Revision as of 07:13, 17 October 2018

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.

Charaterization of promoter with fluorescent intensity measurement

P_asr is reported to be induced in acidic condition. We think that it can be used to report the abnormal acidity of the medium. We thus determine to measure the fluorescent intensity in a short period of time. We first incubated the bacteria to log phase (within 2 hour) with LB medium. We then centrifuged the broth and suspended the pellet with pH modified M9 medium (the pH value is modified with 1M HCl). We then took the sample and incubate in the 96 well and measure its fluorescent intensity for every 3 minutes. We found out that the promoter P_asr will be induced at the pH value below four within 30 minutes. The different fluorescent intensity can be observed within 30 minutes. The fluorescent had the peak at pH value of 4.25.

Fig 2. The data shows the fluorescent intensity (absorbance: 485 nm, excitation: 535 nm) expressed by Pasr in different pH. Sequence and Features