Introduction

Modulation of Selection Stringency

The directed evolution method PACE is an enormously powerful tool to improve and alter activities of different kinds of proteins for industrial, research or therapeutic applications. In some cases, a radically modified or highly specific new activity is demanded, but as a certain basal activity of the unevolved protein is necessary to propagate the selection phage (SP) with the respective accessory plasmid (AP) this task remains highly challenging. Phages encoding the unevolved protein of interest often display no or only low activity for geneIII expression on their AP. If they cannot propagate sufficiently, the phages in the lagoon are washed-out before they were able to gain favorable mutations. To decrease the stringency of an initial selection, intermediate substrates and constructs can be used as evolutionary stepping stones RN120. The difficulty consists in the fact that these evolutionary stepping stones are often not obvious or barely accessible RNRN46.
An easier and generally applicable approach for selection stringency modulation is a carefully regulated provision of ProteinIII independent of the favored evolving activity. This allows faint active or inactive variants to propagate in the lagoon and to accumulate mutations through evolutionary drift. Some of these mutations improve the evolving protein, which is coupled to the propagation abilities of the phages and enables phages with beneficial variants to persist higher selection pressure. The general principle of stringency modulation via controlled geneIII expression was proved by a small molecule-controlled selection stringency modulator engineered by Carlson et. al. 2014 using anhydrotetracycline (ATc) as geneIII inducer. They demonstrated that an inactive starting phage library can propagate with the addition of ATc and that a decrease of the ATc concentration leads to a selective enrichment of active mutants RN46. As exogenous chemical inducers are often limited due to transport process delay, cause toxicity or show a lack of reversibility of gene expression, we designed a novel optogenetic modulator of selection stringency CG01 CG02 CG03.

Motivation

The modulation of selection stringency is an essential tool for PACE and PREDCEL based directed evolution. As we ourselves often struggled with phage wash-out during the initial selection phases we consider the provision of a non-toxic, rapidly delivered and reversible modulator of the selection stringency as highly important for the scientific community. Our Opto Select System enables an easy adaption of selection pressure to the fitness of the evolving gene pool and minimizes the experimental effort of protein optimization. General rules: if html tags (the things starting with "<" and ending with ">") occur, the whole block in which they occur, have to be inside the following (replacing 'content') content Pages on the wiki can be edited or created by going the URL where the page shall be and choosing wiki tools -> Edit/Create Simply paste the text there and save the page. Images can be uploaded by choosing 'upload files' under 'wiki tools'. Filenames shall be T--Heidelberg--Team_Heidelberg_2017_real-name.png If a file was uploaded, a page showing the file occurs, click on 'original file' and take the URL you are getting to. This URL is the image URL used to embed the image. An editor that matches braces is pretty useful. Check if the braces of main are close properly and the chances are good thta the page will work. headers can be written as follows:

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