Difference between revisions of "Part:BBa K911009:Experience"

 
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===Applications of BBa_K911009===
 
===Applications of BBa_K911009===
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Our initial construct used pSPAK as our inducible (i.e. parameter sensitive) promotor. The construct we made can be seen below:
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[[Image:Ratiometrica.png|center]]
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Initial assays were performed with just the construct and no induction of the promotor in order to check the reliability of our ratiometric output. These initial assays gave promising results; YFP increased dramatically during exponential phase, as did CFP to a lesser extent. Given that CFP was not being driven by IPTG, it was not surprising that this should be the case. The OD600/YFP ratio also settled down to a constant value for all cultures at the end of exponential phase, indicating that our YFP channel was a reliable indicator of cell activity in constant conditions.
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However, as we moved on to checking our construct worked for measuring the concentration of IPTG, we noticed some problems. While our construct did produce a constant ratio of CFP/YFP in our cultures during steady state (representative samples of this ratio taking can be seen), the ratio during exponential phase changed rapidly. We have reason to believe that this may have been due to the short amount of time between induction and the end of exponential phase (~4hours, as opposed to a more ideal 15 or so). In this short time span, the fluorescent proteins already present in the cells may not have had time to 'dilute' out upon successive cell divisions.
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[[Image:GradientComparison.jpg|left|300px|thumb|Graphs of YFP normalised fluorescence vs. CFP normalized fluorescence for three different IPTG concentrations. Note the extreme linearity, indicating that our internal ratiometric control is working.]]
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Nevertheless, we hypothesized that the ratio during steady state may be a good indicator of IPTG concentration. To determine this ratio, we assumed that accumulation of fluorescent proteins during steady state was constant, as production was constant and degradation in this time period was negligible. Making these assumptions, simply taking the gradient of the linear regression of the CFP intensity vs. YFP intensity gave a readout of the
  
 
===User Reviews===
 
===User Reviews===

Revision as of 02:43, 2 November 2012

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K911009

Our initial construct used pSPAK as our inducible (i.e. parameter sensitive) promotor. The construct we made can be seen below:

Ratiometrica.png

Initial assays were performed with just the construct and no induction of the promotor in order to check the reliability of our ratiometric output. These initial assays gave promising results; YFP increased dramatically during exponential phase, as did CFP to a lesser extent. Given that CFP was not being driven by IPTG, it was not surprising that this should be the case. The OD600/YFP ratio also settled down to a constant value for all cultures at the end of exponential phase, indicating that our YFP channel was a reliable indicator of cell activity in constant conditions.

However, as we moved on to checking our construct worked for measuring the concentration of IPTG, we noticed some problems. While our construct did produce a constant ratio of CFP/YFP in our cultures during steady state (representative samples of this ratio taking can be seen), the ratio during exponential phase changed rapidly. We have reason to believe that this may have been due to the short amount of time between induction and the end of exponential phase (~4hours, as opposed to a more ideal 15 or so). In this short time span, the fluorescent proteins already present in the cells may not have had time to 'dilute' out upon successive cell divisions.

Graphs of YFP normalised fluorescence vs. CFP normalized fluorescence for three different IPTG concentrations. Note the extreme linearity, indicating that our internal ratiometric control is working.

Nevertheless, we hypothesized that the ratio during steady state may be a good indicator of IPTG concentration. To determine this ratio, we assumed that accumulation of fluorescent proteins during steady state was constant, as production was constant and degradation in this time period was negligible. Making these assumptions, simply taking the gradient of the linear regression of the CFP intensity vs. YFP intensity gave a readout of the

User Reviews

UNIQd38caad05ac6cd4e-partinfo-00000000-QINU UNIQd38caad05ac6cd4e-partinfo-00000001-QINU