Difference between revisions of "Team:NAWI Graz/pHPlasmid"

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Revision as of 13:48, 29 October 2017

pH PLASMID


Figure 1: The pH-controller for the pH inducible promoter. A 3D printed ground plate was set up with two peristaltic pumps and a H-bridge motor control module. Two lab bottles filled with 1M HCL and 1M NaOH can be placed on the sockets. In combination with the pH-Sensor (DFRobot), it was possible to set the pH value of the reactor medium to specific pH values.

Figure 1: The pH-controller for the pH inducible promoter. A 3D printed ground plate was set up with two peristaltic pumps and a H-bridge motor control module. Two lab bottles filled with 1M HCL and 1M NaOH can be placed on the sockets. In combination with the pH-Sensor (DFRobot), it was possible to set the pH value of the reactor medium to specific pH values.


asr Promoter

[asr promoter ph]
The asr promoter was first described by Suziedeliene et al. in 1999 1 . 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. (2003). This allowed us to express our fluorescence protein mCardinal by shifting the cells to acid LPM media with pH 5,5 to 4,5. Best expression was achieved when pH was  5.0???
[asr 'timeline']
Our construct still includes a TEV-site in combination with the F-degron, this allows fast degeneration by TEV-protease but due to changes in our project design is no longer needed. In addition, mCardinal contains a His-tag to enable to control expression independent of fluorescence measurements.

asr Promoter

Alx was first described in 1990 by Bingham et al3. They created over 93.00 operon fusion with lacZ and screened those for increased activity at pH 8.5. The locus they found was named alx. In 2009 the function of alx was characterised by Nechooshtan et al4. They showed that the 5’ part of alx mRNA regulates translation by forming secondary structures. High pH leads to pausing in transcription of this mRNA part which leads to a different secondary structure allowing the ribosom to bind the RBS. Under neutral conditions the transcription is not stopped and secondary structures disable the ribosom to bind the RBS. This mechanism makes alx the first discovered pH-responsive riboregulatory gene.
We used this regulatory unit to express mNeonGreen under alkaline conditions. To increase expression an extra RBS was added after the riboswitch, leading to a constitutive expression of mNeonGreen. Hence, we used our constructed without the extra RBS to get pH depended expression but also showed that the riboswitch really is the regulatory part of this system.
[alx 'timeline']
Our construct still includes a TEV-site in combination with the F-degron, this allows fast degeneration by TEV-protease but due to changes in our project design is no longer needed. In addition, mNeonGreen contains a FLAG-tag to enable to control expression independent of fluorescence measurements.