Bacteriophage M13
It is a commonly used model system for studying viral biology, protein engineering, and biotechnology applications.
Bacteriophage M13 has a single-stranded DNA genome and a rod-shaped capsid structure.
Its small size, ease of manipulation, and ability to display foreign peptides on its surface make it a versatile tool for research.
Optimized protocols and methods for working with Bacteriaphage M13 can enhance reproducibility, accuracy, and efficiency in your research endeavors.
Most cited protocols related to «Bacteriophage M13»
For the synthesis of DNs, 10 nM p7308 scaffold was mixed with tenfold excess of staples in TE buffer (5 mM Tris and 1 mM EDTA) containing 10–20 mM MgCl2. The amount of MgCl2 varied with the structure: DN
Most recents protocols related to «Bacteriophage M13»
Example 2
Bovine serum albumin (BSA), erbB2 extracellular domain (HER2) and streptavidin (100 μl of 2 μg/ml) were separately coated on Maxisorp 96 well plates. After blocking with 0.5% Tween-20 (in PBS), biotinylated and non-biotinylated hu4D5Fabv8-ThioFab-Phage (2×1010 phage particles) were incubated for 1 hour at room temperature followed by incubation with horseradish peroxidase (HRP) labeled secondary antibody (anti-M13 phage coat protein, pVIII protein antibody).
Standard HRP reaction was carried out and the absorbance was measured at 450 nm. Thiol reactivity was measured by calculating the ratio between OD450 for streptavidin/OD450 for HER2. A thiol reactivity value of 1 indicates complete biotinylation of the cysteine thiol. In the case of Fab protein binding measurements, hu4D5Fabv8 (2-20 ng) was used followed by incubation with HRP labeled goat polyclonal anti-Fab antibodies.
Example 4
FACS Screening:
In order to estimate the infection ability of the virus-like model, the plaque assay was used to determine the titer of the produced Model-S since it is the gold standard for titer measurement. The displayed phages Model-S from different dilution ratios were employed to infect E. coli TG1. After that, LB solid culture media with kanamycin was applied to provide a space for cultivation. Moreover, the mixtures were cultivated at 37 °C overnight. The plates were collected, and all the plaques were counted to obtain the titer of Model-S the following day.
The molecular interaction instrument ForteBio Octet K2 (SARTORIUS, Göttingen, Germany) was used to assess the combination between the produced Model-S and the corresponding anti-SARS-CoV-2 spike protein antibody (Rabbit pAb). The interaction between these biomolecules could be monitored and collected through the shift of the probe surface reflection interference spectrum following the thin film interferometry technology. The process, including association, dissociation, and regeneration, was performed repeatedly according to the preprogramming. After the measurement, the association constant (ka) and dissociation constant (kd) were obtained. Moreover, the affinity constant could be calculated.
After production and verification, the two virus-like models: Model-N [45 (link)] and Model-S, were prepared. Accompanied by these two models (as biothreats), M13 phage, bovine serum albumin (BSA), ovalbumin (OVA), commercial N protein, commercial S1 protein, commercial S2 protein, and E. coli TG1 were used as control samples for further study. All selected samples are related to the actual SARS-CoV-2 virus. The produced Model-S and Model-N are the substitutions of SARS-CoV-2 in this research, which could be regarded as the combination of M13 phage (main body) and N/S protein (p3 site). The M13 phage used in the phage display to synthesize the models is a kind of virus that belongs to the virus family, as SARS-CoV-2 does. BSA and OVA are common proteins for scientific research. In addition, the N protein, S1 protein, and S2 protein are the structural proteins of SARS-CoV-2. Furthermore, as common pathogen types, both bacteria and viruses can cause the outbreak of large-scale infectious diseases. Thus, E. coli TG1 was also included for identification research since one of the hosts of the M13 phage is E. coli TG1, and it was also used to produce the models. Therefore, the nine selected samples are more or less related to the SARS-CoV-2 virus. The higher the relation between the sample and the SARS-CoV-2 virus (or its substitution), the more difficult it might be to distinguish. In general, the nine selected samples, including viruses, proteins, and bacteria, might be representative of the research. The recognition of the Model-S and Model-N among those related samples could provide a possibility for the nondestructive detection and identification of targeted biothreats. Therefore, the nine samples were prepared for spectroscopy analysis.
After searching the gene corresponding to the S protein of SARS-CoV-2 at the National Center for Biotechnology Information (NCBI) website (Gene ID: 43740568), it was modified by adding SfiI and NotI restriction digest sites on both sides. After the regular PCR amplification and double endonuclease digestion by SfiI/NotI enzyme, the inserted fragment was prepared. The dosage and duration conditions were in accordance with the previous work, as shown in
When the vector pHB was digested by the same enzymes as well, the two parts were constructed and combined with recombinant vector pHB-S produced. The experimental conditions followed the same protocol preparing pHB-N, as presented in
Then, it was transformed into the competent E. coli TG1. After cultivation, M13 phages were added to infect TG1, and the synthesized Model-S was finally produced after filtrations. The specific cultivation process was described before [45 (link)].
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More about "Bacteriophage M13"
This single-stranded DNA (ssDNA) phage is known for its ability to infect the common bacterium Escherichia coli (E. coli), making it a valuable tool for scientists.
One of the key advantages of Bacteriophage M13 is its small size and ease of manipulation, which allows for efficient genetic engineering and display of foreign peptides on its surface.
This feature has led to its widespread use in applications such as phage display, where the phage's surface is modified to present a diverse library of peptides or proteins for screening and selection purposes.
The M13 phage is often used in conjunction with other laboratory tools and reagents, such as 96-well plates, which provide a convenient format for high-throughput experiments.
Additionally, the M13KE and M13KO7 helper phages are commonly utilized to assist in the production and purification of recombinant M13 phage particles.
Researchers working with Bacteriophage M13 may also encounter the use of E. coli clone n 29664, a specific bacterial strain commonly used as a host for M13 phage propagation.
Other related materials, such as TMB (3,3',5,5'-Tetramethylbenzidine) for colorimetric assays, T4 DNA ligase for genetic manipulations, and antibodies like Anti-M13 bacteriophage antibody and Anti-mouse IgG-R antibody, are often employed in experiments involving the M13 phage system.
By leveraging the insights and techniques associated with Bacteriophage M13, researchers can enhance the reproducibility, accuracy, and efficiency of their research endeavors, leading to valuable advancements in fields ranging from viral biology to biotechnology applications.