Difference between revisions of "Part:BBa K861169:Experience"
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| + | <p align="justify">In a cell, the total amount of ATP, ADP and AMP molecules remains constant. Low glucose concentration results in high activity of adenylate cyclase converting ATP into cAMP, who binds and converts cAMP receptor protein (abbreviated as CRP) to DNA-binding configuration. Conversely, when glucose concentration gets high, more ATP and less cAMP will be produced, resulting in low DNA-binding activity of CRP.</p> | ||
| + | <p align="justify">We embed gene cI of lambda phage(BBa_P0451) downstream promoter PcstA (BBa_K118011) activated by the binding of CRP, and genes of red fluorescence protein(RFP, BBa_I13507) respectively downstream the promoter BBa_R0051 repressed by protein cI. In this way we construct an indirect regulation pathway with sensus glucose, transcription activator CRP and transcription repressor cI. If the device works as design, output of RFP will be increased following the elevation of glucose concentration, and vice versa.</p> | ||
| + | <p align="center" <img src="https://static.igem.org/mediawiki/2012/3/38/Cellulose_1.png" width="500" | ||
| + | height="421" hspace="2" vspace="1" align="middle" /></p> | ||
| + | <p align="justify"><strong>Method</strong></p> | ||
| + | <p align="justify"><strong>Construction of plasmid for indirect regulation pathway</strong></p> | ||
| + | <p align="justify">In this experiment, RFP reported the function of the indirect regulation pathway.</p> | ||
| + | <p align="justify">K861173: BBa_I13507, an mRFP generator with RBS and terminator, was embedded after CRP activated promoter K118011.</p> | ||
| + | <p align="justify">K861172: BBa_P0451, a cI generator with RBS and terminator, was embedded after promoter BBa_K118011 activated by CRP.</p> | ||
| + | <p align="justify">K861169:? K861172 and I763007, a cI repressed RFP generator, were assembled .</p> | ||
| + | <p align="justify">K861174: BBa_K137115, constitutively expressing cI generator with promoter, RBS and terminator, was assembled to I763007.</p> | ||
| + | <p align="justify">All new composite parts mentioned above were transformed to competent cells of Escherichia coli str. DH5a. All positive clones are validated using PCR, restriction enzyme digestion and DNA sequencing.</p> | ||
| + | <p align="justify"><strong>Cell culture fluorescence measurement</strong></p> | ||
| + | <p align="justify">Minimal medium with different concentration of glucose(1mM, 4mM, 10 mM , 20 mM , 50 mM ,100 mM) were transferred into a 96-well plate, 200 μL for each well. Then each well was inoculated with 2 μL of seed liquor which was activated overnight in M9 minimal medium with 50mM glucose at 37℃. The wells without inoculation were regarded as blank controls to revise the results. Under each condition, three parallel samples were setted. The plate was incubated at 37℃, 150rpm. Cell culture fluorescence was recorded on a SpectraMax M2 plate reader (Molecular Devices). Excitation at 584 nm and emission at 607 nm were used. All fluorescence was normalized with cell density by measuring the absorbance at 600 nm.</p> | ||
| + | <p align="justify"><strong>Capturing fluorescent image </strong></p> | ||
| + | <p align="justify">Cell morphology was observed through fluorescence microscope, and the image of bacteria with of each glucose concentration were captured. To know more about these images, please click on Here.</p> | ||
| + | <p align="justify"><strong>Fluorescent analysis of cyto-imaging</strong></p> | ||
| + | <p align="justify">A program named FANCY was ?designed to recognize single cell and calculate the fluorescence strength according to the images. For more information, please click Here.</p> | ||
| + | <p align="justify"><strong>Results</strong></p> | ||
| + | <p align="justify">Purified plasmids constructed before were digested with XbaI and PstI for confirmation. The agarose gel electrophoresis showed that the lengths were correct. At last, the plasmids were sent for sequencing. Results showed no mutation.</p> | ||
| + | <p align="justify">In the cell culture fluorescence measurement experiment, fluorescence of BBa_K861173 decreased coordinating with glucose concentration, while BBa_K861169 was reverse.The fluorescence of BBa_K861174 was too low to record, so we do not show it here. All of the results coincided with expected results indicating that we have successfully constructed the promoter which was activated by high concentration of glucose.</p> | ||
| + | <p align="justify">Fluorescent images indicated that all cells were growing normally, because the size and morphology were both the same as cells in LB medium. The fluorescence of the cells in the images show the same discipline with results from the fluorescence measurement experiments. <br> | ||
| + | The results of FANCY are showed as bellow, which conforms well the results that showed above.</p> | ||
| + | <p align="justify"><img name="" src="https://static.igem.org/mediawiki/igem.org/9/9c/Indirect_Fig_1.png" width="506" height="409" alt=""></p> | ||
| + | <p align="justify"><img name="" src="https://static.igem.org/mediawiki/igem.org/d/dc/Indirect_Fig_2.png" width="500" height="220" alt=""></p> | ||
| + | <p align="justify"><img name="" src="https://static.igem.org/mediawiki/igem.org/2/21/Indirect_Fig_3.png" width="506" height="189" alt=""></p> | ||
| + | <p align="justify"><img name="" src="https://static.igem.org/mediawiki/igem.org/c/c3/Indirect_Fig_4.png" width="500" height="226" alt=""></p> | ||
| + | <p align="justify"> </p> | ||
| + | <p align="justify"><strong>Discussion </strong><br> | ||
| + | </html) | ||
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Revision as of 00:53, 27 September 2012
This experience page is provided so that any user may enter their experience using this part.
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how you used this part and how it worked out.
Applications of BBa_K861169
In this design, we used promoter PcstA and an intermediate regulator cI, as PcstA is activated by CRP in low glucose concentration, whereas we need a device boosted by high concentration of glucose. As you can see, in this pathway, protein cI will be expressed when CRP is activated under low glucose concentration.The large amount of cI repressed the promoter heavily. On the contrary, when glucose concentration is high, the promoter is derepressed. We made RFP as reporter and certify that the design works well via two different methods.
Fluorescence measurement In the first method, Fluorescence intensity of cell culture was directly read from a plate reader. And all data is normalized by OD600. In this figure, the fluorescence of RFP generator promoted by PcstA decreased when concentration of glucose rose, meanwhile, the fluorescence of the indirect regulatory device increased with the glucose concentration, indicating that the device works as expected.
Image https://static.igem.org/mediawiki/2012/3/38/Cellulose_1.png
The second method is practiced for single cells. The fluorescence pictures of different glucose concentration were captured by fluorescence microscope. Also, a program named FANCY was designed to recognize single cell and calculate the fluorescence strength in the images. As the result shows, it conformed well to that of the first method.
Image https://static.igem.org/mediawiki/2012/3/38/Cellulose_1.png
All these results indicated that the device we designed can meet the demand, promoters activated by glucose.
User Reviews
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