Difference between revisions of "Part:BBa K911009:Experience"
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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: | Our initial construct used pSPAK as our inducible (i.e. parameter sensitive) promotor. The construct we made can be seen below: | ||
| − | [[Image:Ratiometrica.png|center]] | + | [[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 | + | 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 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. | ||
| + | </gallery> | ||
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. | 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. | ||
| − | [[Image:GradientComparison.jpg|left| | + | [[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 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|center|900px|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 preferred 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. Notice the tick shape of the data. The raw CFP data and the normalized CFP data give approximately what you would expect - a sharp rise at low IPTG concentrations, followed by a more modest rise as IPTG concentrations increase further. However, if we take the gradient of the graph of YFP vs CFP as a measure of IPTG concentration, then you would expect higher IPTG concentrations to give lower gradients - the opposite trend is observed for the majority of the data! This warrants further investigation, particularly to see if this kind of trend can be reproduced in better controlled conditions. Unfortunately, due to difficulties in our medium production, we were unable to produce any more data that could be usefully analysed. | ||
| + | |||
| + | We also attempted to switch our pSPACK promotor out and replace it with the fluoride riboswitch, as shown below. Unfortunately, the small size of the riboswitch made it very hard to insert with Gibson assembly (we suspect that it may have been fully degraded by the T5 exonuclease before insertion), and as such our attempts were not successful. | ||
| − | + | [[Image:FRSplusRatiometrica.png|600px|center|thumb|The construct that we hoped to make and test with fluoride.]] | |
===User Reviews=== | ===User Reviews=== | ||
Latest revision as of 03:24, 2 November 2012
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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:
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.
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.
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 preferred 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. Notice the tick shape of the data. The raw CFP data and the normalized CFP data give approximately what you would expect - a sharp rise at low IPTG concentrations, followed by a more modest rise as IPTG concentrations increase further. However, if we take the gradient of the graph of YFP vs CFP as a measure of IPTG concentration, then you would expect higher IPTG concentrations to give lower gradients - the opposite trend is observed for the majority of the data! This warrants further investigation, particularly to see if this kind of trend can be reproduced in better controlled conditions. Unfortunately, due to difficulties in our medium production, we were unable to produce any more data that could be usefully analysed.
We also attempted to switch our pSPACK promotor out and replace it with the fluoride riboswitch, as shown below. Unfortunately, the small size of the riboswitch made it very hard to insert with Gibson assembly (we suspect that it may have been fully degraded by the T5 exonuclease before insertion), and as such our attempts were not successful.
User Reviews
UNIQ72d28e094e84e7a1-partinfo-00000001-QINU UNIQ72d28e094e84e7a1-partinfo-00000002-QINU