PHAGE DISPLAY LIBRARY GENERATION

          A 3-step summary of a peptide library. DNA segments are inserted into plasmids and transformed into bacteria. With the addition of modified bacteriophages, the plasmids are packaged into functional bacteriophages where they express a protein of interest from the plasmid. Together, these unique bacteriophages constitute a phage library.
DESIGNED BY ERIN LEMIEUX


Scientists insert variable DNA sequences coding for their proteins of interest into plasmids that carry a phage coat protein gene, an antibiotic resistance gene, and a packaging signal. Then they transform the plasmids into a bacterial vector and infect it with a helper phage that supplies the other necessary viral proteins. This generates the phage library, where each virion expresses versions of the protein of interest on its coat protein and carries its genetic sequence in its genome. 

AFFINITY PURIFICATION

          A 3-step summary of affinity purification. The phage library is introduced to a target ligand that is presented on a solid surface. Phages expressing a protein that recognize this ligand bind and are retained in a subsequent wash step that removes unbound phages. Bound phages are eluted and kept.
DESIGNED BY ERIN LEMIEUX


In the next step, researchers isolate the phages expressing surface proteins using a surface ligand binding assay. The final eluted phages may bear a mix of different surface protein epitopes. 

PHAGE AMPLIFICATION

          A 3-step summary of phage amplification. The eluted phages are introduced to bacteria and the modified helper phage to propagate more plasmid-containing bacteriophages. These are re-introduced to the surface-bound ligand for additional assessment of binding capacity. Bound phages are again retained and eluted.
DESIGNED BY ERIN LEMIEUX


In the amplification step, researchers propagate the eluted phages in a bacterial culture, and may run additional rounds of affinity purification. 

CLONE ISOLATION

          A 2-step summary of clone isolation. Eluted phages are eventually retained and used to infect bacteria without the aid of the helper phages to propagate them. The plasmid-containing bacteria are cultured on agar plates to select for plasmid-containing colonies. The plasmids are isolated from some of these colonies and can be used for sequence analysis, monoclonal antibody production, or other protein analyses.
DESIGNED BY ERIN LEMIEUX


Researchers infect bacteria with the selected phages and culture them on plates containing antibiotics. In the final step, they pick resistant colonies to isolate the plasmids, which are then sequenced and cloned into the desired protein production vectors for various applications.

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