Team:Wash U/Biological Parts
From 2009.igem.org
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'''Problem:''' In a typical reactor, cells at the surface absorb more than enough light to saturate their | '''Problem:''' In a typical reactor, cells at the surface absorb more than enough light to saturate their | ||
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photosynthetic apparatus, transmitting less energy to deeper layers. Cells operating past the saturation point | photosynthetic apparatus, transmitting less energy to deeper layers. Cells operating past the saturation point | ||
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waste incident photons by non-photochemical quenching and possibly undergo photodamage. For wild type cells, the | waste incident photons by non-photochemical quenching and possibly undergo photodamage. For wild type cells, the | ||
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“saturation curve” is assumed to be approximately the same for all cells in all layers, regardless of their | “saturation curve” is assumed to be approximately the same for all cells in all layers, regardless of their | ||
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incident light intensity. This means that layers of cells on the exterior of the reactor nearest a light source | incident light intensity. This means that layers of cells on the exterior of the reactor nearest a light source | ||
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receive an overabundance of photons and in turn block the interior layers from receiving enough light. In an | receive an overabundance of photons and in turn block the interior layers from receiving enough light. In an | ||
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optimal reactor, all layers would operate near their respective saturation points to maximize the photosynthetic | optimal reactor, all layers would operate near their respective saturation points to maximize the photosynthetic | ||
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channeling of incident light energy. | channeling of incident light energy. | ||
The total saturation curve for wild type R. Sphaeroides was fit with a nonlinear least squares regression of the | The total saturation curve for wild type R. Sphaeroides was fit with a nonlinear least squares regression of the | ||
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form in Equation A. Data points were generated from calculating absorbance as the negative logarithm of the ratio | form in Equation A. Data points were generated from calculating absorbance as the negative logarithm of the ratio | ||
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of the absolute irradiance detected on the next layer to incident absolute irradiance on a layer of cells. A | of the absolute irradiance detected on the next layer to incident absolute irradiance on a layer of cells. A | ||
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logistic form was chosen to account for the diminishing returns to absorption of further photons past a threshold | logistic form was chosen to account for the diminishing returns to absorption of further photons past a threshold | ||
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operating capacity of the photosynthetic apparatus. (1) | operating capacity of the photosynthetic apparatus. (1) | ||
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'''Simulating our Mutant's advantage in a Bioreactor''' | '''Simulating our Mutant's advantage in a Bioreactor''' | ||
For our mutant cells, the LH2 saturation curve for each layer scales as a function of light intensity. This | For our mutant cells, the LH2 saturation curve for each layer scales as a function of light intensity. This | ||
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predicted behavior in the mutant is due to negative regulation of LH2 complex production as incident light | predicted behavior in the mutant is due to negative regulation of LH2 complex production as incident light | ||
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intensity increases. The scalar of the magnitude of the saturation curve was altered according to a predicted | intensity increases. The scalar of the magnitude of the saturation curve was altered according to a predicted | ||
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exponential curve of LH2 production in repsone to changes in incident light. It was assumed that the system could | exponential curve of LH2 production in repsone to changes in incident light. It was assumed that the system could | ||
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vary expression levels such that at high light intensities, the saturation curve is scaled to 25% of that of the | vary expression levels such that at high light intensities, the saturation curve is scaled to 25% of that of the | ||
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wild type. At low light intensities, LH2 production was assumed to have the potential to be up-regulated to 150% | wild type. At low light intensities, LH2 production was assumed to have the potential to be up-regulated to 150% | ||
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of wild type expression levels. | of wild type expression levels. | ||
The advantage this mutant would confer stems from the adaptive nature of the saturation curve heights. Cells | The advantage this mutant would confer stems from the adaptive nature of the saturation curve heights. Cells | ||
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receiving the most light on the outside of the bioreactor saturate at low absorption levels. This allows more | receiving the most light on the outside of the bioreactor saturate at low absorption levels. This allows more | ||
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light to transmit to further layers, which have elevated saturation curves due to lower incident light. | light to transmit to further layers, which have elevated saturation curves due to lower incident light. | ||
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- Light intensity at next layer is given by transmittance from previous layer (assumes no backscattering).<br> | - Light intensity at next layer is given by transmittance from previous layer (assumes no backscattering).<br> | ||
- Total energy funneled to photosynthetic pathways is estimated as the sum of light absorbed by each layer. This | - Total energy funneled to photosynthetic pathways is estimated as the sum of light absorbed by each layer. This | ||
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generalizes to the optical density measurement of cell culture density. | generalizes to the optical density measurement of cell culture density. | ||
- The constant wild type saturation curve inherently includes both LH2 and LH1 contributions to absorbance. The | - The constant wild type saturation curve inherently includes both LH2 and LH1 contributions to absorbance. The | ||
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mutant's variable saturation curve only accounts for LH2 absorbance, since this is the only complex under the | mutant's variable saturation curve only accounts for LH2 absorbance, since this is the only complex under the | ||
