Difference between revisions of "Part:BBa K3098021"

Revision as of 13:46, 21 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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE 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.

BBa_K3098021_pOmpC_2%2Bribo.png

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.

BBa_K3098021_theophylline%2Bribo.png

Functional Parameters

chassis

E.coli BL21

@@ Line 3: / Line 3: @@
 <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 osmolarity, EnvZ is more active, creating more OmpR-P. OmpR-P then binds to the low-affinity OmpR operator sites upstream of ompC.
+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, this promoter can be used not only for its osmosensing function, in some other condition, it is also be 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.
+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>
@@ Line 16: / Line 16: @@
 ===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 are id 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.
+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>
@@ Line 30: / Line 30: @@
 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  ranged 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 by osmotic pressure for the promoter pOmpC.
+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>