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Phage display cycle. 1) fusion proteins for a viral coat protein + the gene to be evolved (typically an antibody fragment) are expressed in bacteriophage. 2) the library of phage are washed over an immobilised target. 3) the remaining high-affinity binders are used to infect bacteria. 4) the genes encoding the high-affinity binders are isolated.
The first step is to have phage display libraries prepared. This involves inserting foreign desired gene segments into a region of the bacteriophage genome, so that the peptide product will be displayed on the surface of the bacteriophage virion. The most often used are genes pIII or pVIII of bacteriophage M13. [5]
The T7 promoter region allows large-scale in vitro T7 transcription to transcribe the DNA library into an mRNA library, which provides templates for the in vitro translation reaction later. The ribosomal binding site in the 5’-untranslated region (5’ UTR) is designed according to the in vitro translation system to be used.
The 'helper' phage infects the bacterial host by first attaching to the host cell's pilus and then, after attachment, transporting the phage genome into the cytoplasm of the host cell. Inside the cell, the phage genome triggers production of single stranded phagemid DNA in the cytoplasm. This phagemid DNA is then packaged into phage particles.
Phage libraries are also stored and screened more easily than cosmid libraries. Target DNA: the genomic DNA to be cloned has to be cut into the appropriate size range of restriction fragments. This is usually done by partial restriction followed by either size fractionation or dephosphorylation (using calf-intestine phosphatase) to avoid ...
Phage typing is based on the specific binding of phages to antigens and receptors on the surface of bacteria and the resulting bacterial lysis or lack thereof. [4] The binding process is known as adsorption. [5] Once a phage adsorbs to the surface of a bacteria, it may undergo either the lytic cycle or the lysogenic cycle. [6]
Phage-assisted continuous evolution (PACE) is a phage-based technique for the automated directed evolution of proteins. It relies on relating the desired activity of a target protein with the fitness of an infectious bacteriophage which carries the protein's corresponding gene.
The RNA-dependent RNA polymerase that replicates both the positive and negative RNA strands is a complex of four proteins: the catalytic beta subunit (replicase, ) is encoded by the phage, while the other three subunits are encoded by the bacterial genome: alpha subunit (ribosomal protein S1), gamma subunit (), and delta subunit ().