Latest revision as of 03:00, 2 November 2017

Blue light-induced promotor for application in Phage assisted continous evolution (PACE)

This part consists of a Blue light-induced promotor, flanked by two homology regions for the usage due to the cloning standard of the iGEM Team Heidelberg 2017 (http://2017.igem.org/Team:Heidelberg/RFC). Figure one gives a short overview of our standard. Our BioBricks from the registry can easily be used for the assembly of blasmid with the standard (Fig.: 2).

Figure 1: In our cloning standard, compatible building blocks are defined by specific functionalities. They are flanked by defined homology regions, indicated by numbers, which are necessary for the assembly of the APs with the Gibson method. This results in a highly customizable plasmid, composed of the desired origin of replication, an antibiotic resistance (4-5), a bicistronic operon with geneIII (2-3)and the desired reporter (3-4), which can be activated by any promoter (1-2)and a second expression cassette for additional genes that are necessary for the respective circuit (1-5).

Figure 2: Compatibility of our cloning stadard with the RFC10;Any AP building block can be cloned into RFC[10] standard by inserting BglII sites between the homology regions and the biobrick prefix or suffix, respectively. To use such a part for AP assembly, it has to be digested with BglII. The resulting fragment should be purified and can subsequently used for Gibson assembly with other parts.

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 <x-ref>CG04</x-ref>. 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 <x-ref>CG04</x-ref>. 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 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> RN46</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. 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.

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.

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

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.