Difference between revisions of "Part:BBa K2348000"
(→Functional Parameters) |
(→Functional Parameters) |
||
| (30 intermediate revisions by 2 users not shown) | |||
| Line 1: | Line 1: | ||
| − | |||
__NOTOC__ | __NOTOC__ | ||
| − | <partinfo>BBa_K2348000 short</partinfo>< | + | <partinfo>BBa_K2348000 short</partinfo><br> |
| − | Alx was first described in 1990 by Bingham et al<sup>1</sup>. They created over | + | Alx was first described in 1990 by Bingham et al<sup>1</sup>. They created over 93000 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 characterized 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 a pause in transcription of this mRNA part which leads to a different secondary structure, allowing the ribosome to bind the RBS. Under neutral conditions, the transcription is not stopped and secondary structures disable ribosome binding to RBS. This mechanism makes alx the first discovered pH-responsive riboregulatory gene. |
| − | + | ||
===Usage and Biology=== | ===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 | + | 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 construct without the extra RBS to get pH-dependent expression. But this showed that the riboswitch is in fact the regulatory part of this system. |
<!-- --> | <!-- --> | ||
| − | <span class='h3bb'>Sequence and Features</span> | + | <br><br><b><span class='h3bb'>Sequence and Features</span></b> |
<partinfo>BBa_K2348000 SequenceAndFeatures</partinfo> | <partinfo>BBa_K2348000 SequenceAndFeatures</partinfo> | ||
| Line 15: | Line 13: | ||
<partinfo>BBa_K2348000 parameters</partinfo> | <partinfo>BBa_K2348000 parameters</partinfo> | ||
<!-- --> | <!-- --> | ||
| − | To prove the alx promoter and riboswitch can be used to control pH dependent expression we created | + | To prove the alx promoter and riboswitch can be used to control pH dependent expression, we created a working construct [https://parts.igem.org/Part:BBa_K2348012 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-terminally and for detection on the 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 | + | 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 grow and then inoculated fresh media with adjusted pH (pH 7, 8 and 8.5) to an OD<sub>600</sub> of 0.2. After 20 min and 40 min 1 ml of each culture was taken and diluted to the lowest OD<sub>600</sub> of the three samples and then fluorescence and OD<sub>600</sub> was measured with the plate reader (n=3). |
| − | https://static.igem.org/mediawiki/2017/6/66/Alx_expression_results.png | + | |
| + | https://static.igem.org/mediawiki/2017/6/66/Alx_expression_results.png <br> | ||
| + | <font size="-2"><b>Figure 1. Alx controlled mNeonGreen Expression: </b>Culture media was bufferd to pH 7, 8 and 8.5 and inoculated to an OD<sub>600</sub> of 0.2. After 20 and 40 minutes, aliquotes where taken and fluorescence (extinction 490 nm, emission 520 nm) and OD<sub>600</sub> were measured with the plate reader (n=3).</font><br> | ||
| + | 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 the mNeonGreen plasmid (data not shown) show a high basic fluorescence, leading to the conclusion that the cells naturally show emission at 520 nm. Still, fluorescence is increased twofold at pH 8.5 after 20 and 40 minutes and fluorescence is significantly increased at pH 8 at both measuring points.<br> | ||
| + | <br><font size="-2"><sup>1</sup>Bingham, R. J., Hall, K. S. & Slonczewski, J. L. Alkaline induction of a novel gene locus, alx, in <i>Escherichia coli</i>. Journal of Bacteriology 172, 2184–2186 (1990). | ||
| + | |||
| + | <sup>2</sup>Nechooshtan, G., Elgrably-Weiss, M., Sheaffer, A., Westhof, E. & Altuvia, S. A pH-responsive | ||
| + | riboregulator. Genes & Development 23, 2650–2662 (2009). </font> | ||
Latest revision as of 21:31, 31 October 2017
alx - alkaline regulated riboswitch
Alx was first described in 1990 by Bingham et al1. They created over 93000 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 characterized by Nechooshtan et al2. They showed that the 5’ part of alx mRNA regulates translation by forming secondary structures. High pH leads to a pause in transcription of this mRNA part which leads to a different secondary structure, allowing the ribosome to bind the RBS. Under neutral conditions, the transcription is not stopped and secondary structures disable ribosome binding to 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 construct without the extra RBS to get pH-dependent expression. But this showed that the riboswitch is in fact 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 a 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-terminally and for detection on the 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 grow and then inoculated fresh media with adjusted pH (pH 7, 8 and 8.5) to an OD600 of 0.2. After 20 min and 40 min 1 ml of each culture was taken and diluted to the lowest OD600 of the three samples and then fluorescence and OD600 was 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 490 nm, emission 520 nm) and OD600 were 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 the mNeonGreen plasmid (data not shown) show a high basic fluorescence, leading to the conclusion that the cells naturally show emission at 520 nm. Still, fluorescence is increased twofold at pH 8.5 after 20 and 40 minutes and fluorescence is significantly increased at pH 8 at both measuring points.
1Bingham, R. J., Hall, K. S. & Slonczewski, J. L. Alkaline induction of a novel gene locus, alx, in Escherichia coli. Journal of Bacteriology 172, 2184–2186 (1990).
2Nechooshtan, G., Elgrably-Weiss, M., Sheaffer, A., Westhof, E. & Altuvia, S. A pH-responsive riboregulator. Genes & Development 23, 2650–2662 (2009).