Difference between revisions of "Part:BBa K3098021"
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<partinfo>BBa_K3098021 short</partinfo> | <partinfo>BBa_K3098021 short</partinfo> | ||
| − | The promoter pOmpC is positively regulated, OmpR-controlled promoter (from Part: <html><a href="https://parts.igem.org/Part:BBa_R0082">BBa_R0082</a></html> ). This promoter is taken from the upstream region of OmpC. Phosphorylated OmpR binds to the three operator sites and activates transcription. Its expression is related to the osmotic pressure. In nature, this promoter is upstream of the OmpC porin gene. The regulation of OmpC is determined by the EnvZ-OmpR osmosensing machinery. EnvZ phosphorylates OmpR to OmpR-P. At high | + | The promoter pOmpC is positively regulated, OmpR-controlled promoter (from Part: <html><a href="https://parts.igem.org/Part:BBa_R0082">BBa_R0082</a></html> ). This promoter is taken from the upstream region of OmpC. Phosphorylated OmpR binds to the three operator sites and activates transcription. Its expression is related to the osmotic pressure. In nature, this promoter is upstream of the OmpC porin gene. The regulation of OmpC is determined by the EnvZ-OmpR osmosensing machinery. EnvZ phosphorylates OmpR to OmpR-P. At high osmotic pressure, EnvZ is more active, creating more OmpR-P. OmpR-P then binds to the low-affinity OmpR operator sites upstream of ompC. |
| − | But we found that | + | But we found that this promoter can be used not only for its osmosensing function, in some other condition, it is also used as a part of a system, such as the red light system ( from Part: <html><a href="https://parts.igem.org/Part:BBa_K1017301">BBa_K1017301</a></html> ). In such condition, we hope to get rid of the influence of the osmosis, thus, we create a new part by adding a riboswitch after the promoter, to realize another kind of control via theophylline. |
| + | <br> | ||
| + | |||
| + | [[File:BBa_K3098021_riboswitch.png|thumb|600px|center|'''Fig.Mechanism of riboswitch controlled by theophylline.''']] | ||
===Usage and Biology=== | ===Usage and Biology=== | ||
| − | + | <p align="left"> | |
| − | This part better to be expressed in DH5α, because the EnvZ-OmpR is a protein naturally expressed in K-12 strain. And the unprocessed state of this part is off, because when there | + | This part is better to be expressed in DH5α, because the EnvZ-OmpR is a protein naturally expressed in K-12 strain. And the unprocessed state of this part is off, because when there is no theophylline, the riboswitch is turned off. When you want it to be on and start to express the target gene, you have to add the theophylline to open the riboswitch. Thus we achieved the tightly control of this promoter via a riboswitch. |
| − | + | </p> | |
<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
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| − | === | + | ===Results=== |
<partinfo>BBa_K3098021 parameters</partinfo> | <partinfo>BBa_K3098021 parameters</partinfo> | ||
| − | + | <p align="left"> | |
Two experiments were did to confirm the tightly control of this new part. | Two experiments were did to confirm the tightly control of this new part. | ||
| − | < | + | <br> |
| + | Firstly, transform the plasmids of pOmpC with a sfGFP and pOmpC+ribo with a sfGFP into DH5α. When the OD is between 0.6~0.8, we move them into different LB fluid medium had different osmotic pressure in order to verified the close effect of riboswitch. The result (Fig.1) is that the pOmpC with riboswitch had lower fluorescence value than pOmpC. What’s more, the amplitude of variation of pOmpC is greater than pOmpC with ribo, which means that adding the riboswitch can decrease the influence of osmotic pressure on the promoter pOmpC. | ||
| + | </p> | ||
| + | |||
| + | |||
| + | <center> | ||
| + | https://static.igem.org/mediawiki/parts/2/2c/BBa_K3098021_pOmpC_2%2Bribo.png | ||
| + | </center> | ||
| + | |||
| + | <br> | ||
| + | <p align="left"> | ||
| + | Under the best osmosis condition of pOmpC + ribo (0.1% NaCl), Theophylline is added to the final concentration from 0mM - 8mM. When the concentration of theophylline between 0 -2 mM, the promoter pOmpC with riboswitch did not open, but when it came to 4mM, the fluorescence value increased significantly. | ||
| + | <br> | ||
| + | </p> | ||
| + | <center> | ||
| + | https://static.igem.org/mediawiki/parts/f/fb/BBa_K3098021_theophylline%2Bribo.png | ||
| + | </center> | ||
| + | ===Functional Parameters=== | ||
| + | <partinfo>BBa_K3098024 parameters</partinfo> | ||
Latest revision as of 01:00, 22 October 2019
Omp Promoter + riboswitch/sfGFP
The promoter pOmpC is positively regulated, OmpR-controlled promoter (from Part: BBa_R0082 ). This promoter is taken from the upstream region of OmpC. Phosphorylated OmpR binds to the three operator sites and activates transcription. Its expression is related to the osmotic pressure. In nature, this promoter is upstream of the OmpC porin gene. The regulation of OmpC is determined by the EnvZ-OmpR osmosensing machinery. EnvZ phosphorylates OmpR to OmpR-P. At high osmotic pressure, EnvZ is more active, creating more OmpR-P. OmpR-P then binds to the low-affinity OmpR operator sites upstream of ompC.
But we found that this promoter can be used not only for its osmosensing function, in some other condition, it is also used as a part of a system, such as the red light system ( from Part: BBa_K1017301 ). In such condition, we hope to get rid of the influence of the osmosis, thus, we create a new part by adding a riboswitch after the promoter, to realize another kind of control via theophylline.
Usage and Biology
This part is better to be expressed in DH5α, because the EnvZ-OmpR is a protein naturally expressed in K-12 strain. And the unprocessed state of this part is off, because when there is no theophylline, the riboswitch is turned off. When you want it to be on and start to express the target gene, you have to add the theophylline to open the riboswitch. Thus we achieved the tightly control of this promoter via a riboswitch.
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 822
Results
Two experiments were did to confirm the tightly control of this new part.
Firstly, transform the plasmids of pOmpC with a sfGFP and pOmpC+ribo with a sfGFP into DH5α. When the OD is between 0.6~0.8, we move them into different LB fluid medium had different osmotic pressure in order to verified the close effect of riboswitch. The result (Fig.1) is that the pOmpC with riboswitch had lower fluorescence value than pOmpC. What’s more, the amplitude of variation of pOmpC is greater than pOmpC with ribo, which means that adding the riboswitch can decrease the influence of osmotic pressure on the promoter pOmpC.
Under the best osmosis condition of pOmpC + ribo (0.1% NaCl), Theophylline is added to the final concentration from 0mM - 8mM. When the concentration of theophylline between 0 -2 mM, the promoter pOmpC with riboswitch did not open, but when it came to 4mM, the fluorescence value increased significantly.
Functional Parameters
| chassis | E.coli BL21 |