Used strains and expression plasmids.
E.coli Nissle 1917 (EcN) [16] served as the parental strain for the construction of the new EcN(T7) strain. Test expressions were performed using E. coli BL21(DE3), EcN and EcN(T7) (Table S1). All plasmids used (Table S2) were verified via sequencing (GATC Eurofins genomics, Cologne; Seq-IT, Kaiserslautern).
Construction of the expression cassette.
Genomic DNA from E. coli BL21(DE3) was used as a template to obtain the T7-RNA polymerase gene. All oligonucleotides used in the construction are listed in the Supporting Information (Table S3). Via two PCR reactions overlapping primers (lac-op-fwd, lacUV5-HindIII-fwd) were used to add the lac operator and lacUV5 promoter to the 5’-end of the T7 RNA polymerase gene to allow for isopropyl-β-D-thiogalactopyranoside (IPTG) inducible gene expression. A kanamycin resistance cassette, with flanking FRT sites, was amplified from pKD13 and ligated via a SalI restriction site to the 3’-end of the T7-RNA polymerase gene in order to generate a selection marker for the following homologous recombination reaction. The thus obtained lacUV5-T7-FRT-kan-FRT (T7/Kan) fragment was purified via gel extraction and blunt end cloned into the plasmid pYP168 [17] via a SmaI restriction site. This products (pUC-T7-FRT-kan) was then used as a PCR template to add 50 bp of homologous sequences from the malEFG operon of E. coli Nissle 1917 (T7-mal-fwd and T7-mal-rev) to the expression cassette at both ends. The fragment was purified via agarose gel extraction.
Chromosomal insertion of T7-RNA polymerase via homologous recombination.
For the insertion of the T7/Kan expression cassette into EcN, the λ-Red recombinase system was used as described previously (Fig. 1) [18, 19]. The insertion cassette was introduced via site-specific homologous recombination into the malEFG operon of E. coli Nissle 1917 (oligonucleotides T7-mal-fwd & T7-mal-rev). Insertion of the resistance cassette and loss of malEFG operon was verified by plating transformation reactions onto MacConkey agar plates containing 1% maltose and 50 µg/ml kanamycin. The kanamycin cassette was removed via flanked FRT recombination sites to obtain a markerless mutant using pCP20 [20]. The correct insertion of the T7 RNA polymerase gene was verified via sequencing (Eurofins Genomics).
Test production of T7-RNA-Polymerase induced protein production.
T7 based expression vectors from our lab collection (See Table S2) were transformed into BL21(DE3) as a positive control, EcN as a negative control and into the newly constructed EcN(T7) strain. Test productions were performed in 50 ml LB medium containing the appropriate antibiotics and 100 mM sorbitol. For Rdms_O216K LB high salt medium (0.5 M NaCl) without sorbitol was used. Cultures were incubated at 37 °C up to an OD600 of 0.7 for BL21(DE3) and OD600 of 1.2 for EcN and EcN(T7). Before induction, cultures were cooled down to 17 °C. Expression was induced by adding 0.5 mM IPTG for pACYC-rdmS_O216K and 0.1 mM IPTG for pTD-ho1, pET-cph1 and pACYC-ho1-pcyA to the culture. Expression of bphP was induced with 200 ng/ml anhydrotetracycline. For the production of phycocyanobilin (PCB) and biliverdin (BV) for the holo-phytochrome increasing amounts of hemin (in DMSO) were added in 2 h after induction. Cultures were incubated at 17°C, 160 rpm overnight, harvested for 10 min, 9000 rpm (Sorvall LYNX 6000, F14 rotor), 4 °C and stored at -20 °C. Samples were taken before induction and after overnight incubation and were diluted to an OD600 of 0.5. Cell pellets were disrupted by sonification and separated via SDS-PAGE. Subsequently, the separated proteins were either stained with Coomassie Brilliant Blue or detected using a Western blot with the appropriate antibodies.
Production and purification of recombinant produced StrepII-tagged proteins.
For purification of RdmS_O216K, production was conducted in 2 l LB high salt medium. Cells were incubated as described before [21]. Immediately after induction, 10 µM hemin was added for the production of the holo-protein. Cell pellets were suspended in buffer W (100 mM Tris/HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 10% glycerol) and 1 mM DTT, 0.25 mM 4-(2-aminoethyl) benzene-sulfonyl fluoride and spatula tip of DNase I and lysozyme were added. Cells were incubated on ice for 30 min and disrupted via a Microfluidizer LM20 (Microfluidics Corp., Westwood, MA, USA) at 15,000 PSI. Cell debris were removed via centrifugation at 4°C and 19,000 rpm for 1 h (Sorvall LYNX 6000, T29 rotor). A Strep-Tactin chromatography column (IBA GmbH, Göttingen), equilibrated with buffer W, was used for affinity chromatography. Unwanted proteins were removed by washing with 10 column volumes of buffer W. Elution of StrepII-tagged proteins was performed with buffer E (buffer W containing 2.5 mM D-desthiobiotin). Elution fractions containing the desired protein were dialyzed against 20 mM TES buffer, pH 8.0, containing 100 mM KCl and 10% glycerol. Proteins were concentrated using Amicon concentrator devices (molecular weight cut-off 100,000; Merck).
UV-visible spectroscopy.
UV-vis spectroscopy was performed using an 8453 UV-visible spectrophotometer (Agilent Technologies). Heme spectra were taken at room temperature in 20 mM TES buffer pH 8.0 containing 100 mM KCl and 10% glycerol. Spectra were taken from 350-700 nm under oxidizing conditions. Phytochrome spectra were taken at 25°C using cell-free lysates (100 mM Tris/HCl pH 8.0, 150 mM NaCl, 1 mM EDTA). The BphP samples were incubated for 3 min with red light at 690 nm for Pfr spectra and 3 min with far-red light at 750 nm for Pr spectra as described previously [22]. The Pfr spectra were subtracted from the Pr spectra to obtain the red/infrared induced difference spectra. For Cph1, difference spectra were obtained in a similar way, expect that the red and far-red light filters of 630 and 730 nm were used, respectively [23]. To analyze the saturation of Cph1 with its chromophore, difference spectra were measured again after 30 min incubation with 40 µM phycocyanobilin for 30 min at room temperature.