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 chose the promoter of YcgF/E as blue light sensor and Cph8 as red light sensor. In the beginning, we also thought about using ccaR/ccaS as green-light sensor (instead of either YcgF/E or Cph8). The reason we decided to use a combination of the red and blue light sensor systems, is 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 and cloning has been completed while characterization 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 chose the promoter of YcgF/E as blue light sensor and Cph8 as red light sensor. In the beginning, we also thought about using ccaR/ccaS as green-light sensor (instead of either YcgF/E or Cph8). The reason we decided to use a combination of the red and blue light sensor systems, is 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 and cloning has been completed while characterization 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.