Difference between revisions of "Part:BBa K2348001"
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The asr promoter was first described by Suziedeliene et al.<sup>1</sup> 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. | The asr promoter was first described by Suziedeliene et al.<sup>1</sup> 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.<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. <br><br> | 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. <br><br> | ||
| − | <b>Additional information</b> | + | <b>Additional information</b><br> |
This part actually is a composite part containing the asr promoter and mCardinal. Additionally, between promoter and cardinal an additional RBS, a TEV-site and a f-degron are inserted. Also, mCardinal is 6xHis-taged and the part ends with a T7-terminator. Because the part does not work when build as Biobrick due to the Biobrick scar this part is listed as basic part.<br> | This part actually is a composite part containing the asr promoter and mCardinal. Additionally, between promoter and cardinal an additional RBS, a TEV-site and a f-degron are inserted. Also, mCardinal is 6xHis-taged and the part ends with a T7-terminator. Because the part does not work when build as Biobrick due to the Biobrick scar this part is listed as basic part.<br> | ||
TEV-site and f-degron allow a fast and controllable degradation of proteins and in combination with the fast maturation times of our fluorescence proteins, allow a fast colour change. This enables shorter time spans between signals when controlling the robot’s movement. Due to a change of our reactor design this feature is no longer needed because the pH is only changed in a small amount of media which is discarded after measurement. | TEV-site and f-degron allow a fast and controllable degradation of proteins and in combination with the fast maturation times of our fluorescence proteins, allow a fast colour change. This enables shorter time spans between signals when controlling the robot’s movement. Due to a change of our reactor design this feature is no longer needed because the pH is only changed in a small amount of media which is discarded after measurement. | ||
Revision as of 17:11, 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.
Additional information
This part actually is a composite part containing the asr promoter and mCardinal. Additionally, between promoter and cardinal an additional RBS, a TEV-site and a f-degron are inserted. Also, mCardinal is 6xHis-taged and the part ends with a T7-terminator. Because the part does not work when build as Biobrick due to the Biobrick scar this part is listed as basic part.
TEV-site and f-degron allow a fast and controllable degradation of proteins and in combination with the fast maturation times of our fluorescence proteins, allow a fast colour change. This enables shorter time spans between signals when controlling the robot’s movement. Due to a change of our reactor design this feature is no longer needed because the pH is only changed in a small amount of media which is discarded after measurement.
The 6xHis-tag allows detection of mNeonGreen on protein level and the T7-terminator stops translation. The part itself was created using extension PCR and overlap PCR
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)