy-number plasmid pCR4-TOPO (in contrast to the low-copy-number plasmid pBR322 used in the original system), to obtain plasmid pNJB7. Furthermore, a new helper phage, named Rnano3, was constructed. The helper phage Rnano3, in contrast to the original helper phage R474 used for short phage production (Specthrie et al., 1992), is not only a helper but also a phage display vector. Rnano3 was designed for display of proteins as fusions with phage protein pIII that is present in up to five copies at one end of the Ff-derived particles. Infection of cells containing plasmid pNJB7 with the helper phage/vector Rnano3 resulted in production of the 50 nm × 6 nm particles (Ff-nano or microphage; Figure 2) and full-length phage.


Ff-nano, short functionalized nanorods derived from Ff (f1, fd, or M13) filamentous bacteriophage.

Sattar S, Bennett NJ, Wen WX, Guthrie JM, Blackwell LF, Conway JF, Rakonjac J - Front Microbiol (2015)

The system for production of functionalized Ff-nano.Escherichia coli cells containing the Ff-nano production plasmid pNJB7 were infected with the helper phage Rnano3FnB containing the coding sequence of a “probe” or “detector” protein fused to pIII. Upon infection, pII from the helper phage induces positive strand replication from the pNJB7 Ff-nano origin of replication and also provides all other phage proteins and assembly machinery for production of the Ff-nano particles. All five copies of pIII are fusions to the probe (only three copies of pIII fusions are shown).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4403547&req=5

Figure 1: The system for production of functionalized Ff-nano.Escherichia coli cells containing the Ff-nano production plasmid pNJB7 were infected with the helper phage Rnano3FnB containing the coding sequence of a “probe” or “detector” protein fused to pIII. Upon infection, pII from the helper phage induces positive strand replication from the pNJB7 Ff-nano origin of replication and also provides all other phage proteins and assembly machinery for production of the Ff-nano particles. All five copies of pIII are fusions to the probe (only three copies of pIII fusions are shown).
Mentions: In this work we have constructed an Ff-derived phage display system for production of functionalized nanorods (50 nm × 6 nm) we named Ff-nano (Figure 1). In the first stage, we modified a setup for production of short phage originally published by Specthrie et al. (1992) to increase the amount (per cell) of the 221-nt circular ssDNA available as a template for assembly of the short particles. This was achieved by inserting the 303-nt short-phage (microphage or Ff-nano) origin of replication (Supplementary Figure S1) into the high-copy-number plasmid pCR4-TOPO (in contrast to the low-copy-number plasmid pBR322 used in the original system), to obtain plasmid pNJB7. Furthermore, a new helper phage, named Rnano3, was constructed. The helper phage Rnano3, in contrast to the original helper phage R474 used for short phage production (Specthrie et al., 1992), is not only a helper but also a phage display vector. Rnano3 was designed for display of proteins as fusions with phage protein pIII that is present in up to five copies at one end of the Ff-derived particles. Infection of cells containing plasmid pNJB7 with the helper phage/vector Rnano3 resulted in production of the 50 nm × 6 nm particles (Ff-nano or microphage; Figure 2) and full-length phage.

Bottom Line: In contrast to standard Ff-derived vectors that replicate in E. coli and contain antibiotic-resistance genes, Ff-nano are protein-DNA complexes that cannot replicate on their own and do not contain any coding sequences.These nanorods show an increased resistance to heating at 70(∘)C in 1% SDS in comparison to the full-length Ff phage of the same coat composition.We demonstrate that functionalized Ff-nano particles are suitable for application as detection particles in sensitive and quantitative "dipstick" lateral flow diagnostic assay for human plasma fibronectin.

View Article: PubMed Central - PubMed

Affiliation: Institute of Fundamental Sciences, Massey University Palmerston North, New Zealand.

ABSTRACT
F-specific filamentous phage of Escherichia coli (Ff: f1, M13, or fd) are long thin filaments (860 nm × 6 nm). They have been a major workhorse in display technologies and bionanotechnology; however, some applications are limited by the high length-to-diameter ratio of Ff. Furthermore, use of functionalized Ff outside of laboratory containment is in part hampered by the fact that they are genetically modified viruses. We have now developed a system for production and purification of very short functionalized Ff-phage-derived nanorods, named Ff-nano, that are only 50 nm in length. In contrast to standard Ff-derived vectors that replicate in E. coli and contain antibiotic-resistance genes, Ff-nano are protein-DNA complexes that cannot replicate on their own and do not contain any coding sequences. These nanorods show an increased resistance to heating at 70(∘)C in 1% SDS in comparison to the full-length Ff phage of the same coat composition. We demonstrate that functionalized Ff-nano particles are suitable for application as detection particles in sensitive and quantitative "dipstick" lateral flow diagnostic assay for human plasma fibronectin.

No MeSH data available.


Related in: MedlinePlus