Optimizing protein V untranslated region sequence in M13 phage for increased production of single-stranded DNA for origami.
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ABSTRACT:
SUBMITTER: Lee BY
PROVIDER: S-EPMC8216270 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Background. How host-symbiont interactions coevolve between mutualism and parasitism depends on the ecology of the system and on the genetic and physiological constraints of the organisms involved. Theory often predicts that greater reliance on horizontal transmission favors increased costs of infection and may result in more virulent parasites or less beneficial mutualists. We set out to understand transitions between parasitism and mutualism by evolving the filamentous bacteriophage M13 and its host Escherichia coli. Results. The effect of phage M13 on bacterial fitness depends on the growth environment, and initial assays revealed that infected bacteria reproduce faster and to higher density than uninfected bacteria in 96-well microplates. These data suggested that M13 is, in fact, a facultative mutualist of E. coli. We then allowed E. coli and M13 to evolve in replicated environments, which varied in the relative opportunity for horizontal and vertical transmission of phage in order to assess the evolutionary stability of this mutualism. After 20 experimental passages, infected bacteria from treatments with both vertical and horizontal transmission of phage had evolved the fastest growth rates. At the same time, phage from these treatments no longer benefited the ancestral bacteria. Conclusions. These data suggest a positive correlation between the positive effects of M13 on E. coli hosts from the same culture and the negative effects of the same phage toward the ancestral bacterial genotype. The results also expose flaws in applying concepts from the virulence-transmission tradeoff hypothesis to mutualism evolution. We discuss the data in the context of more recent theory on how horizontal transmission affects mutualisms and explore how these effects influence phages encoding virulence factors in pathogenic bacteria.
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Project description:Phage display (PD) is a tool for developing new molecules to control pathogens. Peptides selected by PD are commonly synthesised and tested, but the use of phage M13 displaying the selected peptides as a direct biding in the intestinal tract has not yet been tested. This study evaluated whether phage M13 can remain viable in the chicken gastrointestinal tract and whether it causes injury or humoral immune response. We inoculated phage M13 or E. coli ER2738 (ECR) infected with M13 into birds at different ages. We found the virus in faeces at 5 or 13 days after inoculation, just when it infected the ECR. The presence of phage M13 or ECR did not result in gut injuries and had no impacts on weight gain and bird health. Furthermore, the levels of IgY were similar in all treatments, which indicates that the virus can be used in chicken until 42 days without being recognised by the immune system. This work provides a scientific basis for the use of PD as a tool in numerous applications to control different pathogens.
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Project description:Scaffolded DNA origami is a widely used technology for self-assembling precisely structured nanoscale objects that contain a large number of addressable features. Typical scaffolds are long, single strands of DNA (ssDNA) that are folded into distinct shapes through the action of many, short ssDNA staples that are complementary to several different domains of the scaffold. However, sources of long single-stranded DNA are scarce, limiting the size and complexity of structures that can be assembled. Here we demonstrated that dsDNA (double-stranded DNA) scaffolds can be directly used to fabricate integrated DNA origami structures that incorporate both of the constituent ssDNA molecules. Two basic principles were employed in the design of scaffold folding paths: folding path asymmetry and periodic convergence of the two ssDNA scaffold strands. Asymmetry in the folding path minimizes unwanted complementarity between staples, and incorporating an offset between the folding paths of each ssDNA scaffold strand reduces the number of times that complementary portions of the strands are brought into close proximity with one another, both of which decrease the likelihood of dsDNA scaffold recovery. Meanwhile, the folding paths of the two ssDNA scaffold strands were designed to periodically converge to promote the assembly of a single, unified structure rather than two individual ones. Our results reveal that this basic strategy can be used to reliably assemble integrated DNA nanostructures from dsDNA scaffolds.
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Project description:BackgroundMetal borides are a class of inorganic solids that is much less known and investigated than for example metal oxides or intermetallics. At the same time it is a highly versatile and interesting class of compounds in terms of physical and chemical properties, like semiconductivity, ferromagnetism, or catalytic activity. This makes these substances attractive for the generation of new materials. Very little is known about the interaction between organic materials and borides. To generate nanostructured and composite materials which consist of metal borides and organic modifiers it is necessary to develop new synthetic strategies. Phage peptide display libraries are commonly used to select peptides that bind specifically to metals, metal oxides, and semiconductors. Further, these binding peptides can serve as templates to control the nucleation and growth of inorganic nanoparticles. Additionally, the combination of two different binding motifs into a single bifunctional phage could be useful for the generation of new composite materials.ResultsIn this study, we have identified a unique set of sequences that bind to amorphous and crystalline nickel boride (Ni3B) nanoparticles, from a random peptide library using the phage display technique. Using this technique, strong binders were identified that are selective for nickel boride. Sequence analysis of the peptides revealed that the sequences exhibit similar, yet subtle different patterns of amino acid usage. Although a predominant binding motif was not observed, certain charged amino acids emerged as essential in specific binding to both substrates. The 7-mer peptide sequence LGFREKE, isolated on amorphous Ni3B emerged as the best binder for both substrates. Fluorescence microscopy and atomic force microscopy confirmed the specific binding affinity of LGFREKE expressing phage to amorphous and crystalline Ni3B nanoparticles.ConclusionsThis study is, to our knowledge, the first to identify peptides that bind specifically to amorphous and to crystalline Ni3B nanoparticles. We think that the identified strong binding sequences described here could potentially serve for the utilisation of M13 phage as a viable alternative to other methods to create tailor-made boride composite materials or new catalytic surfaces by a biologically driven nano-assembly synthesis and structuring.
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