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.³. 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 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.

Alx was first described in 1990 by Bingham et al⁴. 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 al⁵. 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.

This part actually is a composite part containing the alx promoter and riboswitch and mNeonGreen. Additionally, between riboswitch and mNeonGreen a TEV-site and a F-degron are inserted. Also, mNeonGreen is FLAG-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.
The function of TEV-site and F-degron is explained above. The FLAG-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.