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Revision as of 17:42, 1 November 2017
Optogenetics
Modulator of selection stringency
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 stonesAn 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
Optogenetic Tools
In the past ten years the prospects of light-regulated systems rapidly expanded and became a powerful application tool in cell biology, neuroscience, and medical research. Optogenetic tools enable the regulation of biological systems in a non-invasive and reversible manner. In contrast to widely used chemical triggers, light can be applied with high spatial and temporal precision, and does not cause unintended side effects or off-target effects, which are common for most chemical inducers. Furthermore, photosensitive proteins can be easily regulated by light intensity and duration of light illuminationMotivation
The modulation of selection stringency is an essential requirement for PACE and PREDCEL based directed evolution. As we ourselves often struggled with phage wash-out during the initial selection phases, we considered the provision of a non-toxic, rapidly delivered and reversible modulator of the selection stringency as highly important for the scientific community. Our OptoSELECT system enables an easy and prompt adaption of selection pressure to the fitness of the evolving gene pool and minimizes the experimental effort of protein optimization using PACE and PREDCEL.Design of OptoSELECT
The OptoSELECT System composes of the previously described, blue light-dependent transcription factor EL222 and two bidirectional geneIII expression cassettes: the blue light induced pBLind-gIII cassette and the blue light repressed Ppsp-EL222-BR-gIII cassette (Fig: 2). pBLind is a synthetic, light-inducible promoter based on the luxI promoter. The lux box, a 20-bp inverted repeat from the luxI promoter, is replaced by the 18-bp EL222 binding regionTo provide a complementary promoter system that allows an increase of selection pressure upon blue light irradiation, we designed the hybrid Psp-EL222-BR promoter. Therefore, the phage-shock-protein promoter (Ppsp), which is induced by infection with filamentous phages
Results
lection stringency can be easily modulated using the plasmids AP_light and AP_dark. AP_light contains the blue light induced pBLind-gIII expression cassette, which consists of geneIII under control of a modified luxI promoter with an EL222 binding region. In the dark state EL222 cannot bind to the DNA and the transcription of geneIII is repressed. Upon blue light irradiation EL222 undergoes a conformational change and binds to the EL222 binding region. This interaction recruits the RNA polymerase and activates the transcription of geneIII. AP_dark contains the blue light repressed Psp-EL222-BR-gIII cassette. After phage infection the psp promoter is activated and initiates the expression of geneIII. In the dark state this process can proceed without hindrance. Upon blue light irradiation EL222 binds adjacent to the 1+ transcription initiation site and inhibits the expression of geneIII.Outlook
lection stringency can be easily modulated using the plasmids AP_light and AP_dark. AP_light contains the blue light induced pBLind-gIII expression cassette, which consists of geneIII under control of a modified luxI promoter with an EL222 binding region. In the dark state EL222 cannot bind to the DNA and the transcription of geneIII is repressed. Upon blue light irradiation EL222 undergoes a conformational change and binds to the EL222 binding region. This interaction recruits the RNA polymerase and activates the transcription of geneIII. AP_dark contains the blue light repressed Psp-EL222-BR-gIII cassette. After phage infection the psp promoter is activated and initiates the expression of geneIII. In the dark state this process can proceed without hindrance. Upon blue light irradiation EL222 binds adjacent to the 1+ transcription initiation site and inhibits the expression of geneIII.Large header
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