Latest revision as of 14:23, 12 October 2023

The asr promoter is a pH-responsive promoter.

This part contains the asr promoter with its native RBS. The asr promoter is a pH-responsive promoter native to E. coli. It induces transcription in acidic conditions (~pH 5.5).

Usage and Biology

As of yet, the function of the asr gene, or “acid-shock RNA” gene, and the mechanism responsible for its induction are still unclear. However, Lien et al. have taken significant steps toward characterizing the gene. They propose that asr encodes a periplasmic or outer-membrane protein. Knockout experiments illustrated that the PhoBR operon plays a significant role in activating the asr gene. They demonstrated through mobility shift electrophoresis that the PhoB protein binds to the promoter region of asr. By analyzing the sequence of the asr promoter region, they revealed that it contains a sequence similar to that of the Pho box, which is a consensus sequence known to bind the PhoB protein. The Pho box can be found in the promoter regions of other PhoB-regulated genes.

NCKU_Tainan 2018

Improve the Characterization of BBa_K1231000

The asr promoter was first described by Suziedeliene et al. in 1999. They showed that asr is induced under low pH which is about pH 4.8, and it is controlled by the phoBR system. From the article they have published, the promoter is named as acid shock RNA (asr) promoter due to the RNA that has been transcribed after putting the E. coli into a low pH condition.

In 2007 Ogasawara et al2. found out that there is another regulatory system that controlling asr transcription by using SELEX to find the binding sequences of PhoQP-RstBA. Hence the asr promoter is directly controlled by two different systems, the PhoBR system activated through low inorganic phosphate and the RstAB system sensing the pH while it is controlled by PhoQP-system activated by low Mg²⁺ concentrations.

Our team have cloned this gene and also a sfGFP gene downstream of this promoter which could express green fluorescent once the promoter has been activated. In conclusion, we could monitor the pH in the surrounding medium in our device at any time by observing the color change of the medium.

Our constructed biobrick: https://parts.igem.org/Part:BBa_K2762014

Charaterization of promoter with fluorescence intensity measurement

Asr promoter (P_asr) is reported to be induced under acidic condition. It can be used as a reporter when the medium turns acidic. We thus measure the fluorescence intensity in a short period of time. We first incubated the bacteria to log phase (about 2 hours) in Luria-Bertani (LB) medium. We then centrifuged the broth and resuspended the pellet using M9 medium with different pH value (pH 4, 4.25, 4.5, 4.75, 5, 5.5, 6 and 7; the pH value is adjusted with 1M HCl). We then incubated it in the 96 well plate and measured its fluorescence intensity (absorbance: 485 nm, excitation: 535 nm) every 3 minutes for 30 minutes. The difference in fluorescence intensity can be observed within 30 minutes.

Results

Fig 2. The data shows the fluorescence intensity expressed by P_asr in different pH.

Based on the data above, we found out that P_asr will be induced about pH 4. Also, the fluorescence intensity had the peak at pH value of 4.25. We could conclude that P_asr is an acidic promoter. The result shows that P_asr constructed pH sensing system can be used as an alert under low pH. When the medium turns acidic, fluorescence can be easily observed. We believe that this system can also be applied to a various bio-detection system.

Sequence and Features

2020 XHD-Wuhan-China’s Characterization of BBa_K1231000

1. Aim of experiment

Based on K1231000 part, a genetic circuit Pasr-B0034-amilCP was constructed to characterize the function of this Pasr promoter.

2. Methods

2.1 Construction of Pasr-pSB1C3 We construct the following gene circuit based on the principles of synthetic biology, as shown in Figure 1.

Figure 1. constitution of Pasr-amilCP gene circuit.

Replicate the Pasr promoter from the Pasr-pUC19 plasmid, use homologous recombination to obtain the recombinant plasmid Pasr-pSB1C3, and verify the length of the recombinant plasmid to ensure the success of the recombinant plasmid through PCR and enzyme digestion.

2.2 AmilCP expression under the control of K1231000 induced by different pH

The plasmid Pasr-pSB1C3 containing the K1231000 promoter was transformed into E. coli DH5α strain. Culture the DH5α bacteria containing the recombinant plasmid Pasr-pSB1C3 to the logarithmic pHase in LB medium, then take 1000ul centrifugation, discard the supernatant, and resuspend the bacterial solution in 200ul M9 medium. M9 medium is adjusted with HCL for pH, Respectively, pH=4.5, 5, 7, and each group of pH gradient has 3 biological replicates. Regarding the spotting process, add 200ul to each hole, and the pH is 4.5, 5, and 7, respectively. The first three wells are samples, and the last three wells are M9 medium corresponding to the pH (as a negative control). The concentration of amilCP was measured by microplate reader. The measurement interval is the first hour, once every 3 minutes; the second hour, once every 10 minutes; the third hour, once every 20 minutes; the fourth hour, the fifth hour, once every 30 minutes. The concentration of amilCP (OD580) and OD600 value are measured simultaneously.

