Journal of Experimental Microbiology and Immunology (JEMI) Vol. 18: 121 – 128 Copyright © April 2014, M&I UBC Attempted construction of a soxR::PsoxS::lacZ recombinant pBR322 plasmid as a sensitive reporter of internal reactive oxygen species in Escherichia coli DH5α Dominique Haag, Tony Hui, Seo Young Kim, Jane Nguyen Department Microbiology & Immunology, University of British Columbia This study attempted to engineer an internal reactive oxygen species probe using lacZ expression driven by the soxS promoter in response to intracellular superoxide presence being sensed by SoxR. Using polymerase chain reaction amplification, soxR fused to the promoter of soxS (PsoxS) and lacZ amplicons were obtained. Double restriction digests and sticky-end ligations were then performed in an attempt to clone soxR::PsoxS::lacZ into the pBR322 plasmid vector. Following ligation, transformation of competent Escherichia coli DH5α by calcium chloride-heatshock transformation was performed, and resulting transformants were screened for the soxR::PsoxS::lacZ::pBR322 construct. Even though the amplicons were made, a successful ligation was not achieved. The lack of success in obtaining the desired construct prevented further examination of the potential of the proposed reactive oxygen species probe. - Reactive oxygen species (ROS), such as superoxide (O2 ) compared to the previously studied katG::luxCDABE and hydrogen peroxide (H2O2), are radicals that can be probe (9). generated by various mechanisms including exposure to A possible reason for the lack of sensitivity of the UV-A, paraquat, and side reactions involving quinones katG::luxCDABE probe used in a previous study may be a from the electron transport chain (1,2,3). Excessive levels consequence of luminescence activity inhibited by the of ROS are potentially harmful towards microorganisms growth media (9). We decided to create a new probe by due to the indiscriminate reactivity of ROS with many using lacZ instead of luxCDABE,, in pursuit of generating cellular processes within the cell including protein an even more sensitive ROS probe. lacZ is a gene for β- oxidation (1,3,4). As such, many bacteria have developed galactosidase (LacZ), an enzyme that hydrolyzes β- mechanisms in order to prevent the accumulation of ROS galactosides into monosaccharides. When 4- within the cell, particularly the OxyR and Sox regulons. methylumbeliferryl-β-D-galactopyranoside (MUG), a The OxyR regulon is involved in the sensing and chromogenic substrate, is cleaved into 4- metabolism of H2O2 within Escherichia coli. The OxyR methylumbelliferone (MUB) and galactose, a regulon uses OxyR as the ROS sensor to detect chromophore is produced that excites at 390 nm producing intracellular levels of H2O2 (5,6). Upon detection of H2O2, fluorescent emission at 460 nm (13). Because the β- OxyR induces the expression of both katG and katE which galactosidase assay used in conjunction with MUG has encode HPI and HPII, respectively (7). HPI and HPII are been shown to have a detection limit of 2 pg of β- catalases that catalyze the conversion of H2O2 into water galactosidase, this new probe should be more sensitive and molecular oxygen (8). In previous studies, the limited than the luxCDABE gene cluster which has been shown to sensitivity of a katG promoter based probe led to the have its luminescent activity reduced by growth investigation of alternative candidates of ROS sensors, media(14). which incurred the investigation of using SoxR as the The purpose of this study was to use polymerase chain sensor for internal ROS (9). reaction (PCR) to amplify soxR fused to the soxS promoter The Sox regulon uses the SoxR homodimer as the ROS (abbreviated in this report as soxR::PsoxS) and lacZ from E. - sensor to detect intracellular levels of O2 (10). Upon coli MG1655 genome in order to construct a recombinant - detection of O2 , two [2Fe–2S] are oxidized activating soxR::PsoxS::lacZ probe. This recombinant probe was to be SoxR homodimer (7). In both the reduced and oxidized used in future studies to determine the sensitivity and states, SoxR binds upstream of the soxS promoter, but only viability of its use as an internal ROS probe that would be the oxidized state is able to increase the expression of expressed from the recombinant pBR322 plasmid vector SoxS which in turn upregulates the expression of several once transformed into E. coli DH5α. - genes involved in O2 metabolism including a manganese superoxide dismutase (11). Previous studies have shown MATERIALS AND METHODS - that soxS upregulation in the presence of O2 is greater than Bacterial strains and growth conditions. E. coli strain MG1655 the upregulation of katG in the presence of H2O2 (12). was used to amplify genes of interest for constructing our Because of the higher level of constitutive expression and plasmid, and the E. coli strain DH5α was used to transform our - construct, as it lacks an active