Team:SDU-Denmark/project-t

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(Bacterial flagellar motility)
(Photosensor)
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In our system we want to be able to control the amount of flow in the channel, through a remote signal. The signal we have chosen is light since we want to avoid altering the chemical composition of the fluid running through the channel. Having looked at previous iGEM work on light sensitive systems which have all been focused on transcriptional regulation, we realized that we would need a different approach for the fast response times our system requires. We have therefore focused our work on proteorhodopsins that integrate into the chemotaxis pathway, giving us very fast responses to light stimulation. <br><br>
In our system we want to be able to control the amount of flow in the channel, through a remote signal. The signal we have chosen is light since we want to avoid altering the chemical composition of the fluid running through the channel. Having looked at previous iGEM work on light sensitive systems which have all been focused on transcriptional regulation, we realized that we would need a different approach for the fast response times our system requires. We have therefore focused our work on proteorhodopsins that integrate into the chemotaxis pathway, giving us very fast responses to light stimulation. <br><br>
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Our construct centers around a synthetic protein created by Spudich ''Et al.''. It is a fusion chimeric protein that consists of an archaeal proteorhodopsin SRII (Sensory Rhodopsin II) and its transducer protein HtrII, both from ''Natronomonas pharaonis''. These are coupled to a Tar domain from a transmembrane receptor from ''Salmonella enterica''. The Tar domain is the part of the receptor that couples with CheA and CheW, and although it is taken from a different species, it has been shown to work in ''E. coli'' as well '''''REF'''''. In the construct we are working with light acts as an '''''attractant''''', reducing the tumbling rate upon illumination (see picture). This might help us to control our pumping power, by decreasing the fraction of bacteria tumbling in the channel by increasing light stimulus, thus promoting linear drive. The photosensor should be most active in light with a wavelength of about 500nm, according to the original article. We have used DNA sent to us from the original authors to isolate the coding sequence for the protein generator.<br><br>
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Our construct centers around a synthetic protein created by Spudich ''et al.''[[https://2010.igem.org/Team:SDU-Denmark/project-t#References 9]]. It is a fusion chimeric protein that consists of an archaeal proteorhodopsin SRII (Sensory Rhodopsin II) and its transducer protein HtrII, both from ''Natronomonas pharaonis''. These are coupled to a Tar domain from a transmembrane receptor from ''Salmonella enterica''. The Tar domain is the part of the receptor that couples with CheA and CheW, and although it is taken from a different species, it has been shown to work in ''E. coli'' as well [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 10]]. In the construct we are working with light should act as an attractant, reducing the tumbling rate upon illumination (see picture). This might help us to control our pumping power, by decreasing the fraction of bacteria tumbling in the channel by increasing light stimulus, thus promoting linear drive. The photosensor should be most active in light with a wavelength of about 500nm, according to the original article. We have used DNA sent to us from the original authors to isolate the coding sequence for the protein generator.<br><br>
Note that although the bacteria will be stationary in our system, since they are glued to the inner surface of the flowchannel, our construct in reality confers a phototactic ability to ''E. coli''.
Note that although the bacteria will be stationary in our system, since they are glued to the inner surface of the flowchannel, our construct in reality confers a phototactic ability to ''E. coli''.

Revision as of 19:37, 26 October 2010