3. Result

We use the plasmid Pasr-pSB1C3 as a template, and then use PCR to obtain the corresponding Pasr+amilCP fragment (860bp), as shown in Figure 2. The length of the PCR fragment is consistent with expectations. Subsequently, the Pasr-pSB1C3 recombinant plasmid was digested with xbal, as shown in Figure 3, and the result of digestion was also consistent with expectations. All this proves the success of the recombinant plasmid.

Figure 2. The electropHerogram of the Pasr+amilCP fragment after PCR.

Figure 3. ElectropHoresis of Pasr-pSB1C3 plasmid after digestion with xbal.

The successfully recombined plasmid Pasr-pSB1C3 was transformed into E.coli DH5α strain, cultured in LB medium to logarithmic pHase, then transferred to M9 medium corresponding to pH, and continuously cultured in microplate reader for 5 hours. As shown in Figure 4, the final pH=4.5, pH=5 group of bacteria liquid showed obvious blue, pH=7 group did not show blue, indicating that Pasr has a stronger promoting ability under acid induction.

Figure 4. Characterization of the color of bacterial liquid in different pH gradients.

The normalized concentration of amilCP is OD580 divided by OD600. As shown in Figure 5, the concentration of amilCP is the highest in the condition of pH=4.5, followed by the condition of pH=5, and in the condition of neutral (pH=7) lowest.

Figure 5. In different pH gradients (4.5, 5, 7), the corresponding amilCP concentration of culture for 1 hour.

The concentration of amilCP was measured several times during the continuous culture for 5 hours. As shown in Figure 6, in the first 5 minutes, E. coli was in the adaptation stage from LB to M9 medium, so the concentrationt of amilCP decreased. In 5-10 minutes, E. coli has relatively adapted to the M9 medium, its life activities have become active, and the concentrationt of amilCP has been relatively increased. The follow-up results showed that only under acidic conditions (pH=4.5, pH=5), the concentrationt of amilCP gradually increased, and under the condition of pH=4.5, the concentrationt of amilCP increased the fastest, amilCP The concentrationt reaches the maximum under the condition of pH=4.5. Under neutral (pH=7) conditions, the concentrationt of amilCP is gradually decreasing. This proves that Pasr is an acidic promoter, which has a significant activation effect under low pH conditions. After the time reaches 1 hour, the concentration of amilCP gradually decreases, which may be due to the exhaustion of nutrients in the medium, and the life activities of bacteria gradually weaken.

Figure 6. The change trend of amilCP concentration within 5 hours under different pH gradients (4.5, 5, 7).

4. Conclusion

The plasmid Pasr-pSB1C3 based on the K1231000 is sensitive to pH. Under the condition of pH=4.5, the expression of amilCP is the highest. The results indicate that the Pasr promoter is activated by low pH. We use amilCP to characterize the capability of Pasr promoters, and we can observe color changes with the naked eyes. The Pasr-amilCP system can be used as a biosensor for detecting environmental pH. The detection method is convenient and the result is intuitive.

5. References

Liene E S, Lis K S, Garbenciute V, et al. The Acid-Inducible asr Gene in Escherichia coli: Transcriptional Control by the pHoBR Operon[J]. Journal of Bacteriology, 1999, 181(7): 2084-2093.

Ogasawara H, Hasegawa A, Kanda E, et al. Genomic SELEX Search for Target Promoters under the Control of the PHoQP-RstBA Signal Relay Cascade[J]. Journal of Bacteriology, 2007, 189(13): 4791-4799.

Jilin_China 2021

Charaterization of Pasr promoter with fluorescence intensity measurement

This year, based on the previous part BBa_K123100 (Northwestern, 2013), we characterized this part again and added new documentation to it. We focused on testing the sensitivity and durability of Pasr promoter and characterized the Pasr-sfGFP (BBa_K2762014) part by cloning this part into backbone pUC57(Figure.1A). When the OD600 value of the bacteria reached approximately 0.6, sterile H₂SO₄ or NaOH was added to the medium to adjust the pH. The change of fluorescence intensity under different pH over time was measured. As shown in the figure, fluorescence was the highest at the pH of 5. Moreover, within a certain period of time, Pasr continued to play a role. Thus, we suppose that Pasr can be used to monitor acidity in the culture medium. Pasr can be used to monitor acidity in the culture medium and achieve cell self-responsive acid requlation when it connects downstream to other genes. We think it can be applied to the design of many sustainability genetic circuits.

