Difference between revisions of "Part:BBa K2348000"
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Alx was first described in 1990 by Bingham et al<sup>1</sup>. 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 al<sup>2</sup>. 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. | Alx was first described in 1990 by Bingham et al<sup>1</sup>. 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 al<sup>2</sup>. 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. | ||
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===Usage and Biology=== | ===Usage and Biology=== | ||
Revision as of 21:34, 30 October 2017
alx - alkaline regulated riboswitch
Alx was first described in 1990 by Bingham et al1. 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 al2. 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.
Usage and Biology
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 this showed that the riboswitch really is the regulatory part of this system.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 312
Functional Parameters
To prove the alx promoter and riboswitch can be used to control pH dependent expression we created our working construct BBa_K2348012. This part contains the alx promoter and riboswitch controlling expression of mNeonGreen. To enhance degradation of mNeoGreen when needed, a TEV-side and f-degron are added N-terminal and for detection on protein level it contains a C-terminal FLAG-tag.
With this construct we showed that the alx promoter and riboswitch are able to regulate mNeonGreen expression when pH is shifted to pH 8 and above. To do so we let bacteria grown and then inoculated fresh media with adjusted pH (pH 7,8 and 8,5) to an OD600 of 0,2. After 20min and 40min 1ml of each culture were taken and diluted to the lowest OD600 of the three samples and then fluorescence and OD600 where measured with the plate reader (n=3).
Figure 1. Alx controlled mNeonGreen Expression: Culture media was bufferd to pH 7, 8 and 8,5 and inoculated to an OD600 of 0,2. After 20 and 40 minutes aliquotes where taken and fluorescence (Extinction 490nm, Absorbance 520nm) and OD600 where measured with the plate reader.(n=3)
As shown in figure 1. Expression of mNeonGreen is increased at pH 8 and 8,5 compared to standard pH 7. Cultures at ph 7 but also cultures without mNeonGreen as plasmid (data not shown) show a high basic fluorescence leading to the conclusion that the cells on their own show emission at 520nm. But still fluorescence is increased 2 fold at pH 8,5 after 20 and 40 minutes and fluorescence is significant increased at pH 8 at both measuring points.