Optogenetical AND-Gate
In order to be able to adress our immobilized cells in a defined spatiotemporal manner, we require two signals which will be irradiated into the gel block from two different directions. For this purpose, we will introduce an optogenetical AND-Gate into the bacteria. This logical gate is based on amber stop-codon suppression via the non-canonical tRNA supD. A light sensitive promoter induces the expression of a mRNA coding for a T7-polymerase, which can only be translated by ribosomes if the correct amber tRNA is present. The tRNA is expressed by a second light-sensitive promoter. Thus, only cells which receive both signals produce the desired substances.
Light Sensory Domains
We have three candidates which can be used as light sensitive promoters: the promoter of YcgF/E as blue light sensor, Cph8 as red light sensor, and ccaR/ccaS as green-light sensor. At this point, we are inclined to use a combination of the red and blue light sensor systems, because of the great difference between the absorbtion maxima of the light sensory domains. We expect that this assembly will be the one with the lowest amount of unspecific gene expression which could occur due to overlapping of the absorption spectrums of the sensory domains.
Progress
Construct design has been completed while cloning is still in progress. Further more, we found a suitable solid matrix which is, on the one hand, clear enough to be penetrated by light beams without refracting them and, on the other hand, solidifies at a temperature that is low enough to allow for cell immobilization without harming the bacteria.
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Optogenetical AND-Gate
In order to be able to adress our immobilized cells in a defined spatiotemporal manner, we require two signals which will be irradiated into the gel block from two different directions. For this purpose, we will introduce an optogenetical AND-Gate into the bacteria. This logical gate is based on amber stop-codon suppression via the non-canonical tRNA supD. A light sensitive promoter induces the expression of a mRNA coding for a T7-polymerase, which can only be translated by ribosomes if the correct amber tRNA is present. The tRNA is expressed by a second light-sensitive promoter. Thus, only cells which receive both signals produce the desired substances.
Light Sensory Domains
We have three candidates which can be used as light sensitive promoters: the promoter of YcgF/E as blue light sensor, Cph8 as red light sensor, and ccaR/ccaS as green-light sensor. At this point, we are inclined to use a combination of the red and blue light sensor systems, because of the great difference between the absorbtion maxima of the light sensory domains. We expect that this assembly will be the one with the lowest amount of unspecific gene expression which could occur due to overlapping of the absorption spectrums of the sensory domains.
Progress
Construct design has been completed while cloning is still in progress. Further more, we found a suitable solid matrix which is, on the one hand, clear enough to be penetrated by light beams without refracting them and, on the other hand, solidifies at a temperature that is low enough to allow for cell immobilization without harming the bacteria.