Figure 1.Pasr-sfGFP induction at different concentrations of pH. (A). Constitution of Pasr-sfGFP gene circuit. (B). Pasr sensitivity at different pH of culture medium. The designed construct was transformed into E. coli BL21. When the OD600 value of the bacteria reached approximately 0.6, sterile H₂SO₄ or NaOH was added to the medium to adjust the pH respectively. The fluorescence intensity of sfGFP was detected at the indicated time and normalized by the OD600 value. The experiment was performed three times in triplicate. *, P < 0.05 from respective control using Student’s t test.

iGEM23_HZAU-China

This is a promoter that is activated in an acidic environment. It is the promoter of the acid shock protein ASP in E. coli, and its sequence contains a RstA box that can be bound by phosphorylated RstA to enhance its promoter activity in a dose-dependent manner[1]. The team obtained the corresponding promoter fragment from the E. coli MG1655 genome by PCR using a high-fidelity DNA polymerase. 

Usage

The promoter of the acid shock protein ASP in E.coli often functions under acidic stress conditions. Since its sequence contains a series of binding sites for regulatory proteins such as the RstA box, its activity can be well artificially regulated and altered. Therefore, it can be well utilized in synthetic biology circuit design. Some experimental teams have combined pH sensing with toxin expression to create an effective bacterial containment system [2]. We believe the functionality of this promoter in responding to environmental pH changes could provide some reference meaning to projects detecting changes in environmental pH conditions. 

Test Method Design

Charaterization of Pasr with fluorescence intensity measurement

To validate the obtained promoter activity and its activation range in response to pH, we connected Amcyan protein after this promoter and introduced it into E. coli BL21(DE3). When E. coli grew to OD600=0.6, we used HCl aqueous solution and NaOH aqueous solution to adjust the pH and built a gradient from pH 8 to pH 3. Fluorescence intensity (Exλ:453nm Emλ:486nm) was measured every 30 minutes for 5 hours while OD600 was measured simultaneously. The final fluorescence intensity divided by OD600 gave the relative fluorescence intensity. 

Test Protocol

    1) Methods of molecular cloning and transformation are described above. Transform this plasmid into E. coli BL21. Then spread it onto an LB medium plates with 50 μg/mL kanamycin and incubate overnight at 37 ℃ in an incubator.

    2) Pick four colonies from the same plate as parallel repeats. Each colony is inoculated on two identical media with 5mL LB medium containing 50 μg/mL kanamycin and cultured at temperatures(36 ℃) respectively while shaking at 200 rpm .

    3) Measure the OD₆₀₀ value of the resuspending culture media in an automatic microplate reader (SynergyH1 hybrid multimodal reader)until the OD₆₀₀ is in the range of 0.4 and 0.6.

    4) Use HCl aqueous solution and NaOH aqueous solution to adjust the pH of the medium, and construct a gradient from pH 8 to pH 3.

    5) Samples were grouped and incubated continuously at 37 ° C while shaking at 200 rpm for four hours.

    6) Fluorescence intensity (Ex.lamda.: 453 nm, Em.lamda.: 486 nm) and OD₆₀₀ were measured continuously using a an automatic microplate reader (Synergy H1 hybrid multimodal reader). At the end of the detection, the measured fluorescence intensity was divided by OD₆₀₀ to obtain the relative fluorescence intensity, and the results were analyzed by plotting.

Result

Analysis of the experimental results

As shown in Figure 1., Pasr has almost no expression under pH 7-8 conditions, but begins low-dose expression at pH 6 and gradually increases as the pH decreases, reaching a peak at pH = 5, then maintaining a relatively low expression level as the pH continues to decline. As shown in Figure 2.,We selected the final values for differential analysis, which showed extremely significant differences between pH 6, pH 5 and the control group. This experiment validated that this promoter has almost no expression under pH 7-8 conditions, consistent with our project design expectations. Pasr can be used to activate the suicide circuit in the gastric environment of cats to cause engineered bacteria death, thus avoiding potential hazards from leakage. This promoter can be used to respond to changes in environmental pH and has some reference value for projects with corresponding environmental condition changes.

Reference

[1] Ogasawara H, Hasegawa A, Kanda E, Miki T, Yamamoto K, Ishihama A. Genomic SELEX search for target promoters under the control of the PhoQP-RstBA signal relay cascade. J Bacteriol. 2007 Jul;189(13):4791-9.

[2] Stirling F, Naydich A, Bramante J, Barocio R, Certo M, Wellington H, Redfield E, O'Keefe S, Gao S, Cusolito A, Way J, Silver P. Synthetic Cassettes for pH-Mediated Sensing, Counting, and Containment. Cell Rep. 2020 Mar 3;30(9):3139-3148.e4.