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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 phages<x-ref>CG06</x-ref>, 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 <x-ref>Carlson.2014</x-ref>. 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. <i>E. coli</i>, 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. | 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 phages<x-ref>CG06</x-ref>, 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 <x-ref>Carlson.2014</x-ref>. 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. <i>E. coli</i>, 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. | ||
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Revision as of 23:19, 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
![](
https://static.igem.org/mediawiki/2017/c/cc/T--Heidelberg--Team_Heidelberg_2017_EL222cystalstrucpymol2.png)
Figure 1: Crystal Structure of EL222
The light-inducible transcription factor EL222 is classified into three parts: the LOV domain (blue), the conector helix (grey) and the HTH domain (red). The flavinmononucleotide is depicted in green.
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 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
![](
https://static.igem.org/mediawiki/2017/d/da/T--Heidelberg--Team_Heidelberg_2017_CG_OptoPartyPartyParty.png)
Figure 2: Expression Cassetts of OptoSELECT
![](
https://static.igem.org/mediawiki/2017/6/6f/T--Heidelberg--Team_Heidelberg_2017_CGOptoSelectSystem.png)
Figure 2: Expression Cassetts of OptoSELECT
Selection 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.
Results
AP_light: Testing of the pBLind-gIII Expression Cassette
A phage propagation assay was performed to investigate the impact of blue light irradiation on the propagation of geneIII-deficient M13 phages containing EL222 in a culture transformed with the pBLind-gIII cassette of or OptoSELECT system. As we speculated that light might influence the fitness of E. coli and therefore phage propagation, we created a phage carrying the gene for a truncated, non-binding version of EL222. Both phages were used in this propagation assay. An E. coli culture transformed with AP_light was infected with either SP Opto EL222 containing the gene of a functional EL222 protein or phages with a truncated version of EL222 (107 PFU/ml). The cultures were split and cultivated in the dark or under blue light illumination pulses (15 s ON, 45 s OFF; 3 W/m2) for 3 h at 37 °C. Afterwards, one tenth of the culture volume was used to infect a fresh AP_light culture of OD600 0.6, which was again cultivated for 3 h either in the dark or upon blue light irradiation. These steps were repeated three times (cultivation time: 4 x 3 h). Cultures for phage propagation testing under the respective conditions were performed in duplicates. Sample were taken from the last cultures and a plaque assay was performed. The phage titer of the respective cultures was calculated and plotted in the bar chart below (Fig: 3). In this experiment a more than 3-fold increased phage titer of SP Opto EL222 under blue light irradiation compared to the dark state was demonstrated, whereas the non-binding variant exhibited no significant difference.![](
https://static.igem.org/mediawiki/2017/1/16/T--Heidelberg--Team_Heidelberg_2017_AP-light-results.png)
Increase of Phage Propagation under Blue Light using pBLind-geneIII Cassette
Phage titers of SP Opto EL222 and a non-binding variant propagated on AP_light in the dark and upon blue light irradiation after four passages were determined by plaque assays. Host cell cultures infected with SP Opto EL222 and cultured upon blue light irradiation demonstrated a more than 3-fold higher phage titer than the culture cultivated in the dark (left side). The infection with phages containing the non-binding variant of EL222 exhibited no significant difference in phage titer between a cultivation in the dark or under blue light (right side). It was notable that for this variant the phage titer was slightly decreased upon light irradiation. The respective plaque assays are shown below the bar chart.
AP_dark: Testing of the Psp-EL222-BR-gIII Expression Cassette
To determine the influence of light and dark states on the SP Opto EL222 phage propagation with AP_dark, a phage propagation assay similar to AP testing of AP_light was performed. As previous experiments demonstrated a significantly increased phage propagation compared to AP_light, the cultivation time per cycle was reduced to one hour. All other parameters of the propagation assay remained. Samples were taken, and phage titer were determined by plaque assays. Tests with the SP non-binding EL222 variant demonstrated much higher phage titer, which were not influenced by light. After one hour of incubation, cultures incubated in the dark demonstrated a SP Opto phage titer nearly twice as high as the respective sample cultivated upon blue light. Two hours and passages later, there was no significant difference between the phage titer of cultures cultivated upon light or in the dark.![](
https://static.igem.org/mediawiki/2017/f/fa/T--Heidelberg--Team_Heidelberg_2017_CG_AP_dark.png)
Increase of Phage Propagation in the Dark using Ppsp-EL222-BR-geneIII Cassette
Phage titers of SP Opto EL222 propagated on AP_dark upon blue light irradiation and in the dark after one and three hours of cultivation (two passages) were determined by plaque assays. After one hour of cultivation host cell cultures infected with SP Opto EL222 and cultured in the dark nearly demonstrated a phage titer twice as high as the culture cultivated upon light irradiation (left side). Two hours later, this result could not be confirmed as the phage titer of cultures cultivated upon blue light irradiation and in the dark were similar (right side).
Outlook
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https://static.igem.org/mediawiki/2017/e/e3/T--Heidelberg--Team_Heidelberg_2017_CG_OptoPartyParty.png)
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