Revision as of 14:12, 27 October 2020

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.Reference

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.