Part:BBa_K5332001

Reactive oxygen species promoter

Usage and Biology

ROS, or reactive oxygen molecules, can accumulate abnormally in certain environments and affect the occurrence of diseases such as IBD. In order to design a promoter that can start expression in an environment with high reactive oxygen concentrations, we selected the promoter of the KatG gene that can respond to reactive oxygen stress. Referring to the relevant literature on the experimental analysis of reactive oxygen stress response of katG gene alone, it can be seen that the expression of katG increased gradually at first, and then declined after simulation is saturated. The initial upregulation is the result of increased H2 O2 output by aerobic metabolism under favorable conditions. The activity of peroxidase, a gene expression product, also increased first and then remained stable. This mechanism can reduce the extra energy expenditure of cells. Therefore, the katG promoter is suitable for designing elements to regulate the expression of target genes in a spatiotemporal specific manner.

Brief introduction of katG

KatG, a gene encoding hydroperoxidase in bacteria, is regulated by a variety of signaling molecules. At present, most studies focus on the influence of its mutation on bacterial resistance and its upstream pathway. Hydrogen peroxide is one of the reactive oxygen species (ROS). In aerobic breathing organisms, it is transferred from a single electron to oxygen to form a superoxide (O^2-), which is then catalyzed by superoxide dismutase. There are genes in Escherichia coli that can directly respond to excess H₂O₂, such as oxyR, which encodes that oxyR regulator can bind to katG promoter after oxidation to promote katG expression and transcription.

The reason for choosing the promoter of gen katG

Expression of katG during proliferation of Escherichia coli

All the RNA mentioned in this paper was extracted by thermal phenol method, and the data source of MPCR was obtained by RT-MPCR.

The Strain UC574 (arg56 nad113 araD81) derived from E. coli K-12 was considered as the parental wild type.) After overnight culture in LB broth, part of the medium was inoculated in fresh medium diluted and cultured on a shaking table at 150rpm at 37℃. After 1.5, 2, 3, 5, 7, and 12h after inoculation, the same amount of bacterial liquid was taken out and cooled to 0℃ rapidly, and the growth of E. coli was detected by OD600 measurement, and the RNA content of katG and the activity of catalase of the product were determined. In the figure, the dark column indicates catalase activity, and the white column indicates the RNA content expressed by katG.

With the proliferation of E. coli, the concentration of H₂O₂ produced in the medium will also gradually increase due to the aerobic respiration of the bacteria. Therefore, the RNA content expressed by katG and catalase activity also increased during the rise of OD600 measurement curve. When the number of bacteria became stable, the RNA content decreased and the catalase activity remained unchanged (Fig.1). This suggests that the promoter of katG can accurately respond to the regulation of ROS stress response pathway.

Fig.1. Peroxidase activity during course of growth of wild-type bacteria in nutrient LB medium.

Comparison of katG promoter responsiveness with other genes

Since the oxyR regulator also regulates dps, gorA, and ahpCF genes, the following is a comparison by detecting the expression of four genes in the same H2O2 environment at the same time.

Wild-type E. coli cultured overnight in M9 base medium were diluted in fresh medium and incubated in a shaking bed at 37°C and 150 rpm. OD600 values were measured at regular intervals. When the OD600 value reached 0.2, half of the culture was taken out and H2O2 was added to make a solution with a final concentration of 10 M. The other half of the culture served as a control group. Samples were collected immediately after addition of H₂O₂ (within 1min) and after exposure for 5, 10, 15, and 20 min. Samples were frozen with liquid nitrogen, RNA was purified and measured. The fluorescence signal of each PCR product was compared with that of the reference control gene gapA, and the value of the treated sample was divided by the corresponding control value as the data source in the figure. All genes were analyzed, but only those that indicated a statistically significant (P < 0.05) increase was observed over a given time period.

When H₂O₂ was added, all four genes were expressed, katG and dps were the most, while gorA and ahpCF were less expressed. Subsequently, the expression of all genes decreased with the passage of time, and only a small amount of katG was still expressed at the 20th minute. Compared with other genes, the promoter of katG can not only rapidly respond to upstream signals and cause high gene expression, but also has higher sensitivity when H₂O₂ content decreases over time (Fig.2).

Fig.2. The expression of four different genes in response to the ROS.

To observe and compare intuitively, we tested the corresponding strength of the promoters of katG, dps, and gorA in the process of IBD occurrence. Using mathematical modeling, we can obtain the response activity strength of promoters from the katG, dps, and gorA in the high ROS environment caused by IBD over time, as shown in Fig.3.

Compared with the dps and gorA promoters, the activity strength of the katG promoter is much higher, and the response time is also obviously longer. This further verifies that the katG promoter has good application prospects for regulating the target gene under the condition of ROS as upstream signal molecules.

Fig.3. The promotor activity within the IBD progression.

References

1 Brieger K, Schiavone S, Miller FJ Jr, Krause KH. Reactive oxygen species: from health to disease. Swiss Med Wkly. 2012 Aug 17;142:w13659. doi: 10.4414/smw.2012.13659. PMID: 22903797.

2 Storz G, Tartaglia LA, Ames BN. The OxyR regulon. Antonie Van Leeuwenhoek. 1990 Oct;58(3):157-61. doi: 10.1007/BF00548927. PMID: 2256675.

3 Pomposiello PJ, Demple B. Redox-operated genetic switches: the SoxR and OxyR transcription factors. Trends Biotechnol. 2001 Mar;19(3):109-14. doi: 10.1016/s0167-7799(00)01542-0. PMID: 11179804.

4 Tao K. In vivo oxidation-reduction kinetics of OxyR, the transcriptional activator for an oxidative stress-inducible regulon in Escherichia coli. FEBS Lett. 1999 Aug 20;457(1):90-2. doi: 10.1016/s0014-5793(99)01013-3. PMID: 10486570.

5 Michán C, Manchado M, Dorado G, Pueyo C. In vivo transcription of the Escherichia coli oxyR regulon as a function of growth phase and in response to oxidative stress. J Bacteriol. 1999 May;181(9):2759-64. doi: 10.1128/JB.181.9.2759-2764.1999. PMID: 10217765; PMCID: PMC93716.

information

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 182
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 182
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 182
Illegal BglII site found at 253
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 182
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 182
1000
COMPATIBLE WITH RFC[1000]