Difference between revisions of "Part:BBa K568001"

Latest revision as of 19:36, 1 November 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. Cells need to be transfected with another plasmid, containing a T7 promoter followed by a reporter.

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 T7 polymerase 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

Induction with red light at 705 nm

Induction with blue light at 465 nm

Shut down of constant red light signalling with 650 nm

Experimental Testing

The part was tested using the following setup:

K568001 (in vector pSB1C3) and Part:BBa_I746907 (as a GFP-based reporter, in vector pSB6A1) were electroporated into e. coli CP919. A clone from the transformation was picked and incubated over night in LB (Amp, Cam) under red light (650 nm) to shut down the and-gate. The next day, the culture was diluted with LB (Amp, Cam) to an initial OD600 of approx. 0.2, and divided into three parts. These three groups were incubated under three different lighting conditions:

in the dark
under red (705 nm) and blue (465 nm) light (using LEDs)
under the lamp of the incubator (white light).

The expression of GFP was measured using a GFP assay as follows: Over the next 6 hours, the OD600 and the GFP-fluorescence was measured (time between measurements: 1 hour). 16 hours after the last measurement, the terminal OD600 and GFP-fluoresence were measured. Also, serveral measurements of LB-Medium as blank were done. To get information about the "fluorescence per cell", the following formula was used:

"Fluorescence per cell" = (fluorescence(sample) - fluorescence(blank)) / (OD600(sample) - OD600 (blank))

Unfortunately the results we obtainend indicate that the AND Gate does not work as expected. The reason can be the red light sensor which seems not to be light sensitive. For further information see Results in Team TU Munich 2011 wiki and experience section in part BBa_K322127.

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K568001 short</partinfo>
-This logical gate is based on amber stop-codon suppression via the non-canonical tRNA supD(Ba_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(<a href="https://parts.igem.org/Part:BBa_K568000">aaaaaa</a>) is induced. Thus, only cells which receive both signals produce the desired substances.
+This logical gate is based on amber stop-codon suppression via the non-canonical tRNA supD (<html><a href="https://parts.igem.org/Part:BBa_K228001">BBa_K228001</a></html>). The blue light sensor (<html><a href="https://parts.igem.org/Part:BBa_K238013">BBa_K238013</a></html>) induces the expression of a mRNA coding for a T7-polymerase with an amber mutation (<html><a href="https://parts.igem.org Part:BBa_K228000">BBa_K228000</a></html>), that can only be translated by ribosomes if the correct supD tRNA is present. The supD tRNA is expressed when the red light sensor (<html><a href="https://parts.igem.org/Part:BBa_K568000">BBa_K568000</a></html>) is induced. Thus, only cells which receive both signals produce the desired substances. Cells need to be transfected with another plasmid, containing a T7 promoter followed by a reporter.
+<html><img src="https://static.igem.org/mediawiki/2011/c/cd/TumDesign2.jpg"></html>
 ===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 T7 polymerase is only induced when both wavelengths hit bacteria with the part.
 <span class='h3bb'>Sequence and Features</span>
 <partinfo>BBa_K568001 SequenceAndFeatures</partinfo>
 ===Functional Parameters===
 <partinfo>BBa_K568001 parameters</partinfo>
+Induction with red light at 705 nm
+Induction with blue light at 465 nm
+Shut down of constant red light signalling with 650 nm
+===Experimental Testing===
+The part was tested using the following setup:
+K568001 (in vector pSB1C3) and [[Part:BBa_I746907]] (as a GFP-based reporter, in vector pSB6A1) were electroporated into ''e. coli'' CP919. A clone from the transformation was picked and incubated over night in LB (Amp, Cam) under red light (650 nm) to shut down the and-gate. The next day, the culture was diluted with LB (Amp, Cam) to an initial OD600 of approx. 0.2, and divided into three parts. These three groups were incubated under three different lighting conditions:
+*in the dark
+*under red (705 nm) and blue (465 nm) light (using LEDs)
+*under the lamp of the incubator (white light).
+The expression of GFP was measured using a GFP assay as follows:
+Over the next 6 hours, the OD600 and the GFP-fluorescence was measured (time between measurements: 1 hour). 16 hours after the last measurement, the terminal OD600 and GFP-fluoresence were measured. Also, serveral measurements of LB-Medium as blank were done. To get information about the "fluorescence per cell", the following formula was used:
+"Fluorescence per cell" = (fluorescence(sample) - fluorescence(blank))  /  (OD600(sample) - OD600 (blank))
+Unfortunately the results we obtainend indicate that the AND Gate does not work as expected. The reason can be the red light sensor which seems not to be light sensitive. 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>.