Difference between revisions of "Part:BBa K2348001"
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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.<sup>2</sup>. 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. | 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.<sup>2</sup>. 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. | ||
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===Usage and Biology=== | ===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. | |
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<span class='h3bb'><b>Sequence and Features</b></span><br> | <span class='h3bb'><b>Sequence and Features</b></span><br> | ||
Revision as of 17:13, 1 November 2017
asr - acid induced promoter
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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)