Difference between revisions of "Part:BBa K568001"

Revision as of 20:17, 20 September 2011

Optogenetical AND-Gate red/blue light

This logical gate is based on amber stop-codon suppression via the non-canonical tRNA supD (BBa_K228001). The blue light sensor (BBa_K238013) induces the expression of a mRNA coding for a T7-polymerase with an amber mutation (BBa_K228000), that can only be translated by ribosomes if the correct supD tRNA is present. The supD tRNA is expressed when the red light sensor (BBa_K568000) is induced. Thus, only cells which receive both signals produce the desired substances.

Usage and Biology

If the part is hit by both far red light (705 nm) and blue light (465 nm) beams, the AND-Gate is turned on. 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 the supD t-RNA. Blue light leads to dimerisation of YcgF and binding of the repressor YcgE. The formation of the YcgE-YcgF complex leads to the unbinding of YcgE from the YcgZ promoter which activates the transcription of T7ptag (T7 polymerase with the amber stop codon mutation) if enough supD tRNA is available.

This AND-gate should ensure that expression of T7ptag is only induced when both wavelengths hit bacteria with the part.

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
Illegal AgeI site found at 3817
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 3820

Functional Parameters

n/a	Optogenetical AND-Gate red/blue light

@@ Line 8: / Line 8: @@
 ===Usage and Biology===
-The red light sensor is active in the ground state, so the signalling needs to be shut down. This can be achieved by irradiation with light of 650 nm.
 If the part is hit by both far red light (705 nm) and blue light (465 nm) beams, the AND-Gate is turned on.
-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 the supD t-RNA. This signal transduction pathway is supposedly also responsible for sensing of aspartate, a substance which is present in LB as well as other common media for E. coli. Hence a strain with EnvZ knockout is desireable. For this the strain CP919 (V1012) can be used. Blue light leads to dimerisation of YcgF and binding of the repressor YcgE. The formation of the YcgE-YcgF complex leads to the unbinding of YcgE from the YcgZ promoter which activates the transcription of T7ptag (T7 polymerase with the amber stop codon mutation) if enough supD tRNA is available.
+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 the supD t-RNA.
+Blue light leads to dimerisation of YcgF and binding of the repressor YcgE. The formation of the YcgE-YcgF complex leads to the unbinding of YcgE from the YcgZ promoter which activates the transcription of T7ptag (T7 polymerase with the amber stop codon mutation) if enough supD tRNA is available.
 This AND-gate should ensure that expression of T7ptag is only induced when both wavelengths hit bacteria with the part.