Genetic variation is generated using error-prone polymerases on the phage vectors, and over time the protein accumulates beneficial mutations.
The lagoon contains M13 bacteriophage vectors carrying the gene of interest (known as the selection plasmid, or SP), as well as host E. coli cells that allow the phage to replicate.
Hence, a fresh supply of E. coli cells is constantly present in the lagoon, but phage can only be retained via sufficiently fast replication.
[3] Hence, the better the activity of the protein, the higher the rate of pIII production, and the more infectious phage are generated for that particular gene.
Using error-prone polymerases (encoded on the mutagenesis plasmid, or MP), genetic variation is introduced into the protein gene portion of the phage vectors.
This is done by linking undesired activity to the production of non-functional pIII, which decreases the amount of infectious phage made.
Activity of an aaRS is linked to pIII production by the addition of a TAG stop codon in the middle of gIII.
Using this system, aaRSs were evolved that utilize non-canonical amino acids p-nitro-phenyalanine, iodophenylalanine, and Boc-lysine.
[6] This method was used to evolve Bacillus thuringiensis endotoxin variants that can overcome insect toxin resistance.
If the base editor is able to correct the error, functional T7 polymerase is produced, allowing production of pIII.
A general PACE scheme. MP stands for mutagenesis plasmid, and encodes the necessary proteins for introducing mutations into SP. SP stands for selection plasmid, and encodes the gene of the M13 bacteriophage minus gIII, as well as the gene of interest. AP stands for accessory plasmid, and contains gIII, as well as a method for inducing transcription of gIII.
A scheme for evolving polymerase promoter specificity using PACE. T7 RNAP containing mutations unfavorable to T3 promoter binding results in no gIII transcription. However, mutations that allow for the binding to a T3 promoter lead to increased gIII transcription.
A scheme of using PACE to evolve protease activity. When the T7 RNAP and the T7 lysozyme are linked, transcription of gIII is blocked. When the protease is active for the cleavage site, the T7 polymerase is liberated, allowing for transcription of gIII.
A scheme of using PACE to evolve orthogonal aminoacyl-tRNA synthetases. Without synthetase activity, T7 RNAP cannot be fully translated due to the presence of an Amber stop codon. Upon introduction of a functioning synthetase for the Amber codon tRNA, this Amber stop codon now encodes a non-canonical amino acid, allowing for the transcription of the full T7 RNAP and hence allowing gIII transcription.
A scheme for using PACE to evolve protein-protein interactions. The target protein, in purple, is fused to a DNA binding protein, in blue. The evolving protein, in red, is bound to a functioning RNAP, in green. By having the DNA binding protein bind upstream of the gIII promoter, more favorable binding between the target and evolving protein leads to higher transcription of gIII.
A scheme for the engineering of higher soluble expression. POI = protein of interest. When the POI is properly folded, the T7n portion is exposed, allowing for the binding to the T7c portion to form a fully functional T7 RNAP. This T7 RNAP can then transcribe gIII. If the POI is misfolded, then the T7 RNAP doesn't form, leading to no gIII transcription.
Evolution of higher activity deoxyadenosine deaminases using PACE. Here, the T7 RNAP gene contains two stop codons, both containing adenosine nucleotides. Without deoxyadenosine deaminase activity, the resulting T7 RNAP is truncated and hence nonfunctional. However, with a functional deoxyadenosine deaminase, the stop codons are converted into amino acid encoding codons, which allows for the production of functional T RNAP, which can go on to transcribe gIII.