β-galactosidase. Both strains were induction of the Sox regulon in response to O2 in comparison to the OxyR regulon, we attempted to obtained from the MICB 421 Culture Collection in the Microbiology and Immunology Department at University of construct a plasmid with a SoxR sensor, and soxS promoter British Columbia, then streaked onto a Luria-Bertani (LB) agar driven LacZ expression as a more sensitive ROS probe plate and were grown overnight (18 hours) in a shaking incubator 121 Journal of Experimental Microbiology and Immunology (JEMI) Vol. 18: 121 – 128 Copyright © April 2014, M&I UBC FIG 1. Cloning strategy. (a) pBR322 was first cut with EcoRI and SalI to excise and inactivate the Tetracycline resistance gene. soxR::PsoxS and lacZ was then PCR amplified from E. coli MG1655 genome with the generated restriction sites. A 3-way ligation joined all the pieces together at the compatible sticky ends to form a 7.4 kb construct. SoxR homodimers in the host cell cytoplasm would detect internal ROS and activate soxS, which would then drive transcription of lacZ. (b) A close-up view of the cloned soxR fused to the soxS promoter, including the engineered restriction sites, the soxR regulation binding site and soxS native Shine-Dalgarno site. at 37°C at 200 RPM. Growth in broth was measured as optical coli strain MG1655, known to have a full lac operon and density (OD660). soxR::PsoxS gene. We extracted genomic DNA from E. coli Plasmid selection, isolation, and primer construction. E. coli MG1655 using DNeasy kit (Qiagen) kit. The PCR conditions MG1655 genomic sequences were obtained from were found to be the same for both the soxR::PsoxS and lacZ www.ecocyc.com and primers were manually designed to match amplification. We used final concentrations of 1X PCR Reaction genomic lacZ and soxR::PsoxS sequences. EcoRI and SalI Buffer (Invitrogen), 2 units of Taq polymerase (Thermo restriction sites were added onto the 5’ end of the forward and scientific), 2.5 mM of MgCl2 (Fermentas), 200 μM of dNTP reverse primers, respectively. Primers were ordered Integrated (Invitrogen), 1 μM of primer from IDT of each forward and DNA Technologies (IDT) for synthesis. pBR322 plasmid was reverse primers, 50 ng to 100 ng of E. coli MG1655 genomic isolated from E. coli DH5α host strain from the MICB 421 culture DNA template, and sterile distilled water were added to bring the collection and also from Invitrogen, supplied at 500 ng/μl. We PCR reaction up to a final volume of 25 μl. The amplification grew the strains overnight and used the GeneJET Plasmid times were to first denature the DNA at 94°C for 5 minutes, then Purification System (Thermo Scientific) to extract pBR322 for repeat the following steps 35 times: 94°C for 45 seconds, 64°C use. for 45 seconds, 72°C for 3.5 minutes for lacZ or 1.0 minute for soxR::PsoxS, and a final extension at 72°C at 10 minutes for both TABLE 1. 5’3’ Sequences of forward and reverse primers used to lacZ and soxR::PsoxS. After PCR amplification, the PCR amplify soxR::PsoxS and lacZ from E. coli MG1655 genomic DNA. products were loaded into a 1.2 % agarose gel (UltraPure agarose Engineered restriction sites are underlined and are flanked at the 5’ select agar, Invitrogen) with 6X loading dye (Thermo scientific) end by 5 extra basepairs to allow for restriction enzyme recognition and run for 45 minutes at 120 Volts. Products were visualized by and cleavage. soaking the agarose gel in an ethidium bromide bath (0.5 μg/ml) Gene Forward Primer Reverse Primer for 15 minutes. Later, the agarose gels were visualized under the lacZ 5’ - 5’ – Alpha Innotech Corporation, MultiImage Light Cabnet. The GGCGCCCATGGCT CCGGCGTCGACTT Alpha Imager software was used to optimize and adjust the ATGACCATGATTA ATTTTTGACACCA pictures for visualization. CG – 3’ GACC - 3’ Sequencing of PCR products. lacZ and soxR::PsoxS PCR soxR:: PsoxS 5’ - 5’ – products were sequenced using the protocol provided by CGGCGGAATTCGA GCGGCCCATGGAT Genewiz. We made a pre-mixed tube containing PCR products GAAAGACAAAGAC CTGCCTCTTTTCAG and primers with final concentrations of 20 ng/μl for soxR::P CGGA - 3’ TGT – 3’ soxS and 60 ng/μl for lacZ PCR products with 25 pmol of forward soxR:: PsoxS 5’- 5’ – primers in a 15 μl tube before sequencing the PCR fragments with and partial lacZ AAGGGAATAAGGG TGTAAAACGACGG Genewiz. Analysis of sequencing data was performed using CGACACG – 3’ CCAGTGA- 3’ Snapgene Viewer (www.snapgene.com). Purification of PCR amplified fragments and digestion. Successfully amplified soxR::PsoxS and lacZ PCR products were Polymerase chain reaction amplification of soxR::PsoxS and purified using a PCR purification kit (Purelink PCR Purification lacZ. To amplify the soxR::PsoxS and lacZ genes needed to make our construct, we used polymerase chain reaction (PCR) on E. Kit, Invitrogen). Concentrations and purity of isolated PCR 122 Journal of Experimental Microbiology and Immunology (JEMI) Vol.