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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

Ff-nano resistance to heating in SDS. (A) Intact (undamaged) particles and free ssDNA released from particles by the heat/SDS-treatment. Free ssDNA was visualized by staining the gel with ethidium bromide. Bands corresponding to the intact particles were visualized after soaking the gel in 0.4 M NaOH (see Materials and Methods for details). Gel sections containing the bands corresponding to the free ssDNA and intact full-length and Ff-nano particles are boxed. (B) Free ssDNA only, released by the heat/SDS treatment (visualized by direct ethidium bromide staining of the gel prior to NaOH treatment).
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Figure 3: Ff-nano resistance to heating in SDS. (A) Intact (undamaged) particles and free ssDNA released from particles by the heat/SDS-treatment. Free ssDNA was visualized by staining the gel with ethidium bromide. Bands corresponding to the intact particles were visualized after soaking the gel in 0.4 M NaOH (see Materials and Methods for details). Gel sections containing the bands corresponding to the free ssDNA and intact full-length and Ff-nano particles are boxed. (B) Free ssDNA only, released by the heat/SDS treatment (visualized by direct ethidium bromide staining of the gel prior to NaOH treatment).

Mentions: In the course of Ff-nano analyses, we observed that the standard protocol for Ff phage in vitro disassembly, heating in a buffer containing 1% (34 mM) ionic detergent sodium dodecyl sulfate (SDS) for 5 min at 70°C, was not efficient in releasing ssDNA from the Ff-nano particles (data not shown). This indicated that the Ff-nano particles could be more stable to heat/SDS treatment than the full length helper phage, even though both are assembled within the same E. coli cell. To test this hypothesis, a time-course experiment of heat exposure was used to monitor disassembly of full-length helper phage and Ff-nano isolated from the same culture using differential PEG precipitation and preparative agarose electrophoresis, as described in the previous section. Approximately 2 × 1012 Ff-nano or 1 × 1011 full-length phage were heated at 70°C in the presence of 1% SDS for a period from 5 to 20 min; one sample was also incubated at 100°C for 5 min. Disassembly of the helper (full-length) phage and Ff-nano particles was monitored by agarose gel electrophoresis (Figure 3). Released ssDNA was directly visualized by staining with ethidium bromide (Figure 3B), while the ssDNA that remained encapsidated inside the intact Ff-derived particles (resistant to heat/SDS treatment) was not detectable by direct staining. In order to visualize the SDS-resistant intact particle bands after electrophoresis, the coat proteins were dissociated from ssDNA in situ by soaking the gel in an alkaline buffer (0.4 M NaOH), followed by neutralization and re-staining of the gel by ethidium bromide (Figure 3A).


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)

Ff-nano resistance to heating in SDS. (A) Intact (undamaged) particles and free ssDNA released from particles by the heat/SDS-treatment. Free ssDNA was visualized by staining the gel with ethidium bromide. Bands corresponding to the intact particles were visualized after soaking the gel in 0.4 M NaOH (see Materials and Methods for details). Gel sections containing the bands corresponding to the free ssDNA and intact full-length and Ff-nano particles are boxed. (B) Free ssDNA only, released by the heat/SDS treatment (visualized by direct ethidium bromide staining of the gel prior to NaOH treatment).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Ff-nano resistance to heating in SDS. (A) Intact (undamaged) particles and free ssDNA released from particles by the heat/SDS-treatment. Free ssDNA was visualized by staining the gel with ethidium bromide. Bands corresponding to the intact particles were visualized after soaking the gel in 0.4 M NaOH (see Materials and Methods for details). Gel sections containing the bands corresponding to the free ssDNA and intact full-length and Ff-nano particles are boxed. (B) Free ssDNA only, released by the heat/SDS treatment (visualized by direct ethidium bromide staining of the gel prior to NaOH treatment).
Mentions: In the course of Ff-nano analyses, we observed that the standard protocol for Ff phage in vitro disassembly, heating in a buffer containing 1% (34 mM) ionic detergent sodium dodecyl sulfate (SDS) for 5 min at 70°C, was not efficient in releasing ssDNA from the Ff-nano particles (data not shown). This indicated that the Ff-nano particles could be more stable to heat/SDS treatment than the full length helper phage, even though both are assembled within the same E. coli cell. To test this hypothesis, a time-course experiment of heat exposure was used to monitor disassembly of full-length helper phage and Ff-nano isolated from the same culture using differential PEG precipitation and preparative agarose electrophoresis, as described in the previous section. Approximately 2 × 1012 Ff-nano or 1 × 1011 full-length phage were heated at 70°C in the presence of 1% SDS for a period from 5 to 20 min; one sample was also incubated at 100°C for 5 min. Disassembly of the helper (full-length) phage and Ff-nano particles was monitored by agarose gel electrophoresis (Figure 3). Released ssDNA was directly visualized by staining with ethidium bromide (Figure 3B), while the ssDNA that remained encapsidated inside the intact Ff-derived particles (resistant to heat/SDS treatment) was not detectable by direct staining. In order to visualize the SDS-resistant intact particle bands after electrophoresis, the coat proteins were dissociated from ssDNA in situ by soaking the gel in an alkaline buffer (0.4 M NaOH), followed by neutralization and re-staining of the gel by ethidium bromide (Figure 3A).

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