Revision as of 07:21, 25 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.