Team:Aachen/Project/2D Biosensor
From 2014.igem.org
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We are able to detect IPTG, 3-oxo-C{{sub|12}} HSL and ''Pseudomonas aeruginosa''. To prove that the sensor constructs produce the flourescence signal and not the medium or ''E. coli'' in its own we have [http://parts.igem.org/Part:BBa_B0015 B0015] in NEB as a negativ control for IPTG, HSL and ''Pseudomonas aeruginosa'' induction. | We are able to detect IPTG, 3-oxo-C{{sub|12}} HSL and ''Pseudomonas aeruginosa''. To prove that the sensor constructs produce the flourescence signal and not the medium or ''E. coli'' in its own we have [http://parts.igem.org/Part:BBa_B0015 B0015] in NEB as a negativ control for IPTG, HSL and ''Pseudomonas aeruginosa'' induction. | ||
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{{Team:Aachen/Figure|Aachen_B0015_IPTG_HSL_Pseudomonas.png|title=Negativ control |subtitle=B0015 in NEB as negativ control induced with A) 0.2 µl of 100 mM IPTG, image taken after 2.5 h; B) 0.2 µl of 500 µg/ml HSL (3-oxo-C12), image after 2.5 h; C) with 5 spots of ''Pseudomonas aeruginosa'' on the left and one big spot on the right, image taken after 2 h|width=900px}} | {{Team:Aachen/Figure|Aachen_B0015_IPTG_HSL_Pseudomonas.png|title=Negativ control |subtitle=B0015 in NEB as negativ control induced with A) 0.2 µl of 100 mM IPTG, image taken after 2.5 h; B) 0.2 µl of 500 µg/ml HSL (3-oxo-C12), image after 2.5 h; C) with 5 spots of ''Pseudomonas aeruginosa'' on the left and one big spot on the right, image taken after 2 h|width=900px}} | ||
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The measured fluorescence again showed a distinct signal on the induced chip (bottom) compared to the uninduced chip (top). The fluorescence clearly starts in the middle of the chip (point of induction) and then extends outwards, still showing an ever increasing signal of fluorescence. The base level of fluorescence is attributed to leakiness of the promoter and general background fluorescence of growing ''E. coli'' cells. In the induced chip (bottom), the background fluorescence is a lot lower than in the uninduced chip (top) because the signal masks the noise. The difference between the induced and uninduced chips indicates a clear response to the HSL and a proof for the ability of our sensor chip design to detect the HSL produced by ''Pseudomonas aeruginosa''. | The measured fluorescence again showed a distinct signal on the induced chip (bottom) compared to the uninduced chip (top). The fluorescence clearly starts in the middle of the chip (point of induction) and then extends outwards, still showing an ever increasing signal of fluorescence. The base level of fluorescence is attributed to leakiness of the promoter and general background fluorescence of growing ''E. coli'' cells. In the induced chip (bottom), the background fluorescence is a lot lower than in the uninduced chip (top) because the signal masks the noise. The difference between the induced and uninduced chips indicates a clear response to the HSL and a proof for the ability of our sensor chip design to detect the HSL produced by ''Pseudomonas aeruginosa''. | ||
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=== Detecting IPTG with Sensor Chips === | === Detecting IPTG with Sensor Chips === | ||
{{Team:Aachen/FigureFloat|Aachen_I746909_slower_reduced.gif|title=IPTG inducible superfolder GFP (I746909) in sensor chips|subtitle=IPTG inducible superfolder GFP (I746909) is induced with IPTG (2 µl, 100mM) on the right chip with a non induced chip on the left|width=480px}} | {{Team:Aachen/FigureFloat|Aachen_I746909_slower_reduced.gif|title=IPTG inducible superfolder GFP (I746909) in sensor chips|subtitle=IPTG inducible superfolder GFP (I746909) is induced with IPTG (2 µl, 100mM) on the right chip with a non induced chip on the left|width=480px}} | ||
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This video shows the construct [http://parts.igem.org/Part:BBa_I746909 I746909] from the 2007 iGEM Team Cambridge. This BioBrick is a producer of superfolder GFP under the control of a T7 promoter. It was introduced into BL21(DE3) cells making the expression IPTG inducible through the T7 RNA Polymerase encoded in the genome of BL21(DE3) under the control of a lacI promoter. | This video shows the construct [http://parts.igem.org/Part:BBa_I746909 I746909] from the 2007 iGEM Team Cambridge. This BioBrick is a producer of superfolder GFP under the control of a T7 promoter. It was introduced into BL21(DE3) cells making the expression IPTG inducible through the T7 RNA Polymerase encoded in the genome of BL21(DE3) under the control of a lacI promoter. | ||
The left chip does not show visible fluorescence and the right chip exhibits a strong fluorescence signal showing clearly the ability of the sensor chip technology to detect IPTG. On top of that, the fluorescence response is strong enough to be detected and analyzed by the measurement device WatsOn. | The left chip does not show visible fluorescence and the right chip exhibits a strong fluorescence signal showing clearly the ability of the sensor chip technology to detect IPTG. On top of that, the fluorescence response is strong enough to be detected and analyzed by the measurement device WatsOn. | ||
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===Detecting the 3-oxo-C{{sub|12}} HSL with K131026 in our Sensor Chips with WatsOn=== | ===Detecting the 3-oxo-C{{sub|12}} HSL with K131026 in our Sensor Chips with WatsOn=== | ||
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The results clearly demonstrate our ability to detect ''Pseudomonas aeruginosa'' with our measurement device WatsOn. On the induced chip a definite fluorescence response is visible in response to ''Pseudomonas aeruginosa''. The fluorescence eminates outward from the induction point and shows a significant difference to the non induced chip. Therefore detection of ''Pseudomonas aeruginosa'' is possible with our sensor chip technology in our measurement device ''WatsOn''! | The results clearly demonstrate our ability to detect ''Pseudomonas aeruginosa'' with our measurement device WatsOn. On the induced chip a definite fluorescence response is visible in response to ''Pseudomonas aeruginosa''. The fluorescence eminates outward from the induction point and shows a significant difference to the non induced chip. Therefore detection of ''Pseudomonas aeruginosa'' is possible with our sensor chip technology in our measurement device ''WatsOn''! | ||
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=== Comparing the REACh Construct with K731520 and I746909 === | === Comparing the REACh Construct with K731520 and I746909 === | ||
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More information about the kinetic differences between these construct in our sensor chips, look under | More information about the kinetic differences between these construct in our sensor chips, look under | ||
[https://2014.igem.org/Team:Aachen/Project/FRET_Reporter The REACh Construct] Achievements. | [https://2014.igem.org/Team:Aachen/Project/FRET_Reporter The REACh Construct] Achievements. | ||
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{{Team:Aachen/BlockSeparator}} | {{Team:Aachen/BlockSeparator}} |
Revision as of 15:26, 17 October 2014
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