Difference between revisions of "Part:BBa M36900:Experience"
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| − | In experimentation of this part's performance, our goals were to establish whether or not the device at hand would perform as a functional salt sensor in eukaryotic HeLa cells, by quantifying fluorescence level outputs actuated by a coupled actuator for various levels of salt stress input, both in terms of stress duration and NaCl concentration in the solution. At all experimental conditions (40,80,120 micromolar NaCl solution x 35, 70 min exposure time) our transformed HeLa cells exhibited significantly larger levels of fluorescence after salt stress exposure, as compared to the mock-transfected cells exposed to identical environment. However, | + | ---- |
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| + | In experimentation of this part's performance, our goals were to establish whether or not the device at hand would perform as a functional salt sensor in eukaryotic HeLa cells, by quantifying fluorescence level outputs actuated by a coupled actuator for various levels of salt stress input, both in terms of stress duration and NaCl concentration in the solution. At all experimental conditions (40,80,120 micromolar NaCl solution x 35, 70 min exposure time) our transformed HeLa cells exhibited significantly larger levels of fluorescence after salt stress exposure, as compared to the mock-transfected cells exposed to identical environment. However, in terms of the direction of this trend, an unexpected behavior is present: Output fluorescence levels appear to decline as, opposed to rise, with increased salt stress input, according to an image-by-image Image-J fluorescence analysis. Interestingly however, there is a significant main effect of stress exposure time on fluorescence levels at the 70-min level, with the transfected 70-minute conditions showing significantly more Comet expression relative to their respective mock condition. The significance was only trend-level at the 35-minute level. | ||
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| + | Future analysis of fluorescence with higher statistical power under a cell-by-cell basis with the same software is highly recommended to confirm the trend with the fluorescence output. Furthermore, a real-time PCR would be a beneficial next step in order to quantify the DREB1A mRNA as opposed to merely test for its existence; potential scenarios may include the existence of a 'saturation point' for the DREB1A gene with respect to the NaCl binding substrate. Alternatively, DREB1A mRNA may not have failed to be translated into the protein form to subsequently attach to the rd29A promoter. In order to test for the latter possibility, a product assay for the DREB1A protein would be a valid future step. A more dynamic range of salt stress conditions may also prove beneficial in detecting the difference between auto-fluorescing dead cells (excessive salt stress) and lives cells actually expressing the Comet protein in response to moderate salt stress. Ultimately, an appropriate actuator responsible for inducing salt tolerance will be installed downstream of this sensor in order for the device to be applied against problems such as hypertension. Prior to that however, additional steps are required in order to characterize and adjust the DREB1A sensor for application inside the human body. | ||
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Please see the attached .pdf for details on device performance as a dose response curve over two ranges of mercury ion concentration. In the graphs, red bars represent growth of cells with the BBa_M36567 construct and blue bars represent growth of normal E.coli K12 cells with no mercury reduction plasmid | Please see the attached .pdf for details on device performance as a dose response curve over two ranges of mercury ion concentration. In the graphs, red bars represent growth of cells with the BBa_M36567 construct and blue bars represent growth of normal E.coli K12 cells with no mercury reduction plasmid | ||
Revision as of 03:32, 11 December 2014
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.
Barcode # (glycerol stock): 133018572
Plasmid Name: NeginandAbbey
Antibiotic Resistance: Ampicillin
DNA 2.0 Gene #: 193570
Organism Expressed in HeLa
Sensor
In experimentation of this part's performance, our goals were to establish whether or not the device at hand would perform as a functional salt sensor in eukaryotic HeLa cells, by quantifying fluorescence level outputs actuated by a coupled actuator for various levels of salt stress input, both in terms of stress duration and NaCl concentration in the solution. At all experimental conditions (40,80,120 micromolar NaCl solution x 35, 70 min exposure time) our transformed HeLa cells exhibited significantly larger levels of fluorescence after salt stress exposure, as compared to the mock-transfected cells exposed to identical environment. However, in terms of the direction of this trend, an unexpected behavior is present: Output fluorescence levels appear to decline as, opposed to rise, with increased salt stress input, according to an image-by-image Image-J fluorescence analysis. Interestingly however, there is a significant main effect of stress exposure time on fluorescence levels at the 70-min level, with the transfected 70-minute conditions showing significantly more Comet expression relative to their respective mock condition. The significance was only trend-level at the 35-minute level.
Future analysis of fluorescence with higher statistical power under a cell-by-cell basis with the same software is highly recommended to confirm the trend with the fluorescence output. Furthermore, a real-time PCR would be a beneficial next step in order to quantify the DREB1A mRNA as opposed to merely test for its existence; potential scenarios may include the existence of a 'saturation point' for the DREB1A gene with respect to the NaCl binding substrate. Alternatively, DREB1A mRNA may not have failed to be translated into the protein form to subsequently attach to the rd29A promoter. In order to test for the latter possibility, a product assay for the DREB1A protein would be a valid future step. A more dynamic range of salt stress conditions may also prove beneficial in detecting the difference between auto-fluorescing dead cells (excessive salt stress) and lives cells actually expressing the Comet protein in response to moderate salt stress. Ultimately, an appropriate actuator responsible for inducing salt tolerance will be installed downstream of this sensor in order for the device to be applied against problems such as hypertension. Prior to that however, additional steps are required in order to characterize and adjust the DREB1A sensor for application inside the human body.
Please see the attached .pdf for details on device performance as a dose response curve over two ranges of mercury ion concentration. In the graphs, red bars represent growth of cells with the BBa_M36567 construct and blue bars represent growth of normal E.coli K12 cells with no mercury reduction plasmid
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