Team:Austin Texas/photocage

From 2014.igem.org

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After this exposure, the cells were grown for an additional [1 hour?], allowing the newly decaged T7 RNAP to transcribe the GFP reporter gene, which ultimately results in fluorescence.
After this exposure, the cells were grown for an additional [1 hour?], allowing the newly decaged T7 RNAP to transcribe the GFP reporter gene, which ultimately results in fluorescence.
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=Discussion=
 
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The data from our photocage project provides strong evidence that we have, in fact, replicated the light-activated protein expression system that Chou et al. had constructed.
 
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From the data, we are able to observe that GFP expression has a positive correlation with the amount of time that it is irradiated by light. Although the difference in fluorescence between 0 minutes and 1 minute appears to be negligible, there was a clear change in fluorescence at the 5 minute, 15 minute, and 30 minute time intervals. Although the increases in fluorescence are clearly present, there are still a few problems regarding the system. For one, the level of background expression of the uninduced T7 RNAP is relatively high. The uninduced caged T7 RNAP has roughly 25 times as much expression as the measured negative control. The relatively high level of fluorescence may be explained by the efficient nature of T7 RNAP. Even if a few polymerases were "decaged", they would have 16 hours to bind to the promoter and transcribe the RNA transcript for GFP. In order to decrease the level of background, a second amber stop codon was mutated at position _______. 
 
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The replication of such a system provides the foundation for potential projects for our future iGEM teams. By replacing the GFP reporter with coding sequences for other proteins, ....
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=Discussion=
 +
 
 +
The data from our photocage project provides strong evidence that we have, in fact, replicated the light-activated protein expression system that Chou et al. had constructed.
 +
From the data, we are able to observe that GFP expression has a positive correlation with the amount of time that it is irradiated by light. Although the difference in fluorescence between 0 minutes and 1 minute appears to be negligible, there was a clear change in fluorescence at the 5 minute, 15 minute, and 30 minute time intervals.
 +
 +
Although the increases in fluorescence are clearly present, there are still a few problems regarding the system. For one, the level of background expression of the uninduced T7 RNAP is relatively high. The uninduced caged T7 RNAP has roughly 25 times as much expression as the measured negative control. The relatively high level of fluorescence may be explained by the efficient nature of T7 RNAP. Even if a few polymerases were "decaged", they would have 16 hours to bind to the promoter and transcribe the RNA transcript for GFP. In order to decrease the level of background, a second amber stop codon was mutated into _______. 
 +
 
 +
The replication of such a system provides the foundation for potential projects for our future iGEM teams. By replacing the GFP reporter with coding sequences for other proteins, we will be able to find novel ........  

Revision as of 01:58, 15 October 2014