Team:Aachen/Project/2D Biosensor
From 2014.igem.org
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<br>On the molecular side, we use the '''[https://2014.igem.org/Team:Aachen/Project/FRET_Reporter REACh construct]''' to transform regular ''E. coli'' cells into ''Cellocks''. | <br>On the molecular side, we use the '''[https://2014.igem.org/Team:Aachen/Project/FRET_Reporter REACh construct]''' to transform regular ''E. coli'' cells into ''Cellocks''. | ||
- | {{Team:Aachen/Figure|Aachen 17-10-14 The basics of quorum sensing ipo.png||title=The principle of quorum sensing|subtitle=Microorganisms can sense the presence of their own kind based on quorum sensing which is a form of chemical communication. Depending on their cell density, quorum sensing allows these cells to activate or deactivate certain gene expression cascades for a specific function. (Waters and Bassler, 2005).|width= | + | {{Team:Aachen/Figure|Aachen 17-10-14 The basics of quorum sensing ipo.png||title=The principle of quorum sensing|subtitle=Microorganisms can sense the presence of their own kind based on quorum sensing which is a form of chemical communication. Depending on their cell density, quorum sensing allows these cells to activate or deactivate certain gene expression cascades for a specific function. (Waters and Bassler, 2005).|width=900px}} |
Our '''sensor cells, ''Cellocks'', are immobilized in agar chips'''. To make the chips, we mix the ''Cellocks'' with liquid LB agar. | Our '''sensor cells, ''Cellocks'', are immobilized in agar chips'''. To make the chips, we mix the ''Cellocks'' with liquid LB agar. | ||
In the course of our project, we designed a casting mold specifically for the production of our agar chips. When the agar has cooled down, the chips are cut out of the mold and are ready to use. Storage of the readily usable sensor chips is possible for up to 2 days at 4 °C when using LB medium or up to 5 days if TB medium is used. A detailed description of the sensor chip manufacturing can be found in our [https://2014.igem.org/Team:Aachen/Notebook/Protocols/detection Protocols] section. | In the course of our project, we designed a casting mold specifically for the production of our agar chips. When the agar has cooled down, the chips are cut out of the mold and are ready to use. Storage of the readily usable sensor chips is possible for up to 2 days at 4 °C when using LB medium or up to 5 days if TB medium is used. A detailed description of the sensor chip manufacturing can be found in our [https://2014.igem.org/Team:Aachen/Notebook/Protocols/detection Protocols] section. | ||
- | {{Team:Aachen/Figure|Aachen 14-10-14 Flowsheet OD-device part1 ipo.png|title=Assay to detect ''P. aeruginosa'' using ''Cellock Holmes''|subtitle=This flow sheet shows the procedure to sample and detect ''P. aeruginosa'': A sampling chip is briefly put onto the potentially contaminated surface, added onto one of our sensor chips and inserted into ''WatsOn''.|width= | + | {{Team:Aachen/Figure|Aachen 14-10-14 Flowsheet OD-device part1 ipo.png|title=Assay to detect ''P. aeruginosa'' using ''Cellock Holmes''|subtitle=This flow sheet shows the procedure to sample and detect ''P. aeruginosa'': A sampling chip is briefly put onto the potentially contaminated surface, added onto one of our sensor chips and inserted into ''WatsOn''.|width=900px}} |
Using ''Cellock Holmes'', we developed a simple assay to detect ''P. aeruginosa''. | Using ''Cellock Holmes'', we developed a simple assay to detect ''P. aeruginosa''. | ||
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* Third, the two layered chip-stack is put into a petri dish which is inserted into our measurement device [https://2014.igem.org/Team:Aachen/Project/Measurement_Device ''WatsOn''] for evalutation. | * Third, the two layered chip-stack is put into a petri dish which is inserted into our measurement device [https://2014.igem.org/Team:Aachen/Project/Measurement_Device ''WatsOn''] for evalutation. | ||
- | {{Team:Aachen/Figure|Aachen 14-10-14 Flowsheet OD-device part2 ipo.png|title=Mode of action inside ''WatsOn''.|subtitle=Chips are incubated at 37 °C to stimulate cell growth and then illuminated with blue light to excite fluorescence. A picture is taken and analyzed for fluorescence signals using the software ''Measurarty''.|width= | + | {{Team:Aachen/Figure|Aachen 14-10-14 Flowsheet OD-device part2 ipo.png|title=Mode of action inside ''WatsOn''.|subtitle=Chips are incubated at 37 °C to stimulate cell growth and then illuminated with blue light to excite fluorescence. A picture is taken and analyzed for fluorescence signals using the software ''Measurarty''.|width=900px}} |
Inside ''WatsOn'', the chips are incubated at 37 °C and the sampled populations of microorganisms attached on the sampling chip start to grow and multiply. During incubation the chips can be '''illuminated with blue light''' at any time, and a '''photo of the chips''' is taken. The '''software ''Measurarty''''' then analyzes any fluorescent signal. ''P. aeruginosa'' secrets an increasing number of quorum sensing molecules that are recognized by ''Cellocks'', thereby producing a fluorescence signal. For detection of ''P. aeruginosa'', we focused on a quorum sensing molecule called N-3-oxo-dodecanoyl-L-homoserine lactone (for short: 3-oxo-C<sub>12</sub>-HSL), which is involved in virulence regulation of ''P. aeruginosa'' (Jimenez, Koch, Thompson et al., 2012). The incorporation of the 3-oxo-C<sub>12</sub>-HSL detection system into the ''Cellocks'' is explained in detail in the [https://2014.igem.org/Team:Aachen/Project/FRET_Reporter REACh Construct] section. | Inside ''WatsOn'', the chips are incubated at 37 °C and the sampled populations of microorganisms attached on the sampling chip start to grow and multiply. During incubation the chips can be '''illuminated with blue light''' at any time, and a '''photo of the chips''' is taken. The '''software ''Measurarty''''' then analyzes any fluorescent signal. ''P. aeruginosa'' secrets an increasing number of quorum sensing molecules that are recognized by ''Cellocks'', thereby producing a fluorescence signal. For detection of ''P. aeruginosa'', we focused on a quorum sensing molecule called N-3-oxo-dodecanoyl-L-homoserine lactone (for short: 3-oxo-C<sub>12</sub>-HSL), which is involved in virulence regulation of ''P. aeruginosa'' (Jimenez, Koch, Thompson et al., 2012). The incorporation of the 3-oxo-C<sub>12</sub>-HSL detection system into the ''Cellocks'' is explained in detail in the [https://2014.igem.org/Team:Aachen/Project/FRET_Reporter REACh Construct] section. |
Revision as of 15:21, 17 October 2014
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