Part:BBa_K2348001

asr - acid induced promoter
Available through the twin part BBa_K1231000
The asr promoter was first described by Suziedeliene et al.¹ in 1999. They showed that asr is induced through low pH, about 4.8, and that the promoter is controlled by the phoBR system. They also named the asr promoter, because of RNA they found after shifting E. coli to low pH conditions and therefore named the RNA they found and its corresponding promoter acid shock RNA (asr). In 2007 Ogasawara et al 2 . found a second regulatory system controlling asr transcription by SELEX search for PhoQP-RstBA binding sequences. 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. RstAB itself is controlled by PhoQP-system activated by low Mg 2+ concentrations. This complex regulatory mechanism for this small promoter amazed us and provided us with an interesting challenge to get expression going. Because of the two regulatory systems only becoming active when Mg2+ or Pi are low expression could not be done in LB-media. Also, our M9 media used for expression for the thermos project did not work because as it seems both systems must be active to activated asr transcription and M9 still contains Mg2+. To solve this problem, we used the LPM media described by Suziedeliene et al.². This allowed us to express our fluorescence protein mCardinal by shifting the cells to acid LPM media with pH 5.0 and 4,5. Best expression was achieved at pH 5.0.

Usage and Biology

We used this regulatory unit to express Cardinal under acid conditions. To increase expression, an extra RBS was added after the promoter.
Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Functional Parameters

To test acid induced expression of mCardinal controlled by the asr promoter, our asr-mCardinal construct, we tried the same protocol we used for the alx-mNeonGreen but with pH 7.0, 5.0 and 4.5. Unfortunately this did not work out because, as we discovered later, expression works but mCardinal, a fluorescence protein with extreme fast maturation time, is designed for expression in mammalian¹ cells and does not show any fluorescence when expressed in E. coli. Therefore we investigated the expression on protein level with Western blotting. To do so we cultivated bacteria containing the construct overnight in LPM with pH 7.0 and used this culture to inoculate 100 ml LPM with pH 7.0, 5.0 and 4.5 to an OD₆₀₀ of 0.1. The cultures were incubated at 37°C. Before inoculating, after 1.5, 2.0 and 2.5 hours 3 OD units were taken of each culture, pelleted and cooled on ice. After all samples were collected they were prepared for SDS gel electrophoresis by sonicating, mixing with loading buffer and heating to 95°C. Samples and pageruler protein standard were separated on a SDS gel and blotted on a nitrocellulose membrane. After washing and incubating with anti-6xHis antibodies tagged with horse radish peroxidase, detection was performed with Thermo Scientific^TM SuperSignal^TM West Pico Chemiluminescent Substrate.
Fig. 1. Asr controlled expression of mCardinal Overlay of the membrane showing the PageRuler prestained protein Ladder (S) and a picture showing the signal of the horse radish peroxidase substrate. Lanes from left to right, protein ladder, inoculation culture with pH 7, the three samples, pH 7.0, 5.0, 4.5, after 1.5 h (t1), after 2 h (t2) and after 2.5 h (t3).

All detected signals seen in Fig. 1 are the same height as the 30 kDa band of the standard, correlating with the size of mCardinal plus TEV-site, f-degron and the 6xHis-tag, with an approximate weight of 29.9 kDa. There is no signal in any sample with pH 7.0 leading to the conclusion that asr is not active at pH 7.0. At pH 5.0 and 4.5 there are bands of different intensity at all time points, hence asr is active at these pH and mCardinal is expressed. This is proof that our construct does work. Also expression is higher at pH 5.0 than 4.5 especially after 2h (t2) and 2.5 h (t3). To verify this results and use the construct for our robot a different fluorescence protein can be inserted in the construct and the new construct could be tested and used like the alx construct above.

¹Suziedeliené, E., Suziedélis, K., Garbenciūté, V. & Normark, S. The acid-inducible asr gene in Escherichia coli. Transcriptional control by the phoBR operon. Journal of bacteriology 181, 2084–2093 (1999).
²Ogasawara, H. et al. Genomic SELEX search for target promoters under the control of the PhoQP-RstBA signal relay cascade. Journal of bacteriology 189, 4791–4799 (2007)