Difference between revisions of "Part:BBa K568000"

Latest revision as of 19:37, 1 November 2011

Red light sensor

Single red light inducible construct without downstream genes. This part can be cloned upstream of any desired gene product.

Usage and Biology

Red light induces the autophosphorylation at the cytosolic site of cph8. This leads to phosphorylation of OmpR which subsequently binds to OmpC promotor and enables transcription of a following gene. This part needs an EnvZ deficient strain such as CP919 to function properly, as EnvZ pathway is supposedly also responsible for sensing of aspartate.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 7
Illegal NheI site found at 30
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 644
Illegal XhoI site found at 1786
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 971
1000
COMPATIBLE WITH RFC[1000]

Functional Parameters

n/a	Red light sensor

Induction with red light at 705 nm. Since OmpR is expressed phosphorylated and therefore gene expression is induced in the dark, red light signalling can be shut down with 650 nm.[1]

Experimental Testing

We performed Miller Assays, using this part and its motherpart BBa_K322127 and a part containing cph8 (kindly provided by Team Uppsala-Sweden 2011) as a negative control. We tested these three parts in E. coli CP919 (part BBa_V1012) at 37°C, with irradiation at 650 nm ("shut down wavelength") and at 705 nm ("induction wavelength") as well as in the dark. The E. coli CP919 genome carries OmpC-LacZ fusion, which is why we expected the strain to express LacZ after irradiation with 705 nm. However, the data obtained did not match our expectations. Additional testing with GFP in JT2 cells indicated, that this sensor is not sensitive to red light only but works light independet. For further information see Results in Team TU Munich 2011 wiki and experience section in part BBa_K322127.

@@ Line 11: / Line 11: @@
 <span class='h3bb'>Sequence and Features</span>
 <partinfo>BBa_K568000 SequenceAndFeatures</partinfo>
 ===Functional Parameters===
 <partinfo>BBa_K568000 parameters</partinfo>
-Induction with red light at 705 nm.
+Induction with red light at 705 nm. Since OmpR is expressed phosphorylated and therefore gene expression is induced in the dark, red light signalling can be shut down with 650 nm.[1]
+===Experimental Testing===
-Since OmpR is expressed phosphorylated and therefore gene expression is induced in the dark, red light signalling can be shut down with 650 nm.[1]
+We performed Miller Assays, using this part and its motherpart BBa_K322127 and a part containing cph8 (kindly provided by Team Uppsala-Sweden 2011) as a negative control. We tested these three parts in ''E. coli'' CP919  (part BBa_V1012) at 37°C, with irradiation at 650 nm ("shut down wavelength") and at 705 nm ("induction wavelength") as well as in the dark. The ''E. coli'' CP919 genome carries OmpC-LacZ fusion, which is why we expected the strain to express LacZ after irradiation with 705 nm. However, the data obtained did not match our expectations.
+Additional testing with GFP in JT2 cells indicated, that this sensor is not sensitive to red light only but works light independet. For further information see <html><a href="http://2011.igem.org/Team:TU_Munich/lab/results">Results</a></html> in Team TU Munich 2011 wiki and experience section in part <html><a href="https://parts.igem.org/Part:BBa_K322127">BBa_K322127</a></html>.