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light-sensing regulation. To account for the component of absorbance provided by LH1, the proportion of total | light-sensing regulation. To account for the component of absorbance provided by LH1, the proportion of total | ||
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optical density due strictly to LH1 was investigated by comparing the growth of wild type and LH2-knockout | optical density due strictly to LH1 was investigated by comparing the growth of wild type and LH2-knockout | ||
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(dbcOmega) cultures. It is evident that by day three the proportion of Optical Density accounted for by LH1 | (dbcOmega) cultures. It is evident that by day three the proportion of Optical Density accounted for by LH1 | ||
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absorption converges to a value near 0.2. In other words, at the phase the layers of cells have grown in the model, | absorption converges to a value near 0.2. In other words, at the phase the layers of cells have grown in the model, | ||
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20% of total optical density can be attributed to LH1. To account for this, the absorption in the mutant was | 20% of total optical density can be attributed to LH1. To account for this, the absorption in the mutant was | ||
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divided by the factor (1-0.2) = 0.8. Then, the total optical density of the mutant cultures reflects total | divided by the factor (1-0.2) = 0.8. Then, the total optical density of the mutant cultures reflects total | ||
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absorption by both LH1 and LH2. | absorption by both LH1 and LH2. | ||
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'''Model Assumptions''' <br> | '''Model Assumptions''' <br> | ||
- Light intensity at next layer is given by transmittance from previous layer (assumes no backscattering).<br> | - Light intensity at next layer is given by transmittance from previous layer (assumes no backscattering).<br> | ||
- Total energy funneled to photosynthetic pathways is estimated as the sum of light absorbed by each layer. This | - Total energy funneled to photosynthetic pathways is estimated as the sum of light absorbed by each layer. This | ||
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generalizes to the optical density measurement of cell culture density. <br> | generalizes to the optical density measurement of cell culture density. <br> | ||
- The constant wild type saturation curve inherently includes both LH2 and LH1 contributions to absorbance. The | - The constant wild type saturation curve inherently includes both LH2 and LH1 contributions to absorbance. The | ||
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mutant's variable saturation curve only accounts for LH2 absorbance, since this is the only complex under the | mutant's variable saturation curve only accounts for LH2 absorbance, since this is the only complex under the | ||
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light-sensing regulation. To account for the component of absorbance provided by LH1, the proportion of total | light-sensing regulation. To account for the component of absorbance provided by LH1, the proportion of total | ||
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optical density due strictly to LH1 was investigated by comparing the growth of wild type and LH2-knockout | optical density due strictly to LH1 was investigated by comparing the growth of wild type and LH2-knockout | ||
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(dbcOmega) cultures. It is evident that by day three the proportion of Optical Density accounted for by LH1 | (dbcOmega) cultures. It is evident that by day three the proportion of Optical Density accounted for by LH1 | ||
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absorption converges to a value near 0.2. In other words, at the phase the layers of cells have grown in the model, | absorption converges to a value near 0.2. In other words, at the phase the layers of cells have grown in the model, | ||
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20% of total optical density can be attributed to LH1. To account for this, the absorption in the mutant was | 20% of total optical density can be attributed to LH1. To account for this, the absorption in the mutant was | ||
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divided by the factor (1-0.2) = 0.8. Then, the total optical density of the mutant cultures reflects total | divided by the factor (1-0.2) = 0.8. Then, the total optical density of the mutant cultures reflects total | ||
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absorption by both LH1 and LH2. <br> | absorption by both LH1 and LH2. <br> | ||
- The model was revised upon gathering optical density data from the five layers of the bioreactor setup. (See | - The model was revised upon gathering optical density data from the five layers of the bioreactor setup. (See | ||
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Results Figure 2a.) In the wild type, the optical density of the first flask of cells was much lower than | Results Figure 2a.) In the wild type, the optical density of the first flask of cells was much lower than | ||
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predicted, a phenomenon that was attributed to photodamage of the cells due to exposure to a large quantity of | predicted, a phenomenon that was attributed to photodamage of the cells due to exposure to a large quantity of | ||
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light past the saturation point of the LH2 complexes. In the optical density data for the flasks, both the | light past the saturation point of the LH2 complexes. In the optical density data for the flasks, both the | ||
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dbcOmega knockout and the wild type logistically grew to an absorbance value of 1. This gave reason to put a hard | dbcOmega knockout and the wild type logistically grew to an absorbance value of 1. This gave reason to put a hard | ||
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limit of 1 on the first flask's potential optical density. Any light left over from this cutoff was transmitted to | limit of 1 on the first flask's potential optical density. Any light left over from this cutoff was transmitted to | ||
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the next layer, as evidenced by in the increased growth of the second wild type flask in the Optical Density data. | the next layer, as evidenced by in the increased growth of the second wild type flask in the Optical Density data. | ||
- The response curve for the coefficient of saturation for the mutant due to changes in light intensity was modeled | - The response curve for the coefficient of saturation for the mutant due to changes in light intensity was modeled | ||
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as an inverse exponential form. In other words, the system reacts to increasing light intensity by exponentially | as an inverse exponential form. In other words, the system reacts to increasing light intensity by exponentially | ||
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tapering the coefficient of saturation. | tapering the coefficient of saturation. | ||
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Revision as of 03:16, 22 October 2009