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

Revision as of 03:12, 2 November 2012

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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:

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 YFP/OD600 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.

Graph of YFP/OD600 and time during exponential phase. Note that the OD600/YFP ratio reaches a constant value near the end of exponential phase, indicating that it is a good indicator of cell activity - this ratio gives a measure of the YFP per cell, which is constant.
Graph of YFP activity against OD600. Note the steep gradient of the data during exponential phase.
Graph of CFP activity against OD600. As there is some transcriptional leak through of the pSPACK promotor, it is not surprising to see some relationship between CFP and cell count. However, the gradient is much shallower, indicating much poorer transcription.

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 which could be used to form a callibration curve. Our attempts at doing this are shown in boxplot form - we have also provided the raw CFP readout and the CFP readout adjusted against OD600 for reference.

Boxplots of callibration data produced from our construct when induced at different concentrations of IPTG.

The first thing to notice about this boxplot is that our data does not seem to be very reliable. Especially at the lowest IPTG concentrations, the 25 percentile bars overlap large chunks of the data. This indicates that this technique of taking the ratio of CFP to YFP during steady state phase is flawed. We would have prefered to have used a more advanced technique, however this would have required us to use the data from exponential phase and, for the reasons discussed above, this was not possible.

Secondly, and possibly more interestingly, the trend in this box plot actually appears to be going in the opposite direction to what would be expected.

User Reviews

UNIQ61d81eae0ab925eb-partinfo-00000001-QINU UNIQ61d81eae0ab925eb-partinfo-00000002-QINU

@@ Line 9: / Line 9: @@
 [[Image:Ratiometrica.png|center|700px]]
-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.
+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 YFP/OD600 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.
 <gallery widths=400px heights=300px perrow=2 caption=''Results of inital plate reader assay.''>
-Image:YFPoverOD600vstime.png|Graph of OD600/YFP and time during exponential phase. Note that the OD600/YFP ratio reaches a constant value near the end of exponential phase, indicating that it is a good indicator of cell activity - this ratio gives a measure of the YFP per cell, which is constant.
+Image:YFPoverOD600vstime.png|Graph of YFP/OD600 and time during exponential phase. Note that the OD600/YFP ratio reaches a constant value near the end of exponential phase, indicating that it is a good indicator of cell activity - this ratio gives a measure of the YFP per cell, which is constant.
 Image:OD600againstYFP.png|Graph of YFP activity against OD600. Note the steep gradient of the data during exponential phase.
 Image:OD600againstCFP.png|Graph of CFP activity against OD600. As there is some transcriptional leak through of the pSPACK promotor, it is not surprising to see some relationship between CFP and cell count. However, the gradient is much shallower, indicating much poorer transcription.
@@ Line 21: / Line 21: @@
 [[Image:GradientComparison.jpg|left|400px|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.]]
-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
+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 which could be used to form a callibration curve. Our attempts at doing this are shown in boxplot form - we have also provided the raw CFP readout and the CFP readout adjusted against OD600 for reference.
+[[Image:Boxplots.png|right|400px|thumb|Boxplots of callibration data produced from our construct when induced at different concentrations of IPTG.]]
+The first thing to notice about this boxplot is that our data does not seem to be very reliable. Especially at the lowest IPTG concentrations, the 25 percentile bars overlap large chunks of the data. This indicates that this technique of taking the ratio of CFP to YFP during steady state phase is flawed. We would have prefered to have used a more advanced technique, however this would have required us to use the data from exponential phase and, for the reasons discussed above, this was not possible.
+Secondly, and possibly more interestingly, the trend in this box plot actually appears to be going in the opposite direction to what would be expected.
 ===User Reviews===