Difference between revisions of "Part:BBa K3771048"

Latest revision as of 12:49, 2 October 2024

PsoxS

Description

The soxS gene is one component of the soxRS regulon (Fig. 1), which is important for E. coli to sense and respond to the oxidants. The soxS promoter (P_soxS) is regulated by SoxR and SoxS protein. SoxR protein binds to the site between the -10 and -35 elements of the soxS promoter. The distance between the ends of the -10 and -35 hexamers of the soxS promoter is 19 bp, which is different from 17±1 bp spacers for most E. coli promoters. Therefore, transcription of soxS is low without oxidized SoxR^[1]. Oxidized SoxR induces target gene expression through distorting soxS promoter (Fig. 2)^[2]. In addition, SoxS protein serves as a negative autoregulator and diminishes soxS transcription^[3,4].

Fig. 1. Transcription unit of the soxRS regulon in E. coli K-12^[5].

Fig. 2. The mechanism of SoxR activation by superoxide^[1]. The SoxR dimer has two forms, reduced and oxidized, both of them can bind with DNA. After SoxR is oxidized by oxidative stress, its conformation changes, modifies the local DNA topology at the promoter, and compensates for a dysfunctional spacing between promoter elements, and hence increasing the transcription of soxS^[1].

Usage and Biology

After conducting colony PCR from the E.coli MG1655, the P_soxS fragment can be amplified from the chromosome and the experiment result can be checked by agarose gel electrophoresis. The result is shown in Fig. 3. The part has been confirmed by sequencing and has no mutations.

Fig. 3. The electrophoresis result of P_soxS fragment from the chromosome PCR. M: Marker; Lane 1: soxS promoter (P_soxS) (220 bp).

The promoter strength of P_soxS is determined by the expression level of the reporter, sfGFP, under oxidative stress. While using hydrogen peroxide (H₂O₂) as the inducer, the strength of P_soxS shows no significant difference under different concentrations of hydrogen peroxide (Fig. 4). However, P_soxS is well induced by using paraquat (PQ), which is a commonly used agent to induce oxidative stress for bacteria (Fig. 5 & 6).

Fig. 4. Relative fluorescence intensity of P_soxS after 4.5-hour incubation with hydrogen peroxide in various concentrations.

Fig. 5. Relative fluorescence intensity of P_soxS after 4.5-hour incubation with paraquat in low concentrations.

Fig. 6. Relative fluorescence intensity of P_soxS after 4.5-hour incubation with paraquat in high concentrations.

References

1. Pomposiello PJ, Demple B. Redox-operated genetic switches: the SoxR and OxyR transcription factors. Trends Biotechnol. 2001;19(3):109-114. doi:10.1016/s0167-7799(00)01542-0

2. Koo MS, Lee JH, Rah SY, et al. A reducing system of the superoxide sensor SoxR in Escherichia coli. EMBO J. 2003;22(11):2614-2622. doi:10.1093/emboj/cdg252

3. Nunoshiba T, Hidalgo E, Li Z, Demple B. Negative autoregulation by the Escherichia coli SoxS protein: a dampening mechanism for the soxRS redox stress response. J Bacteriol. 1993;175(22):7492-7494. doi:10.1128/jb.175.22.7492-7494.1993

4. Seo SW, Kim D, Szubin R, Palsson BO. Genome-wide Reconstruction of OxyR and SoxRS Transcriptional Regulatory Networks under Oxidative Stress in Escherichia coli K-12 MG1655. Cell Rep. 2015;12(8):1289-1299. doi:10.1016/j.celrep.2015.07.043

5. Keseler IM, Mackie A, Santos-Zavaleta A, et al. The EcoCyc database: reflecting new knowledge about Escherichia coli K-12. Nucleic Acids Res. 2017;45(D1):D543-D550. doi:10.1093/nar/gkw1003

Characterization by 2024 iGEM-NJTech-China

Usage and Biology

In the site of intestinal inflammation, there is a high concentration of nitric oxide (NO), which we selected as the signaling trigger for the therapeutic system. We chose the SoxR/SoxS oxidative stress-responsive promoter as the regulatory element. SoxR is a transcription factor containing a [2Fe-2S] cluster, while SoxS is a promoter sequence. In a high NO and reactive oxygen species (ROS) environment, SoxR binds to specific sequences of the SoxS promoter and undergoes a conformational change due to oxidative stress. This change causes SoxS to bend (with a bending angle of approximately 65°), bringing the -10 and -35 regions of the promoter closer together, facilitating the binding of RNA polymerase (RNAP) and other transcription factors, thereby regulating the expression of downstream genes.

Figure 1-1 Mechanism of SoxR protein regulating gene transcription

In our study of the SoxR/SoxS system, we determined that the optimal final concentration of sodium nitroprusside (SNP, an NO donor) for induction is 100 μM. However, there is a lack of research on the induction temperature. Therefore, we aimed to supplement this data, hoping it would guide future studies. Additionally, we explored whether the promoter's functionality meets expectations under physiological conditions at 37°C. We designed and constructed the plasmid pET29a-J23119-RBS-SoxR-T7-pSoxS-RBS-eGFP-T7 (BBa_K5322037) and measured the fluorescence intensity per unit OD using a microplate reader to analyze promoter expression at different induction temperatures (20°C, 25°C, 30°C, 37°C, and 40°C).

Figure 1-2 pET29a-J23119-RBS-SoxR-T7-pSoxS-RBS-eGFP-T7

We selected samples to be taken every 4 hours for detection, with each measurement conducted in triplicate. Using a microplate reader, we measured OD₆₀₀ and fluorescence intensity (FL). After processing the data, we obtained the fluorescence intensity per unit OD₆₀₀ (FL/OD₆₀₀), with the results presented in the following figure.

Figure 1-3 Growth curve and fluorescence intensity per unit OD

Figure 1-4 Fluorescence intensity per unit OD

References

1. Holden, E. R., & Webber, M. A. (2020). MarA, RamA, and SoxS as Mediators of the Stress Response: Survival at a Cost. Frontiers in microbiology, 11, 828. https://doi.org/10.3389/fmicb.2020.00828.

2. Pomposiello, P. J., & Demple, B. (2000). Identification of SoxS-regulated genes in Salmonella enterica serovar typhimurium. Journal of bacteriology, 182(1), 23–29. https://doi.org/10.1128/jb.182.1.23-29.2000.

3. Nunoshiba, T., Hidalgo, E., Amábile Cuevas, C. F., & Demple, B. (1992). Two-stage control of an oxidative stress regulon: the Escherichia coli SoxR protein triggers redox-inducible expression of the soxS regulatory gene. Journal of bacteriology, 174(19), 6054–6060. https://doi.org/10.1128/jb.174.19.6054-6060.1992

Sequence and Features