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 RN16. The difficulty consists in the fact that these evolutionary stepping stones are often not obvious or barely accessible RN46.
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.

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 illumination CG04. The naturally occuring light activated transcription factor EL222 from the marine bacterium Erythrobacter litoralis HTCC2594 consists of a N-terminal light-oxygen-voltage (LOV) domain, a linker helix and a C-terminal LuxR-type helix-turn-helix (HTH) motif CG04 (Fig: 1). Upon blue light irradiation with a wavelength of 450 nm, an internal flavinmononucleotide-protein adduct is formed, which results in a conformational change of the modular 222 amino acid photosensitive protein. Subsequently, a previously sequestered DNA-binding domain is exposed, which allows the homo-dimerization of EL222, and thus DNA binding. In the dark, the process spontaneously reverses. EL222 is released from the DNA, and deactivates transcription within seconds to minutes CG05.

Motivation

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 region CG04. Upon blue light irradiation, the EL222 dimer binds to its binding region on the DNA and activates transcription by recruiting the RNA polymerase (RNAP). Consequently, the protein expression increase up to 5-fold compared to the dark state CG04. To regulate phage propagation and to adapt selection stringency in a light-dependent manner, geneIII was set under control of pBLind. This plasmid containing the pBLind-gIII expression cassette is further referred as AP_light. PACE and PREDCEL experiments with host cells carrying AP_light display a reduced selection stringency in the initial selection phase by illumination with blue light. As soon as the starting phage library acquires enough favorable mutations to persist higher selection pressure, the light can be switched off and the intensified expression of geneIII is aborted.
To 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 phagesCG06, was combined with an EL222 binding region to repress gene expression post infection in the present of blue light irradiation. A similar promoter was previously engineered consisting of a Psp promoter and a tetracycline binding region RN46. To engineer a novel light-dependent promoter, this Psp-tet promoter was used, but instead of a tetracycline binding region an EL222 binding region (EL222-BR) was inserted adjacent to the 1+ transcription initiation site. The plasmid containing the Ppsp-EL222-BR-gIII expression cassette is referred as AP_dark. E. coli, which are transformed with AP_dark, express geneIII in the dark state only post phage infection. Upon blue light irradiation EL222 binds to the EL222 binding region and inhibits the expression of supplementary geneIII.

Results

Outlook

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