Difference between revisions of "Team:NAWI Graz/pHPlasmid"
| Line 33: | Line 33: | ||
</li> | </li> | ||
<li class="list-group-item "> | <li class="list-group-item "> | ||
| − | <b>asr | + | <b>asr controlled mCardinal: </b> |
<a href=“http://parts.igem.org/Part:BBa_K2348011”> BBa_ K2348011</a> | <a href=“http://parts.igem.org/Part:BBa_K2348011”> BBa_ K2348011</a> | ||
</li> | </li> | ||
| Line 77: | Line 77: | ||
<ul class="list-group"> | <ul class="list-group"> | ||
<li class="list-group-item"> | <li class="list-group-item"> | ||
| − | <b> | + | <b>alx Promoter: </b> |
<a href="http://parts.igem.org/Part: BBa_ K2348000">alx Promoter and Roboswitch</a> | <a href="http://parts.igem.org/Part: BBa_ K2348000">alx Promoter and Roboswitch</a> | ||
</li> | </li> | ||
<li class=" list-group-item "> | <li class=" list-group-item "> | ||
| − | <b> | + | <b>mNeonGreen: </b> |
<a href=“http://parts.igem.org/Part:BBa_K1761003”>mNeonGreen</a> | <a href=“http://parts.igem.org/Part:BBa_K1761003”>mNeonGreen</a> | ||
</li> | </li> | ||
<li class="list-group-item "> | <li class="list-group-item "> | ||
| − | <b> | + | <b>alx controlled mNeonGreen: </b> |
<a href=“http://parts.igem.org/Part:BBa_K2348012”>alx controlled mNeonGreen</a> | <a href=“http://parts.igem.org/Part:BBa_K2348012”>alx controlled mNeonGreen</a> | ||
</li> | </li> | ||
Revision as of 15:22, 30 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: BBa_ K2348001
- mCardinal: BBa_ K2348002
- asr controlled mCardinal: BBa_ K2348011
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???
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 Promoter: alx Promoter and Roboswitch
- mNeonGreen: mNeonGreen
- alx controlled mNeonGreen: alx controlled mNeonGreen
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.
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.
